Abeloff's clinical oncology [6 ed.] 9780323476744, 0323476740, 9780323568159, 0323568157

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Abeloff’s

CLINICAL ONCOLOGY

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Abeloff’s

CLINICAL ONCOLOGY SIXTH EDITION JOHN E. NIEDERHUBER, MD Executive Vice President, Inova Health System President and CEO, Genomics and Bioinformatics Research Institute Fairfax, Virginia; Professor, Department of Public Health Sciences Member, Center for Public Health Genomics University of Virginia School of Medicine Charlottesville, Virginia; Adjunct Professor, Oncology and Surgery The Johns Hopkins University School of Medicine Deputy Director Johns Hopkins Clinical Research Network Baltimore, Maryland

JAMES O. ARMITAGE, MD

JAMES H. DOROSHOW, MD

Joe Shapiro Professor of Medicine University of Nebraska Medical Center Omaha, Nebraska

Bethesda, Maryland

MICHAEL B. KASTAN, MD, PhD

JOEL E.TEPPER, MD

Executive Director, Duke Cancer Institute William and Jane Shingleton Professor, Pharmacology and Cancer Biology Professor of Pediatrics Duke University School of Medicine Durham, North Carolina

Hector MacLean Distinguished Professor of Cancer Research Department of Radiation Oncology UNC Lineberger Comprehensive Cancer Center University of North Carolina School of Medicine Chapel Hill, North Carolina

ABELOFF’S CLINICAL ONCOLOGY, SIXTH EDITION

ISBN: 978-0-323-47674-4

Copyright © 2020 by Elsevier, Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. With respect to any drug or pharmaceutical products identified, readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsibility of practitioners, relying on their own experience and knowledge of their patients, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Previous editions copyrighted 2014, 2008, 2004, 2000, and 1995. Library of Congress Control Number: 2018953655

Executive Content Strategist: Robin Carter Content Development Manager: Laura Schmidt Publishing Services Manager: Catherine Jackson Senior Project Manager: Amanda Mincher Design Direction: Bridget Hoette Printed in China Last digit is the print number: 9 8 7 6 5 4 3 2 1

1600 John F. Kennedy Blvd. Ste 1600 Philadelphia, PA 19103-2899

To my son, Matthew, and my wife, Kathy, who have and continue to make sacrifices so that I might pursue my passions in medicine and research. To my colleagues at the National Cancer Institute, University of Virginia, Johns Hopkins, and across the country, whose selfless dedication to patient care and cancer research is truly an inspiration to all. To the many students who have trained with me over the years, to my patients, and to my colleagues at the Inova Translational Medicine Institute, who have given me the opportunity to have this tremendously rewarding career. Lastly, to Tracey, and to Marty, who, in memory, inspire all who knew them to work a little harder each day toward the elimination of the pain and suffering from this disease. JOHN E. NIEDERHUBER, MD To my wife, Nancy, for her love and support over 49 ½ years. JAMES O. ARMITAGE, MD To my wife, Robin Winkler Doroshow, MD, my classmate and greatest supporter, for her love, dedication, and commitment and for the remarkable joy and caring she brings to her patients and to all around her. To my remarkable daughter, Deborah Doroshow, MD, PhD, who is completing her training for a career in academic oncology; my fondest hope is that you will enjoy sharing with and learning from those you help as much as I have. To my patients and colleagues at the City of Hope and the National Cancer Institute who have all contributed so much of themselves to my continuing education as a physician and investigator, please accept my appreciation and utmost gratitude. JAMES H. DOROSHOW, MD To my wife, Kathy, and my sons, Benjamin, Nathaniel, and Jonathan. You are the lights of my life. I also acknowledge all of my mentors, colleagues, and patients, who have taught me so much. A special note of gratitude goes to Marty Abeloff, a mentor and an inspiring role model for career and for life. MICHAEL B. KASTAN, MD, PhD To my wife, Laurie, who has been my soul mate for many years and has constantly reminded me of life’s priorities. To my family including my daughters, Miriam and Abigail, and my grandchildren, Zekariah, Zohar, Samuel, Marcelo, Jonah, and Aurelio. They have been an inspiration. To my many teachers through the years who have helped define and foster my professional career, but especially Herman Suit and Eli Glatstein. JOEL E. TEPPER, MD

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Memoriam

Martin D. Abeloff, MD (1942-2007)

Martin D. Abeloff, a founding editor of Clinical Oncology, dedicated his life to caring for patients with cancer and to teaching his art to fellows, residents, and students. He was a brilliant and caring clinician, an extremely effective leader, and a beloved mentor to many trainees and young faculty. Marty was born on April 4, 1942, in Shenandoah, Pennsylvania. He received his BA from The Johns Hopkins University in 1963 and his MD from The Johns Hopkins University School of Medicine in 1966. He spent the next year as an intern at the University of Chicago Hospitals and Clinics. His legacy in medicine was established on his return to Baltimore in 1971 as a fellow in clinical oncology. He would spend the rest of his career at The Johns Hopkins Hospital, achieving the rank of Professor of Medicine in 1990. At various times, he served as the fellowship training program director, chief of medical oncology, clinical director of the cancer center, oncologist in chief at The Johns Hopkins Hospital, and in 1992, was appointed the second director of The Johns Hopkins Oncology Center, later renamed, thanks to Marty’s efforts, the Sidney Kimmel Comprehensive Cancer Center. It was during his time as cancer center director that Marty brought to life the idea of a comprehensive, user-friendly textbook of oncology

that would be as valuable to the practicing oncologist as to the primary care physician and physicians-in-training. The first edition of Clinical Oncology was published in 1995 to a gratifying response. It is now established as a cornerstone reference for those caring for patients with cancer. In the sixth edition, we continue Marty’s vision for an ever better, unique, and accessible text so that future generations of oncologists will remember his inspiration and leadership. The editors again dedicate this text, which is already a recognized tangible aspect of his legacy in medicine, as a living memorial to him. Abeloff ’s Clinical Oncology will continue to serve as a reminder to all its users of this extraordinary person and exemplary physician who went before them. John E. Niederhuber, MD James O. Armitage, MD James H. Doroshow, MD Michael B. Kastan, MD, PhD Joel E. Tepper, MD

vii

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Contributors

James L. Abbruzzese, MD, FACP, FASCO, DSc (hon)

Dara L. Aisner, MD, PhD

Duke Cancer Institute Distinguished Professor of Medical Oncology Chief, Division of Medical Oncology, Department of Medicine Associate Director for Clinical Research, Duke Cancer Institute Duke University Medical Center Durham, North Carolina

Associate Professor of Pathology CU Anschutz Medical Campus University of Colorado Aurora, Colorado

Omar Abdel-Wahab, MD

Helene Blum Assistant Professor Department of Radiation Oncology University of Pennsylvania Philadelphia, Pennsylvania

Associate Attending Department of Medicine Leukemia Service Memorial Sloan Kettering Cancer Center New York, New York

Ghassan K. Abou-Alfa, MD Attending Physician Memorial Sloan Kettering Cancer Center Professor of Medicine Weill Cornell Medicine New York, New York

Janet L. Abrahm, MD Professor of Medicine Harvard Medical School Member, Division of Palliative Care Psychosocial Oncology and Palliative Care Dana-Farber Cancer Institute Boston, Massachusetts

Jeffrey S. Abrams, MD Associate Director, Cancer Therapy Evaluation Program Division of Cancer Treatment and Diagnosis National Cancer Institute Rockville, Maryland

Jeremy S. Abramson, MD, MMSc Director, Center for Lymphoma Hematology/Oncology Massachusetts General Hospital Assistant Professor Department of Medicine Harvard Medical School Boston, Massachusetts

Michelle Alonso-Basanta, MD, PhD

Jesus Anampa, MD, MS Assistant Professor Department of Oncology Montefiore Medical Center Albert Einstein College of Medicine Bronx, New York

Megan E. Anderson, MD Assistant Professor Department of Orthopaedic Surgery Harvard Medical School Attending Orthopedic Surgeon Department of Orthopedic Surgery Boston Children’s Hospital Attending Orthopedic Surgeon Department of Orthopedic Surgery Beth Israel Deaconess Medical Center Boston, Massachusetts

Emmanuel S. Antonarakis, MD Associate Professor of Oncology Sidney Kimmel Comprehensive Cancer Center Johns Hopkins University School of Medicine Baltimore, Maryland

Richard Aplenc, MD, PhD Department of Pediatrics Section Chief, Hematologic Malignancies Chief Clinical Research Officer Children’s Hospital of Philadelphia Philadelphia, Pennsylvania

ix

x Contributors

Frederick R. Appelbaum, MD

Karen Basen-Engquist, PhD, MPH

Executive Vice President and Deputy Director Fred Hutchinson Cancer Research Center Professor Division of Medical Oncology University of Washington Seattle, Washington

Professor of Behavioral Science University of Texas MD Anderson Cancer Center Houston, Texas

Luiz H. Araujo, MD, PhD Scientific Director COI Institute for Research and Education Brazilian National Cancer Institute Rio de Janeiro, Brazil

Ammar Asban, MD Surgical Resident Department of Surgery University of Alabama at Birmingham Birmingham, Alabama

Edward Ashwood, MD President and CEO ARUP Laboratories Professor of Pathology University of Utah Salt Lake City, Utah

Farrukh T. Awan, MD, MS Associate Professor of Medicine Hematology The Ohio State University Columbus, Ohio

Juliet L. Aylward, MD Associate Professor of Dermatology University of Wisconsin School of Medicine and Public Health Madison, Wisconsin

Arjun V. Balar, MD Associate Professor of Medicine Division of Hematology/Oncology Director, Genitourinary Cancers Program New York University Perlmutter Cancer Center New York University Langone Medical Center New York, New York

Courtney J. Balentine, MD Assistant Professor of Surgery Dallas VA Hospital University of Texas Southwestern Dallas, Texas

Stefan K. Barta, MD, MS, MRCP(UK) Associate Professor Hematology and Oncology Fox Chase Cancer Center Philadelphia, Pennsylvania

Nancy Bartlett, MD Professor of Medical Oncology Washington University School of Medicine St. Louis, Missouri

Lynda Kwon Beaupin, MD Director, Adolescent and Young Adult Program Roswell Park Cancer Institute Buffalo, New York

Ross S. Berkowitz, MD William H. Baker Professor of Gynecology Department of Obstetrics and Gynecology Harvard Medical School Director of Gynecologic Oncology Department of Obstetrics and Gynecology Brigham and Women’s Hospital Boston, Massachusetts

Donald A. Berry, PhD Professor of Biostatistics Department of Biostatistics The University of Texas MD Anderson Cancer Center Houston, Texas

Therese Bevers, MD Professor of Clinical Cancer Prevention Medical Director, Cancer Prevention Center The University of Texas MD Anderson Cancer Center Houston, Texas

John F. Boggess, MD Professor of Obstetrics and Gynecology University of North Carolina Chapel Hill, North Carolina

Julie R. Brahmer, MD, MSc Professor of Oncology Department of Oncology Johns Hopkins Kimmel Cancer Center Baltimore, Maryland

Janet Brown, MD, FRCP, MSc, MBBS, BSc Professor Academic Unit of Clinical Oncology, Oncology, and Metabolism Weston Park Hospital University of Sheffield Sheffield, United Kingdom

Karen Brown, MD Attending Physician Memorial Sloan Kettering Cancer Center Professor of Clinical Radiology Weill Medical College at Cornell University New York, New York

Powel Brown, MD, PhD Professor and Chairman Clinical Cancer Prevention The University of Texas MD Anderson Cancer Center Houston, Texas

Contributors xi

Ilene Browner, MD

Stephen J. Chanock, MD

Assistant Professor Department of Oncology and Division of Geriatric Medicine The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins and Johns Hopkins Bayview The Johns Hopkins University Baltimore, Maryland

Director Division of Cancer Epidemiology and Genetics National Cancer Institute Bethesda, Maryland

Paul A. Bunn, MD Distinguished Professor of Medical Oncology CU Anschutz Medical Campus University of Colorado Aurora, Colorado

William R. Burns, MD Assistant Professor of Surgery University of Michigan Health System Ann Arbor, Michigan

John C. Byrd, MD Professor of Internal Medicine–Hematology The Ohio State University Columbus, Ohio

Karen Cadoo, MD Attending Medical Oncologist Gynecologic Medical Oncology and Clinical Genetic Services Memorial Sloan Kettering Cancer Center Weill Cornell Medical College New York, New York

David P. Carbone, MD, PhD Professor of Medicine Director, James Thoracic Center James Cancer Center The Ohio State University Medical Center Columbus, Ohio

H. Ballentine Carter, MD Professor of Urology Johns Hopkins University School of Medicine Baltimore, Maryland

Jorge J. Castillo, MD Physician Hematologic Malignancies Dana-Farber Cancer Institute Assistant Professor Harvard Medical School Boston, Massachusetts

Claudia I. Chapuy, MD St. Elizabeth’s Medical Center Dana-Farber Cancer Institute Boston, Massachusetts

Vikash P. Chauhan, PhD Massachusetts Institute of Technology Boston, Massachusetts

Herbert Chen, MD, FACS Chairman and Fay Fletcher Kerner Endowed Chair Department of Surgery University of Alabama at Birmingham Surgeon-in-Chief University of Alabama at Birmingham Health System Birmingham, Alabama

Ronald C. Chen, MD, MPH Associate Professor Department of Radiation Oncology University of North Carolina at Chapel Hill Chapel Hill, North Carolina

Nai-Kong V. Cheung, MD, PhD Enid A. Haupt Endowed Chair in Pediatric Oncology Department of Pediatrics Memorial Sloan-Kettering Cancer Center New York, New York

Jennifer H. Choe, MD, PhD Medical Instructor Division of Medical Oncology Duke Cancer Institute Durham, North Carolina

Michaele C. Christian, MD Cancer Therapy Evaluation Program (Retired) National Cancer Institute Rockville, Maryland

Paul M. Cinciripini, PhD Professor and Chair of Behavioral Science The University of Texas MD Anderson Cancer Center Houston, Texas

Alfred E. Chang, MD

Michael F. Clarke, MD

Hugh Cabot Professor of Surgery University of Michigan Health System Ann Arbor, Michigan

Professor of Medicine Division of Oncology Stanford School of Medicine Palo Alto, California

Eric Chang, MD, FASTRO Professor and Chair of Radiation Oncology Keck School of Medicine of USC Los Angeles, California

Robert E. Coleman, MBBS, MD Academic Unit of Clinical Oncology Weston Park Hospital University of Sheffield Sheffield, United Kingdom

xii Contributors

Robert L. Coleman, MD

Jeffrey Crawford, MD

Professor and Executive Director, Cancer Network Research Department of Gynecologic Oncology and Reproductive Medicine The University of Texas MD Anderson Cancer Center Houston, Texas

Professor of Medicine Division of Medical Oncology Duke Cancer Institute Durham, North Carolina

Adriana M. Coletta, PhD, RD

Kristy Crooks, PhD

Department of Behavioral Science Center for Energy Balance in Cancer Prevention and Survivorship The University of Texas MD Anderson Cancer Center Houston, Texas

Assistant Professor of Pathology CU Anschutz Medical Campus University of Colorado Aurora, Colorado

Jerry M. Collins, PhD

Daniel J. Culkin, MD

Associate Director Division of Cancer Treatment and Diagnosis National Cancer Institute Bethesda, Maryland

Professor Department of Urology University of Oklahoma Health Sciences Center Oklahoma City, Oklahoma

Jean M. Connors, MD

Brian G. Czito, MD

Hematology Division Brigham and Women’s Hospital Dana-Farber Cancer Institute Harvard Medical School Boston, Massachusetts

Professor of Radiation Oncology Duke University Medical Center Durham, North Carolina

Piero Dalerba, MD

Department of Neurosurgery University of North Carolina Chapel Hill, North Carolina

Assistant Professor of Pathology and Cell Biology Assistant Professor of Medicine Division of Digestive and Liver Diseases Columbia University College of Physicians and Surgeons New York, New York

Kevin R. Coombes, PhD

Josep Dalmau, MD, PhD

Professor of Biomedical Informatics The Ohio State University Columbus, Ohio

The University of Texas MD Anderson Cancer Center Houston, Texas

ICREA Research Professor Hospital Clínic/Institut d’Investigació Biomèdica August Pi i Sunyer (IDIBAPS) Barcelona, Spain Adjunct Professor Neurology University of Pennsylvania Philadelphia, Pennsylvania

Mauro W. Costa, MSc, PhD

Mai Dang, MD, PhD

Research Scientist The Jackson Laboratory Bar Harbor, Maine

Instructor in Neurology Children’s Hospital of Philadelphia Philadelphia, Pennsylvania

Anne Covey, MD

Michael D’Angelica, MD

Attending Physician Memorial Sloan Kettering Cancer Center Professor of Radiology Weill Medical College at Cornell University New York, New York

Attending Physician Memorial Sloan Kettering Cancer Center Professor of Surgery Weill Medical College at Cornell University New York, New York

Kenneth H. Cowan, MD, PhD

Kurtis D. Davies, PhD

Director, Fred and Pamela Buffett Cancer Center University of Nebraska Medical Center Omaha, Nebraska

Assistant Professor of Pathology CU Anschutz Medical Campus University of Colorado Aurora, Colorado

Michael Cools, MD

Jorge Cortes, MD

Christopher H. Crane, MD Vice Chairman Attending Physician Memorial Sloan Kettering Cancer Center New York, New York

Contributors xiii

Myrtle Davis, DVM, PhD

James H. Doroshow, MD

Chief, Toxicology and Pharmacology Branch Division of Drug Treatment and Diagnosis National Cancer Institute National Institutes of Health Bethesda, Maryland

Bethesda, Maryland

Nicolas Dea, MD, MSc, FRCSC

Jay F. Dorsey, MD, PhD Associate Professor of Radiation Oncology University of Pennsylvania Philadelphia, Pennsylvania

Spinal Neurosurgeon Clinical Associate Professor Department of Surgery Vancouver General Hospital University of British Columbia Vancouver, British-Columbia, Canada

Marianne Dubard-Gault, MD, MS

Ana De Jesus-Acosta, MD

Associate Professor Department of Pediatrics Harvard Medical School Attending Physician Department of Pediatrics Boston Children’s Hospital Dana Farber Cancer Institute Boston, Massachusetts

Assistant Professor of Oncology Sidney Kimmel Comprehensive Cancer Center The Johns Hopkins University School of Medicine Baltimore, Maryland

Angelo M. DeMarzo, MD, PhD Professor of Pathology Johns Hopkins University School of Medicine Baltimore, Maryland

Theodore L. DeWeese, MD Professor and Director of Radiation Oncology and Molecular Radiation Sciences Johns Hopkins University School of Medicine Baltimore, Maryland

Maximilian Diehn, MD, PhD Associate Professor of Radiation Oncology Stanford University Palo Alto, California

Subba R. Digumarthy, MD Massachusetts General Hospital Boston, Massachusetts

Angela Dispenzieri, MD

Medical Genetics Fellow Department of Medicine Memorial Sloan Kettering Cancer Center New York, New York

Steven G. DuBois, MD, MS

Dan G. Duda, PhD, DMD Associate Professor Harvard Medical School Boston, Massachusetts

Malcolm Dunlop, MD MRC Institute of Genetics and Molecular Medicine The University of Edinburgh Western General Hospital Edinburgh, United Kingdom

Linda R. Duska, MD University of Virginia Health System Emily Couric Clinical Cancer Center Charlottesville, Virginia

Madeleine Duvic, MD

Professor of Medicine and Laboratory Medicine Mayo Clinic Rochester, Minnesota

Professor and Deputy Chairman Department of Dermatology The University of Texas MD Anderson Cancer Center Houston, Texas

Khanh T. Do, MD

Imane El Dika, MD

Assistant Professor of Medicine Harvard Medical School Medical Oncology Dana-Farber Cancer Institute Boston, Massachusetts

Assistant Attending Physician Memorial Sloan Kettering Cancer Center Instructor of Medicine Weill Medical College at Cornell University New York, New York

Konstantin Dobrenkov, MD

Hashem El-Serag, MD, MPH

Clinical Director, Oncology Merck & Company, Inc. Kenilworth, New Jersey

Margaret M. and Albert B. Alkek Chair of the Department of Medicine Professor of Gastroenterology and Hepatology Baylor College of Medicine Houston, Texas

Jeffrey S. Dome, MD, PhD Vice President, Center for Cancer and Blood Disorders Children’s National Medical Center Washington, D.C.

xiv Contributors

Jeffrey M. Engelmann, PhD

Debra L. Friedman, MD, MS

Assistant Professor of Psychiatry and Behavioral Medicine Medical College of Wisconsin Milwaukee, Wisconsin

Vanderbilt-Ingram Cancer Center Nashville, Tennessee

David S. Ettinger, MD, FACP, FCCP

Winchester Hospital North Reading Medical North Reading, Massachusetts

Alex Grass Professor of Oncology The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Hospital The Johns Hopkins University Baltimore, Maryland

Lola A. Fashoyin-Aje, MD, MPH Medical Officer Office of Hematology and Oncology Products Center for Drug Evaluation and Research U.S. Food and Drug Administration Silver Spring, Maryland

Arian F. Fuller, Jr., MD

Lorenzo Galluzzi, PhD Assistant Professor of Cell Biology in Radiation Oncology Weill Cornell Medical College New York, New York

Mark C. Gebhardt, MD

Maisel Professor of Oncology Professor of Internal Medicine University of Michigan Medical School Ann Arbor, Michigan

Frederick W. and Jane M. Ilfeld Professor of Orthopaedic Surgery Harvard Medical School Surgeon-in-Chief Department of Orthopedic Surgery Beth Israel Deaconess Medical Center Orthopedic Surgeon Department of Orthopedics Children’s Hospital Boston, Massachusetts

James M. Ford, MD

Daniel J. George, MD

Professor of Medicine, Pediatrics, and Genetics Division of Oncology and Medical Genetics Stanford University School of Medicine Stanford, California

Professor of Medicine Duke University Medical Center Durham, North Carolina

Wilbur A. Franklin, MD

Assistant Attending Department of Medicine Leukemia Service and Cellular Therapeutics Center Memorial Sloan Kettering Cancer Center Instructor in Medicine Joan and Sanford I. Weill Department of Medicine Weill Cornell Medical College New York, New York

Eric R. Fearon, MD, PhD

Professor Emeritus of Pathology CU Anschutz Medical Campus University of Colorado Aurora, Colorado

Phoebe E. Freer, MD Associate Professor Radiology and Imaging Sciences University of Utah Hospitals/Huntsman Cancer Institute Salt Lake City, Utah

Boris Freidlin, PhD Division of Cancer Treatment and Diagnosis National Cancer Institute Bethesda, Maryland

Alison G. Freifeld, MD Professor of Internal Medicine Infectious Diseases Division University of Nebraska Medical Center Omaha, Nebraska

Terence W. Friedlander, MD Associate Clinical Professor Medicine UCSF Medical Center San Francisco, California

Mark B. Geyer, MD

Amato J. Giaccia, PhD Jack, Lulu, and Sam Willson Professor of Cancer Biology Department of Radiation Oncology Stanford University School of Medicine Stanford, California

Mark R. Gilbert, MD Senior Investigator and Chief Neuro-Oncology Branch National Cancer Institute Bethesda, Maryland

Whitney Goldner, MD Associate Professor of Internal Medicine Division of Diabetes, Endocrinology, and Metabolism University of Nebraska Medical Center Omaha, Nebraska

Contributors xv

Donald P. Goldstein, MD

Missak Haigentz, MD

Professor of Obstetrics, Gynecology, and Reproductive Biology Harvard Medical School Senior Scientist Department of Obstetrics and Gynecology Brigham and Women’s Hospital Boston, Massachusetts

Montefiore Medical Center Bronx, New York

Annekathryn Goodman, MD Massachusetts General Hospital Boston, Massachusetts

Karyn A. Goodman, MD, MS Professor of Radiation Oncology Grohne Chair in Clinical Cancer Research University of Colorado School of Medicine Aurora, Colorado

Kathleen Gordon, MD Medical Director of Ophthalmology IQVIA Co-Chair IQIVA Ophthalmology Center of Excellence Clinical Associate Professor of Ophthalmology University of North Carolina at Chapel Hill Chapel Hill, North Carolina

Laura Graeff-Armas, MD, MS Associate Professor of Internal Medicine Division of Diabetes, Endocrine and Metabolism University of Nebraska Medical Center Omaha, Nebraska

John D. Hainsworth, MD Chief Scientific Officer Sarah Cannon Research Institute Nashville, Tennessee

Benjamin E. Haithcock, MD Associate Professor of Surgery University of North Carolina at Chapel Hill Chapel Hill, North Carolina

Christopher L. Hallemeier, MD Assistant Professor of Radiation Oncology Mayo Clinic Rochester, Minnesota

Samir Hanash, MD, PhD Evelyn & Sol Rubenstein Distinguished Chair for Cancer Prevention Professor of Clinical Cancer Prevention The University of Texas MD Anderson Cancer Center Houston, Texas

Aphrothiti J. Hanrahan, PhD Assistant Lab Member Human Oncology and Pathogenesis Program Memorial Sloan Kettering Cancer Center New York, New York

James Harding, MD

Associate Professor of Surgery Icahn School of Medicine at Mount Sinai New York, New York

Assistant Attending Physician Memorial Sloan Kettering Cancer Center Assistant Professor of Medicine Weill Medical College at Cornell University New York, New York

Stuart A. Grossman, MD

Michael R. Harrison, MD

Professor of Oncology, Medicine, and Neurosurgery The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Medicine The Johns Hopkins University Baltimore, Maryland

Assistant Professor of Medicine Division of Medical Oncology Duke Cancer Institute Durham, North Carolina

Stephan Grupp, MD, PhD

Research Scientist The Jackson Laboratory Bar Harbor, Maine

Alexander J. Greenstein, MD, MPH

Section Chief, Cellular Therapy and Transplant Director, Cancer Immunotherapy Frontier Program CCCR Director of Translational Research Children’s Hospital of Philadelphia Philadelphia, Pennsylvania

Arjun Gupta, MD Assistant Instructor Department of Internal Medicine University of Texas Southwestern Medical Center Dallas, Texas

Irfanullah Haider, MD, MBA Breast Imaging Brigham and Women’s Hospital Boston, Massachussetts

Muneer G. Hasham, PhD

Ernest Hawk, MD, MPH Boone Pickens Distinguished Chair for Early Prevention of Cancer Vice President and Division Head Division of Cancer Prevention and Population Sciences The University of Texas MD Anderson Cancer Center Houston, Texas

Jonathan Hayman, MD Department of Internal Medicine Johns Hopkins Bayview Medical Center Baltimore, Maryland

xvi Contributors

Jonathan E. Heinlen, MD

Clifford A. Hudis, MD

Assistant Professor Department of Urology University of Oklahoma Health Sciences Center Oklahoma City, Oklahoma

Chief Executive Officer American Society of Clinical Oncology Alexandria, Virginia

N. Lynn Henry, MD, PhD

Jeffrey E. Perelman Distinguished Chair Department of Pediatrics Chief, Division of Oncology Pediatrics Children’s Hospital of Philadelphia Philadelphia, Pennsylvania

Associate Professor Internal Medicine University of Utah Salt Lake City, Utah

Joseph Herman, MD Professor and Division Head ad-interim Department of Radiation Oncology The University of Texas MD Anderson Cancer Center Houston, Texas

Brian P. Hobbs, PhD Associate Staff Quantitative Health Sciences and The Taussig Cancer Institute Cleveland Clinic Cleveland, Ohio

Ingunn Holen, BSc, MSc, PhD Oncology University of Sheffield Sheffield, United Kingdom

Leora Horn, MD, MSc Associate Professor of Medicine Medicine–Hematology Oncology Vanderbilt University Nashville, Tennessee

Neil S. Horowitz, MD Department of Obstetrics and Gynecology Division of Gynecologic Oncology Brigham and Women’s Hospital Dana Farber Cancer Institute Boston, Massachusetts

Steven M. Horwitz, MD Associate Attending Department of Medicine, Lymphoma Service Memorial Sloan Kettering Cancer Center Assistant Professor of Clinical Medicine Weill-Cornell Medical College New York, New York

Odette Houghton, MD Associate Professor Department of Ophthalmology Mayo Clinic Scottsdale, Arizona

Scott C. Howard, MD, MSc Professor of Acute and Tertiary Care University of Tennessee Health Sciences Center Memphis, Tennessee

Stephen P. Hunger, MD

†

Arti Hurria, MD

Professor Department of Medical Oncology and Therapeutics Research City of Hope Comprehensive Cancer Center Duarte, California

David H. Ilson, MD, PhD Attending Physician Gastrointestinal Oncology Service Department of Medicine Memorial Sloan-Kettering Cancer Center New York, New York

Annie Im, MD Assistant Professor of Medicine Department of Hematology and Oncology UPMC Hillman Cancer Center Pittsburgh, Pennsylvania

Gopa Iyer, MD Assistant Attending Physician, Genitourinary Oncology Service Department of Medicine Memorial Sloan Kettering Cancer Center New York, New York

Elizabeth M. Jaffee, MD The Dana and Albert “Cubby” Broccoli Professor of Oncology Deputy Director, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Johns Hopkins University School of Medicine Baltimore, Maryland

Reshma Jagsi, MD, DPhil Professor and Deputy Chair Radiation Oncology University of Michigan Ann Arbor, Michigan

Rakesh K. Jain, PhD A.W. Cook Professor of Tumor Biology Department of Radiation Oncology Harvard Medical School Director E.L. Steele Laboratory for Tumor Biology Department of Radiation Oncology Massachusetts General Hospital Boston, Massachusetts †

Deceased.

Contributors xvii

William Jarnagin, MD, FACS

Hagop Kantarjian, MD

Winchester Hospital North Reading Medical North Reading, Massachusetts

The University of Texas MD Anderson Cancer Center Houston, Texas

Aminah Jatoi, MD

Assistant Professor of Surgery University of Pennsylvania Philadelphia, Pennsylvania

Professor of Oncology Mayo Clinic Rochester, Minnesota

Anuja Jhingran, MD

Giorgos Karakousis, MD

Maher Karam-Hage, MD

The University of Texas MD Anderson Cancer Center Houston, Texas

Professor of Behavioral Science The University of Texas MD Anderson Cancer Center Houston, Texas

David H. Johnson, MD

Nadine M. Kaskas, MD

Chairman, Department of Internal Medicine University of Texas Southwestern Medical School Dallas, Texas

Resident Physician Department of Dermatology The Warren Alpert Medical School of Brown University Providence, Rhode Island

Brian Johnston, MD Royal Victoria Hospital Belfast, United Kingdom

Michael B. Kastan, MD, PhD

Center for Cancer Research and Cell Biology School of Medicine, Dentistry, and Biomedical Sciences Queen’s University Belfast Belfast, United Kingdom

Executive Director, Duke Cancer Institute Director, Cancer Immunotherapy Frontier Program William and Jane Shingleton Professor, Pharmacology and Cancer Biology Professor of Pediatrics Duke University School of Medicine Durham, North Carolina

Kevin D. Judy, MD

Nora Katabi, MD

Professor of Neurosurgery Thomas Jefferson University Jefferson Medical College Philadelphia, Pennsylvania

Department of Pathology Memorial Sloan-Kettering Cancer Center New York, New York

Lisa A. Kachnic, MD

Associate Professor of Infectious Disease University of Michigan Ann Arbor, Michigan

†

Patrick G. Johnston, MD

Professor and Chair of Radiation Oncology Vanderbilt University Medical Center Nashville, Tennessee

Orit Kaidar-Person, MD Ramban Medical Center Haifa, Israel

Sanjeeva Kalva, MD, RPVI, FSIR Chief, Interventional Radiology Associate Professor of Radiology University of Texas Southwestern Medical Center Dallas, Texas

Deborah Y. Kamin, RN, MS, PhD Vice President Policy and Advocacy American Society of Clinical Oncology Alexandria, Virginia

†

Deceased.

Daniel R. Kaul, MD

Scott R. Kelley, MD, FACS, FASCRS Assistant Professor of Surgery Division of Colon and Rectal Surgery Mayo Clinic Rochester, Minnesota

Nancy Kemeny, MD Attending Physician Memorial Sloan Kettering Cancer Center Professor of Medicine Weill Medical College at Cornell University New York, New York

Erin E. Kent, PhD, MS Scientific Advisor Outcomes Research Branch Healthcare Delivery Research Program Division of Cancer Control and Population Sciences National Cancer Institute Rockville, Maryland ICF, Inc. Fairfax, Virginia

xviii Contributors

Oliver Kepp, PhD

Daniel A. Laheru, MD

Metabolomics and Cell Biology Platforms Gustave Roussy Cancer Campus Villejuif, France

Ian T. MacMillan Professorship in Clinical Pancreatic Research Department of Medical Oncology The Johns Hopkins University School of Medicine Baltimore, Maryland

Simon Khagi, MD Assistant Professor University of North Carolina School of Medicine Lineberger Comprehensive Cancer Center Chapel Hill, North Carolina

Paul F. Lambert, PhD

Joshua E. Kilgore, MD

Mark Lawler, PhD

Division of Gynecologic Oncology University of North Carolina School of Medicine Chapel Hill, North Carolina

Chair in Translational Cancer Genomics Centre for Cancer Research and Cell Biology School of Medicine, Dentistry and Biomedical Sciences Queen’s University Belfast Belfast, United Kingdom

D. Nathan Kim, MD, PhD Associate Professor Department of Radiation Oncology University of Texas Southwestern Medical Center Dallas, Texas

Bette K. Kleinschmidt-DeMasters, MD Professor of Neurology, Neurosurgery, and Pathology CU Anschutz Medical Campus University of Colorado Aurora, Colorado

Edward L. Korn, PhD Biometric Research Program National Cancer Institute Bethesda, Maryland

Professor of Oncology University of Wisconsin Madison, Wisconsin

Jennifer G. Le-Rademacher, PhD Associate Professor of Biostatistics Health Sciences Research Associate Professor of Oncology Mayo Clinic Rochester, Minnesota

John Y.K. Lee, MD Associate Professor of Neurosurgery University of Pennsylvania Philadelphia, Pennsylvania

Nancy Y. Lee, MD

Guido Kroemer, MD, PhD

Department of Radiation Oncology Memorial Sloan-Kettering Cancer Center New York, New York

Team 11, Centre de Recherche des Cordeliers Paris, France

Susanna L. Lee, MD, PhD

Geoffrey Y. Ku, MD

Massachusetts General Hospital Boston, Massachusetts

Assistant Attending Physician Gastrointestinal Oncology Service Department of Medicine Memorial Sloan Kettering Cancer Center New York, New York

Jonathan E. Leeman, MD Department of Radiation Oncology Memorial Sloan-Kettering Cancer Center New York, New York

Shivaani Kummar, MD

Andreas Linkermann, MD

Professor of Medicine Director, Phase 1 Clinical Research Program Stanford University Palo Alto, California

Department of Internal Medicine III Division of Nephrology University Hospital Carl Gustav Carus at the Technische Universität Dresden Dresden, Germany

Bonnie Ky, MD, MSCE Assistant Professor of Medicine and Epidemiology Division of Cardiovascular Medicine Senior Scholar Center for Clinical Epidemiology and Biostatistics University of Pennsylvania School of Medicine Philadelphia, Pennsylvania

Jinsong Liu, MD, PhD Professor of Pathology The University of Texas MD Anderson Cancer Center Houston, Texas

Simon Lo, MD, FACR Professor and Vice Chair for Strategic Planning Department of Radiation Oncology Professor of Neurological Surgery University of Washington School of Medicine Seattle, Washington

Contributors xix

Jason W. Locasale, PhD

Amit Maity, MD

Associate Professor Department of Pharmacology and Cancer Biology Duke University School of Medicine Durham, North Carolina

University of Pennsylvania Philadelphia, Pennsylvania

Charles L. Loprinzi, MD Regis Professor of Breast Cancer Research Department of Oncology Mayo Clinic Rochester, Minnesota

Maeve Lowery, MD Professor of Translational Cancer Medicine Trinity College Dublin, Ireland

Emmy Ludwig, MD Associate Attending Physician Memorial Sloan Kettering Cancer Center Associate Professor of Medicine Weill Medical College at Cornell University New York, New York

Neil Majithia, MD Mayo Clinic Rochester, Minnesota

Marcos Malumbres, PhD Group Leader Cell Division and Cancer Spanish National Cancer Research Center (CNIO) Madrid, Spain

Karen Colbert Maresso, MPH Program Director Division of Cancer Prevention and Population Sciences The University of Texas MD Anderson Cancer Center Houston, Texas

John D. Martin, PhD The University of Tokyo Tokyo, Japan

Matthew A. Lunning, DO

Koji Matsuo, MD, PhD

Associate Professor of Internal Medicine University of Nebraska Medical Center Omaha, Nebraska

Assistant Professor of Obstetrics and Gynecology University of Southern California Los Angeles, California

Robert A. Lustig, MD

Natalie H. Matthews, MD

Professor of Clinical Radiation Oncology University of Pennsylvania Philadelphia, Pennsylvania

Department of Dermatology The Warren Alpert Medical School of Brown University Providence, Rhode Island

Mitchell Machtay, MD

Lauren Mauro, MD

University Hospitals Cleveland Medical Center Case-Western Reserve University School of Medicine Cleveland, Ohio

Assistant Professor of Medicine George Washington University School of Medicine Washington, D.C.

Crystal Mackall, MD

R. Samuel Mayer, MD, MEHP

Endowed Professor of Pediatrics and Medicine Stanford University Director Stanford Center for Cancer Cell Therapy Director Parker Institute for Cancer Immunotherapy at Stanford Associate Director Stanford Cancer Institute Stanford, California

Associate Professor and Vice Chair for Education Physical Medicine and Rehabilitation The Johns Hopkins University School of Medicine Medical Director, Cancer Rehabilitation Program Physical Medicine and Rehabilitation The Johns Hopkins Hospital Baltimore, Maryland

David A. Mahvi, MD General Surgery Resident Brigham and Women’s Hospital Boston, Massachusetts

David M. Mahvi, MD Professor of Surgery Chief, Surgical Oncology Medical University of South Carolina Charleston, South Carolina

Worta McCaskill-Stevens, MD Chief, Community Oncology and Prevention Trials Research Group Division of Cancer Prevention National Cancer Institute Rockville, Maryland

Megan A. McNamara, MD Assistant Professor of Medicine Department of Medical Oncology Duke University Medical Center Durham, North Carolina

xx Contributors

Neha Mehta-Shah, MD

Jarushka Naidoo, MB, BCH

Assistant Professor Washington University St. Louis, Missouri

Assistant Professor of Oncology The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Hospital Baltimore, Maryland

Robert E. Merritt, MD Director, Thoracic Surgery James Cancer Center The Ohio State University Medical Columbus, Ohio

Amol Narang, MD

Matthew I. Milowsky, MD

Heidi Nelson, MD, FACS, FASCRS

Professor of Medicine Division of Hematology/Oncology UNC Lineberger Comprehensive Cancer Center Chapel Hill, North Carolina

Professor of Surgery Division of Colon and Rectal Surgery Mayo Clinic Rochester, Minnesota

Lori M. Minasian, MD

William G. Nelson, MD, PhD

Deputy Director Division of Cancer Prevention National Cancer Institute National Institutes of Health Bethesda, Maryland

Professor and Director Sidney Kimmel Comprehensive Cancer Center Johns Hopkins University School of Medicine Baltimore, Maryland

Tara C. Mitchell, MD

Clinical Pharmacy Specialist, Pain Management Research Associate, Department of Oncology The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins The Johns Hopkins Hospital Baltimore, Maryland

Assistant Professor of Medicine University of Pennsylvania Philadelphia, Pennsylvania

Demytra Mitsis, MD Medical Oncology and Hematology Fellow Department of Medicine Roswell Park Cancer Institute Buffalo, New York

Michelle Mollica, PhD, MPH, RN

Assistant Professor of Radiation Oncology Johns Hopkins University School of Medicine Baltimore, Maryland

Suzanne Nesbit, PharmD, BCPS, CPE

Mark Niglas, MD, FRCPC Clinical Fellow Department of Radiation Oncology Sunnybrook Health Sciences Centre Toronto, Ontario, Canada

Program Director Division of Cancer Control and Population Sciences Healthcare Delivery Research Program National Cancer Institute Bethesda, Maryland

Tracey O’Connor, MD

Margaret Mooney, MD

Kenneth Offit, MD, MPH

Branch Chief, Clinical Investigations Branch Cancer Therapy Evaluation Program Division of Cancer Treatment and Diagnosis National Cancer Institute Rockville, Maryland

Chief, Clinical Genetics Service Robert and Kate Niehaus Chair in Inherited Cancer Genomics Memorial Sloan Kettering Cancer Center New York, New York

Farah Moustafa, MD

Medical Director OncoMetrix Laboratories Poplar Healthcare Memphis, Tennessee

Department of Dermatology The Warren Alpert Medical School of Brown University Providence, Rhode Island

Lida Nabati, MD Instructor in Medicine Harvard Medical School Senior Physician Dana-Farber Cancer Institute Boston, Massachusetts

Associate Professor of Oncology Department of Medicine Roswell Park Cancer Institute Buffalo, New York

Mihaela Onciu, MD

Contributors xxi

Eileen M. O’Reilly, MD

Steven Z. Pavletic, MD, MS

Winthrop Rockefeller Chair in Medical Oncology Section Head Hepatopancreaticobiliary & Neuroendocrine Cancers Gastrointestinal Oncology Service Associate Director David M. Rubenstein Center for Pancreatic Cancer Research Attending Physician, Member Memorial Sloan Kettering Cancer Center Professor of Medicine Weill Medical College at Cornell University New York, New York

Head, Graft-Versus-Host Disease and Autoimmunity Section Experimental Transplantation and Immunology Branch National Cancer Institute Bethesda, Maryland

Elaine A. Ostrander, PhD

Associate Professor of Pathology CU Anschutz Medical Campus University of Colorado Aurora, Colorado

Chief and Distinguished Investigator Cancer Genetics and Comparative Genomics Branch National Human Genome Research Institute Bethesda, Maryland

Lisa Pappas-Taffer, MD Assistant Professor of Clinical Dermatology University of Pennsylvania Philadelphia, Pennsylvania

Drew Pardoll, MD, PhD Director, Bloomberg~Kimmel Institute for Cancer Immunotherapy Sidney Kimmel Comprehensive Cancer Center Johns Hopkins School of Medicine Baltimore, Maryland

Jae H. Park, MD Assistant Attending Department of Medicine Leukemia Service and Cellular Therapeutics Center Memorial Sloan Kettering Cancer Center Assistant Professor of Medicine Joan and Sanford I. Weill Department of Medicine Weill Cornell Medical College New York, New York

Peter C. Phillips, MD Professor of Neurology and Oncology The Children’s Hospital of Philadelphia Philadelphia, Pennsylvania

Miriam D. Post, MD

Amy A. Pruitt, MD University of Pennsylvania Philadelphia, Pennsylvania

Christiane Querfeld, MD, PhD Chief, Division of Dermatology Director, Cutaneous Lymphoma Program Assistant Professor of Dermatology City of Hope Duarte, California

Vance A. Rabius, PhD Research Director, Tobacco Treatment Program The University of Texas MD Anderson Cancer Center Houston, Texas

S. Vincent Rajkumar, MD Edward W. and Betty Knight Scripps Professor of Medicine Division of Hematology Mayo Clinic Rochester, Minnesota

Anery Patel, MD

Mohammad O. Ramadan, MD

Clinical Instructor Department of Internal Medicine Division of Diabetes, Endocrine, and Metabolism University of Nebraska Medical Center Omaha, Nebraska

Assistant Professor Department of Urology University of Oklahoma Health Sciences Center Oklahoma City, Oklahoma

Anish J. Patel, MD

Assistant Professor of Radiation Oncology, Obstetrics, and Gynecology Stanford University School of Medicine Stanford, California

Assistant Professor of Endocrinology Department of Endocrinology University of Alabama at Birmingham Birmingham, Alabama

Steven R. Patierno, PhD Deputy Director Duke Cancer Institute Professor of Medicine Professor of Pharmacology and Cancer Biology Professor of Community and Family Medicine Duke University Medical Center Durham, North Carolina

Erinn B. Rankin, PhD

Sushanth Reddy, MD Assistant Professor of Surgery Department of Surgery University of Alabama at Birmingham Birmingham, Alabama

xxii Contributors

Michael A. Reid, PhD

Nadia Rosenthal, PhD

Postdoctoral Fellow Department of Pharmacology and Cancer Biology Duke University School of Medicine Durham, North Carolina

Scientific Director The Jackson Laboratory Bar Harbor, Maine Chair, Cardiovascular Science National Heart and Lung Institute Imperial College London London, United Kingdom

Scott Reznik, MD Associate Professor Department of Cardiothoracic Surgery University of Texas Southwestern Medical Center Dallas, Texas

Tina Rizack, MD, MPH Hematologist/Oncologist South County Health Clinical Assistant Professor of Internal Medicine and Obstetrics & Gynecology Alpert Medical School of Brown University Providence, Rhode Island

Jason D. Robinson, PhD Associate Professor of Behavioral Science The University of Texas MD Anderson Cancer Center Houston, Texas

Leslie Robinson-Bostom, MD Senior Attending Department of Dermatology The Warren Alpert Medical School of Brown University Providence, Rhode Island

Carlos Rodriguez-Galindo, MD Departments of Global Pediatric Medicine and Oncology St. Jude Children’s Research Hospital Memphis, Tennessee

Paul B. Romesser, MD Department of Radiation Oncology Memorial Sloan-Kettering Cancer Center New York, New York

Steven T. Rosen, MD Provost & Chief Scientific Officer Director, Comprehensive Cancer Center and Beckman Research Institute Irell & Manella Cancer Center Director’s Distinguished Chair Helen & Morgan Chu Director’s Chair, Beckman Research Institute City of Hope Duarte, California

Myrna R. Rosenfeld, MD, PhD Senior Investigator, Neuroimmunology Institut d’Investigació Biomèdica August Pi i Sunyer (IDIBAPS) Barcelona, Spain Adjunct Professor Neurology University of Pennsylvania Philadelphia, Pennsylvania

Meredith Ross, MD Fellow Department of Internal Medicine Division of Diabetes, Endocrinology, and Metabolism University of Nebraska Medical Center Omaha, Nebraska

Julia H. Rowland, PhD Director, Office of Cancer Survivorship Division of Cancer Control and Population Sciences National Cancer Institute Rockville, Maryland

Anthony H. Russell, MD Massachusetts General Hospital Boston, Massachusetts

Michael S. Sabel, MD, FACS Associate Professor Surgery University of Michigan Ann Arbor, Michigan

Arjun Sahgal, MD, FRCPC Professor of Radiation Oncology and Surgery Deputy Chief, Department of Radiation Oncology Sunnybrook Health Sciences Center University of Toronto Faculty of Medicine Toronto, Ontario

Ryan D. Salinas, MD Resident Physician Department of Neurosurgery University of Pennsylvania Philadelphia, Pennsylvania

Erin E. Salo-Mullen, MS, MPH, CGC Senior Genetic Counselor Clinical Genetics Service Department of Medicine Memorial Sloan Kettering Cancer Center New York, New York

Manuel Salto-Tellez, MD Center for Cancer Research and Cell Biology School of Medicine, Dentistry, and Biomedical Sciences Queen’s University Belfast Belfast, United Kingdom

Contributors xxiii

Sydney M. Sanderson, BS

Konstantin Shilo, MD

PhD Candidate Department of Pharmacology and Cancer Biology Duke University School of Medicine Durham, North Carolina

Department of Pathology James Cancer Center The Ohio State University Medical Columbus, Ohio

John T. Sandlund, MD

Eric Small, MD

Member Department of Oncology St. Jude Children’s Research Hospital Professor of Pediatrics University of Tennessee College of Medicine Memphis, Tennessee

Professor of Medicine University of California, San Francisco San Francisco, California

Victor M. Santana, MD Department of Oncology St. Jude Children’s Research Hospital Memphis, Tennessee

Michelle Savage, MD Department of Clinical Cancer Prevention The University of Texas MD Anderson Cancer Center Houston, Texas

Eric C. Schreiber, PhD Associate Professor Department of Radiation Oncology University of North Carolina School of Medicine Chapel Hill, North Carolina

Lynn Schuchter, MD C. Willard Robinson Professor of Hematology and Oncology Professor of Medicine University of Pennsylvania Philadelphia, Pennsylvania

Liora Schultz, MD Department of Pediatric Oncology, Division of Oncology Stanford University Stanford, California

Michael V. Seiden, MD, PhD Texas Oncology The Woodlands, Texas

Morgan M. Sellers, MD, MS Icahn School of Medicine at Mount Sinai New York, New York

Payal D. Shah, MD Assistant Professor Medicine University of Pennsylvania Philadelphia, Pennsylvania

Jinru Shia, MD Member and Attending Pathologist Memorial Sloan Kettering Cancer Center Professor of Pathology and Laboratory Medicine Weill Medical College at Cornell University New York, New York

Angela B. Smith, MD, MS, FACS Associate Professor of Urology Department of Urology University of North Carolina Chapel Hill, North Carolina

Stephen N. Snow, MD Professor of Dermatology Northwest Permanente Portland, Oregon

David B. Solit, MD Geoffrey Beene Chair Director, Center for Molecular Oncology Member, Human Oncology and Pathogenesis Program Attending Physician, Genitourinary Oncology Service, Department of Medicine Memorial Sloan Kettering Cancer Center New York, New York

Anil K. Sood, MD Professor and Vice Chair Department of Gynecologic Oncology and Reproductive Medicine The University of Texas MD Anderson Cancer Center Houston, Texas

Enrique Soto-Perez-de-Celis, MD International Fellow Department of Medical Oncology and Therapeutics Research City of Hope Duarte, California Researcher in Medical Science Cancer Care in the Elderly Clinic Department of Geriatrics Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran Mexico City, Mexico

Joseph A. Sparano, MD Associate Chairman Department of Oncology Montefiore Medical Center Professor of Medicine and Women’s Health Department of Medicine and Oncology Albert Einstein College of Medicine Bronx, New York

Vladimir S. Spiegelman, MD, PhD Professor of Pediatrics and Pharmacology Department of Pediatrics Pennsylvania State University College of Medicine Hershey, Pennsylvania

xxiv Contributors

Sheri L. Spunt, MD, MBA

James E. Talmadge, PhD

Endowed Professor of Pediatric Cancer Department of Pediatrics Division of Hematology/Oncology Stanford University School of Medicine Stanford, California

Professor of Pathology and Microbiology University of Nebraska Medical Center Omaha, Nebraska

Zsofia K. Stadler, MD Assistant Attending Physician Department of Medicine Memorial Sloan Kettering Cancer Center Assistant Professor of Medicine Weill Cornell Medical College New York, New York

David P. Steensma, MD Institute Physician Department of Medical Oncology Dana-Farber Cancer Institute Associate Professor of Medicine Harvard Medical School Boston, Massachusetts

Richard M. Stone, MD Professor of Medicine Harvard Medical School Chief of Staff Department of Medical Oncology Dana-Farber Cancer Institute Boston, Massachusetts

David T. Teachey, MD Department of Pediatrics Divisions of Hematology and Oncology Children’s Hospital of Philadelphia Philadelphia, Pennsylvania

Catalina V. Teba, MD University Hospitals Cleveland Medical Center Case-Western Reserve University School of Medicine Cleveland, Ohio

Ayalew Tefferi, MD Department of Hematology Mayo Clinic Rochester, Minnesota

Bin Tean Teh, MD, PhD Professor Division of Medical Sciences National Cancer Centre Singapore Professor, Cancer and Stem Cell Biology Program Duke–NUS Medical School Singapore

Joyce M.C. Teng, MD, PhD

Polsinelli PC Washington, D.C.

Associate Professor of Dermatology and Pediatrics Stanford of Medicine Stanford University Palo Alto, California

Kelly Stratton, MD

Joel E. Tepper, MD

Assistant Professor Department of Urology University of Oklahoma Health Sciences Center Oklahoma City, Oklahoma

Hector MacLean Distinguished Professor of Cancer Research Department of Radiation Oncology University of North Carolina School of Medicine University of North Carolina Lineberger Comprehensive Cancer Center Chapel Hill, North Carolina

Steven Kent Stranne, MD, JD

Bill Sugden, PhD Professor of Oncology University of Wisconsin Madison, Wisconsin

Andrew M. Swanson, MD Assistant Professor of Dermatology University of Wisconsin School of Medicine and Public Health Madison, Wisconsin

Martin S. Tallman, MD Chief, Leukemia Service Memorial Sloan-Kettering Cancer Center Professor of Medicine Joan and Sanford I. Weill Department of Medicine Weill Cornell Medical College New York, New York

Premal H. Thaker, MD Professor in Gynecologic Oncology Division of Obstetrics and Gynecology Washington University School of Medicine St. Louis, Missouri

Aaron P. Thrift, PhD Assistant Professor Dan L. Duncan Comprehensive Cancer Center Department of Medicine, Gastroenterology Section Baylor College of Medicine Houston, Texas

Arthur-Quan Tran, MD Division of Gynecologic Oncology University of North Carolina School of Medicine Chapel Hill, North Carolina

Contributors xxv

Grace Triska, MS

Richard L. Wahl, MD

Washington University School of Medicine St. Louis, Missouri

Elizabeth Mallinckrodt Professor and Director Mallinckrodt Institute of Radiology Washington University School of Medicine St. Louis, Missouri

Donald Trump, MD, FACP Chief Executive Officer and Executive Director Inova Schar Cancer Institute Falls Church, Virginia

Kenneth Tsai, MD, PhD Associate Member Anatomic Pathology and Tumor Biology H. Lee Moffitt Cancer Center and Research Institute Tampa, Florida

Chia-Lin Tseng, MD, FRCPC Assistant Professor Radiation Oncologist Sunnybrook Health Sciences Centre Toronto, Ontario, Canada

Diane Tseng, MD, PhD Department of Medicine Division of Oncology Stanford University Stanford, California

Sandra Van Schaeybroeck, MD

Michael F. Walsh, MD, FAAP, FACMG, DABMG Assistant Member Department of Pediatrics and Medicine Divisions of Solid Tumor and Clinical Genetics Memorial Sloan Kettering Cancer Center New York City, New York

Thomas Wang, MD Professor of Surgery Department of Surgery University of Alabama at Birmingham Birmingham, Alabama

Jared Weiss, MD Associate Professor of Medicine Section Chief of Thoracic and Head/Neck Oncology Division of Hematology and Oncology University of North Carolina at Chapel Hill Chapel Hill, North Carolina

Irving L. Weissman, MD

Center for Cancer Research and Cell Biology School of Medicine, Dentistry, and Biomedical Sciences Queen’s University Belfast Belfast, United Kingdom

Director, Institute for Stem Cell Biology and Regenerative Medicine Director, Stanford Ludwig Center for Cancer Stem Cell Research and Medicine Stanford University Palo Alto, California

Brian A. Van Tine, MD, PhD

Shannon N. Westin, MD, MPH

Associate Professor Internal Medicine Washington University in Saint Louis St. Louis, Missouri

Associate Professor of Gynecologic Oncology and Reproductive Medicine The University of Texas MD Anderson Cancer Center Houston, Texas

Erin R. Vanness, MD

Jeffrey D. White, MD

Associate Professor of Dermatology University of Wisconsin School of Medicine and Public Health Madison, Wisconsin

Associate Director, Office of Cancer Complementary and Alternative Medicine Division of Cancer Treatment and Diagnosis National Cancer Institute Bethesda, Maryland

Gauri Varadhachary, MD Professor Medical Director, Gastrointestinal Center Executive Medical Director, Ambulatory Operations Department of Gastrointestinal Medical Oncology The University of Texas MD Anderson Cancer Center Houston, Texas

Richard Wilson, MD Center for Cancer Research and Cell Biology School of Medicine, Dentistry, and Biomedical Sciences Queen’s University Belfast Belfast, United Kingdom

Marileila Varella-Garcia, PhD

Richard J. Wong, MD

Professor of Medicine and Medical Oncology CU Anschutz Medical Campus University of Colorado Aurora, Colorado

Department of Surgery Memorial Sloan-Kettering Cancer Center New York, New York

xxvi Contributors

Gary S. Wood, MD

Timothy Zagar, MD

Professor and Chair of Dermatology University of Wisconsin School of Medicine and Public Health Middleton VA Medical Center Madison, Wisconsin

Northeastern Radiation Oncology Glens Falls, New York

Yaohui G. Xu, MD, PhD Associate Professor of Dermatology University of Wisconsin School of Medicine and Public Health Madison, Wisconsin

Meng Xu-Welliver, MD, PhD Associate Professor of Radiation Oncology James Cancer Center The Ohio State University Medical Center Columbus, Ohio

Shlomit Yust-Katz, MD Professor Davidoff Cancer Center Rabin Medical Center Petah Tikva, Israel

Elaine M. Zeman, PhD Associate Professor Department of Radiation Oncology University of North Carolina School of Medicine Chapel Hill, North Carolina

Tian Zhang, MD Assistant Professor of Medicine Duke University Medical Center Durham, North Carolina

James A. Zwiebel, MD Cancer Therapy Evaluation Program (Retired) National Cancer Institute Rockville, Maryland

Preface

New insights into whole genome sequence variations and the genomic structural alterations associated with cancer, including their downstream effects on protein structure and function, are helping us to define specific communication pathway changes that drive cancer initiation, progression, metastasis, and resistance. We have learned that each individual and each tumor may be unique. Individual physiognomies in terms of path of progression and unique cellular communication pathway alterations are continuing to define the nature of specific cancers and offer greater opportunities for the development of highly prescriptive intervention(s). In addition, we have a much greater understanding of the relationship of the host’s tissues, the patient’s immune system, and the broad tumor microenvironment, to the process of tumor development and progression and their impact on tumor control. This new body of knowledge on how the body’s immune system and the tumor’s microenvironment are altered to support disease growth, invasion, and distant spread is providing opportunities for the development of novel therapeutic interventions. There is exciting new evidence to support the presence of a special subclass of cells within the tumor that has properties of “stemness,” which places them in the key role of maintaining tumor growth and tumor spread. The cumulative effect of these advances— where certain cancers can be prevented and where others will be detected earlier and controlled—promises to be transformative in our effort to conquer cancer. The sixth edition of Abeloff ’s Clinical Oncology incorporates the exciting advances in basic, translational, clinical, and epidemiologic oncology. Each chapter begins with a summary highlighting the key points and comprises a critical analysis of the literature and updated clinical studies—authors present their own opinions in specially identified boxes and algorithms. Despite significant progress, the diagnosis of cancer remains devastating to patients and their families. Our goal is to provide a reference textbook that is the most useful, understandable, attractive, and thorough in presenting the principles of clinical oncology. It is meant to be equally

useful to students and trainees, experts in the various disciplines of oncology, and as a reference text for physicians from other medical disciplines and the various staff who regularly care for patients with cancer. It is our hope that readers will find this scholarly textbook properly balanced between the disciplines of science, clinical medicine, and humanism and that it will serve them well in their efforts to prevent, diagnose, and effectively treat their patients suffering from cancer. The multidisciplinary nature of cancer care is, and will continue to be, reflected in our editors. Experts in cancer biology, surgical oncology, pediatric oncology, radiation oncology, medical oncology, and hematologic malignancies directed the development of the content. Reflecting the multispecialty approach necessary for optimal care of patients, the majority of chapters are the joint product of several of these disciplines. Engaging the very best subject matter authorities was a guiding principle for the editors and we are deeply indebted to our outstanding authors who, in a most diligent and thoughtful way, have brought their knowledge and skills to the sixth edition of Abeloff ’s Clinical Oncology.

ACKNOWLEDGMENTS This sixth edition represents a highly collaborative and dynamic effort between the editors and Elsevier. We are greatly indebted to Laura Schmidt, Kathleen Schlom, and Kristi Batchelor for their creative input and guidance and for turning the principles behind this text into a reality. Finally, we want to express our gratitude to our many contributing authors for their dedication to this project, their generosity of time, and, of course, their very valuable friendship. John E. Niederhuber, MD James O. Armitage, MD James H. Doroshow, MD Michael B. Kastan, MD, PhD Joel E. Tepper, MD

xxvii

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Contents

Part I Science and Clinical Oncology Section A Biology and Cancer 1 Molecular Tools in Cancer Research,  2 Mauro W. Costa, Muneer G. Hasham, and Nadia Rosenthal

2 Intracellular Signaling, 24

Aphrothiti J. Hanrahan, Gopa Iyer, and David B. Solit

3 Cellular Microenvironment and Metastases,  47 Erinn B. Rankin and Amato J. Giaccia

4 Control of the Cell Cycle,  56 Marcos Malumbres

5 Pathophysiology of Cancer Cell Death,  74

Lorenzo Galluzzi, Andreas Linkermann, Oliver Kepp, and Guido Kroemer

6 Cancer Immunology, 84

Diane Tseng, Liora Schultz, Drew Pardoll, and Crystal Mackall

7 Stem Cells, Cell Differentiation, and Cancer,  97

Piero Dalerba, Maximilian Diehn, Irving L. Weissman, and Michael F. Clarke

8 Tumor Microenvironment: Vascular and Extravascular Compartment,  108

Rakesh K. Jain, John D. Martin, Vikash P. Chauhan, and Dan G. Duda

9 Cancer Metabolism, 127

Michael A. Reid, Sydney M. Sanderson, and Jason W. Locasale

Section B Genesis of Cancer 10 Environmental Factors,  139 Steven R. Patierno

11 DNA Damage Response Pathways and Cancer, 154 James M. Ford and Michael B. Kastan

12 Viruses and Human Cancer,  165 Paul F. Lambert and Bill Sugden

13 Genetic Factors: Hereditary Cancer Predisposition Syndromes, 180 Michael F. Walsh, Karen Cadoo, Erin E. Salo-Mullen, Marianne Dubard-Gault, Zsofia K. Stadler, and Kenneth Offit

14 Genetic and Epigenetic Alterations in Cancer, 209 Bin Tean Teh and Eric R. Fearon

Section C Diagnosis of Cancer 15 Pathology, Biomarkers, and Molecular Diagnostics, 225

Wilbur A. Franklin, Dara L. Aisner, Kurtis D. Davies, Kristy Crooks, Miriam D. Post, Bette K. Kleinschmidt-DeMasters, Edward Ashwood, Paul A. Bunn, and Marileila Varella-Garcia

16 Imaging, 254

Richard L. Wahl

Section D Clinical Trials 17 Biostatistics and Bioinformatics in Clinical Trials, 284 Brian P. Hobbs, Donald A. Berry, and Kevin R. Coombes

18 Clinical Trial Designs in Oncology,  296 Edward L. Korn and Boris Freidlin

19 Structures Supporting Cancer Clinical Trials,  308 Jeffrey S. Abrams, Margaret Mooney, James A. Zwiebel, Worta McCaskill-Stevens, Michaele C. Christian, and James H. Doroshow

20 Oncology and Health Care Policy,  317 Steven Kent Stranne, Clifford A. Hudis, and Deborah Y. Kamin

Section E Prevention and Early Detection 21 Discovery and Characterization of Cancer Genetic Susceptibility Alleles,  323 Stephen J. Chanock and Elaine A. Ostrander

xxix

xxx Contents

22 Lifestyle and Cancer Prevention,  337

Karen Basen-Engquist, Powel Brown, Adriana M. Coletta, Michelle Savage, Karen Colbert Maresso, and Ernest Hawk

23 Screening and Early Detection,  375

Therese Bevers, Hashem El-Serag, Samir Hanash, Aaron P. Thrift, Kenneth Tsai, Karen Colbert Maresso, and Ernest Hawk

24 Nicotine Dependence: Current Treatments and Future Directions,  399 Jeffrey M. Engelmann, Maher Karam-Hage, Vance A. Rabius, Jason D. Robinson, and Paul M. Cinciripini

Section F Treatment 25 Cancer Pharmacology,  411 Jerry M. Collins

26 Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents,  420 Khanh T. Do and Shivaani Kummar

27 Basics of Radiation Therapy,  431

Elaine M. Zeman, Eric C. Schreiber, and Joel E. Tepper

28 Hematopoietic Stem Cell Transplantation,  461 Annie Im and Steven Z. Pavletic

29 Gene Therapy in Oncology,  470

James E. Talmadge and Kenneth H. Cowan

30 Therapeutic Antibodies and Immunologic Conjugates, 486

Konstantin Dobrenkov and Nai-Kong V. Cheung

31 Complementary and Alternative Medicine,  500 Jeffrey D. White

Section B Symptom Management 35 Hypercalcemia, 565

Anery Patel, Laura Graeff-Armas, Meredith Ross, and Whitney Goldner

36 Tumor Lysis Syndrome,  572 Scott C. Howard

37 Cancer-Related Pain,  581

Suzanne Nesbit, Ilene Browner, and Stuart A. Grossman

38 Cancer Cachexia,  593

Jennifer G. Le-Rademacher and Aminah Jatoi

39 Nausea and Vomiting,  598 John D. Hainsworth

Section C Treatment Complications 40 Oral Complications,  607

Neil Majithia, Christopher L. Hallemeier, and Charles L. Loprinzi

41 Dermatologic Toxicities of Anticancer Therapy, 621

Natalie H. Matthews, Farah Moustafa, Nadine M. Kaskas, Leslie Robinson-Bostom, and Lisa Pappas-Taffer

42 Cardiovascular Effects of Cancer Therapy,  649 Lori M. Minasian, Myrtle Davis, and Bonnie Ky

43 Reproductive Complications,  665

Demytra Mitsis, Lynda Kwon Beaupin, and Tracey O’Connor

44 Paraneoplastic Neurologic Syndromes,  676 Josep Dalmau and Myrna R. Rosenfeld

45 Neurologic Complications,  688 Shlomit Yust-Katz, Simon Khagi, and Mark R. Gilbert

46 Endocrine Complications,  707

Part II Problems Common to Cancer and Therapy Section A Hematologic Problems and Infections 32 Disorders of Blood Cell Production in Clinical Oncology, 514 Jennifer H. Choe and Jeffrey Crawford

33 Diagnosis, Treatment, and Prevention of Cancer-Associated Thrombosis,  523 Claudia I. Chapuy and Jean M. Connors

34 Infection in the Patient With Cancer,  544 Alison G. Freifeld and Daniel R. Kaul

Donald Trump

47 Pulmonary Complications of Anticancer Treatment, 715 Mitchell Machtay and Catalina V. Teba

Section D Posttreatment Considerations 48 Rehabilitation of Individuals With Cancer,  725 R. Samuel Mayer

49 Survivorship, 732

Julia H. Rowland, Michelle Mollica, and Erin E. Kent

50 Second Malignant Neoplasms,  741 Debra L. Friedman

Contents xxxi

51 Caring for Patients at the End of Life,  751 Lida Nabati and Janet L. Abrahm

Section B Head, Neck, and Eye 64 Ocular Tumors,  968

Section E Local Effects of Cancer and Its Metastasis 52 Acute Abdomen, Bowel Obstruction, and Fistula,  764 William R. Burns and Alfred E. Chang

53 Superior Vena Cava Syndrome,  775

Arjun Gupta, D. Nathan Kim, Sanjeeva Kalva, Scott Reznik, and David H. Johnson

54 Spinal Cord Compression,  786

Mark Niglas, Chia-Lin Tseng, Nicolas Dea, Eric Chang, Simon Lo, and Arjun Sahgal

55 Brain Metastases and Neoplastic Meningitis,  794 Orit Kaidar-Person, Michael Cools, and Timothy Zagar

56 Bone Metastases,  809

Robert E. Coleman, Janet Brown, and Ingunn Holen

57 Lung Metastases,  831

Jonathan Hayman, Jarushka Naidoo, and David S. Ettinger

58 Liver Metastases,  846

David A. Mahvi and David M. Mahvi

59 Malignancy-Related Effusions,  863

Lola A. Fashoyin-Aje and Julie R. Brahmer

Section F Special Populations 60 Cancer in the Elderly: Biology, Prevention, and Treatment,  874

Enrique Soto-Perez-de-Celis and Arti Hurria†

Odette Houghton and Kathleen Gordon

65 Cancer of the Head and Neck,  999 Jonathan E. Leeman, Nora Katabi, Richard J. Wong, Nancy Y. Lee, and Paul B. Romesser

Section C Skin 66 Melanoma, 1034

Tara C. Mitchell, Giorgos Karakousis, and Lynn Schuchter

67 Nonmelanoma Skin Cancers: Basal Cell and Squamous Cell Carcinomas,  1052

Yaohui G. Xu, Juliet L. Aylward, Andrew M. Swanson, Vladimir S. Spiegelman, Erin R. Vanness, Joyce M.C. Teng, Stephen N. Snow, and Gary S. Wood

Section D Endocrine 68 Cancer of the Endocrine System,  1074

Ammar Asban, Anish J. Patel, Sushanth Reddy, Thomas Wang, Courtney J. Balentine, and Herbert Chen

Section E Thoracic 69 Cancer of the Lung: Non–Small Cell Lung Cancer and Small Cell Lung Cancer,  1108 Luiz H. Araujo, Leora Horn, Robert E. Merritt, Konstantin Shilo, Meng Xu-Welliver, and David P. Carbone

61 Special Issues in Pregnancy,  882

70 Diseases of the Pleura and Mediastinum,  1159

62 Human Immunodeficiency Virus (HIV) Infection and Cancer,  894

71 Cancer of the Esophagus,  1174

Tina Rizack and Jorge J. Castillo

Jesus Anampa, Stefan K. Barta, Missak Haigentz, and Joseph A. Sparano

Part III Specific Malignancies Section A Central Nervous System 63 Cancer of the Central Nervous System,  906 Jay F. Dorsey, Ryan D. Salinas, Mai Dang, Michelle Alonso-Basanta, Kevin D. Judy, Amit Maity, Robert A. Lustig, John Y.K. Lee, Peter C. Phillips, and Amy A. Pruitt

†

Deceased.

Orit Kaidar-Person, Timothy Zagar, Benjamin E. Haithcock, and Jared Weiss Geoffrey Y. Ku and David H. Ilson

Section F Gastrointestinal 72 Cancer of the Stomach,  1197

Geoffrey Y. Ku and David H. Ilson

73 Cancer of the Small Bowel,  1211

Morgan M. Sellers and Alexander J. Greenstein

74 Colorectal Cancer,  1219

Mark Lawler, Brian Johnston, Sandra Van Schaeybroeck, Manuel Salto-Tellez, Richard Wilson, Malcolm Dunlop, and †Patrick G. Johnston

xxxii Contents

75 Cancer of the Rectum,  1281

Scott R. Kelley and Heidi Nelson

76 Cancer of the Anal Canal,  1300

Karyn A. Goodman, Lisa A. Kachnic, and Brian G. Czito

77 Liver and Bile Duct Cancer,  1314

Ghassan K. Abou-Alfa, William Jarnagin, Imane El Dika, Michael D’Angelica, Maeve Lowery, Karen Brown, Emmy Ludwig, Nancy Kemeny, Anne Covey, Christopher H. Crane, James Harding, Jinru Shia, and Eileen M. O’Reilly

78 Carcinoma of the Pancreas,  1342

Ana De Jesus-Acosta, Amol Narang, Lauren Mauro, Joseph Herman, Elizabeth M. Jaffee, and Daniel A. Laheru

Section G Genitourinary 79 Cancer of the Kidney,  1361

Megan A. McNamara, Tian Zhang, Michael R. Harrison, and Daniel J. George

87 Gestational Trophoblastic Disease,  1544 Donald P. Goldstein, Ross S. Berkowitz, and Neil S. Horowitz

88 Cancer of the Breast,  1560

N. Lynn Henry, Payal D. Shah, Irfanullah Haider, Phoebe E. Freer, Reshma Jagsi, and Michael S. Sabel

Section I Sarcomas 89 Sarcomas of Bone,  1604

Megan E. Anderson, Steven G. DuBois, and Mark C. Gebhardt

90 Sarcomas of Soft Tissue,  1655 Brian A. Van Tine

Section J Cancer of Undefined Site of Origin 91 Carcinoma of Unknown Primary,  1694

Gauri Varadhachary and James L. Abbruzzese

Section K Pediatrics

80 Carcinoma of the Bladder,  1382

92 Pediatric Solid Tumors,  1703

81 Prostate Cancer,  1401

93 Childhood Leukemia,  1748

Angela B. Smith, Arjun V. Balar, Matthew I. Milowsky, and Ronald C. Chen William G. Nelson, Emmanuel S. Antonarakis, H. Ballentine Carter, Angelo M. DeMarzo and Theodore L. DeWeese

82 Cancer of the Penis,  1433

Jonathan E. Heinlen, Mohammad O. Ramadan, Kelly Stratton, and Daniel J. Culkin

83 Testicular Cancer,  1442

Terence W. Friedlander and Eric Small

Section H Gynecological 84 Cancers of the Cervix, Vulva, and Vagina,  1468 Anuja Jhingran, Anthony H. Russell, Michael V. Seiden, Linda R. Duska, Annekathryn Goodman, Susanna L. Lee, Subba R. Digumarthy, and Arlan F. Fuller, Jr.

85 Uterine Cancer,  1508

John F. Boggess, Joshua E. Kilgore, and Arthur-Quan Tran

86 Carcinoma of the Ovaries and Fallopian Tubes, 1525

Robert L. Coleman, Jinsong Liu, Koji Matsuo, Premal H. Thaker, Shannon N. Westin, and Anil K. Sood

Jeffrey S. Dome, Carlos Rodriguez-Galindo, Sheri L. Spunt, and Victor M. Santana Stephen P. Hunger, David T. Teachey, Stephan Grupp, and Richard Aplenc

94 Childhood Lymphoma,  1765

John T. Sandlund and Mihaela Onciu

Section L Hematological 95 Acute Leukemias in Adults,  1783 Frederick R. Appelbaum

96 Myelodysplastic Syndromes,  1798

David P. Steensma and Richard M. Stone

97 Myeloproliferative Neoplasms,  1821 Ayalew Tefferi

98 Chronic Myeloid Leukemia,  1836

Hagop Kantarjian and Jorge Cortes

99 Chronic Lymphocytic Leukemia,  1850 Farrukh T. Awan and John C. Byrd

100 Hairy Cell Leukemia,  1872

Mark B. Geyer, Omar Abdel-Wahab, Martin S. Tallman, and Jae H. Park

101 Multiple Myeloma and Related Disorders,  1884 S. Vincent Rajkumar and Angela Dispenzieri

Contents xxxiii

102 Hodgkin Lymphoma,  1911

Nancy Bartlett and Grace Triska

103 Non-Hodgkin Lymphomas,  1926 Jeremy S. Abramson

104 Cutaneous T-Cell Lymphoma and Cutaneous B-Cell Lymphoma,  1948 Christiane Querfeld, Steven T. Rosen, and Madeleine Duvic

105 Adult T-Cell Leukemia/Lymphoma,  1965 Matthew A. Lunning, Neha Mehta-Shah, and Steven M. Horwitz

Index 1975

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P A R T

I

SCIENCE AND CLINICAL ONCOLOGY A. BIOLOGY AND CANCER

13. Genetic Factors: Hereditary Cancer Predisposition Syndromes

22. Lifestyle and Cancer Prevention

2. Intracellular Signaling

14. Genetic and Epigenetic Alterations in Cancer

3. Cellular Microenvironment and Metastases

24. Nicotine Dependence: Current Treatments and Future Directions

C. DIAGNOSIS OF CANCER

4. Control of the Cell Cycle

15. Pathology, Biomarkers, and Molecular Diagnostics

5. Pathophysiology of Cancer Cell Death

16. Imaging

6. Cancer Immunology

D. CLINICAL TRIALS

1. Molecular Tools in Cancer Research

7. Stem Cells, Cell Differentiation, and Cancer 8. Tumor Microenvironment: Vascular and Extravascular Compartment 9. Cancer Metabolism B. GENESIS OF CANCER 10. Environmental Factors 11. DNA Damage Response Pathways and Cancer 12. Viruses and Human Cancer

17. Biostatistics and Bioinformatics in Clinical Trials 18. Clinical Trial Designs in Oncology 19. Structures Supporting Cancer Clinical Trials 20. Oncology and Health Care Policy E. PREVENTION AND EARLY DETECTION 21. Discovery and Characterization of Cancer Genetic Susceptibility Alleles

23. Screening and Early Detection

F. TREATMENT 25. Cancer Pharmacology 26. Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents 27. Basics of Radiation Therapy 28. Hematopoietic Stem Cell Transplantation 29. Gene Therapy in Oncology 30. Therapeutic Antibodies and Immunologic Conjugates 31. Complementary and Alternative Medicine

1 

A. BIOLOGY AND CANCER Molecular Tools in Cancer Research Mauro W. Costa, Muneer G. Hasham, and Nadia Rosenthal

S UMMARY

OF

K EY

P OI N T S

• Our understanding and treatment of cancer have always relied heavily on parallel developments in biologic research. Molecular biology provides the basic tools to study genes involved with cancer growth patterns and tumor suppression. An advanced understanding of the molecular processes governing cell

growth and differentiation has revolutionized the diagnosis, prognosis, and treatment of malignant disorders. • This introductory chapter relates basic principles of molecular biology to emerging perspectives on the origin and progression of cancer and explains newly developed laboratory

Since the last edition of this book was published, advances in our understanding of the basic mechanisms of cancer have continued to inform and refine clinical approaches to prevention and therapy. New prognostic and predictive markers derived from molecular biology can now pinpoint specific genetic changes in particular tumors or detect occult malignant cells in normal tissues, leading to improved technologies for tumor screening and early detection. Diagnostic approaches have expanded from morphologic criteria and single-gene analysis to whole-genome technologies and single-cell genomics imported from other biologic disciplines. A new systemic vision of cancer is emerging, in which the importance of individual mutation has been superseded by an appreciation for higher-order organization and individual genetic background, disrupted by complex interactions of disease-associated factors and gene-environmental parameters that affect tumor cell behavior. Results from these cross-disciplinary investigations underscore the complexity of carcinogenesis and have profoundly influenced the design of strategies for both cancer prevention and advanced cancer therapy. This overview will serve as a foundation of conceptual and technical information for understanding the exciting new advances in cancer research described in subsequent chapters. Since the discovery of oncogenes, which provided the first concrete evidence of cancer’s genetic basis, applications of advanced molecular techniques and instrumentation have yielded new insights into normal cell biology. A basic fluency in molecular biology and genetics has become a necessary prerequisite for clinical oncologists because many of the new diagnostic and prognostic tools currently in use rely on these fundamental principles of gene, protein, and cell function.

OUR UNSTABLE HEREDITY Cancer genetics has classically relied on the candidate-gene approach, detecting acquired or inherited changes in specific genetic loci accumulated in a single cell, which then proliferates to produce a tumor composed of its identical clonal progeny. During the early steps of tumor formation, mutations that lead to an intrinsic genetic instability allow additional deleterious genetic alterations to accumulate. These 2

techniques, including whole-genome analysis, expression profiling, and refined genetic manipulation in and use of genetically diverse animal models, providing the conceptual and technical background necessary to grasp the central principles and new methods of current cancer research.

genetic changes confer selective advantages on tumor cell clones by disrupting control of cell proliferation. The identification of specific mutations that characterize a tumor cell has proved invaluable for analyzing the neoplastic progression and remission of the disease. The emergence of cancer cells is a byproduct of the necessity for continuous cell division and DNA replication to maintain organ functionality throughout the life cycle. The highly heterogeneous nature of tumors, each composed of multiple cell types, led to the formulation of the “cancer stem cell” hypothesis, which posits that only a subpopulation of cancer cells is able to maintain self-renewal, unlimited growth, and capacity for differentiation into other, more specialized cancer cell types. Cancer stem cells display bona fide stem cell markers, in contrast to other cancer cells present in the tumor, which do not have tumorigenic potential. In fact, fewer than 1 in 10,000 cells present in human acute myeloid leukemia are capable of reinitiating a new tumor when transplanted into animals. Cancer stem cells have been identified in many solid tumors in the brain, colon, ovaries, prostate, and pancreas, suggesting that more effective cancer therapies would target these self-renewing cells, rather than the tumor as a whole. The cancer stem cell concept differs from the original clonal evolution hypothesis, which states that every cell in a tumor mass is capable of self-renewal and differentiation, and suggests that detecting and targeting subtle genetic and epigenetic differences that distinguish cancer stem cells may provide a more effective avenue to intervention in disease progression. Heterogeneity can also arise as a result of stochastic mutational events that lead to cancer progression. Clastogenic insults to the genome, or genomic instability due to aberrant gene regulation, could lead to loss of heterozygosity (LOH) of tumor suppressor genes such as TP53, RB1, or BRCA, and can also lead to tumor heterogeneity and change in disease progression. Furthermore, activation of DNA or RNA editing enzymes in tumors could lead to kataegis, a DNA hypermutation process, and increase tumor heterogeneity. Although there are molecular biology tools currently available to detect aberrant but stable genomes, the later processes that lead to genomic instability make diagnosis and prognosis more challenging.

Molecular Tools in Cancer Research  •  CHAPTER 1 3

The functional unit of inherited information in DNA, the gene, is most often represented by a discrete section of sequence necessary to encode a particular protein structure. Gene expression is initiated by forming a copy of the gene, messenger RNA (mRNA), constructed base by base from the DNA template by a polymerase enzyme. Once transcribed, an mRNA transcript is modified and the processed product is transported out of the nucleus. In the cytoplasm, proteins are then synthesized, or translated, in macromolecular complexes called ribosomes that read the mRNA sequence and convert the nucleic acid code, based on three-base segments or codons, into a 20–amino acid code to form the corresponding protein. Although these canonic processes drive gene expression in all normal cells, cancer cells defy the rules. For instance, uracils, which are found

DETECTING CANCER MUTATIONS Methods for mutation detection all rely on the manipulation of DNA, the basic building block of heredity in the cell. DNA consists of two long strands of polynucleotides that twist around each other clockwise in a double helix (Fig. 1.1). Nucleic acid bases attached to the sugar groups of each strand face each other within the helix, perpendicular to its axis. These comprise only four bases: the purines adenine and guanine (A and G) and the pyrimidines cytosine and thymine (C and T). During assembly of the double helix, stable pairings of nucleotides from either strand are made between A and T, or between G and C. Each base pair forms one of the billions of rungs in the long, unbroken ladder of DNA forming a chromosome.

Cell nucleus containing 23 pairs of chromosomes

Genes

DNA strand Chromosomes Sugar Bases

Cytosine thymine Bases

Adenine and guanine Phosphate P group

H

H H H

C

P CH2

H H

CH2

H H

P

H

CH2

CH2

P

P

H

H

CH2

H

C

P

P

H

H H

H

C

Figure 1.1  •  DNA structure. Deoxyribonucleic acid (DNA) is the cell’s genetic material, contained in single compacted strands comprising chromosomes

within the cell nucleus. In the DNA double helix, the two intertwined components of its backbone, composed of sugar (deoxyribose) and phosphate molecules, are connected by pairs of molecules called bases. The sequence of four bases (guanine, adenine, thymine, and cytosine) in the DNA helix determines the specificity of genetic information. The bases face inward from the sugar-phosphate backbone and form pairs with complementary bases on the opposing strand for specific recognition. The arrangement of chemical groups is unique for each base pair, allowing base pairs to be specifically targeted by transcription factors, polymerases, restriction enzymes, and other DNA-binding proteins. (From http://www.terrapsych.com/dna.jpg.)

4 Part I: Science and Clinical Oncology

on RNA, can be detected in the DNA of cancer cells because of their high mutation rates. Paradoxically, these deviations from the norm allow the development of molecular biology tools to better diagnose and predict tumor progression.

GENERATING DIVERSITY WITH ALTERNATE SPLICING In higher organisms, most protein coding gene sequences are interrupted by stretches of noncoding DNA sequences, called introns. In the nucleus, these introns are removed after mRNA transcription to produce a continuous chain of coding sequences, or exons, that subsequently undergo translation into protein. The splicing process requires absolute precision because the deletion or addition of a single nucleotide at the splice junction would throw the three-base coding sequence out of frame, or lead to exon skipping or addition, creating abnormal proteins. The dramatic increase in genetic complexity conferred by alternate RNA splicing is underscored by the multiple splice patterns of many medically relevant genes, in which different combinations of exons are chosen for the final mRNA transcript, such that one gene can encode many different proteins (Fig. 1.2). The choice of protein isoform to be expressed from a gene with multiple splicing possibilities is a decision that can be perturbed in disease. Errors in splicing mechanisms have been associated with a large group of cancers. These include mutations in the oncogene p53 in more then 12 different types of cancer, mutL homolog 1 protein (MLH1) mutation in hereditary nonpolyposis colorectal cancer, and several transcription factors and cell signaling and membrane proteins. When mutations in the splicing site lead to insertion of novel sequences in the mRNA, the encoded protein can be used as a potential clinical marker, as seen for the transcription factor NSFR in small cell lung cancer. Owing to their unique expression in cancer cells, these markers can be further explored as new cancer-specific therapeutic targets.

GENOMICS OF CANCER The complete set of DNA sequences carried on all the chromosomes is known as the genome. Although the general map of the genome

Gene

RNA

is shared by all members of a species, the recent sequencing of thousands of individual human genomes has given rise to the new field of genomics, providing us with new tools to reveal the more subtle variations that arise between individuals. These variations are critical, both as a natural engine driving heterogeneity within a species, and as a source of predisposition to cancer types. The most common forms of human genetic variations, or alleles, arise as single-nucleotide polymorphisms (SNPs). Because these allelic dissimilarities are abundant, inherited, and dispersed throughout the genome, SNPs can be used to track racial diversity, personal traits, and susceptibility to common forms of cancer (Fig. 1.3). Commercial entities have developed tools that can detect thousands of SNPs with relatively little sample material. Platforms such as MegaMUGA or GigaMUGA can allow mammalian genetic mapping that can aid in a number of diagnoses and can distinguish between predictive and prognostic markers. How do SNPs arise between individuals? One source of variation in DNA sequence derives from deviations in the strict base-pairing rule underlying the structure, storage, retrieval, and transfer of genetic information. The duplicated genetic information in the two strands of DNA not only permits the repair of a damaged coding sequence but also forms the basis for the replication of DNA. During cell division, polymerase enzymes unwind the DNA strands and copy them, using the base sequences as a template for constructing a new helix so that the dividing cell passes its entire genetic content on to its progeny. Errors in this process are rare, and person-to-person differences comprise only about 0.1% of the human genome. SNPs are inherited if they occur in the germline. Many genetically inherited variations occur in regions that do not encode protein or alter the regulation of nearby genes. Given the disruptive effects even subtle genetic changes may have on cell function, it is important to distinguish SNPs that represent true mutations from benign polymorphisms. Our ability to monitor hundreds of thousands of SNPs simultaneously is one of the most important advances in modern medical genetics. Relatively simple genotyping technologies for SNP detection rely largely on the polymerase chain reaction (PCR). In procedures that use this reaction, two chemically synthesized single-stranded DNA fragments, or primers, are designed to match chromosomal DNA sequences flanking the segment in which an SNP is positioned. With the addition of nucleotide building blocks and a heat-stable DNA polymerase, the

Exon

Exon

Exon

Exon

Exon

1

2

3

4

5

1

2

3

4

5

Alternative splicing

RNA

1

2

3

4

5

1

2

4

5

1

2

Translation

Translation

Translation

Protein A

Protein B

Protein C

3

5

Figure 1.2  •  RNA splicing. Alternate splicing produces multiple related proteins, or isoforms, from a single gene. (From Guttmacher AE, Collins F. Genomic medicine—a primer. N Engl J Med. 2002;347:1512–1520.)

Molecular Tools in Cancer Research  •  CHAPTER 1 5

Primary tumor

Tens to hundreds of changes between primary and secondary tumors

Primary tumor

SNP density

Metastasis

>10 million SNPs between individuals

Metastasis

11p15.5 11p15.4 11p15.3 11p15.2 11p15.1 11p14.3 11p14.2 11p14.1 11p13 11p12 11p11.2 11p11.12 11p11.11 11q11 11q12.1 11q12.2 11q12.3 11q13.1 11q13.2 11q13.3 11q13.4 11q13.5 11q14.1 11q14.2 11q14.3 11q21 11q22.1 11q22.2 11q22.3 11q23.1 11q23.2 11q23.3 11q24.1 11q24.2 11q24.3 11q25

Figure 1.3  •  Determining cancer susceptibility with single-nucleotide polymorphisms (SNPs). Millions of SNPs exist between individuals, as depicted by the red arrows and the SNP density map of human chromosome 11 (right). By contrast, point mutations, deletions, insertions, and rearrangements between normal tissues and tumors or between primary and secondary tumors probably number in the tens to hundreds (or potentially thousands), as depicted by the spectral karyotype image at the bottom of the figure. Because the constitutional genetic polymorphisms are present in all the tissues of the body, it might be possible to distinguish differences in metastatic versus nonmetastatic tumors and in nontumor tissues before they ever happen to develop a solid tumor. (From Hunter K. Host genetics influence tumour metastasis. Nat Rev Cancer. 2006;6:141–146.)

primer pairs, or amplicons, initiate synthesis of new DNA strands, using the chromosomal material as a template. Each successive copying cycle, initiated by “melting” the resulting double-stranded products with heat, doubles the number of DNA segments in the reaction (Fig. 1.4). The technique is exceptionally sensitive; millions of identical DNA copies can be generated in a matter of hours with PCR by using a single DNA molecule as the starting material. Other novel methods for large-scale SNP detection include singlenucleotide primer extension, allele-specific hybridization, oligonucleotide ligation assay, and invasive signal amplification, which detect polymorphisms directly from genomic DNA without the requirement of PCR amplification. The International HapMap Project was established with the objective of identifying those variations (commonly thought to be on the order of 10 million in our genome) in the human population. This project is already in its third phase (HapMap3), now including both SNPs and copy number variations observed in 1184 samples from 11 different human populations. Regardless of the method used to characterize them, the collective SNPs in a selected genomic region characterize a haplotype, or specific combination of alleles at multiple linked genetic loci along a chromosome that are inherited together. Even when the SNPs within a given haplotype are not directly involved in a disease, they provide markers for clonality and for the loss or rearrangement of specific chromosomal segments in growing tumors. In the human nucleus, each of the 23 tightly compacted chromosomes has a characteristic size and structure, and a distinctive

base sequence that carries unique protein coding information. Other noncoding DNA sequences are used for directing the transcription of neighboring genes, through complex regulatory circuits involving protein binding and modification of the DNA itself, or shifting of its chromosomal packaging. Although genomic instability is generally considered a consequence of tumor formation rather than the initial trigger of cancer, the loss, gain, or rearrangement of chromosomal segments through deletion or translocation is a common form of neoplastic mutation, as protein coding segments from different genes are combined or regulatory sequences are brought into new proximity to genes they do not normally control, as seen in chronic myeloid leukemia (CML). In CML, recombination events lead to the fusion of BCR and ABL genes (Philadelphia chromosome). This results in constitutive activation of the fused gene, leading to loss of proliferative control in myeloid cells and consequently cancer. Gross changes in DNA arrangement can be detected by cytogenetic analysis of chromosomal features on metaphase spreads. Although fluorescence in situ hybridization (FISH) provides greater resolution by localizing specific chromosomal DNA sequences corresponding to fluorescently labeled probes (Fig. 1.5), and can be used to track specific alterations in chromosomal structure where known genes are involved, spectral karyotyping (SKY) is a powerful and more general tool that could aid diagnosis of cancer genomes. With each fluorescently labeled chromosome assigned a specific color, translocations and additions are revealed as multicolored chromosomes, or large deletions as pieces of missing chromosomes.

6 Part I: Science and Clinical Oncology

Primers Heat

Cycle 3

Separation of strands

Primers

Cycle 2

Separation of strands

Sequence to be amplified

Separation of strands

Cycle 1

Heat

Heat

Figure 1.4  •  Amplification of DNA by polymerase chain reaction (PCR).

The DNA sequence to be amplified is selected by primers, which are short, synthetic oligonucleotides that correspond to sequences flanking the DNA to be amplified. After an excess of primers is added to the DNA, together with a heat-stable DNA polymerase, the strands of both the genomic DNA and the primers are separated by heating and allowed to cool. A heat-stable polymerase elongates the primers on either strand, thus generating two new, identical double-stranded DNA molecules and doubling the number of DNA fragments. Each cycle takes just a few minutes and doubles the number of copies of the original DNA fragment.

The plethora of data arising from genome-wide association studies using currently available techniques poses particular challenges to cancer researchers. Discerning the causal genetic variants among genotype-phenotype associations requires extensive replication, control for underlying genetic differences in population cohorts, and consistent classification of clinical outcomes. New technologies must be met with equivalently sophisticated and rigorous analytic methodologies for the true genetic cause of cancer to be teased out from our variable and often unstable heredity.

BUILDING GENE LIBRARIES The engineering of genes by recombinant DNA technology evolved from methods initially devised to provide sequences in amounts sufficient for biochemical analysis. The original protocol involves clipping the desired segment from the surrounding DNA and inserting it into a bacterial or viral vector, which is then amplified millions of times in a host bacterium. Using recombinant DNA technology, genetic engineering can routinely produce industrial quantities of pure, clinically useful products in a cost-effective way. For diagnostic purposes, it is easier and faster to amplify a known genomic DNA sequence directly from a patient sample with PCR, but the classic approach is still applied to the construction of recombinant DNA libraries. To be useful, a DNA library must be as complete as possible, with recombinant members, or clones, sufficiently numerous to include all the sequences in an individual genome. For certain kinds of genelinkage analysis that require long, uninterrupted stretches of DNA, special vectors, such as bacterial or yeast artificial chromosomes, can carry foreign DNA fragments of enormous lengths. Chromosomal segments represented in genomic DNA libraries can contain the

9

9

22

22

9

der(9)

22

der(22)

Figure 1.5  •  Detection of chromosomal translocations. Fluorescence in situ hybridization (FISH) technology uses a labeled DNA segment as a probe to search homologous sequences in interphase chromosomes for the t(9;22)(q34;q11) translocation, associated with chronic myeloid leukemia. On the left, patient nuclei were hybridized with probes for chromosome 9 (labeled with SpectrumRed fluorophore) and chromosome 22 (labeled with SpectrumGreen). (Modified from Varella-Garcia M. Molecular cytogenetics in solid tumors: laboratorial tool for diagnosis, prognosis, and therapy. Oncologist. 2003;8:45–58.)

Molecular Tools in Cancer Research  •  CHAPTER 1 7

structure of an entire gene, including the information that regulates its expression, and formed the starting material for sequencing of the human genome. Many cancer-associated genes were originally identified through use of partial DNA libraries, which contain only the DNA sequences transcribed by a particular tissue or type of cell. The starting material in this case is mRNA. For cloning purposes, the enzyme reverse transcriptase can convert mRNA into complementary DNA (cDNA). The number of clones in a cDNA library is much smaller than in a genomic library because a cDNA library represents only the genes expressed by the tissue of interest and contains exclusively the coding portion of genes. For this particular reason, this technique has now become obsolete for organisms whose genome has now been fully sequenced. New advances in PCR chemistry allowed for the direct cloning of increasingly larger cDNA fragments with high specificity and low error rates. Highly accurate PCR technology, coupled with the constantly evolving generation of genomic sequence maps in humans and model organisms, has exponentially expanded the availability of candidate genes to be tested in cancer biology.

LOSING CONTROL OF THE GENOME Mutations that lead to oncogenic transformation of a cell invariably affect the expression of its genetic information that specifies functional products, either RNA molecules or proteins used for various cellular functions. The primary level of gene control is the transcription of DNA into RNA. Gene regulation, or the control of RNA synthesis, represents a complex process that is itself a frequent target of neoplastic mutation. DNA regulatory sequences do not encode a product. However, without them a cell could not coordinate the expression of the hundreds of thousands of genes in its nucleus, select only certain genes for expression, and activate or repress them in response to precise internal or external signals. These control centers of the genome contain binding sites for multiple proteins, called transcription factors, which interact to form regulatory networks controlling gene transcription. Their function can be altered by signals that induce modifications such as phosphorylation, or by interactions with other regulators such as steroid hormones. Many of the cell’s responses to a wide variety of external stimuli, such as neurotransmitters, antigens, cytokines, and growth factors, are mediated through transcription factors binding to DNA regulatory sequences. Certain regulatory DNA sequences common to many genes are positioned upstream of the transcription start site (Fig. 1.6). Collectively called the “promoter” of a gene, these proximal sequences comprise binding sites for the RNA polymerase and its numerous cofactors. Whereas the position of the promoter with regard to the transcription start site is relatively inflexible, other DNA regulatory elements, known as enhancers, occur in unpredictable locations, often at a considerable distance from the genes they control. Some transcription factors bind to particular regions of enhancers and drive their associated genes in many types of cells, whereas others, active in only a limited variety of cells, maintain a tissue-specific pattern of gene expression. Enhancers are often responsible for the aberrant expression of genes induced by chromosomal translocation associated with specific forms of cancer: a normally quiescent gene promoting cell growth that is dislocated to a position near a strong enhancer may be activated inappropriately, resulting in loss of growth control. Enhancers and promoters have been assigned specific roles by means of cell culture assays or in transgenic animals in which putative regulatory DNA sequences are linked to test or “reporter” genes, and are examined for their ability to activate expression of the reporter gene in response to the appropriate signals. Through assessment of the effects of deleting, adding, or changing DNA sequences within the regulatory element, the precise nucleotides that are critical for recognition by transcription factors can be determined.

The interaction between protein and DNA is increasingly used to identify transcription factor binding sites in a regulatory region. Whereas electrophoretic mobility shift assays (EMSAs), or DNA footprinting, were once standard techniques for determining protein-DNA interactions, emerging genome-wide technologies, such as chromatin immunoprecipitation on microarray chip (ChIP-chip) and chromatin immunoprecipitation on sequencing (ChIP-seq), are revolutionizing the way in which we see the interaction of a transcription factor complex with virtually all of its potential genomic targets in a particular cell state. These strategies involve the use of candidate protein–specific antibodies to pull down DNA targets regulated by them. These targets are further identified with the use of microarray ChIP-chip or next generation sequencing ChIP-seq technologies (see Fig. 1.14). Our appreciation of oncogenic perturbations, by mutation of regulatory protein coding genes or by loss of controlled signaling by cell cycle switches or in the target sequences these proteins recognize, has recently extended to include posttranslational modifications that control protein activity, such as phosphorylation, ubiquitylation, and SUMOylation. Tumor-associated changes in these modifications underscore the multiple levels of control necessary to ensure correct gene expression that is so central to the normal function of the cell.

EPIGENETICS AND CANCER Epigenetics refers to general control of gene expression that is inherited during cell division, although not part of the DNA sequence itself. Epigenetic regulation involves changes in chromatin, a higher-order building block of chromosomes that wraps DNA into coils with scaffolding proteins such as histones. Histones are a necessary component of chromosomal compaction, but also play a critical role in gene accessibility (Fig. 1.7). Active genetic loci are associated with loosely configured euchromatin, whereas silent loci are condensed in heterochromatin. The state of chromatin configuration (euchromatin or heterochromatin) both controls and is controlled by patterns of histone modifications such as methylation and acetylation on specific DNA sequences. This pattern relates the underlying genetic information to its higher-order structure that determines whether a particular gene regulatory element is available to transcription factors (on or off status). These epigenetic modifications of the nuclear environment that determine the accessibility of a gene can persist during cell division, because inherited epigenetic patterns provide permanent marks for altered chromatin configuration in daughter cells. The pattern of modifications generated by the epigenetic code rivals the complexity of the DNA code itself. The accessibility of genomic regions can favor mutations. Enzymes such as the APOBEC family exploit this accessibility to induce C to U mutation, which is then converted to T or staggered single-strand breaks. If not rectified, these point mutations or breaks can lead to hypermutations. Kataegis is an example wherein such hypermutation occurs on the BRCA locus, generating neoplasia. Research has linked rearrangement of chromatin and associated DNA methylation with the inactivation of tumor suppressor genes and neoplastic transformation. Defects that could lead to cancer involve perturbations in the “epigenotype” of a particular locus, through the silencing of normally active genes or activation of normally silent genes, associated with changes in DNA methylation, histone modification, and chromatin proteins (Fig. 1.8). Changes in the number or density of heterochromatin proteins associated with cancer-related genes such as EZH2, or of euchromatic proteins such as trithorax in leukemia, can also be associated with abnormal patterns of methylation in gene promoter regions and with higher-order chromosomal structures that are only beginning to be understood. Finally, it is increasingly evident that interactions among the “epigenome,” the genome, and the environment are common targets for mutation and can have profound effects on the gene expression readout of a cancer cell.

8 Part I: Science and Clinical Oncology

Gene structure Enhancer

Promoter

Exon 1

TATAA

Intron 1

Exon 2 Intron 2

GT AG

Exon 3

GT AG

AATAAA

Gene expression

Transcription factors

RNA polymerase Exon 1

Exon 2

Exon 3

Transcription Transcription-initiation complex

5'

3'

Transcript processing

premRNA

RNA-clipping enzyme

AAUAAA 5' cap PolyA tail AAAA... Adenosine-adding enzyme (terminal transferase)

Intron lariat

Nucleus

Splicing

AAAA... Spliceosome

Cytoplasm

Processed transcript

AAAA...

mRNA

Translation into protein

Figure 1.6  •  Mammalian gene structure and expression. The DNA sequences that are transcribed as RNA are collectively called the gene and include exons

(expressed sequences) and introns (intervening sequences). Introns invariably begin with the nucleotide sequence GT and end with AG. An AT-rich sequence in the last exon forms a signal for processing the end of the RNA transcript. Regulatory sequences that make up the promoter and include the TATA box occur close to the site where transcription starts. Enhancer sequences are located at variable distances from the gene. Gene expression begins with the binding of multiple protein factors to enhancer sequences and promoter sequences. These factors help form the transcription-initiation complex, which includes the enzyme RNA polymerase and multiple polymerase-associated proteins. The primary transcript (pre-mRNA) includes both exon and intron sequences. Posttranscriptional processing begins with changes at both ends of the RNA transcript. At the 5′ end, enzymes add a special nucleotide cap; at the 3′ end, an enzyme clips the pre-mRNA about 30 base pairs (bp) after the AAUAAA sequence in the last exon. Another enzyme adds a polyA tail, which consists of up to 200 adenine nucleotides. Next, spliceosomes remove the introns by cutting the RNA at the boundaries between exons and introns. The process of excision forms lariats of the intron sequences. The spliced mRNA is now mature and can leave the nucleus for protein translation in the cytoplasm. (From Rosenthal N. Regulation of gene expression. N Engl J Med. 1994;331:931–932.)

Molecular Tools in Cancer Research  •  CHAPTER 1 9

Nucleosome

DNA

The solenoid

Figure 1.7  •  Chromatin packaging of DNA. The 4 meters of DNA in every human cell must be compressed in the nucleus, reaching compaction ratios

of 1 : 400,000. This is achieved by wrapping the DNA (blue) around histone protein complexes (green), forming nucleosomes connected by a thread of free linker DNA. Each nucleosome, together with its linker, packages about 200 bp (66 nm) of DNA. The nucleosomes are then coiled into chromatin, a rope of nucleoprotein about 30 nm thick (bottom left electron micrograph). To allow DNA to be accessed by transcription and replication apparatus, chromatin is relaxed (bottom right electron micrograph). (Courtesy Jakob Waterborg. www.umkc.edu/sbs/waterborg/chromat/chromatn.html. Copyright 1998 Jakob Waterborg.)

Gene X

Gene X X

Gene Y X

Gene Y

A

Normal

B

Epigenetic lesions

Figure 1.8  •  Gene accessibility through epigenetics. Illustration depicts known and possible defects in the epigenome that could lead to disease. (A) Gene

X is a transcriptionally active gene with sparse DNA methylation (brown circles), an open chromatin structure, interaction with euchromatin proteins (green protein complex), and histone modifications such as H3K9 acetylation and H3K4 methylation (green circles). Gene Y is a transcriptionally silent gene with dense DNA methylation, a closed chromatin structure, interaction with heterochromatin proteins (red protein complex), and histone modifications such as H3K27 methylation (pink circles). (B) The abnormal cell could switch its epigenotype through the silencing of normally active genes or activation of normally silent genes, with the attendant changes in DNA methylation, histone modification, and chromatin proteins. In addition, the epigenetic lesion could include a change in the number or density of heterochromatin proteins in gene X (such as EZH2 in cancer) or euchromatic proteins in gene Y (such as trithorax in leukemia). There may also be an abnormally dense pattern of methylation in gene promoters (shown in gene X ), and an overall reduction in DNA methylation (shown in gene Y ) in cancer. The insets show that the higher-order loop configuration may be altered, although such structures are currently only beginning to be understood.

10 Part I: Science and Clinical Oncology

PROFILING TUMORS Monitoring global gene expression patterns of cells represents one of the latest breakthroughs in developing a molecular taxonomy of cancer. Although classic blotting and probe hybridization techniques (Northern blot) are still a reliable way to monitor expression of individual genes, these techniques have limitations, such as unequal hybridization efficiency of individual probes, sensitivity for low copy or small transcripts, and difficulty in detecting multiple RNAs simultaneously or in simultaneously analyzing a large number of targets. For cancer studies, it is important to be able to compare the expression pattern of all known RNAs, including noncoding RNAs, between cancer cells and normal cells. Thus new genome-wide analytic techniques are the state-of-the-art choice to detect mRNA expression profiles at a single point in time or cell state. Genome-wide profiling of gene expression in tumors delivers an unprecedented view into the biologic processes

Reference RNA

underlying tumor progression by following the changes in a tumor cell’s transcriptional landscape. With reliance on two-color fluorescence-based microarray technology (DNA microarray), simultaneous evaluation of thousands of gene transcripts and their relative expression can provide a snapshot of the “transcriptome,” the full complement of RNA transcripts produced at a specific time during the progression of malignancy. Transcriptional profiling with microarrays typically involves screens of mRNA expression from two sources (such as tumor and normal cells), using cDNA or oligonucleotide libraries that are arranged in extremely high density on microchips. These are probed with a mixture of fluorescently tagged cDNAs generated from the tumor and normal samples, which results in differential staining of each gene spot. The relative intensity of the two different colors reflects the RNA expression level of each gene; this is analyzed with a laser confocal scanner (Fig. 1.9). With microarrays, single genes that constitute diagnostic, Tumors

Tumor RNA

Genes Statistical analysis

cDNA Hybridization of probe to microarray

A

Donor paraffin block

D

Multidimensional-scaling plot

B

C

Recipient paraffin block

E

Tissue microarray

Figure 1.9  •  Microarray-based expression profiling of tumor tissue. (A) Reference RNA and tumor RNA are labeled by reverse transcription with different fluorescent dyes (green for the reference cells and red for the tumor cells) and hybridized to a cDNA microarray containing robotically printed cDNA clones. (B) The slides are scanned with a confocal laser scanning microscope, and color images are generated with RNA from the tumor and reference cells for each hybridization. Genes upregulated in the tumors appear red, whereas those with decreased expression appear green. Genes with similar levels of expression in the two samples appear yellow. Genes of interest are selected on the basis of the differences in the level of expression by known tumor classes (e.g., BRCA1mutation–positive and BRCA2-mutation–positive). Statistical analysis determines whether these differences in the gene expression profiles are greater than would be expected to occur by chance. (C) The differences in the patterns of gene expression between tumor classes can be portrayed in the form of a color-coded plot, and the relations between tumors can be portrayed in the form of a multidimensional-scaling plot. Tumors with similar gene-expression profiles cluster close to one another in the multidimensional-scaling plot. (D) Particular genes of interest can be further studied through the use of a large number of arrayed, paraffin-embedded tumor specimens, referred to as tissue microarrays. (E) Immunohistochemical analyses of hundreds or thousands of these arrayed biopsy specimens can be performed in order to extend the microarray findings. (From Hedenfalk I, Duggan D, Chen Y, et al. Gene expression profiles in hereditary breast cancer. N Engl J Med. 2001;344:539–548.)

Molecular Tools in Cancer Research  •  CHAPTER 1 11

prognostic, or therapeutically relevant markers can be systematically monitored. Alternatively, the entire set of expressed genes can be collectively analyzed through use of powerful statistical methods to classify tumors according to their transcriptional profile. Microarray analysis has already dramatically improved our ability to explore the genetic changes associated with cancer etiology and development and is providing new tools for disease diagnosis and prognostic assessment. For example, DNA microarray analysis of multiple primary breast tumor transcriptomes has revealed reproducible signature expression of 70 associated genes. These markers have been recently cleared by the US Food and Drug Administration (FDA) for PCR–based diagnostics showing that expression analysis of a relative small gene group can predict the prognosis of early stage breast cancers. When applied on a larger scale, these assays can predict response to chemotherapy, or optimize pharmaceutical intervention by targeting therapeutic approaches to specific patient populations and ultimately to individualized therapy. A novel high-throughput approach for global transcriptome analysis has been made possible by advances in strategies that allow mass sequencing of DNA fragments. With this technique, called RNA-seq, it is now possible to obtain a comprehensive and unbiased analysis of all mRNA transcripts present in cells or tissues. (Fig. 1.10). The

2x poly (A) selection

technique relies on the generation of small fragments of cDNA from any RNA sample, followed by sequencing of these expressed tags from one end (single-end sequencing) or both ends (pair-end sequencing), resulting in fragments of 30 to 400 base pairs (bp). The resulting sequences can be then mapped against the known reference genome or transcriptome of a certain species. Unlike microarray analysis of preselected gene sets, RNA-seq allows the unbiased identification of all genes, or even the presence of different isoforms, expressed in the sample, allowing a comprehensive comparison of transcript levels between normal and cancer cells. The technologies just described can also be applied to the analysis of noncoding RNA species. In addition to the 20,000 protein coding transcripts used to classify a wide variety of human tumors, hundreds if not thousands of small, noncoding interference RNA species, with critical functions in multiple biologic processes, have been discovered; many of these RNA species are directly or indirectly involved in the control of cell proliferation. Known as microRNAs (miRNAs), these short transcripts arise from primary genome-encoded transcripts of variable sizes that are processed into 70- to 100-nucleotide hairpinshaped precursors, which are processed into mature miRNAs of 21 to 23 bp RNA molecules (Fig. 1.11). miRNAs function by base-pairing

RPKM

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Figure 1.10  •  Methods for high-throughput transcriptome analyses. (A) Schematics of regular protocol for RNA-seq sample preparation, showing poly-A

tail specific mRNA isolation followed by fragmentation of RNA into smaller regions, further used for cDNA conversion. Polymerase chain reaction (PCR) fragments are then tethered by adaptors, sequenced by synthesis, and aligned to the reference genome or transcriptome to calculate relative prevalence of mRNAs (RPKM). (B) Target fragments can be used to map exon-intron boundaries and thus infer present and quantify different mRNA isoforms in the sample of interest, as shown for the muscle specific gene Myf6 in this example. (C) Data generated with this method can also be compared with analysis of other tissues or samples, allowing assessment of relative quantification of targets, as exemplified here for a highly specific gene (red peaks) for muscle samples. (From Mortazavi A, Williams BA, et al. Mapping and quantifying mammalian transcriptomes by RNA-seq. Nat Methods. 2008;5:621–628.)

12 Part I: Science and Clinical Oncology

Protein-coding gene

MicroRNA gene

Transcription of pri-microRNA Transcription of mRNA

Pri-microRNA OR

Drosha DGCR8 Processing of pri-microRNAs into pre-microRNA

Nucleus

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Pre-microRNA Exportin 5 Transport of pre-microRNAs into the cytoplasm

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Figure 1.11  •  MicroRNA production and gene regulation in animal cells. Mature functional microRNAs of approximately 22 nucleotides are generated

from long primary microRNA (pri-microRNA) transcripts. First, the pri-microRNAs, which usually contain a few hundred to a few thousand base pairs, are processed in the nucleus into stem-loop precursors (pre-microRNA) of approximately 70 nucleotides by the RNase III endonuclease Drosha and DiGeorge syndrome critical region gene 8 (DGCR8). The pre-microRNAs are then actively transported into the cytoplasm by exportin 5 and Ran-GTP and further processed into small RNA duplexes of approximately 22 nucleotides by the Dicer RNase III enzyme and its partner Loqacious (Loqs), a homologue of the human immunodeficiency virus transactivating response RNA-binding protein. The functional strand of the microRNA duplex is then loaded into the RNA-induced silencing complex (RISC). Finally, the microRNA guides the RISC to the target messenger RNA (mRNA) target for translational repression or degradation of mRNA. (Modified from Chen CZ. New Eng J Med. 2005;353:1768–71.)

with target mRNAs to inhibit translation and/or promote mRNA degradation. In the context of cancer, miRNAs may act in concert with other effectors such as p53 to inhibit inappropriate cell proliferation. A global decrease in miRNA levels is often observed in human cancers, indicating that small RNAs may have an intrinsic function in tumor

suppression. The usefulness of monitoring the expression of miRNAs in human cancer is just now being explored, but preliminary findings reveal an extraordinary level of diversity in miRNA expression across cancers, and the large amount of diagnostic information encoded in a relatively small number of miRNAs. Significant technologic

Molecular Tools in Cancer Research  •  CHAPTER 1 13

advances facilitating the profiling of the miRNA expression patterns in normal and cancer tissues hint at the unexpected greater reliability of miRNA expression signatures than the respective signatures of protein coding genes in classifying cancer types. Along with their potential diagnostic value, miRNAs are also being tested for their prognostic use in predicting clinical behaviors of cancer patients. Because probe specificity in miRNA microarray analysis is problematic owing to the small target size, hybridization can be performed first in solution, and then quantified with multicolor flow sorting. Real-time PCR can also be used to quantify specific miRNA sets, or to capture a more detailed picture of their changing expression profiles in tumor progression. Identification of the miRNAs involved in tumor pathogenesis and elucidation of their action in a specific cancer will be the next necessary steps for their manipulation in a therapeutic setting. Advances in this field have revealed that miRNAs are also involved in cancer initiation and progression, and specific modulation of such RNAs may serve as a therapeutic strategy. Inhibition of key miRNAs using antagomirs (a class of chemically modified anti-miRNA oligonucleotides) has been effective in suppressing tumor growth in mouse models. It remains to be seen if these results can be extended to treatment of cancer in the clinic, but interference with miRNA function is an attractive new tool for the development of cancer therapies.

CANCER PROTEOME The term proteome describes the entire complement of proteins expressed by the genome of a cell, tissue, or organism. More specifically, it is used to describe the set of all the expressed proteins at a given time point in a defined setting, such as a tumor. Like RNA transcription, the synthesis of proteins is a highly regulated process that contributes to the specific proteome of a particular cell and can be perturbed in diseases such as cancer. Advances in protein analytic techniques over the last decades have progressed to the point that even small numbers of specific proteins expressed in tissues can be used to predict the prognosis of a cancer. The improvement of protein-based assays has made it possible to identify and examine the expression of most proteins, and to envision large-scale protein analysis on the level of gene-based screens. Various systematic methodologies have contributed to the current explosion of information on the proteome. These are now being compared for their suitability as platforms for the generation of databases on protein structural features, interaction maps, activity profiles, and regulatory modifications. The yeast two-hybrid system has been a popular genetics-based approach for detecting protein-protein interactions inside a cell (Fig. 1.12). One protein fused to the DNA binding domain (bait) and a

DNA-binding domain fused to protein A

A

A

Promoter

Reporter gene Activator region fused to protein B

B

B

Promoter

Reporter gene

Figure 1.12  •  Exploring protein-protein interactions with the yeast two-hybrid

system. Two-hybrid technology exploits the fact that transcriptional activators are modular in nature. Two physically distinct functional domains are necessary to get transcription: a DNA-binding domain that binds to the DNA of the promoter and an activation domain that binds to the basal transcription apparatus and activates transcription. (A) The known gene encoding protein A is cloned into the “bait” vector, fused to the gene encoding a DNA-binding domain from some transcription factor. When placed into a yeast system with a reporter gene, this fusion protein can bind to the reporter gene promoter, but it cannot activate transcription. (B) Separately, a second gene (or a library of cDNA fragments encoding potential interactors), protein B, is cloned into the “prey” vector, fused to an activation domain of a different transcription factor. When placed into a yeast strain containing the reporter gene, it cannot activate transcription because it has no DNA-binding domain. (C) When the two vectors are placed into the same yeast, a transcription factor is formed that can activate the reporter gene if protein B, made by the second plasmid, binds to protein A. (D) Screening a yeast two-hybrid library. The plate on the left holds 96 different yeast strains in patches (or colonies), each of which expresses a different bait protein (top). The plate on the right holds 96 patches, each of the same yeast strain (prey strain) that expresses a protein fused to an activation domain (prey). The plate of bait strains and the plate of prey strains are pressed to the same replica velvet, and the impression is lifted with a plate containing yeast extract peptone dextrose (YPD) medium. After 1 day of growth on the YPD plate, during which time the two strains mate to form diploids, the YPD plate is pressed to a new replica velvet, and the impression is lifted with a plate containing diploid selection medium and an indicator such as X-Gal. Blue patches (dark spots) on the X-Gal plate indicate that the lacZ reporter is transcribed, suggesting that the prey interacts with the bait at that location. (C from http://www.nature.com/…/journal/ v403/n6770/full/403601a0_r.html. D from Bartel PL, Fields S, eds. The Yeast Two-Hybrid System. New York: Oxford University Press; 1997; Finley RL Jr, Brent R: Two-hybrid analysis of genetic regulatory networks. Retrieved from http://www. genetics.wayne.edu/finlab/YTHnetworks.html.)

DNA-binding domain fused to protein A

C

Activator region fused to protein B A

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Transcription

Promoter 96 Bait strains

Reporter gene 1 Prey strain

Replicate velvet Diploids YPD Replicate velvet

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X-Gal

14 Part I: Science and Clinical Oncology

Direct cytotoxicity

Signal perturbation

as phosphorylation, SUMOylation, or ubiquitination. These LC-MS/ MS systems, such as the iTRAQ, allow for a more precise and individualized diagnosis of cancer. Monoclonal antibodies (mAbs) have been a cornerstone of protein analysis in cancer research, and more recently have risen to prominence as cancer therapeutics based on their exquisite specificity for protein targets and their potent interference with protein function. Novel strategies have been developed that target not only antigens highly expressed in cancer cells but also to enhance the innate immune response against cancer cells. These antibodies can act via several mechanisms, including antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CMC), and antibodydependent cellular phagocytosis (ADCP) (Fig. 1.13). Laboratory mice have been the animal model of choice for generating a ready source of diverse, high-affinity and high-specificity mAbs; however, the use of rodent antibodies as therapeutic agents has been restricted by the inherent immunogenicity of mouse proteins in a human setting. The more recent application of transgenic mouse technology to introduce variable regions encoded by human sequences into the corresponding

Bispecific Toxin

BiTE

Radionuclide Drug

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Nonrestricted activation of cytotoxic cells

different protein fused to the activation domain of a transcriptional activator (prey) are expressed together in yeast cells. If the bait and prey interact, transcription of a reported gene is induced and detected typically by a color reaction that reflects the transactivation of the reporter gene, and by proxy, the interaction of the two test proteins. The method can also be used for large-scale protein interactions, determination of RNA-protein interactions, and proteinligand binding. As a complementary proteomics tool, mass spectrometry (MS) is an accurate mass measurement of charged peptides isolated by twodimensional gel electrophoresis, producing a mass-to-charge ratio of charged samples under vacuum that can be used to determine the sequence identity of peptides. Combined with a specific proteolytic cleavage step, mass spectroscopy can be used for peptide mass mapping. Automation of this process has made mass spectroscopy the analytic tool of choice for many proteomics projects. For diagnostic purposes, liquid chromatography and mass spectrometry (LC-MS/MS) have been combined to detect not only a single–amino acid change in the whole proteome, but also posttranslational protein modification such

Tumor cell death

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Introduction of adaptive immune responses Antigen

BiTE

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Immunotoxin

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CD3

Phagocytic APC Perforin and granzymes

Figure 1.13  •  Mechanisms for antibody-based therapies used against cancer cells. Multiple current approaches involve direct cytotoxicity, Fc-mediated immune effector engagement, nonrestricted activation of cytotoxic T cells, and blockade of inhibitory signaling. The diverse spectrum of action of these therapies will allow the inclusion of various anticancer targets in the near future (From Weiner LM, Murray JC, Shuptrine CW. Antibody-based immunotherapy of cancer. Cell. 2012;148:1081–1084.)

Molecular Tools in Cancer Research  •  CHAPTER 1 15

mouse immunoglobulin genes has enabled the generation of “humanized” therapeutic mAbs with reduced immunogenicity. In addition, bispecific antibodies (bsAbs) with dual affinity for tumor antigens, such as TriomAb, have been shown to effectively kill tumor cells by inducing memory T-cell protective immunity. In addition to the expected use of mAbs directed at extracellular epitopes (protein regions recognized by the antibody), evidence from mouse models has raised the possibility of using antibodies targeting intracellular epitopes for anticancer therapies. Targeting such antigens would enrich immunotherapy, allowing the use of tumor-specific intracellular mediators of cell survival and proliferation. Numerous mAb-based agents are currently in trial or in use as therapeutics for cancer, and the potential for further optimization of mAbs through genetic engineering promises to open new avenues for in vivo therapy. A recent advancement in mAb-based cancer therapy is the generation of chimeric antigen receptor (CAR) T lymphocytes to target tumors in vivo. These are effector T-lymphocytes engineered to express a mAb that recognizes specific groups of cancer cells. The receptors are chimeric, composed of engineered molecules from diverse sources. The first generation of CAR-modified T cells (CAR T cells) showed success in preclinical trials and have entered phase I clinical trials in ovarian cancer, neuroblastoma, and various types of leukemia and lymphoma. Newer generations of these therapeutic lymphocytes are currently

Information content

Analysis and visualization

ChlP-Seq

being developed that have increased specificity toward individualized cancers. From an epigenetic perspective, new techniques are enabling the genome-wide characterization of protein-DNA interactions that can uncover novel transcription factor targets, histone modifications, and DNA methylation patterns within a cancer cell. Combining chromatin immunoprecipitation (ChIP) with microarray (ChIP-chip) allows genome-wide screening for the binding position of protein factors to their gene targets. In ChIP-chip assays or ChIP-seq, a cross-linking reagent is applied in vivo to proteins associated with DNA in the nucleus, which then can be coimmunoprecipitated with specific antibodies to the protein under analysis. The bound DNA and appropriate controls are then fluorescently labeled and applied to microscopic slides for microarray analysis, or directly sequenced, rendering a simultaneous profile of all the binding positions of specific proteins in the cancer cell’s genome (Fig. 1.14). The global profiling of promoter occupancy of specific cancers, wherein protein-DNA interaction profiles discriminate patients with tumors from those presenting different clinical outcomes, is a promising predictive method. After a decade of development, proteomics is still primarily a basic research activity, yet in the near future this technology is likely to have a profound impact on medicine. By defining the collective

Peak calling

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Figure 1.14  •  Methods for unbiased identification of transcription factor binding sites. Chromatin immunoprecipitation on sequencing (ChIP-seq) and

chromatin immunoprecipitation on microarray chip (ChIP-chip) can provide location, isolation, and identification of the DNA sequences occupied by specific DNA-binding proteins in cells. Proteins capable of DNA interactions are targeted with specific antibodies. DNA and the associated proteins are cross-linked; DNA is fragmented into 150 to 500 bp and immunoprecipitated. After reversion of the cross-link, DNA is isolated and either mass-sequenced (ChIP-seq) or used as probes in a genomic array (ChIP-chip), and binding sites occupied by the proteins can be identified in the genome. These binding sites may indicate functions of various transcriptional regulators and help identify their target genes during development and disease progression. The types of functional elements identified with these techniques include promoters, enhancers, repressor and silencing elements, insulators, boundary elements, and sequences that control DNA replication. (From Kim TH, Ren B. Annu Rev Genomics Hum Genet. 2006;7:81–102 and Liu et al. BMC Biol. 2010;8:56.)

16 Part I: Science and Clinical Oncology

Human Transcriptional regulatory network

Virus-host network

Drosophila Yeast C. elegans Arabidopsis Metabolic network

Protein-protein interaction

Disease network Alzheimer’s disease Hypertension Atherosclerosis

Pseudohypoaldosteronism

Asthma

Figure 1.15  •  Interactome networks and human disease. Networks are integrated sources of information obtained from biochemical, molecular, proteomic, and other high-throughput analyses. Different networks can be obtained for each organism, organ, or cell. In the first instance, central regulatory “nodes” identify important components in the network. These networks and their data can then be integrated and compared with healthy and disease models, allowing an integrative view of events that is much more powerful than isolated networks. (Modified from Vidal M, Cusick ME, Barabási A. Interactome networks and human disease. Cell. 2011;144:986–998.)

protein-protein interactions in a cancer cell (its “interactome”), functional relationships between disease-promoting genes may be revealed that provide novel candidates for intervention (Fig. 1.15). Networks of disorder-gene associations are already being built that offer a platform for describing all known phenotype and disease-gene associations, often indicating the common genetic origin of many diseases. A precise diagnosis of cancer through use of proteomics can be envisioned, based on highly discriminating patterns of proteins in easily accessible patient samples. Proteomics information also promises to provide sophisticated mathematical models of the molecular events underlying a process as complex as neoplastic transformation, which will capture the dynamics of the disease with unprecedented power.

MODELING CANCER IN VIVO Once the mechanistic underpinnings of a particular cancer have been described, creating an animal model to test that mechanism becomes critical to understanding the pathophysiology and to design therapeutic strategies for treatment. Advances in manipulation of the mouse genome have resulted in more sophisticated models of human cancer. These methodologies can circumvent embryonic death by targeted alteration of gene expression only after a critical period in development, and reduce the complexity of gene functional analysis by restricting its pattern of activation. Inducible gene expression or silencing also allows acute, as opposed to chronic, effects to be assessed. Although species differences in tumor susceptibility and disease remission exist between mouse and man, the tools for genetic manipulation in mouse are superior to those in other mammals, and useful information about the function of oncogenes can be gained by targeted expression of mutant protein products in mouse tissues. A major hurdle in generation of clinically relevant mouse models to develop cancer treatments stems from the lack of patient tailoring. Cancer cells present a highly heterogeneous population that varies with the genetic makeup of the individual patient. This shortcoming has been addressed with the advent of patient-specific avatars, also known as personalized mouse models or patient-derived xenograft (PDX) models (Fig. 1.16). Implanting patient biopsy specimens into immunodeficient mice allows growth of the tumor, generating in vivo precision models without further in vitro manipulation of the tumor tissue. These models show great promise for designing treatment and drug tests that should best target the patient-specific tumor. Most

recently, PDX models have been further optimized with the use of humanized host mice that are modified to contain human immune systems.

TRANSGENIC MODELS OF CANCER Integrating an oncogene that causes malignancy into the genome of a mouse without altering the mouse’s own genes generates a transgenic, cancer-prone mouse that transmits this trait to its offspring with a dominant pattern of inheritance. The technology for producing transgenic mice joins recombinant DNA methodology with standard techniques that are used today by in vitro fertilization clinics, relying on the understanding of mammalian reproduction and the development of protocols to harvest, manipulate, and reimplant eggs and early embryos (Fig. 1.17). The transgene is constructed so that the gene product will be expressed under appropriate spatial and temporal control. In addition to all the standard signals necessary for efficient transcription and translation of the gene, transgenes contain a promoter, or regulatory region, that drives transcription in either a ubiquitous or a tissue-restricted pattern. This requires an extensive knowledge of genetic regulation in the target cells. A recent advance that circumvents this requirement involves embedding the transgene inside another gene locus that is expressed in the desired pattern. Held in a bacterial artificial chromosome (BAC) for easier manipulation, this long stretch of DNA surrounding the host gene is likely to carry all the necessary regulatory information to guarantee a predictable expression pattern of the introduced transgene. The transgene DNA is then injected into the male pronucleus of a fertilized mouse egg, obtained from a female mouse in which hyperovulation has been hormonally induced. The injected eggs are cultured to the two-cell stage and then implanted in the oviduct of another recipient female mouse. Transgenic pups are identified by the presence of the transgene in their genomic DNA (obtained from the tip of the tail and analyzed with PCR assay). Typically, several copies of the transgene are incorporated in a head-to-tail orientation into a single random site in the mouse genome. About 30% percent of the resulting pups will have integrated the transgene into their germline DNA and constitute the founders of the transgenic lines. RNA analysis of their progeny determines the level of transgene expression, and whether the transgene is being expressed in the desired location or at the appropriate time. Given the variability in transgene number and chromosomal location, transgene expression patterns and levels can

Molecular Tools in Cancer Research  •  CHAPTER 1 17

A

B

Figure 1.16  •  Mouse avatar (PDX) models. (A) Patient-derived xenograft (PDX) mice are generated by implanting patient tumors into immunodeficient/ humanized mice. The tumors can then be propagated for several passages in fresh mice for a number of generations. (B) Usually, after the third generation the tumors can be isolated and characterized for further study. These mice can potentially be used for patient drug-specific testing and molecular characterization, therefore allowing for personalization of cancer treatment. (From http://www.the-scientist.com/?articles.view/articleNo/42470/title/My-Mighty-Mouse/.)

diverge considerably among different founder lines carrying the same transgene. In general, transgenesis is optimal for modeling oncogenic mutations that cause a gain of function, producing disease even when they occur in only one of a gene’s two alleles. For example, an activating mutation in a growth factor that causes abnormal cell proliferation can be mimicked by introducing a transgenic version of the mutated growth factor gene under the control of an appropriate regulatory sequence for expression in the tissue of interest. The relative susceptibility of such a transgenic mouse to tumorigenesis can help distinguish between a primary and secondary role of the mutant factor, and established lines of these animals can be used for testing new therapeutic protocols.

CONDITIONAL CONTROL OF ONCOGENE ACTIVATION The genetic construction of cancer-prone transgenic mice with the capacity to induce oncogene expression in vivo provides a new avenue to modeling the role of oncogenes in tumor generation and maintenance. This technology relies on conditional mutagenesis. Producing conditional mutations in mice requires a DNA recombinase enzyme that does not recognize any mouse sequence, but rather targets short, foreign recognition sequences to catalyze recombination between them. By strategic placement of these recognition sequences in appropriate orientations either beside or within a mouse gene, the recombination results in deletion, insertion, inversion, or translocation of associated genomic DNA (Fig. 1.18). Two recombinase systems are currently in use: the Cre-loxP system from bacteriophage P1, and the Flp-FRT system from yeast. The 34 bp loxP or FRT recognition sequences do not occur in the mouse genome, and both Cre and Flp recombinases function autonomously, without the need for cofactors. Cre- or Flpmediated recombination is not distance or cell-type dependent, and can occur in proliferating or differentiated tissues.

The general scheme involves two mouse lines, one carrying the recombinase either as a transgene driven by inducible regulatory elements or knocked into one allele of a gene expressed in the desired tissue. The other mouse line harbors a modified gene target including recognition sequences. Mating the two lines results in progeny carrying both the target gene and the recombinase, which interacts with the target gene only in the desired tissue. A popular conditional methodology is based on the activation of nuclear hormone receptors to control gene expression. Two current systems involve activation of a mammalian estrogen receptor, estrogen analogue 4-hydroxy-tamoxifen, or an insect hormone receptor with the corresponding ligand ecdysone. Although several variations on these hormone-receptor systems are currently in use, the underlying principle is the same. The Cre recombinase gene, or another regulatory protein, such as a transcription factor, is fused with the ligand-binding domain (LBD) from a nuclear hormone receptor protein. The resulting chimeric transgene is placed under the control of a promoter that directs expression to the tissue of interest, and transgenic animals are generated. In the absence of the hormone or an analogue, the fusion protein accumulates in the desired tissue but is rendered inactive through its association with resident heat shock proteins. Hormone, administered either systemically or topically, binds to the LBD moiety of the fusion protein, dissociates it from the heat shock protein, and allows the transcriptional regulatory component to find its natural DNA targets and promote lox-P mediated recombination, or in the case of an inducible transcription factor, activate expression of the corresponding genes. If the LBD is fused to a recombinase, administration of hormone leads to the rearrangement of target sequences. This reaction is not reversible, but lends additional temporal control over recombinase-based mutation. If the LBD is fused to a transcription factor, removal of hormone leads to inactivation of the fusion protein and gene downregulation. Another inducible method in use is the tetracycline (tet) regulatory system. In the classic design (tTA or tet-off ), a fusion protein

18 Part I: Science and Clinical Oncology

Promoter

5' UT

Coding region 2' UT

Figure 1.17  •  Generation of transgenic mice. The transgene containing

3' Flanking region

the DNA sequences necessary for the expression of a functional protein is injected into the male (larger) pronucleus of uncleaved fertilized eggs through a micropipette. The early embryos are then transferred into the reproductive tract of a mouse rendered “pseudopregnant” by hormonal therapy. The resulting pups (founders) are tested for incorporation of the transgene by assaying genomic DNA from their tails. Founder animals that have incorporated the transgene (+) are mated with nontransgenic mice, and their offspring are mated with each other to confirm germline integration and to establish a line of homozygous transgenic mice. Several transgenic lines that have incorporated different numbers of transgenes at different integration sites (and thus express various amounts of the protein of interest) are usually studied. UT, Untranslated. (From Schuldiner AR. Transgenic animals. N Engl J Med. 1996;334:653–655.)

Transgene

Collection of fertilized eggs from a superovulated donor mouse

Cell type specific promoter

Cre

loxP

Target gene

loxP

X

Injection of transgene into male pronucleus of uncleaved fertilized egg

Transfer of early embryos into reproductive tract of a pseudopregnant mouse

A

Special cell type

CMV-β actin promoter

–

βgeo

– +

+

loxP –

All other cells

3PA Cre

EGFP loxP

CMV-β actin promoter

+ Assay of genomic DNA from tails of founder animals for incorporation of the transgene

Cre

Cre

B

EGFP loxP

Figure 1.18  •  Conditional mutagenesis schemes. (A) Two mouse lines are

Sequential matings to determine germline integration Study of phenotype

required for conditional gene deletion: first, a conventional transgenic mouse line with Cre targeted to a specific tissue or cell type; and second, a mouse strain that embodies a target gene (endogenous gene or transgene) flanked by two loxP sites in a direct orientation (“floxed gene”). Recombination (excision and consequently inactivation of the target gene) occurs only in those cells expressing Cre recombinase. Hence, the target gene remains active in all cells and tissues that do not express the Cre recombinase. (B) The Z/EG double reporter system. These transgenic mice constitutively express lacZ under the control of the cytomegalovirus enhancer/chicken actin promoter. Expression is widespread, with notable exceptions being liver and lung tissue. Expression is observed throughout all embryonic and adult stages. When crossed with a Cre recombinase-expressing strain, lacZ expression is replaced with enhanced green fluorescent protein expression in tissues expressing Cre. This double reporter system makes it possible to distinguish a lack of reporter expression from a lack of Cre recombinase expression while providing a means to assess Cre excision activity in live animals and cells. (A Courtesy Kay-Uwe Wagner, National Institutes of Health; B from Novak A, Guo C, Yang W, Nagy A, Lobe CG. Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis. 2000;28:147–155.)

Molecular Tools in Cancer Research  •  CHAPTER 1 19

combining a bacterial tet repressor and a viral transactivation domain drives expression of the target transgene by binding to upstream tet operator sequences flanking the transgene transcription start site. In the presence of the antibiotic inducer, the fusion protein is dissociated from the operator sequences, inactivating the transgene. In a complementary design, called reverse tetracycline-controlled transactivator (rtTA or tet-on), structural modification of the tet repressor makes the antibiotic an active requirement for binding of the fusion protein to the operator sequences, such that its administration activates transgene expression at any time during the life span of the mouse, whereas withdrawal results in downregulation of the gene. It is important that the transgene integrate into a genomic locus that permits proper tTA or rtTA regulation so that the system exhibits minimal “intrinsic leakiness” and good antibiotic responsiveness. Conditional expression systems have already been developed to generate hematopoietic, leukemogenic, and lymphomagenic mutations in the mouse, as well as solid tumors. These inducible cancer models can be exploited to identify oncogenic signals that influence host-tumor interactions, to establish the role of a given oncogenic lesion in advanced tumors, and to evaluate therapies targeted toward cancer-causing mutations. Potential clinical application of inducible systems include targeting virally delivered transgene expression to malignant tissues by the use of specific inducible regulatory elements, restricting the expression of transgenes exclusively to affected tissues, and increasing the therapeutic index of the vectors, particularly in the context of solid tumors. In all cases, a basic knowledge of the specific mutations involved in the molecular genetics of malignancies is required because it is often unclear that the causal mutation underlying the genesis of neoplasia continues to play a central role in the progression to the fully transformed state. This is particularly important in modeling cancers characterized by genetic plasticity, wherein drug resistance can arise subsequent to primary tumor formation.

MODELS OF RECESSIVE GENE MUTATIONS IN CANCER In contrast to dominantly acting oncogenes, recessive genetic disorders, such as loss-of-function mutations in tumor suppressor genes, require both copies (alleles) of a gene to be inactivated. The methods needed to produce animal models of recessive genetic disease differ from those used in studying dominant traits. Gene knockout technology has been developed to generate mice wherein one allele of an endogenous gene is removed or altered in a heritable pattern (Fig. 1.19). Gene disruption or replacement is first engineered in pluripotential cells, termed embryonic stem cells (ESCs), which are genetically altered by introduction of a replacement gene that is inactive or mutant. To reduce random integration of the foreign DNA, the replacement gene is embedded into a long stretch DNA from its native locus in the mouse, which targets the recombination event to the homologous position in the ESC genome. Inclusion of selectable markers along with the replacement gene allows selection of the cells in which homologous recombination has taken place. Site-specific recombinase systems combined with gene targeting techniques in ESCs can also be used to induce recessive single point mutations or site-specific chromosomal rearrangements in a tissue- and time-restricted pattern. In a variation on this theme called knockin, a foreign gene, such as one encoding a marker or a mutated gene, can be placed in the locus of an endogenous gene. The engineered ESCs are then microinjected into the cavity of an intact mouse blastocyst sufficiently early in gestation that they can, in principle, populate all the tissues of the developing chimeric embryo. This is rarely the case, so contribution of ESCs to the resulting animal is most often assessed with use of ESCs and blastocysts whose genes for coat color differ. If the ESCs contribute to the germ cells of the founder mouse, their entire haploid genome can be passed on to subsequent generations. Through mating together of subsequent progeny of the founder mouse, both alleles of the mutated gene can be passed to a single animal.

Overlapping genetic functions can also be defined by crossbreeding mice with mutations in different genes. In this way it is possible to study the combinatorial effects of oncogene and tumor suppressor gene mutations. Several caveats are important in considering the use of knockout technology in modeling cancer. Most knockouts generate loss-offunction (null) germline mutations. Inactivation of widely expressed genes with multiple functions may have complex phenotypes. Conversely, if the functions of two genes overlap, a mutation in one of the genes may not produce an abnormal phenotype, owing to compensation by the unaltered partner. Perhaps the greatest drawback of conventional knockout technology derives from the disruption of gene function at the earliest stage of its expression. If the gene has a vital developmental role, the identification of functions later in development can be occluded. Therefore, although the generation of a null mutation is an excellent starting point for analysis, it is far from being functionally exhaustive. For these reasons, conditional mutagenesis is the emerging method of choice for the elucidation of the gene functions that exert pleiotropic effects in a variety of cell types and tissues throughout the life of the animal, which is particularly relevant for the generation of mouse models of adult-onset diseases such as cancer. Use of recombinase-mediated gene mutation as described earlier for conditional transgenesis, conditional knockout mutations can be designed to disrupt the function of a target gene in a specific tissue (spatial control) and/or life stage (temporal control). Depending on the design of the experiment, recombinase action can delete an entire gene, remove blocking sequences to induce gene expression, or rearrange chromosomal segments. With the advent of recent internationally coordinated systematic mutagenesis programs aiming to place a conditional inactivating mutation in each of the 20,000 genes in the mouse genome, the possibilities for modeling cancer are limited only by a researcher’s choice of the gene loci to test. The constantly evolving techniques for gene manipulation in vivo constitute a major advance in cancer research. Genetically modified mice are of great value in dissecting the pathogenesis of many tumor types. In some knockout studies, the phenotype of the mutated gene is anticipated by prior knowledge of the gene’s function. However, unexpected mutant phenotypes may help clarify the mechanism of the underlying neoplasia. Pharmacologic manipulation of transgenic, knockout, diversified animal models of cancer will prove useful in screening therapeutic agents with potential for study in clinical trials. Therapy involving gene or cell replacement can be also tested in genetically engineered disease models.

EXPLOITING MOUSE DIVERSITY FOR CANCER RESEARCH A novel in vivo tool has emerged that aids in understanding the etiology of cancers, by more accurately reflecting the broad genetic variability in the human population. Cancer research performed with mice has largely focused on a few individual highly inbred strains with limited genetic diversity, which would equate to single individuals in the population. Yet drugs designed to treat one individual are often not effective in other patients. The Collaborative Cross (CC) was created to provide mouse models that better represent the diversity seen in natural human populations while still retaining the broad power of genetic analysis seen in mice. The CC resource is a large panel of recombinant inbred (RI) strains generated by randomly mixing the genetic diversity of eight extant inbred mouse lines, and can be used to test the impact of treatments in a diverse genetic pool akin to the human population (Fig. 1.20). A related resource, the Diversity Outcross (DO), offers higher mapping resolution by randomized outcrossing of partially inbred CC strains, which segregates the same allelic variants but embeds them in a distinct population architecture in which each

20 Part I: Science and Clinical Oncology

Embryonic stem cell Tumor suppressor gene 5' Homologous region Intron

3' Homologous region Cellular gene

Embryonic stem cell culture

pgk-neo

pgk-tk

Plasmid DNA Knockout vector

Homologous recombination

Cellular gene replaced

Selection by neomycin and ganciclovir

Injection of embryonic stem cells into host blastocyst

Implantation of chimeric blastocyst in foster mother

Germline offspring

Chimeric offspring

Figure 1.19  •  Gene knockout strategies. Embryonic stem cells (upper left panel) contain the tumor suppressor cellular gene (upper right panel), which

consists of exon 1 (olive green, a 5′ noncoding region), an intron, and exon 2 (red, a protein coding region, and yellow, a 3′ noncoding region). A knockout vector—consisting of a collinear assembly of a DNA flanking segment 5′ to the cellular gene (blue), the phosphoglycerate kinase–bacterial neomycin gene (pgk-neo, violet), a 3′ segment of the cellular gene (yellow), a DNA flanking segment 3′ to the cellular gene (green), and the phosphoglycerate kinase–viral thymidine kinase gene (pgk-tk, orange)—is created and introduced into the embryonic–stem cell culture. Double recombination occurs between the cellular gene and the knockout vector in the 5′ homologous regions and the 3′ homologous regions (dashed lines), resulting in the incorporation of the inactive knockout vector, including pgk-neo but not pgk-tk, into the cellular genomic locus of the embryonic stem cell. The presence of pgk-neo and the absence of pgk-tk in these replaced genes will allow survival of these embryonic stem cells after positive-negative selection with neomycin and ganciclovir. The clone of mutant embryonic stem cells is injected into a host blastocyst, which is implanted into a pseudopregnant foster mother and subsequently develops into a chimeric offspring (bottom). The contribution of the embryonic stem cells to the germ cells of the chimeric mouse results in germline transmission of the embryonic stem cell genome to offspring that are heterozygous for the mutated tumor suppressor allele. The heterozygotes are mated to produce mutant, cancer-prone mice homozygous for tumor suppressor deficiency. (Modified from Mazjoub JA, Muglia LJ. Knockout mice. N Engl J Med. 1996;334:904–906.)

Molecular Tools in Cancer Research  •  CHAPTER 1 21 Founder strains

Diversity Outbred (DO)

A/J C57BL/6J

Outbreeding

129S1/SvImJ NOD/ShiLtJ Collaborative Cross (CC) Inbreeding

NZO/H1LtJ CAST/EiJ PWK/PhJ WSB/EiJ

A

B Figure 1.20  •  Generation and characteristics of Collaborative Cross (CC) and Diversity Outbred (DO) mice. (A) Each CC line originates from intercrosses obtained from eight founder lines. Individual unique independent line is inbred for at least 15 generations, creating individual CC lines. Together, those lines represent a much broader genetic variation when compared with the parental lines and therefore are a better representation of natural populations but contain a high degree of homozygosity. Random mating from early 144 CC crosses leads to a highly genetic heterogeneous outbred population that more closely resembles the diversity found in human populations. (B) Images of mice representing CC lines. (From Centre for Diabetes Research, Harry Perkins Institute of Medical Research, Nedlands 6009, W.A Australia” and “School of Medical and Health Sciences, Edith Cowan University, Joondalup W.A. 6027 Australia.)

animal has a high degree of heterozygosity and carries a unique combination of alleles. These diversified mice have been shown to be a powerful tool in determining the etiology of cancers. In a recent study, analysis of mice generated by crossing CC strains with a mouse line carrying a mutated tumor suppressor APCmin showed that colon cancer frequency and spectrum varied predictably with genetic background. Identification of these genetic modifiers of cancer genesis or suppression would inform the design of novel mouse cancer models, potentially yielding genetic markers that can predict human cancers.

FUTURE VIEW Recent discoveries that cancer stem cells share essential signaling nodes with normal stem cells suggest that targeting critical steps in these pathways may lead to improved alternative cancer therapies. However, every human tumor is subtly different. We are now fast approaching a new era in medicine: creating “tailored” treatments to individual tumors by obtaining integrative “personal omics profiles” (Fig. 1.21). The generation of novel mouse strains that represent the diversity seen in human populations will likely lead to a more tailored approach in understanding cancer diversity and therefore will allow the development of more efficient treatments. Understanding of underlying molecular biologic principles of malignancy, with pathophysiologic consequences, will generate an invaluable resource for resolution of complex genetics of tumor

formation and holds great promise for improved treatment of human cancer. Chemotherapy still has numerous side effects. As a number of oncologists may testify, patients sometimes forgo treatment because of its toxic nature. In a number of cases of chronic lymphocytic leukemia, if the symptoms of the disease are “under control,” treatment is not prescribed. Therefore it is important to find alleviation strategies and cures that minimize the side effects. The goal should be to cure the patient without devastating the person. One future avenue, apart from finding treatment regimens that reduce side effects, is the discovery of chemotherapies with minimal side effects. The nature of several generations of chemotherapy was to attack cellular processes, such as DNA replication, metabolism, and cell division, with brute force, and by trial and error find a balance between alleviating the neoplastic growth and not interfering with the normal processes. It may be time to rethink that premise. We now see examples wherein a less potent chemotherapy elicits a similar effect on a cancer as a potent one, although the less potent compound may take longer to achieve its effect. However, because of its low potency, the side effects are minimal. Therefore, in the future, the success of a therapy needs to be measured not only in terms of how quickly a patient is cancer free, but how well the patient is during and after the treatment. With emerging technologies we will be able to fine-tune current successful therapies so that treatment is not so burdensome. In any field of medicine, resistance to the therapy occurs. Evolutionary processes show that predation leads to natural selection of species with mutations that avoid their elimination. Similarly, in cancers, during the predation—chemotherapy—clones arise that become resistant to the therapy. With emerging technologies such as single-cell deep sequencing, we will be able to not only detect the rare subclone that could give rise to resistance, but also predict the probability of the development of resistance by sequencing certain markers. For instance, clones with mutations in DNA repair pathways have a higher probability for genomic instability, which is a precursor for the rise of resistant clones. While we pursue new technologies to diagnose patients and understand the molecular nature of the cancers, an emerging trend will be to reexamine previous formulated hypotheses or treatments that could not be tackled before because of the lack of technologies or resources. For instance, it has been long thought that genetic variation plays an important role in cancer treatment. This premise was observed in human clinical trials, wherein conditions in some populations were refractory to certain treatments. In the future, we would be able to predict the extent to which a treatment would either work or fail in certain population by using diverse mice, and to map alleles that confer resistance or susceptibility of a tumor before reaching human clinical trials. Another example is processes that lead to neoplasticity, such as LOH, which have been studied in cell lines that are homogeneous in nature. Attempts to use primary cells were not possible because of polyclonality of cells and the short life span of tumor cells in vitro. With all the aforementioned technologies, we now can, and need to, reexamine older hypotheses with the appropriate type of primary cells. We will probably uncover novel processes of cancer that were masked, and resolve decades-old controversies and competing hypotheses, leading to better understanding and cures for cancer. In the future, fields of medicine will continue to marry and merge; this has been exemplified in numerous examples, some mentioned in this chapter—for instance, the use of viruses to deliver chemotherapy, or the use of engineered T cells to attack cancer. As emerging molecular tools uncover novel processes in different fields of medicine, the cross talk between oncology and these other fields will continue, enabling discovery of new avenues to alleviate or even cure cancers.

22 Part I: Science and Clinical Oncology

SAMPLE TYPE

METHOD

ANALYSES

Whole-genome sequencing

Variant calling/phasing Heteroallelic and variant expression

Whole-transcriptome sequencing (mRNA and miRNA)

PBMC

RNA editing

Serum

A

Proteome profiling

Variant confirmation in RNA and protein

Untargeted proteome profiling

Quantitative differential expression and dynamics

Targeted proteome profiling (cytokines)

Quantitative expression

Metabolome profiling

Dynamics

AutoAntibodyome profiling

Differential reactivity

Medical/lab tests

Glucose, HbA1c, CRP, Telomere length

3

Integrated personal OMICS

Quantitative differential expression & dynamics

4

5

2

Serpina 1 E366K

RNA edits

6

1

Heteroallelic SNVs

7

Protein-downregulated (HRV vs healthy)

Y

Protein-upregulated (HRV vs healthy) 8

RNA-downregulated (HRV vs healthy)

X

RNA-upregulated (HRV vs healthy) 22

9

Indels

21

SV-duplications 10

20

SV-deletions

19

Chr. ideogram

18

11 17

B

14 16

Chr. number

12

15

14

13

Figure 1.21  •  Integrative personal omics profiles (iPOP). (A) Integrative analysis of iPOP experimental design, indicating tissues and techniques analyzed in healthy and diseased individuals. (B) Circos plot summarizing iPOP. From outer to inner rings: chromosome ideogram; genomic data (pale blue ring), structural variants (deletions [blue tiles], duplications [red tiles]), indels (green triangles); transcriptomic data (yellow ring); proteomic data (light purple ring). (Modified from Chen R, Mias GI, et al. Personal omics profiling reveals dynamic molecular and medical phenotypes. Cell. 2012;148:1293–1307.)

Molecular Tools in Cancer Research  •  CHAPTER 1 23

SUGGESTED READINGS Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular Biology of the Cell. 4th ed. London, UK: Taylor and Francis Group; 2002. Chen CZ. MicroRNAs as oncogenes and tumor suppressors. New Eng. J. Med. 2005;353:1768–1771. Feinberg AP. Phenotypic plasticity and the epigenetics of human disease. Nature. 2007;447:433–440. Frese KK, Tuveson DA. Maximizing mouse cancer models. Nat Rev Cancer. 2007;7:654–658. Goh KI, Cusick ME, Valle D, Childs B, Vidal M, Barabasi AL. The human disease network. Proc Natl Acad Sci USA. 2007;104:8685–8690.

Hunter K. Host genetics influence tumour metastasis. Nat Rev Cancer. 2006;6:141–146. Malaney P, Nicosia SV, Davé V. One mouse, one patient paradigm: new avatars of personalized cancer therapy. Cancer Lett. 2014;344:1–12. Pecorino L. Molecular Biology of Cancer: Mechanisms, Targets, and Therapeutics. USA: Oxford University Press; 2005. Svenson KL, Gatti DM, Valdar W, Welsh CE, Cheng R, Chelser EJ, et al. High resolution genetic mapping using the mouse diversity outbred population. Genetics. 2012;190:437–447.

The Complex Trait Consortium. The Collaborative Cross, a community resource for the genetic analysis of complex traits. Nat Genet. 2004;36:1133–1137. Weinberg RA. Biology of Cancer. Garland Science; 2006. Rosenthal N, Brown S. The mouse ascending: perspectives for human-disease models. Nat Cell Biol. 2007;9:993–999. Wu J, Smith LT, Plass C, Huang TH. ChIP-chip comes of age for genome-wide functional analysis. Cancer Res. 2006;66:6899–6902.

2 

Intracellular Signaling Aphrothiti J. Hanrahan, Gopa Iyer, and David B. Solit

S UMMARY

OF

K EY

P OI N T S

• Ligand binding and activation of cell surface and internal receptors trigger the activation and/or suppression of signaling cascades that regulate diverse cellular processes including cell growth, proliferation, survival, and invasion, among others. • Multiple nodes within these intracellular signaling networks are genetically and epigenetically altered in human cancers, leading to constitutive pathway activation or suppression. • Some cancers are dependent on genomic alterations in oncogenes or tumor suppressor genes for their

growth and survival, a phenomenon known as oncogene addiction. • Drugs that selectively target mutated proteins critical for the maintenance of the transformed phenotype have shown unprecedented clinical activity in genetically defined cancer subsets. • Precision medicine refers to the use of genetic and epigenetic information unique to an individual cancer patient to develop treatment regimens that target the driver oncogenes and tumor suppressors responsible for tumor progression. Potential challenges to the application of this approach include

The underlying basis of the cancer phenotype is deregulated cell growth, which stems from two main hallmarks of cancer: uncontrolled proliferation, and loss of programmed cell death (enhanced survival). In normal cells, these processes are tightly controlled through integration of signaling cascades that translate extracellular and intracellular cues into specific output responses. These signaling pathways are often initiated on binding of ligand to the extracellular domain of a receptor, followed by recruitment of adaptor proteins or kinases that activate an intracellular cascading network of protein and lipid intermediaries that ultimately produce a cellular response. In normal cells, the specificity, amplitude, and duration of signaling are tightly regulated, and these constraints are often abrogated in human cancers. Investigation of the signal transduction pathways that regulate normal cellular functions has revealed that key components of these networks are commonly altered in cancer cells by mutation, amplification and deletion, chromosomal translocation, overexpression, or epigenetic silencing. These alterations lead to activation or suppression of signaling cascades that underlie the various hallmarks of the cancer phenotype. This chapter reviews the major signal transduction cascades, with a focus on those that are frequently altered in human cancers. Individual sections highlight signaling intermediaries that have been validated as drug targets in patients with cancer. Table 2.1 summarizes actionable gene-level and mutation-level alterations in cancer and the drugs that are currently approved by the US Food and Drug Administration (FDA) for treatment, that are recommended standard of care biomarkers, or that have promising clinical or preclinical efficacy.1 24

the current inability to directly inhibit some oncogenic proteins (i.e., mutant KRAS), the development of drug resistance, technical hurdles posed by limited tissue availability for prospective molecular characterizing, and intratumoral and lesion-to-lesion genomic heterogeneity. • Routine genomic analysis of tumors or tumor-derived cell free DNA in plasma is now a component of standard care in an increasing number of cancer types, with the results used to guide treatment selection.

RECEPTOR TYROSINE KINASE SIGNALING The receptor tyrosine kinases (RTKs) comprise a family of transmembrane (TM) cell surface receptors that transduce extracellular signals internally to promote growth and survival and/ or to regulate other cellular phenotypes.2,3 Members of this protein family share a similar modular domain structure. Growth factors bind to the extracellular ligand-binding domain of RTKs and induce dimerization of two receptor monomers, juxtaposing the intracellular tyrosine kinase domains of each monomer.4 This results in transphosphorylation of tyrosine residues within the cytoplasmic domains of the RTK dimer. Following transphosphorylation, a variety of intracellular proteins are recruited to the activated RTK through Src homology 2 (SH2) domains that recognize the phosphotyrosine plus a specific amino acid sequence motif C-terminal to the tyrosine residues.5,6 Over 117 SH2 domains have been characterized, each with unique phosphotyrosine sequence specificities.7 Each domain is part of a larger adaptor protein involved in transducing extracellular signals to activate, or in some cases suppress, specific intracellular signaling cascades. Thus the complement of signaling pathways that a given RTK regulates is dictated by the profile of phosphorylated tyrosine residues plus flanking amino acids within their intracellular domains.8,9 However, more than one adaptor protein can often recognize individual context-dependent phosphotyrosine motifs within an RTK, underscoring how this system is designed to provide both specificity and diversity of intracellular signaling. Text continued on p. 29

Intracellular Signaling  •  CHAPTER 2 25

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer Gene

Variant

Cancer Type

ABL1

BCR-ABL1 fusion

ALL

AKT1

E17K

ALK

Fusions Oncogenic mutations Fusions

ARAF ATM BRAF

L1196M, L1196Q R1275Q S214A S214C N2875K, R3008C, truncating mutations V600D, V600E, V600G, V600K, V600M, V600R

BRCA1

V600E, V600K Fusions K601E L597Q, L597R, L597S, L597V D594E, D594N, G466V, G469A, G469V, G596C KIAA1549-BRAF Fusion L597Q, L597V Oncogenic mutations

BRCA2

Oncogenic mutations

CDK4

Amplification

CDKN2A

Oncogenic mutations

EGFR

729_761del, 729_761indel, L858R

E709_T710delinsD, E709K, G719A, G719C, G719D, G719S, A763_Y764insFQEA, L747P, A750P, A763_Y764insFQEA, L833V, L861Q, L861R, S768I, EGFR-KDD T790M 762_823ins 762_823ins, G719A, L861R, S768I

Drug

Dasatinib Imatinib Dasatinib CML Imatinib Nilotinib Breast AZD5363 Ovarian AZD5363 All Tumors ARQ 751 NSCLC Alectinib Ceritinib Crizotinib NSCLC Brigatinib Soft tissue sarcoma Ceritinib Crizotinib NSCLC Brigatinib Embryonal tumor Crizotinib Histiocytosis Sorafenib NSCLC Sorafenib Prostate cancer Olaparib Melanoma Cobimetinib + vemurafenib Dabrafenib Dabrafenib + trametinib Vemurafenib Histiocytosis Vemurafenib NSCLC Dabrafenib Dabrafenib + trametinib Vemurafenib Colorectal Binimetinib + cetuximab + encorafenib Panitumumab + vemurafenib Colorectal Fluorouracil + radiation + trametinib Melanoma Trametinib Ovarian Paclitaxel + selumetinib Melanoma Trametinib Melanoma Trametinib Melanoma Trametinib Soft tissue sarcoma Sorafenib + temsirolimus Melanoma BGB659 Ovarian Niraparib Rucaparib Olaparib Ovarian Niraparib Rucaparib Olaparib Dedifferentiated liposarcoma Abemaciclib, palbociclib Well-differentiated Abemaciclib, palbociclib liposarcoma

Evidencea 1 1 1 1 1 3 3 4 1 1 1 1 2 2 3 4 3 3 4 1 1 1 1 2 2 2 2 3 3 4 1 3 3 3 4 4 4 1 1 2 1 1 2 2 2

NSCLC

Letrozole + palbociclib Palbociclib Afatinib Erlotinib Gefitinib Osimertinib Afatinib, erlotinib, gefitinib

4 4 1 1 1 4 1

NSCLC NSCLC NSCLC

Osimertinib EGF816 AP32788

1 4 4

Breast Esophagogastric NSCLC

Continued

26 Part I: Science and Clinical Oncology

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer—cont’d Evidencea

Gene

Variant

Cancer Type

Drug

ERBB2

Amplification

Breast

All liquid tumors Leukemia GIST Thymic tumor

Ado-trastuzumab emtansine Lapatinib Lapatinib + trastuzumab Pertuzumab + trastuzumab Trastuzumab Trastuzumab Neratinib Neratinib Lapatinib AP32788 Cisplatin AZD9496, fulvestrant GDC-0810 GSK126 Tazemetostat AZD4547 Debio1347 Ponatinib Debio1347 JNJ-42756493 Debio1347 JNJ-42756493 BGJ398 Debio1347 Debio1347 JNJ-42756493 Debio1347 JNJ-42756493 Debio1347 JNJ-42756493 Debio1347 JNJ-42756493 Debio1347 JNJ-42756493 Debio1347 Sorafenib AG-120 BAY1436032 CB-839 AG-221 Ruxolitinib Sunitinib Sunitinib

1 1 1 1 1 1 3 3 3 4 3 3 4 4 4 3 3 4 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 3 3 4 4 3 3 1 2

GIST

Imatinib

1

GIST

Regorafenib

1

GIST

Dasatinib

2

GIST

Nilotinib

2

GIST Thymic tumor Melanoma

Sorafenib Sorafenib Imatinib

2 2 2

EZH2

Oncogenic mutations V659E E770_K831indel, E770_K831ins Oncogenic mutations Oncogenic mutations D538G, Y537S Oncogenic mutations

FGFR1

Amplification

FGFR2

BCR-FGFR1 fusion Fusions

ERCC2 ESR1

Esophagogastric Breast Breast NSCLC NSCLC Bladder Breast Breast Diffuse large B-cell lymphoma Lung squamous cell carcinoma Leukemia Adrenocortical carcinoma Bladder Cholangiocarcinoma Endometrial

FGFR3

Fusions

Adrenocortical carcinoma Bladder Glioma

G370C, G380R, K650E, K650M, K650N, K650Q, K650R, K650T, R248C, S249C, S371C, Y373C FLT3 IDH1

Y572_Y630ins R132C, R132G, R132H, R132Q, R132S

IDH2 JAK2 KIT

R140Q, R172G, R172K, R172M, R172S PCM1-JAK2 fusion 449_514mut, 550_592mut, A502_Y503dup, D579del, D820G, E554_K558del, H697Y, K550_W557del, K558delinsNP, L576P, P551_M552del, V555_L576del, V560D, V560del, V654A 449_514mut, 550_592mut, D419del, D579del, E554_I571del, E554_K558del, E554_V559del, F522C, I563_L576del, I653T, K550_W557del, K558N, K558_E562del, K558_V559del, K558delinsNP, K642E, L576P, M541L, M552_W557del, N564_Y578del, N822H, N822Y, P573_D579del, P577_ W582delinsPYD, P838L, Q556_K558del, T417_D419delinsI, T417_D419delinsRG, T574insTQLPYD, V530I, V555_L576del, V555_V559del, V559C, V559D, V559G, V559_V560del, V559del, V560D, V560G, V560del, V569_L576del, W557G, W557R, W557_K558del, Y553N, Y553_K558del, Y570H, Y578C 449_514mut, 550_592mut, D820G, D820Y, K550_W557del, K558delinsNP, N822K, V560D D816F, D816Y, D820G, D820Y, L576P, N822I, V559D, V560G, W557_K558del D816V, D820A, D820G, D820Y, K642E, L576P, V555_L576del, V559C, V559D, V654A, W557_K558del D820A, D820E, D820G, D820Y, K642E, N505I, P577_D579del, V559D, W557_K558del K642E, L576P, V559A

Bladder Breast AML AML All tumors

Intracellular Signaling  •  CHAPTER 2 27

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer—cont’d Gene

Variant

Cancer Type

Drug

KRAS

Wild type

Colorectal

Cetuximab Panitumumab Regorafenib Cabozantinib + panitumumab Panitumumab + regorafenib Pembrolizumab alpelisib + binimetinib Cobimetinib + GDC-0994 Atezolizumab + cobimetinib Fluorouracil + radiation therapy + trametinib Abemaciclib, PD0325901 + palbociclib, palbociclib, ribociclib, ribociclib + trametinib Binimetinib + erlotinib Binimetinib, selumetinib, trametinib Docetaxel + trametinib Cobimetinib, selumetinib, trametinib Cobimetinib, selumetinib, trametinib Cobimetinib, selumetinib, trametinib Cobimetinib, selumetinib, trametinib DS-3032b RG7112 SAR405838 Crizotinib Cabozantinib Capmatinib Crizotinib Cabozantinib Crizotinib Cabozatinib Capmatinib Everolimus Everolimus, rapamycin, temsirolimus Trametinib Trametinib Binimetinib PLX3397 Atezolizumab + cobimetinib Binimetinib Binimetinib + ribociclib Radioiodine uptake therapy + selumetinib Fluorouracil + radiation therapy + trametinib Larotrectinib Entrectinib Entrectinib Larotrectinib Entrectinib Larotrectinib

Oncogenic mutations

All tumors Colorectal NSCLC

MAP2K1

Oncogenic mutations

Histiocytic disorder Low-grade serous ovarian Melanoma NSCLC

MDM2

Amplification

Liposarcoma

MET

963_D1010splice, 981_1028splice, X1006_splice, X1007_ splice, X1008_splice, X1009_splice, X1010_splice, X963_ splice Amplification

NSCLC

D1010H, D1010N, D1010Y

NSCLC RCC NSCLC

MTOR

E2014K C1483F, F1888L, L2230V, S2215F, T1977K

Bladder RCC (clear cell)

NF1

Oncogenic mutations

NRAS

Oncogenic mutations

Glioblastoma Melanoma Neurofibroma Neurofibroma Colorectal Melanoma Thyroid Colorectal

NTRK1

Fusions

NTRK2

Fusions

All tumors Salivary gland Salivary gland

NTRK3

Fusions

Salivary gland

Evidencea 1 1 1 4 4 4 4 4 4 4 4

4 4 4 3 3 3 3 3 3 4 2 3 3 2 2 2 3 3 3 4 4 4 4 4 3 3 3 3 4 3 3 3 3 3 3 Continued

28 Part I: Science and Clinical Oncology

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer—cont’d Gene

Variant

PDGFRA

FIP1L1-PDGFRA fusion Fusions

PDGFRB

PIK3CA

Cancer Type

Leukemia Myelodysplasia Myeloproliferative Neoplasm 560_561insER, A633T, C450_K451insMIEWMI, C456_N468del, GIST C456_R481del, D568N, D842I, D842_H845del, D842_ M844del, D846Y, E311_K312del, G853D, H650Q, H845Y, H845_N848delinsP, I843del, N659K, N659R, N659S, N848K, P577S, Q579R, R748G, R841K, S566_E571delinsR, S584L, V469A, V536E, V544_L545insAVLVLLVIVIISLI, V561A, V561D, V561_I562insER, V658A, W559_R560del, Y375_K455del, Y555C, Y849C, Y849S D842V GIST Fusions Dermatofibrosarcoma Protuberans Myelodysplasia Myeloproliferative neoplasm Oncogenic mutations Breast

All tumors Endometrial Ovarian

PTCH1

Truncating mutations

Embryonal tumor Skin cancer, nonmelanoma

PTEN

Oncogenic mutations

All tumors

RAF1 RET

S257L Fusions

Endometrial Prostate Lung adenocarcinoma NSCLC

ROS1

Fusions D2033N

NSCLC

Drug

Evidencea

Imatinib Imatinib Imatinib Imatinib

1 1 1 2

Dasatinib Imatinib

2 1

Imatinib Imatinib Alpelisib Alpelisib + fulvestrant Buparlisib Buparlisib + fulvestrant Copanlisib Fulvestrant + taselisib GDC-0077 Serabelisib Taselisib Alpelisib + everolimus Alpelisib + letrozole, Alpelisib + letrozole + ribociclib Alpelisib + LJM716 + trastuzumab Alpelisib + olaparib, buparlisib + olaparib AZD5363 + fulvestrant AZD8835 + fulvestrant MLN0128 + serabelisib ARQ 751 AZD5363 + olaparib GDC-0077 Alpelisib + fulvestrant Buparlisib + fulvestrant Fulvestrant + taselisib Alpelisib + fulvestrant Buparlisib + fulvestrant Fulvestrant + taselisib Sonidegib Sonidegib Vismodegib ARQ 751 AZD5363 + olaparib AZD8186 Gedatolisib + palbociclib GSK2636771 LY3023414 Olaparib Enzalutamide + LY3023414 Sorafenib Cabozantinib Vandetanib Crizotinib Cabozantinib

1 1 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3 4 4 4 4 4 4 4 4 4 2 3 1 3

Intracellular Signaling  •  CHAPTER 2 29

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer—cont’d Gene

Variant

Cancer Type

Drug

TSC1

Oncogenic mutations

TSC2

Oncogenic mutations

CNS RCC CNS

Everolimus Everolimus Everolimus

Evidencea 2 2 2

a

Levels of evidence: • Level 1: FDA-recognized biomarker predictive of response to an FDA-approved drug in this indication • Level 2: Standard care biomarker predictive of response to an FDA-approved drug in this indication or another indication; including those recommended by NCCN, but not FDA recognized as standard of care • Level 3: Evidence of clinical activity in this indication, or another indication • Level 4: Preclinical or biologic evidence of activity ALL, Acute lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic myelogenous leukemia; CNS, central nervous system; FDA, US Food and Drug Administration; GIST, gastrointestinal stromal tumor; NCCN, National Comprehensive Cancer Network; NSCLC, non-small cell lung cancer; RCC, renal cell carcinoma. Data modified from oncokb.org1; Chakravarty D, Gao J, Phillips S Kundra R, Zhang H, Wang J. OncoKB: a precision oncology knowledge base. JCO Precis Oncol. Published online May 16, 2017.

Recruitment of signaling intermediaries to the plasma membrane facilitates their interaction with membrane-bound proteins responsible for stimulating a diverse array of downstream pathways (Fig. 2.1). As an example, the lipid kinase phosphatidylinositol 3-kinase (PI3 kinase), described in more detail in a later section, recognizes and binds to a pattern of phosphorylated tyrosine residues present within multiple activated RTKs through the SH2 domain located in its p85 regulatory subunit. Binding of the p85 regulatory subunit in turn results in activation of its kinase activity. Approximately 20 classes of RTKs have been defined based on growth factor specificity. This section will focus on those RTK classes for which specific cancer therapies exist or are in development.

Epidermal Growth Factor Receptor Signaling Historically, the growth factors that stimulate RTKs were first discovered, followed by the structural and functional characterization of the RTKs themselves.10,11 Epidermal growth factor (EGF) was initially purified from mouse submaxillary glands in 1962 by Stanley Cohen and was found to stimulate premature eyelid opening and incisor eruption, phenotypes that suggested a role for EGF in the regulation of cellular proliferation.12 In 1978, the epidermal growth factor receptor (EGFR) was identified as the cell surface binding site for EGF.13 Over the next several years, tyrosine phosphorylation was identified in cells, followed by the discovery that the viral Src oncogene, which induces transformation of cells in vitro, is itself a tyrosine kinase, underscoring the potential importance of tyrosine phosphorylation for oncogenesis.14,15 Once the complete sequence of the EGFR protein was elucidated in the 1980s,16 the amino acid sequence of the receptor cytoplasmic domain was found to be similar to Src, suggesting that EGFR also possessed tyrosine phosphorylation activity. The connection between RTK activation and oncogenesis was further solidified when the amino acid sequence of EGFR was found to be homologous to the avian erythroblastosis virus erbB oncogene, which, when infected into chicken red blood cell precursors, is sufficient to induce erythroleukemia.17,18 The erbB oncogene encodes a TM protein that lacks the extracellular ligand binding domain of EGFR but possesses a cytoplasmic kinase domain that, when expressed in cells, can signal in a growth factor–independent manner. Subsequent studies have since identified within human cancers numerous alterations of EGFR and other RTKs that enhance proliferation without the need for growth factor stimulation. The EGFR class of RTKs comprises four receptor proteins encoded by four genes (in parentheses): EGFR (ERBB1), HER2/Neu (ERBB2), HER3 (ERBB3), and HER4 (ERBB4). EGFR binds to and is activated by a number of ligands, including EGF, transforming growth factor–α (TGF-α), HB-EGF, amphiregulin, betacellulin, epiregulin, and

epigen.19–21 Growth factor binding promotes either homodimerization or heterodimerization with other HER family members, followed by transphosphorylation.22 A ligand for HER2 has not yet been identified; instead, HER2 is activated through heterodimer formation with one of the other three ligand-bound receptors.23 Notably, HER2 is the preferred dimerization partner for EGFR, and EGFR-HER2 heterodimers are more stable than EGFR homodimers, remaining at the cell surface for a longer duration and undergoing endocytosis at a lower rate than EGFR homodimers.24,25 Furthermore, HER2 reduces the dissociation rate of EGF from EGFR, allowing for a more sustained period of EGF-induced signaling.19 The EGFR and HER2 components of the EGFR-HER2 heterodimer are also more likely to be recycled back to the cell surface than EGFR homodimers, which are more readily targeted for degradation.26 In addition, HER2-HER3 heterodimers possess the most potent mitogenic activity among the heterodimer and homodimer HER kinase combinations.27 In contrast to the other HER kinase family members, HER3 does not have intrinsic kinase activity and preferentially forms heterodimers with HER2.28 The ligands for HER3 and HER4 are the neuregulins, including heregulin. A number of tumor types frequently exhibit alterations within the EGFR family of RTKs.21 Sustained activation of these pathways can result in oncogene- or pathway-addicted tumors, and selective HER kinase inhibitors are now a component of the standard treatment of several malignancies. Alterations that affect RTK activity include mutations that result in constitutive activation of the tyrosine kinase; overexpression of the receptor, often due to gene amplification; and elevated levels of RTK ligands that stimulate signaling. EGFR mutations are found in 10% to 25% of non–small cell lung cancers (NSCLCs), with variation in the frequency of such alterations influenced by ethnicity and geographic location; in-frame microdeletions in exon 19 and point mutations in exon 21 (most commonly L858R or L861Q) or exon 18 (G719X) comprise over 80% of these alterations.29–31 In glioblastoma multiform (GBM), EGFR mutations, indels (including the EGFRvIII variant in which exons 2 to 7 of the extracellular domain are deleted, generating a ligand-independent, activated protein), amplification, splice variants, and rearrangements occur in 57% of tumors.32–34 However, because of the heterogeneity of GBM tumors, targeting EGFR is complicated; EGFR alteration is often concurrent with amplification or mutation of another RTK such as PDGFR, MET, or FGFR, or the presence of EGFRvIII on extrachromosomal DNA, or activation of IDH1.35–38 Overexpression of wild-type EGFR as a result of gene amplification has been observed in NSCLC and breast, gastric, colorectal, and head and neck cancers, and less commonly in other tumor types.39–41 Up to 30% of breast cancers display overexpression of HER2, which is an unfavorable prognostic factor, and therapy for these

30 Part I: Science and Clinical Oncology Growth Factor Ligand

oncogene tumor suppressor

Receptor Tyrosine Kinase

P Shc P Grb2

Ras-GDP Sos

NF1

Ras-GTP

Vermurafenib Dabrafenib

PLCε

Raf

P P

RalA/B

RalGDS

MEKK1/NF-κB

TIAM1

Trafficking/ proliferation p85

PIK3CAp110α

P P

Trametinib

MEK

Selumetinib Cobimetinib

CDC42/RAC P P

SCH772984 BVD-523

ERK

DEL-22379

DUSPs

AKT

NF- κB/actin

mTORC1 P p90RSK translation

NF- κB, Myt-1, GSK3, PP-1

P Fos/Jun/etc.

Proliferation/growth Negative feedback

Figure 2.1  •  Canonical Ras/MAPK signaling pathway. Ras proteins cycle between GDP-bound inactive and GTP-bound active states. Ras is often activated in response to ligand-specific binding to its cognate receptor. Ras can also be activated via intracellular cross talk. This schematic depicts the classic Ras/MAPK signal transduction cascade. Growth factor stimulation induces receptor tyrosine kinase (RTK) dimerization and autophosphorylation of tyrosine residues located within the intracellular domain of the receptor. These phosphorylated tyrosine residues serve as docking sites for scaffold proteins that facilitate activation of intracellular signaling cascades. For example, the adaptor protein Grb2, via its SH3 domain, recruits the Ras GEF (guanine-nucleotide exchange factor) SOS. Colocalization of SOS and Ras facilitates substitution of GTP for GDP and thus Ras activation. Active GTP-bound Ras binds and recruits the Raf (A-, B-, and C-Raf ) serine/threonine kinases to the plasma membrane and facilitates their activation. Active Raf in turn phosphorylates and activates MEK, which in turn phosphorylates and activates ERK. ERK phosphorylates substrates in the cytoplasm (p90RSK) and in the nucleus (Jun, Fos, Ets-2, Elk-1, CREB1, AP-1, ATF-2, among others), which regulate cell proliferation and survival. Ras interacts with more than 20 effector proteins, including the p110α subunit of PI3 kinase, RasGDS, PLCε, and TIAM1, which in sum control transcription, translation, vesicular trafficking, cell cycle progression, cytoskeletal changes, metabolic processes, immune inflammatory responses, and survival. Induction of Ras signaling also upregulates negative feedback elements that inhibit the pathway (e.g., DUSPs and SPROUTYs/SPREDs). Several proteins within the Ras signaling cascade are proto-oncogenes (green) and tumor suppressors (red) that are mutated, amplified, or deleted in many cancers. A number of selective inhibitors of Ras effectors have been tested as anticancer therapies. Examples include kinase inhibitors, which selectively target B-Raf and its downstream effectors MEK and ERK (see red boxes).

ERBB2-amplified breast cancers is now distinct from that of breast cancers with normal HER2 expression levels.21,42 ERBB2 amplification is also a driving event in gastric and to a lesser extent in bladder, endometrial, and cervical cancer.43–45 More recently, activating mutations and in-frame insertions/indels in ERBB2 were found to occur in 1% to 2% of all cancer patients, most commonly in patients with bladder cancer.44 Mutations in ERBB2 localize to either the extracellular domain, where they are presumed to promote dimer formation, or the kinase domain.46,47 In addition, activating mutations in ERBB3 have also been identified in bladder, colon, and gastric cancers.48,49 Numerous targeted agents have been developed that selectively inhibit EGFR-induced signaling (see Table 2.1 and Fig. 2.2).21,50 Cetuximab, a chimeric monoclonal antibody that binds to the extracellular domain of EGFR and competitively inhibits ligand binding, thereby preventing receptor activation, is approved for the treatment

of KRAS wild-type colorectal and head and neck cancers.51–56 Panitumumab, another human monoclonal anti-EGFR antibody, is also approved for KRAS wild-type metastatic colorectal cancer.56 The first-generation reversible EGFR tyrosine kinase inhibitors gefitinib and erlotinib, as well as the second-generation irreversible inhibitor afatinib, are FDA approved for the treatment of NSCLC, with greatest efficacy in patients with EGFR mutations or in-frame deletions.57–60 A second site mutation in EGFR (T790M) is a common mechanism of acquired resistance to first generation EGFR inhibitors. Osimertinib (AZD9291), a third-generation EGFR inhibitor, is highly active in patients with NSCLC in which resistance is mediated by the EGFR T790M mutation and is now FDA approved for this indication.61,62 The development of osimertinib and the fourth-generation EGFR inhibitor EAI04563 highlights how studies of acquired resistance can lead to the rational development of more effective kinase inhibitors.

Intracellular Signaling  •  CHAPTER 2 31

XL147 Buparlisib Alpelisib (α-specific) AZD8186 (β-specific) Copanlisib (α/δ-specific) Idelalisib (δ-specific)

AZD5363 Afureser­b

AKT

PIP2/3 P P

PI3Kp110α

PTEN PIP3

PDK1

P

mTORC2

SGK

Bad P NF-κB

GSK3β

P

P Cyclin D1

p85

IRS1

RasGTP CDC42/RAC NF-κB/ac­n

RHEB-GDP TSC1 TSC2

apoptosis

EGFR:Cetuximab EGFR/HER2:Lapa­nib, Trastuzumab

Receptor Tyrosine Kinase

PIP2

P IκB

Growth Factor Ligand

P P

P P

EGFR:gefi­nib, erlo­nib, osimer­nib EGFR/HER2:lapa­nib, afa­nib, nera­nib Other: ima­nib, alec­nib, larotrec­nib, etc

Dual PI3K-mTOR inhibitors: dactolisib, voxtalisib

RHEB-GTP

P

mTOR inhibitors: rapamycin, everolimus, temsirolimus, AZD8055, RapaLink

mTORC1

PRAS40 degrada­on

P 4EBP1

P p70S6K P

Protein transla­on

S6

P FOXO1/4

Arrest/Apoptosis oncogene tumor suppressor

prolifera­on and survival

Figure 2.2  •  PI3K/mTOR signaling pathway. The PI3 kinase family proteins are lipid kinases that transduce signals from receptor tyrosine kinases (RTK)

and G protein–coupled receptors to intracellular cascades that control proliferation, survival, and other cellular phenotypes. As an example, growth factor binding causes receptor dimerization and subsequent phosphorylation of tyrosine residues in the intracellular domain of the receptor. These tyrosine phosphorylation sites serve as docking sites for the p85 regulatory subunit of PI3 kinase and adaptor proteins such as IRS-1 in the case of signaling induced by the insulin/ IGF1 receptors. This results in allosteric activation of the catalytic subunit of PI3 kinase, which converts PIP2 to PIP3. PIP3 recruits PDK1 and AKT to the membrane via their pleckstrin homology (PH) domains. Colocalization of PDK1 and AKT results in phosphorylation (on threonine 308) and activation of AKT. Phosphorylation of AKT on Ser473 by mTORC2 is required for full activation of AKT. Activated AKT phosphorylates several effectors, including GSK3β, Bad, PRAS40, IκB, the FOXO1/4 transcription factors, and TSC2. AKT phosphorylation of TSC2, which is bound to TSC1, inhibits the GTPase function of this complex, thereby allowing activation of Rheb and subsequent activation of mTORC1. In turn, mTORC1 phosphorylates p70S6 kinase (p70S6K) and 4EBP1, an inhibitor of the eIF4E component of the cap-dependent translation initiation complex. p70S6K and mTOR also function to negatively regulate the pathway by initiating the phosphorylation and inhibition of IRS-1. PI3 kinases can also signal to other effectors such as Rac/CDC42 and the serum-glucocorticoid kinase (SGK) family to promote cellular survival, motility, and cytoskeletal rearrangement. Many components of the PI3 kinase signaling pathway are mutationally altered in cancer (oncogenes in green, tumor suppressors in red). A variety of compounds have been developed that selectively inhibit PI3 kinase signaling components. US Food and Drug Administration (FDA)–approved drugs and novel inhibitors in clinical testing that target RTKs, PI3 kinase, mTOR kinase, and AKT are highlighted in the red boxes.

ERBB2 amplification strongly correlates with HER2 protein overexpression, and the presence of either marker predicts for trastuzumab response in certain cancers.64 Trastuzumab, a humanized antibody that binds to the extracellular domain of HER2, has been FDA approved for the treatment of breast65–71 and esophagogastric72 cancers displaying HER2 overexpression. In patients with breast and gastric cancers, trastuzumab has modest activity when administered as singleagent therapy and is most commonly used in combination with chemotherapy.72–74 The combination of docetaxel, trastuzumab, and pertuzumab,75 an antibody that binds to a different HER2 epitope (the dimerization domain) than trastuzumab and results in impaired dimer formation, is also approved for breast cancer.76–78

Although the introduction of trastuzumab has resulted in a significant improvement in the survival of patients with HER2overexpressing breast cancers, drug resistance remains a major clinical problem. Potential resistance mechanisms include concomitant overexpression of other HER kinase family members and/or ligands, PTEN loss, and the expression of a truncated HER2 protein lacking the extracellular antibody binding site.79 Additional HER2-directed agents include the tyrosine kinase inhibitors lapatinib and neratinib (see Table 2.1). Lapatinib is FDA approved for use in combination with capecitabine in patients with HER2-overexpressing advanced or metastatic breast cancer that has progressed on prior therapy with trastuzumab and certain classes of chemotherapy.80 The combination

32 Part I: Science and Clinical Oncology

of lapatinib and trastuzumab is also FDA approved in HER2-amplified breast cancer.81–83 Lapatinib also received accelerated approval for use in combination with the aromatase inhibitor letrozole.84 Clinical efficacy has been reported with lapatinib in HER2-mutant NSCLC.85,86 The irreversible pan-HER kinase inhibitor neratinib has shown promising clinical activity in patients with ERBB2-amplified and ERBB2-mutant breast tumors, but also other cancer types.46,87–93

alectinib and ceritinib that are either more potent or more selective for ALK have significant clinical activity in patients with acquired resistance to crizotinib and are FDA approved for this indication.116,117 In addition, brigatinib, a dual inhibitor of ALK and EGFR, was granted accelerated FDA approval in 2017 for patients with metastatic NSCLC and in patients with ALK alterations who progressed on crizotinib.118

Insulin, Insulin-Like Growth Factor-1 Receptor Signaling, ALK, and ROS1

Platelet-Derived Growth Factor Receptor, KIT, and FLT-3 Signaling

The insulin and insulin-like growth factor 1 (IGF1) receptor family is dysregulated in multiple malignancies.94 The insulin receptor exists as two isoforms encoded by splice variants of the same gene.95 Each isoform can dimerize with the other (forming hybrid dimers) or with itself.96 The IGF1 receptor (IGF1R) can dimerize with either of the insulin receptor isoforms or with itself, resulting in six different dimer combinations.96 The insulin receptor is stimulated by insulin or insulin-like growth factor 2 (IGF2), whereas IGF1R can be activated by either IGF1 or IGF2. Both of these latter ligands can stimulate IGF1R in an autocrine fashion or can be elaborated from distant sites.97,98 Circulating IGF binding proteins have a similar affinity for IGF1 and 2 as IGF1R does and therefore compete for binding to both ligands, thus titrating the amount of free ligand available for IGF1R stimulation.99 IGF binding protein proteases provide an additional mechanism for controlling ligand levels by increasing the half-life of free ligand available for receptor binding.100 After ligand binding, IGF1R dimerizes and undergoes transphosphorylation, leading to activation of downstream signaling pathways, including both the Ras-Raf-MAPK and the PI3 kinase-AKT-mTOR cascades (see individual sections later in the chapter; see also Fig. 2.1).101 Specifically, insulin receptor substrate 1 (IRS1) binds to a phosphotyrosine motif on IGF1R via its SH2 domains and is phosphorylated by IGF1R.102 It subsequently recruits PI3 kinase to the plasma membrane, which converts phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3), subsequently resulting in AKT and mTOR pathway activation. Activating mutations of IGF1R do not appear to be common in human cancer. However, amplification of the IGF1R gene locus has been identified in some colon, pancreas, and lung cancers. Sarcomas often exhibit either increased expression of the IGF1 and IGF2 ligands or decreased IGFBP-3 expression (Ewing sarcoma), which results in increased IGF1 levels in the tumor microenvironment.103 Gastrointestinal stromal tumors (GISTs) lacking c-KIT and platelet-derived growth factor receptor (PDGFR) mutations also commonly harbor IGF1R amplification.104 AMG479, a monoclonal human antibody targeting IGF1R, has shown promising antitumor activity in patients with Ewing sarcoma.103 Activating kinase domain point mutations and gene rearrangements of the insulin receptor family members anaplastic lymphoma kinase (ALK) and ROS1 play driving roles in many cancers, most notably lymphomas, neuroblastoma, NSCLC, and thyroid cancer.105–108 Chromosomal translocations involving ALK and at least 22 5′ fusion partners have been identified,108 which dictate spatial and temporal expression of the ALK fusions, and likely their function and tumorigenic potential.105 In NSCLC, the EML4 gene is the preferred translocation partner, resulting in the expression of an EML4-ALK fusion protein in 4% to 6% of patients.109,110 Notably, EML4-ALK fusions are found in a mutually exclusive pattern with EGFR kinase domain mutations, suggesting that they have overlapping downstream effects. ROS1 gene rearrangements are also found in a minority of NSCLC patients with binding partners including SLC34A2 and CD74.106,111,112 Crizotinib, an inhibitor of the ALK, ROS1, and MET tyrosine kinases (see Table 2.1), is now FDA approved for use in NSCLC patients with ALK or ROS1 fusions (see Table 2.1),113–115 although acquired resistance mutations in ALK (of note, C1156Y and the gatekeeper mutation L1196M) commonly develop. Newer ALK inhibitors including

Platelet-derived growth factor (PDGF) is the ligand for PDGFRs, which stimulate the proliferation and migration of mesenchymal cells, such as oligodendrocyte precursors, vascular smooth muscle cells, and pericytes during embryonic development.119 PDGF signaling is also implicated in organ development, including lung and intestinal epithelial folding and glomerular capillary tuft formation. Furthermore, PDGFs promote angiogenesis, wound healing, and erythropoiesis.120 Aberrations in the PDGFR pathway result in uncontrolled proliferation and enhanced angiogenesis. Four isoforms of PDGF have been identified: PDGFA, PDGFB, PDGFC, and PDGFD.121 These isoforms are activated by proteolytic cleavage and assemble into five homodimeric or heterodimeric combinations that bind to and stimulate either PDGFRα or PDGFRβ. PDGFRα homodimers inhibit chemotaxis, whereas PDGFRβ homodimers and α/β heterodimers stimulate chemotaxis within fibroblasts and smooth muscle cells.122 Angiogenic endothelial cells recruit PDGFRβ-positive pericytes to cover blood channels and aid in their maturation and stabilization through secretion of PDGFβ.123 Following dimerization and transphosphorylation, PDGFRs activate signal transduction pathways through recruitment of adaptor proteins containing SH2 domains, most notably the Grb2 protein, which in turn binds the guanine nucleotide exchange factor (GEF) Sos, which subsequently activates Ras.124,125 In addition, phosphorylated tyrosine residues serve as docking sites for SH2 domain–containing kinases, including PI3 kinase, phospholipase-Cγ, and Src, as well as the tyrosine phosphatase SHP2 and the STAT transcription factor family.125 Alterations in PDGFR signaling in cancer include excess autocrine secretion of PDGF (glioblastoma, sarcomas), gain-of-function mutations that cause constitutive tyrosine kinase activation (GISTs),126 translocation of either the PDGF or PDGFR gene (dermatofibrosarcoma protruberans, chronic myelomonocytic leukemia, hypereosinophilic syndrome),127–129 and PDGFR gene amplification (glioblastoma).130 PDGFRα mutations are found in approximately 10% of KIT wild-type GISTs and are sensitive to imatinib, a tyrosine kinase inhibitor of KIT, BCR-ABL, and PDGFRs, which is standard of care in this setting (see Table 2.1).122,126,131 The D842V mutation comprises approximately two-thirds of PDGFRα activating mutations, and confers resistance to imatinib. Notably, the second-generation inhibitor dasatinib is effective in preclinical models of imatinib-resistant GIST.126,132,133 Dermatofibrosarcoma protuberans is a rare, low-grade cutaneous sarcoma that harbors a chromosome 17;22 translocation that fuses portions of the COL1A1 (collagen 1A1) gene and PDGFB, resulting in overexpression of PDGF-β and subsequent stimulation of PDGFR signaling.122 Twenty other fusions partners have been identified in PDGFβ rearrangements, including ETV6 and EBF1. Imatinib has shown significant benefit in patients with recurrent or metastatic dermatofibrosarcoma protuberans, myelodysplasia, and myeloproliferative neoplasms and is FDA approved for these indications.134–138 The KIT gene is a member of the type III RTK family, which includes PDGFR and FLT3 (see later).139–141 It was first identified as the human homologue of the viral oncogene v-Kit responsible for the Hardy-Zuckerman IV feline sarcoma virus.142 Mutation of KIT or its ligand, stem cell factor (SCF),143–146 in mice induces coat color abnormalities (“white spotting”), anemia, and mast cell deficiencies, suggesting that it plays a role in hematopoiesis and melanogenesis.147,148 Furthermore, the KIT protein was discovered as a cell surface receptor

Intracellular Signaling  •  CHAPTER 2 33

in acute myeloid leukemia (AML).149 KIT expression is mainly restricted to mast cells, hematopoietic cells, germ cells, melanocytes, and the interstitial cells of Cajal (ICCs) in the gut.150,151 SCF and KIT integrate signals that lead to mitogen-activated protein kinase (MAPK), PI3K, and SRC pathway activation, and mediate critical survival and proliferation cues to distinct hematopoietic lineages, including the bone marrow and progenitor cells. Hot-spot mutations in exons 9 and 11 of KIT have been identified in several tumor types, including GIST, melanomas, and germ cell tumors.146,152–156 In GIST, 85% of tumors have activating KIT mutations that drive the transformation of precursors of ICCs.153 Imatinib157–161 and sunitinib158,162,163 inhibit KIT and PDGFR, among other kinases, and are FDA approved for use in patients with KIT mutant GIST (see Table 2.1). Regorafenib is approved for patients with imatinib- or sunitinib-refractory GIST.164 Imatinib has also been shown to induce tumor regression in patients with KIT-mutant or KIT-amplified melanoma.165,166 Second site mutations in KIT, typically in exon 17, are a mechanism of acquired resistance to imatinib therapy in patients with GIST.167 Novel agents that retain activity in the setting of an exon 17 KIT mutation are now in clinical testing (clinical trial NCT02401815).168 New areas of investigation into mutant KIT therapies in GIST include blocking mutant KIT subcellular localization to the Golgi150 and combination of FGFR3 and KIT inhibitors to quench pathway cross talk.169 The FMS-like tyrosine kinase 3 receptor (FLT3), a third member of the RTK class that includes PDGFR and KIT, is involved in the development of normal hematopoietic cells. It contains an extracellular region composed of five immunoglobulin (Ig) domains, TM and juxtamembrane domains, and two cytoplasmic tyrosine kinase domains that transmit proliferative signals through the RAS/MAPK, PI3K/AKT, and STAT5 pathways.170 Two main FLT3 alterations are common in hematopoietic malignancies, namely in approximately 30% of AMLs.171 First, internal tandem duplication (ITD) within exons 14 and 15 of the FLT3 gene (FLT-ITD) interferes with the negative regulatory function of the juxtamembrane segment.172 This duplication results in ligand-independent activation of FLT3 and is associated with a poor prognosis in patients with AML. Second, kinase domain mutations at or near D835 in the activation loop of FLT3 disrupt autoinhibitory interactions and render the kinase open and active.173 The clinical activity of FLT3 inhibitors has been modest to date, although responses appear to be more common in patients with FLT3/ITD AML.171 TAK-659, a reversible dual Syk/Flt inhibitor, showed early clinical activity in numerous lymphoma subtypes and AML.174,175 Sorafenib has been shown in preclinical in vitro studies, mouse models, and in a phase I study of AML patients to reduce leukemia burden and block signaling selectively in FLT3-ITD versus FLT-wt settings.176 Interesting to note, resistance to FLT3 inhibition in such patients is associated with selection for secondary mutations within the tyrosine kinase domain of FLT3, suggesting a central role of FLT3 in AML pathogenesis.171

Fibroblast Growth Factor Receptor Signaling Fibroblast growth factor receptors (highly conserved FGFR1, FGFR2, FGFR3, and FGFR4; and FGFRL1/FGFR5, which lacks a kinase domain) comprise a family of RTKs that regulate cell proliferation, differentiation, and migration as well as selective apoptosis during embryogenesis. The FGFRs are composed of an extracellular ligandbinding domain, a hydrophobic TM region, and an intracellular tyrosine kinase domain.177 The extracellular domain is organized into three Ig domains; differential splicing of the second half of the third Ig domain dictates tissue-specific expression of the receptor. Fibroblast growth factors (FGFs) are protein ligands that bind to the extracellular domain of the FGFRs in combination with specific heparan sulfate glycosaminoglycans inducing FGFR dimerization and transphosphorylation of intracellular tyrosine residues. Eighteen FGFs have been identified, and specificity for FGFRs is based on numerous

factors, including tissue-specific FGF ligand and receptor expression, the presence of cell surface molecules that facilitate the interaction between individual FGF ligands and receptors, and the differential binding capability of the ligands themselves for specific FGFRs.178 Subsequent stimulation of the tyrosine kinase domain leads to phosphorylation and activation of multiple downstream signaling proteins in the same manner as described earlier for other RTKs. Unique to the FGFR signaling complex is FGFR substrate 2 (FRS2), an adaptor protein that binds to specific phosphotyrosines on the intracellular domain of active FGFR dimers.179 FRS2 is itself phosphorylated by FGFRs and serves as a docking site for the Grb2-Sos adaptor complex, which activates the Ras/Raf/MAPK pathway. Phosphorylated FRS2 also recruits Grb2-associated binding protein 1 (GAB1), which activates PI3 kinase. In addition, phospholipase-Cγ binds to phosphorylated FGFR dimers via an SH2 domain, leading to its activation and the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) to form inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). Germline mutations of the FGFR genes are the basis of a spectrum of skeletal developmental disorders that are thought to derive from premature differentiation and growth restriction of chondrocytes resulting from dysregulated FGFR pathway activation.180,181 FGFR signaling is dysregulated in cancer by multiple mechanisms including mutational or translocation-induced activation of FGFRs, gene amplification of receptors, and abnormal ligand regulation.182 For example, autocrine and paracrine FGF ligand secretion with resultant pathway activation has been reported to occur in a subset of melanomas and prostate cancers, respectively.183,184 FGFR1 amplification occurs in approximately 17% of squamous cell lung cancers and 6% of small cell lung cancers (SCLCs).185 Approximately 10% of diffuse-type, aggressive gastric cancers display FGFR2 gene amplification, and cell lines with this amplification show ligand-independent pathway activation and sensitivity to selective FGFR inhibitors.186,187 Whereas FGFR1 mutations are rather rare, FGFR2 mutations are found in approximately 10% of endometrial cancers.188,189 FGFR3 mutations occur in up to 75% of non–muscle invasive bladder cancers and 15% of patients with advanced urothelial tumors.44,179,189,190 Activating mutations within FGFR3 result in constitutive receptor dimerization and subsequent signaling. Unlike EGFR-activating mutations, which predominantly affect the tyrosine kinase domain of the receptor, FGFR3 mutations are commonly located within the extracellular domain (R248, S249) and TM segment (G370, Y373) and promote ligand-independent receptor dimerization through formation of an aberrant disulfide bridge between two receptor monomers.191 Chromosomal rearrangement of FGFRs have been identified using next-generation sequencing. Up to 15% of multiple myelomas harbor an intergenic 4;14 translocation between the FGFR3 gene and the Ig heavy chain locus, which places FGFR3 expression under the highly active heavy chain promoter.192,193 More recently, translocations involving FGFR2 have been reported in cholangiocarcinoma and more rarely in other cancers, whereas FGFR3 fusions are most common in glioblastoma and bladder cancers but also are found rarely in other solid tumor types.194,195 The FGFR3-TACC3 constitutively active fusion protein has been characterized to induce aneuploidy by disrupting proper chromosomal segregation.196 Multiple FGFR inhibitors are currently being tested in early-phase clinical trials, but the majority of these compounds are multitargeted tyrosine kinase inhibitors, many of which also potently inhibit members of the VEGFR and PDGFR families. The close structural similarity between these RTKs has made development of FGFR-selective inhibitors challenging, although several such drugs are now in early clinical testing, such as BGJ398, AZD4547, JNJ-42756493, and Debio1347 (see Table 2.1).179,182,197–200 On-target hyperphosphatemia resulting from FGFR1 inhibition is a primary toxicity with this class of agents, suggesting that the development of isoform-selective FGFR inhibitors may be a more rational approach for patients whose tumors are driven by mutations or translocations in FGFR2 and FGFR3. FGFRs are located on the cell surface and thus may also be susceptible to monoclonal antibody

34 Part I: Science and Clinical Oncology

mediated inhibition similar to trastuzumab-mediated inhibition of HER2. FGFR ligand traps are also in development.182

RET Signaling The RET (Rearranged During Transfection) gene encodes three alternatively spliced isoforms (RET9, RET43, and RET51) which are TM RTKs that contain four cadherin-like extracellular repeats important for dimerization, a cysteine-rich juxtamembrane region critical for ligand binding and conformation, and an intracellular kinase domain.201 RET9 and RET51 are highly conserved in all vertebrates and play important roles in the normal development and maintenance of many tissues, including the kidney, spermatogonial stem cells, and the enteric nervous system.201–203 RET is expressed predominantly on the surface of neural crest tissues, and glial-derived neurotrophic factors (GDNFs) such as neurturin, artemin, and persephin serve as ligands for RET. GDNFs initially bind to their cognate coreceptors, the GDNF receptors (GFPα1 to GFPα4), on the cell surface, which recruits RET into lipid raft membrane domains and causes conformational changes via the cadherin-like moieties, dimer formation, and then subsequent transphosphorylation of tyrosine residues and kinase activation.204 Y1062 is the common docking site for all three RET isoforms and serves to recruit many adapters including SHC1, FRS2, IRS1/2, DOK, and JNK.201 Phosphorylation of Y752 and Y928 binds STAT3, whereas other phosphorylated residues are recognized by Src, resulting in activation of focal adhesion kinase (FAK), which promotes cell migration and metastatic spread. In addition, the MAP kinase, PI3 kinase/AKT, and phospholipase-Cγ pathways can be activated by RET to promote cellular proliferation and survival.204 Germline loss-of-function RET mutations occur in Hirschsprung and CAKUT (congenital anomalies of the kidney and urinary tract) disease, which causes abnormalities of the developing gut and kidneys, respectively. Conversely, germline activating RET mutations are the basis for the multiple endocrine neoplasia type 2 (MEN2) syndromes. Patients with MEN2 develop familial medullary thyroid carcinomas and other cancers.205 MEN2A is mainly driven by mutations in six cysteine resides in the RET extracellular domain (C609, C611, C618, C620, C630, and C634), whereas the kinase domain mutations M918T or A883F are associated with MEN2B. Sporadic medullary thyroid carcinomas are much more common, and up to 60% of such tumors harbor somatic mutations in RET, notably G691S, which are thought to be a driver alteration in this disease.206 Furthermore, RET gene rearrangements with numerous fusion partners, including CCDC6 and NCOA4, termed RET-PTC1 and RET-PTC3, respectively, occur in 20% to 40% of papillary thyroid carcinomas (PTCs) and often occur as a consequence of high doses of radiation.207 RET inhibitors have shown significant antitumor activity in patients with medullary thyroid cancer. Vandetanib, an oral inhibitor of RET, EGFR, and VEGFR, is FDA approved for the treatment of patients with advanced medullary thyroid cancer.208 A randomized, placebocontrolled phase III study of cabozantinib, an oral, multitargeted TKI that inhibits RET, VEGFR2, and MET, was also recently conducted in patients with unresectable, locally advanced, or metastatic medullary thyroid carcinoma.209 This trial documented a statistically significant improvement in median progression-free survival with cabozantinib as compared with placebo (11.2 months versus 4.2 months in placebo arm, P < .0001). More recently, cabozantinib was FDA approved for the treatment of renal cell carcinomas that progressed on antiangiogenic therapy, although the activity of cabozantinib in this context may not be attributable to its inhibition of RET. Several RET inhibitors have, however, shown promising clinical activity in patients with NSCLC treated with RET fusions (see Table 2.1).210

Vascular Endothelial Growth Factor Signaling Six vascular endothelial growth factor (VEGF) ligands have been identified: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental

growth factors 1 and 2.211 VEGF-A has four isoforms produced by alternative gene splicing, with the 165–amino acid length isoform playing a central role in tumor angiogenesis.212 Specifically, VEGF-A enhances vascular permeability and stimulates endothelial cell proliferation, resulting in new blood vessel formation. Vascular endothelial growth factor receptors (VEGFR1 to VEGFR3) are RTKs that possess a modular structure consisting of an extracellular domain with seven Ig-like regions, a TM domain, and an intracellular tyrosine kinase domain.211 VEGF-A, VEGF-B, and placental growth factor all bind VEGFR1 (also known as FLT1), but the exact role of VEGFR1 in tumor angiogenesis has yet to be fully elucidated. In some settings it may act as a decoy receptor that prevents ligandmediated stimulation of VEGFR2 (also known as FLK1/KDR).213 VEGFR2 has been implicated in the development of vasculature during development and is considered the primary receptor through which VEGF exerts its angiogenic effects in endothelial cells.213,214 Binding of ligand to VEGFR2 results in receptor dimerization and transphosphorylation followed by activation of multiple mitogenic signal transduction cascades.215,216 More recently, VEGF has been implicated in many angiogenesis-independent roles including regulation of immune cells in the tumor microenvironment, fibroblasts in the tumor stroma, and cancer stem cells.217 VEGF can also bind and signal through a class of TM glycoprotein coreceptors called neuropilins (NRP1 and NRP2), which are found on tumor cells and can signal along many oncogenic axes including Hedgehog and JNK.217 The complex network of cross talk among VEGFs, VEGFRs, and canonic oncogenic pathways makes VEGF and VEGFR critical but elusive targets in cancer therapy. Targeted therapies that inhibit VEGF signaling include antibodies that bind circulating ligand and RTK inhibitors. The humanized monoclonal antibody bevacizumab binds to free VEGF, thereby preventing its association with VEGFRs. This antibody has been FDA approved for use in combination with chemotherapy for patients with several cancers, including metastatic colorectal218 and nonsquamous NSCLCs.211,219 Bevacizumab also has activity in patients with glioblastoma220 and metastatic renal cell carcinoma, where it is often used in combination with interferon-α (IFN-α).221 In addition, ramucirumab is a VEGFR2-directed antibody that has received FDA approval with or without chemotherapy in several cancers.222 Sorafenib, sunitinib, pazopanib, and axitinib are multitargeted tyrosine kinase inhibitors with nanomolar potency for VEGFR2. Sunitinib is used in the treatment of patients with metastatic renal cell carcinoma, GISTs, and pancreatic neuroendocrine tumors.223 Sorafenib has been approved for the treatment of liver and renal cell cancers.224,225 Although these agents inhibit multiple kinases, their antitumor effects have been attributed primarily to their antiangiogenic activity. More recently, the tyrosine kinase inhibitor pazopanib was approved for the initial treatment of metastatic renal cell carcinoma and in cytokine-pretreated patients,226 and axitinib227 was approved in the second-line setting following failure of prior systemic therapy. Lenvatinib, a multitargeted RTK inhibitor that inhibits VEGFR1, VEGFR2, and VEGFR3, has received recent FDA approval for both thyroid cancer (as monotherapy)228 and renal cell carcinoma in combination with everolimus.229 Despite widespread activity in preclinical models, antiangiogenic therapies have shown disappointing activity in several tumor types. A number of resistance mechanisms have been hypothesized to explain the lack of broader clinical activity, including the activation of redundant signaling pathways that promote angiogenesis; the recruitment by tumors of bone marrow–derived endothelial progenitor cells; increased pericyte density around existing blood vessels, which enhances vascular growth and survival; and the ability of tumor cells to invade surrounding stroma to co-opt additional blood supply.230 A better understanding of these resistance mechanisms may lead to the development of more effective antiangiogenic therapies in the future.

Intracellular Signaling  •  CHAPTER 2 35

Hepatocyte Growth Factor Receptor Signaling The hepatocyte growth factor receptor (HGFR or MET) is encoded by the MET gene.231,232 Both MET and its ligand hepatocyte growth factor/scatter factor (HGF/SF) are expressed as immature precursors that require proteolytic cleavage.233 The MET extracellular domain consists of an alpha subunit connected by a disulfide bridge to a TM beta subunit, and contains a Sema domain, a PSI domain, and four IPT domains.234,235 The intracellular portion of the receptor contains a juxtamembrane region that harbors a serine residue (Ser 975) that inhibits RTK activity on phosphorylation, as well as a tyrosine kinase domain with Y1234 and Y1235 acting as key sites of autophosphorylation required for activation.235 A tyrosine residue at position 1003, proximal to the tyrosine kinase domain, serves as an interaction site for the ubiquitin ligase CBL, which marks the receptor for endocytosis and degradation.236,237 C-terminal residues Y1349 and Y1356 represent docking sites for adaptor proteins.238 On binding of HGF/SF to the extracellular portion of MET, receptor dimerization occurs, followed by transphosphorylation. A number of adaptor proteins then bind to phosphorylated tyrosine residues, including Grb2 and GAB1, phospholipase-C (PLC), and SRC, which promotes the activation of the MAP kinase and PI3 kinase/AKT signaling pathways.239,240 MET can also activate RAC1/CDC42 and p21-activated kinase (PAK1), both of which regulate cytoskeletal proteins and integrin expression and activation, and thus cell migration.238,239 MET also plays an important role in driving epithelial-to-mesenchymal transition (EMT) cell migration during embryo development, and organ regeneration.238,241 Dysregulation of MET signaling can occur through multiple mechanisms, including activating point mutations (often in the kinase domain; prevalent in lung cancer), exon skipping events, receptor overexpression, and upregulation of HGF, which can activate MET in an autocrine and/or paracrine manner.238,240,242–246 Germline mutations of MET are found in patients with hereditary papillary renal cell carcinomas, and MET overexpression is observed in a significant proportion of sporadic papillary cancers as well as collecting duct carcinomas.247 Multiple other malignancies exhibit aberrations in MET signaling, including lung, breast, pancreatic, colon, and gastric cancers. Amplification of MET is associated with a worse prognosis in lung and gastric cancers, whereas expression of MET or HGF is an unfavorable prognostic biomarker in liver, kidney, colorectal, and gastric cancers.248 Recurrent somatic splice site alterations involving MET exon 14 (METex14) have been identified in lung cancer. These mutations result in exon skipping, loss of the juxtamembrane CBL E3-ubiquitin ligase-binding site, diminished receptor turnover, and ultimately, MET activation.243,249,250 These exon 14 MET mutations are mutually exclusive with activating mutations in EGFR and KRAS as well as ALK, ROS1, and RET fusions, and treatment of patients with exon 14 MET splice variants with a MET kinase inhibitor is now considered to be a standard treatment option (see Table 2.1).210 Several RTKs have also been shown to activate MET, including EGFR, HER2, and IGF1R. For example, EGFR activation can stimulate MET signaling, and resistance to EGFR inhibitors in some lung cancers has been shown to stem from coactivation of MET in the setting of gene amplification.251 Inhibitors of MET signaling have been in development for a number of years. Therapeutic strategies for targeting MET activation in cancer patients include antibodies that target the extracellular domain of the receptor, antibodies that bind to and thus sequester circulating HGF, and small-molecule tyrosine kinase inhibitors that selectively target MET or are multi-kinase MET inhibitors.248 Durable responses to crizotinib and cabozantinib, multikinase MET inhibitors, have been reported in patients with NSCLC with MET splice mutants or MET amplification (see Table 2.1).252,253 Cabozantinib is also now standard of care in MET-amplified renal cell carcinoma.254,255 Combination therapies to combat MET reactivation after EGFR kinase inhibitor therapy suggest an improvement in progression-free survival.256

Tropomyosin Receptor Kinases/Neurotrophic Tyrosine Kinase First cloned as an oncogenic fusion partner of the tropomyosin receptor kinases and subsequently characterized for their role in neural differentiation and survival in the peripheral and central nervous systems, the TRKA/B/C family of RTKs, encoded by the neurotrophic tyrosine kinase (NTRK) genes 1 to 3 (NTRK1/2/3), respectively, integrate ligand stimulation from nerve growth factor, brain-derived neurotrophic factor, neurotrophins 3 to 6 with downstream activation of PI3K, phospholipase-C (PLC)-gamma, and MAPK signaling.257–261 Chromosomal rearrangements involving the tropomyosin receptor kinases (TrkA/NTRK1, TrkB/NTRK2, and TrkC/NTRK3) were recently shown to occur at high frequency in several rare cancer types including mammary secretory carcinoma of the breast and congenital-infantile fibrosarcoma.262–264 NTRK fusions also occur at low frequency in a broad range of more common adult solid tumors.260,265 These NTRK fusions induce ligand-independent constitutive kinase activity, resulting in upregulation of canonic downstream signaling pathways involved in growth and survival. TPM3, LMNA, MPRIP, TRIM24, ETV6, and PPL, among others, have been identified as fusions partners with the NTRK genes.266,267 Dramatic and durable clinical responses have recently been reported in patients with NTRK fusions, with first- and secondgeneration TRK inhibitors such as larotrectinib (LOXO-101), entrectinib, and LOXO-195 (see Table 2.1).268–273 Notably, clinical activity was observed in both adult and pediatric patients and was independent of site of tumor origin.264

G PROTEIN–COUPLED RECEPTOR SIGNALING G protein–coupled receptors (GPCRs) are seven TM domain–containing proteins that transduce ligand-specific signals across the plasma membrane to mediate numerous physiologic processes including sensory perception, immunologic responses, neurotransmission, weight regulation, and cardiovascular activity.274 GPCRs also regulate basic cellular functions including growth, motility, differentiation, and gene transcription. The GPCR family comprises more than 800 receptors, which are the targets of over 30% of all FDA-approved drugs, although few to date have found a role as anticancer therapies.275,276 Given that GPCRs activate many of the signaling cascades that are deregulated in human cancer, it is not surprising that studies have implicated GPCRs in cancer initiation and progression.277–279 GPCRs can be categorized into five or six families, depending on the nomenclature used.280 In a more recent phylogenetic classification, the five major families are represented by the acronym GRAFS: Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2, and Secretin.281 The Rhodopsin family of receptors (also referred to as class A GPCRs) is the largest class, with more than 670 members. Crystallization of the bovine rhodopsin receptor in 2000 provided the first high-resolution insight into the structure of GPCRs.282 In general, Rhodopsin family receptors have short N-termini. Included in this family are the α group (histamine, dopamine, serotonin, adrenoceptors, and muscarinic, prostanoid, and cannabinoid receptors), the β group (endothelin, gonadotropin-releasing hormone, and neuropeptide Y), the γ group (opioid, somatostatin, and angiotensin), and the δ group (P2RYs, glycoprotein-binding FSHR/TSHR/LHCGR, PARs, and olfactory receptors).283 The 15 Secretin receptors (class B) all have conserved cysteines in the first and second extracellular loop, and most have three cysteine bridges in the N-termini. This class includes the calcitonin-like, corticotrophin-releasing hormone, glucagon-like, gastric inhibitory polypeptide, growth hormone–releasing hormone, adenylate cyclase– activating polypeptide, parathyroid hormone, secretin, and vasoactive intestinal peptide receptors.284 The Adhesion family (also included in class B, according to another classification system) has distinctly long N-termini, and only 3 of 33 receptors in the family have known ligands (epidermal growth factor-like module containing mucin-like

36 Part I: Science and Clinical Oncology

receptors EMR2, EMR, EMR4).285 The Glutamate receptor family (class C) binds ligands in their N-termini, which have a complex two-domain folded structure bridged by disulfide bonds. Common receptors in this family of 22 include the glutamate, GABAB, calciumsensing, sweet and umami taste (TAS1R1–TAS1R3), and GPCR6 receptors.286,287 The Frizzled receptors (FZD1–FZD10 and SMO; see later for in depth discussion of signaling) bind Wnt ligands in the extracellular domain region containing nine conserved cysteine residues.288 The Taste2 receptors (25 members of T2R, bitter taste) have varying sequence homologies that likely allow the sensing of thousands of distinct bitter tastes.289,290 Overall, GPCRs share the seven–TM domain structure, but have different regions of conservation. All of the families include orphan receptors, which are related by sequence and structure but have no identified ligand to date. All GPCRs have seven α-helical domains that weave through the plasma membrane and are interconnected by flexible extracellular and intracellular segments, thus engendering the synonyms serpentine or heptahelical receptors. The specificity of biologic response initiated by each GPCR depends on (1) ligand recognition; (2) the distinct structure of each receptor class and subclass; and (3) ligand-directed binding of specific cytosolic enzymes and adapters that initiate a plethora of intracellular signaling cascades (general and cancer-specific examples are discussed further later). The GPCR Network was created in 2010 to tackle the challenge of delineating the structure and function of this diverse class of receptors.291 Operationally, ligand binding on the GPCR extracellular surface induces a conformational change in the receptor, mainly via TM helices TM3, TM5, and TM6, which creates a deep pocket in the intracellular face of the receptor.292–294 This cleft enables binding and activation of heterotrimeric G proteins, which consist of an inactive, GDP-bound Gα subunit and a Gβγ-subunit dimer, which act as a molecular switch.295,296 Activated GPCRs promote GDP for GTP exchange on the Gα nucleotide binding site.297 This GTP-bound Gα dissociates from Gβγ and the receptor, and then both activated subunits go on to initiate signaling cascades. There are four members of the Gα family, Gαs, Gαi/o, Gαq/11, and Gα12/13, which can be further subtyped and can each stimulate several downstream effectors. Moreover, each GPCR can couple to multiple Gα family members, thus generating a complex pattern of intracellular signaling. Classic GPCR activation of Gαs stimulates adenylyl cyclase, which generates the second messenger 3′-5′-cyclic adenosine monophosphate (cAMP).298,299 cAMP activates multiple downstream effectors including cAMP-gated ion channels; Epac, a GEF for Rap1/2 (which functions in cell adhesion and junction formation); and protein kinase A (PKA).274,295,300–303 cAMP binds to PKA regulatory subunits, releasing catalytic subunits and triggering the activation of cytosolic and nuclear substrates, including the transcription factor CREB (cAMP response element binding protein), which can induce proliferation and differentiation, among other phenotypes, depending on cell origin.304–306 Gαs also activates the Src tyrosine kinase and the GTPase activity of tubulin.295,300 GPCR-coupled Gαi/o typically works in opposition to Gαs by inhibiting adenylyl cyclase and decreasing cAMP levels.274,307 In addition, some Gαi/o isoforms can signal to K+ and Ca+ channels, increase cGMP phosphodiesterases, interact with Rap1GAP1, and cross talk to the MAP kinase pathway (as described later in the chapter).274 Members of the Gαq/11 family activate phospholipase-Cβ, which catalyzes the hydrolysis of PIP2 to yield IP3 and DAG.308 IP3 mobilizes calcium from intracellular stores, whereas DAG activates some isoforms of protein kinase C (PKC).309,310 Both Gαq/11 and Gα12/13 activate a variety of RhoGEFs (p115-RhoGEF, PDZ-RhoGEF, LARG, Lbc, AKAP-Lbc) and thus regulate Rho activity (mainly RhoA) and its contribution to actin stress fiber formation, cell shape and polarity, cell adhesion and migration, gene transcription, and cell cycle progression.311 In addition, Gα12/13 activates the Na+/H− exchanger, inducible nitric oxide synthase, phospholipase D, E-cadherin, radixin, and protein phosphatase 5. Gβγ signaling is equally complex, as the active dimer

stimulates G protein–regulated inwardly rectifying K+ channels adenylyl cyclase (types II, IV, VII), PLCβ, PI3Kγ, Src, and GPCR kinases (GRKs; see later). It inhibits adenylyl cyclase (type I), some Ca+ channels, and calmodulin; stabilizes Gα in the GDP-bound, inactive state; and helps specify coupling to the proper Gα member.274,295,296,300 Gα signaling is switched off by a family of GTPase activating proteins called Regulators of G-protein Signaling, or RGS proteins, which enhance the intrinsic rate of GTP hydrolysis by greater than 1000-fold.312,313 Some RGS proteins enhance signaling through RhoGEFs and act as scaffolds for other signaling cascades (e.g., tethering to Raf and MEK2). The GPCR effectors PKA and PKC also contribute to receptor inhibition by phosphorylating their cognate-activated GPCR and thereby uncoupling and inactivating Gαs/Gαq in a classic negative feedback loop.314,315 GPCRs can also function via G protein–independent mechanisms by binding to the arrestin family of cytosolic adapter proteins.316,317 GPCR-coupled arrestins have pleiotropic cellular roles including (1) dampening G-protein signaling by scaffolding enzymes that degrade G-protein second messengers; (2) desensitizing receptors by binding GPCR kinase (GRK)–phosphorylated GPCRs and sterically blocking access to further Gα subunits; (3) mediating GPCR trafficking and endocytosis to clathrin-coated pits; and (4) acting as a scaffold for multiple MAP kinase cascades. For example, MEK1 is engaged by a tethered complex of Raf-1, ERK2, and β-arrestin to facilitate mitogenic signaling.318,319 GPCRs are well established drug targets for antihistamine, antacid, cardiovascular, and antipsychotic drugs; pain suppressants; and antihypertension therapies.275,276 Although less well appreciated as drug targets in cancer, there is increasing evidence that dysregulated GPCR signaling contributes to cancer initiation and progression. For example, in 1986 the wild-type MAS1 gene, which encodes the MAS GPCR, was reported to induce transformation by coupling to the small G protein Rac.320,321 Large-scale deep sequencing efforts have revealed that GPCR mutations occur in approximately 20% of all cancers. Receptors for thyroid-stimulating hormone (TSHR), Hedgehog (Smoothened receptor [SMO]), glutamate (GRM), the adhesion family, lysophosphatidic acid (LPA), and sphingosine-1-phosphate (SIP) are the most frequent GPCRs altered in cancer.277,312,313,322 G proteins themselves are also mutated in cancer. In particular, recurrent mutations in GNAS (which encodes Gαs) have been identified in thyroid and pituitary tumors, as well as mutations in GNAQ and GNA11 in melanoma of the eye and skin, respectively.322 Hot spot resides that disrupt GTPase activity and render the protein constitutively active have been identified in GNAS at positions R201 and Q227, in GNAQ at R183, and in GNA11 at Q209. Numerous inhibitors of GPCR signaling are also being studied in the clinic including BKT-140/ BL-8040 in pancreatic adenocarcinoma and blood cancers (target: CXCR4 receptor), and CXCR2 ligands (target: CXCR2 receptor).278 Detailed later are a few specific examples of GPCRs that have been shown to play a role in cancer initiation and/or progression. A connection between GPCRs and EGFR signaling has been established in both normal cell physiology and in colon, lung, breast, ovarian, prostate, and head and neck cancer development.323–326 Ligand-bound GPCRs activate Src, PKC, Ca+ channels, and PKA intermediaries, which stimulate proteolytic cleavage and release of membrane-tethered growth factors that bind and thereby transactivate EGFR. Specifically, estrogen binding to the GPCR GRP30 facilitates matrix metalloproteinase–2 (MMP2) and MMP9-mediated cleavage of the growth factor precursor pro-heparin-binding-EGF (pro-HB-EGF), thus initiating HB-EGF–mediated EGFR transactivation.324 Through a related mechanism, LPA-, SIP- and thrombin-activated GPCRs transactivate EGFR in breast cancer cells via growth factor shedding of tumor necrosis factor–α (TNF-α) through the action of TACE/ ADAM17 zinc-dependent proteases.326 Thrombin-mediated N-terminal cleavage and activation of proteinase-activated receptor 1 (PAR1), which can act through EGFR, has been found to promote metastasis and invasiveness in melanoma, breast, colon, and prostate cancers.278,279

Intracellular Signaling  •  CHAPTER 2 37

Intriguingly, MMP1 was found to function similarly to thrombin in activating PAR1 and promoting breast cancer tumorigenesis and invasion.327,328 This cross talk between GPCRs and EGFR provides a rationale for the combinatorial use of GPCR agonists/antagonists and EGFR inhibitors in patients with EGFR-driven lung and colorectal cancers. Aberrant GPCR signaling through Gα12/13 also contributes to tumorigenesis by enhancing cancer cell migration, invasion, angiogenesis, and metastasis.329 Ligand-activated LPA, PAR1, SIP, thromboxane A2 (TP), CXC chemokine (CXCR4), and prostaglandin E2 (PGE2) receptors couple to Gα12/13 and RhoGEFs to hyperactivate RhoA (see earlier), which elicits these progression-associated phenotypes in glioma, melanoma, lung, breast, and ovarian cancers.278,279 Overexpression of Gα12/13 and RhoA in breast, prostate, and colon cancers also promotes metastasis by decreasing cell adhesion.311 RhoGEF inhibitors; RhoGTPase inhibitors; inhibitors of prenylation, which could indirectly impair proper Rho localization (statins, farnesyl/ geranylgeranyl transferase inhibitors); and inhibitors of kinases downstream of Rho (ROCK, LIMK, MRCK, PAK) have all been developed and tested in biochemical, cell line, and mouse experiments. However, a clear benefit to the use of such compounds in cancer patients has yet to be established.330 Two other prominent examples of dysregulated GPCR signaling in human cancer are the Hedgehog/Smoothened and Wnt/Frizzled signaling pathways.277,278,331 Both pathways, along with Notch, also play critical roles in cell fate and have been implicated in the underlying pathway cross talk that is key to cancer stem cells.332 Secreted Hedgehog (Hh) ligands (first identified based on their roles in normal development and stem cell homeostasis, with Sonic Hedgehog [SHH] being the most ubiquitous) bind to the 12-pass TM receptor Patched (PTCH), which relieves its repression of the GPCR Smoothened (SMO).333,334 Activated Smoothened couples to Gαi and Gα12, which regulate the glioma-associated oncogene homologue (GLI) transcription factors, which in turn regulate proliferative, survival, and differentiation signals involving cyclin D1, myc, BCL2 and the Forkhead transcription factors, to name a few.335,336 Mutations in SHH, PTCH, and SMO are found in patients with inherited and sporadic basal cell carcinomas337,338 and ameloblastomas,339 whereas overexpression of Hh ligands has been shown to result in hyperactivation of the pathway in breast, colon, prostate, and pancreatic ductal adenocarcinomas331,340 Vismodegib and sonidegib, both selective Smoothened receptor inhibitors, are FDA approved for the treatment of advanced basal cell carcinomas (see Table 2.1).341–344 Several other Hh/SMO inhibitors are currently being tested in patients with cancer, mostly for advanced and/or metastatic solid tumors.278,345,346 The secreted Wnt glycolipoprotein ligands activate the single TM low-density lipoprotein–related coreceptors LRP5/6 and the GPCR-like TM protein Frizzled (Fz), which are phosphorylated and likely couple to Gαq and Gαo, respectively, to activate the cytoplasmic scaffold Dishevelled.347–349 Dishevelled in turn inhibits the β-catenin degradation complex (which consists of APC, axin, CKIα and GSK-3β, and the E3 ubiquitin ligase β-TrCP).350 This results in accumulation of β-catenin, which translocates to the nucleus where it induces TCF/ LEF-mediated transcription of genes important for cell differentiation and proliferation, including myc, cyclin D1, VEGF, FGF4/18, E-cadherin, COX-2, and members of the Wnt cascade itself.349,351,352 Noncanonic Wnt/Fz pathways include signaling to the transcription factors NFAT via Gαq/i/Gβγ/PLC/PKC/Ca+ (Wnt-calcium pathway) and AP1 via Rho/Rac/JNK (planar cell polarity pathway).277,353 These pathways regulate cell polarity and migration and are implicated in cancer metastasis.353 Aberrations in canonical Wnt signaling promote tumorigenesis in melanoma and colon, liver, ovarian, and prostate cancers.354,355 Specifically, loss-of-function mutations or truncations in APC and AXIN1/2 and gain-of-functions mutations in β-catenin are found in almost all colorectal cancers, with APC alteration found in over 85%.349,356 Germline mutations in the APC gene are also the basis for the inherited cancer predisposition syndrome familial

adenomatous polyposis (FAP).357 Efforts to develop selective inhibitors of Wnt signaling are ongoing and will be aided by current endeavors to crystallize members of the Wnt cascade.354,358 Of note, nonsteroidal antiinflammatory drugs (NSAIDs) have shown some promise in modulating Wnt signaling, likely by inhibiting the Wnt-output gene COX-2 or by enhancing E-cadherin signaling.349,359 COX-2 inhibitors have shown efficacy in reducing the risk of polyps in patients with FAP.358,360

CYTOKINE RECEPTOR SIGNALING Cytokines are protein and glycoprotein ligands secreted by immune cells; they initiate diverse and often opposing effects based on target cell lineage. Processes regulated by cytokine signaling include cell proliferation, differentiation, survival, inflammation, angiogenesis, antiviral activity, and modulation of immune function. Cytokines signal in an autocrine and/or paracrine fashion and can be subclassified by protein structure into four families, which total over 100 members: hematopoietins, IFNs, chemokines, and the TNF superfamily. The hematopoietin family consists of interleukins (IL-1 to IL-31), growth hormone, prolactin, erythropoietin, thrombopoietin, leptin, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, and a few others.361 The majority bind to either class I or II cytokine receptors, which are single TM glycoproteins that lack kinase activity and diverge in their extracellular domains in order to specify ligand binding.362,363 Class I receptors function as a cluster of two or three subunits that each have two sets of conserved cysteine pairs and a WSXWS motif in their external domains. Class II receptors lack the WSXWS motif and one of the class I conserved cysteine pairs, but contain conserved proline, tryptophan, and an additional two conserved cysteine residues. Ligand binding causes aggregation of the γ-chain (also called γc, CD132) which is common to many cytokines, and the β-chain (also known as IL-2Rβ, IL-15Rβ, or CD122).364,365 In the case of specific cytokines, such as IL-2, association with a third subunit, the α chain (also called IL-2Rα, CD25, or Tac antigen), allows for high-affinity ligand binding.366,367 The IFN family members are divided into type I, II, and III classes.368,369 Most IFNs are type I and can be further subtyped.370 All type I IFNs bind the type I IFN receptor, which consists of two subunits (IFNAR1 and IFNAR2). The sole type II IFN, IFN-γ, binds the type II IFN receptor, which is composed of the IFNGR1 and IFNGR2 subunits. Both types of IFN receptors belong to the class II cytokine receptor family. The IFN family also includes a third branch, the IFN-like molecules, IFN-λ1 (IL-29), IFN-λ2 (IL-28A), and IFN-λ3 (IL-28B), which display some structural overlap with ILs and the antiviral properties of IFNs, and bind a distinct receptor made up of IFNLR1/IL-28Rα and IL-10Rβ.371 Given that cytokine receptors are promiscuous and bind multiple cytokine ligands, there is a high degree of redundancy in the output profiles of individual cytokines. This redundancy serves to amplify and sustain signaling downstream of these transitory stimuli. Hematopoietin or IFN binding induces oligomerization of cytokine receptor subunits and autophosphorylation and activation of the JAK (Janus activated kinase) family of intracellular tyrosine kinases (TYK2, JAK1–JAK3), which are constitutively bound to box I and box II α-helix motifs in the receptor cytoplasmic tail.372 Activated JAK proteins phosphorylate tyrosine residues on the cytokine receptor chains, which classically bind the STAT (signal transducer and activator of transcription) family of transcription factors (STAT1–STAT6).361,373 Docking of STATs facilitates their phosphorylation by JAKs, which in turn causes STAT dimerization, nuclear translocation, and alterations in gene transcription.374 There is also cross talk between STAT signaling and the nuclear factor–κB (NF-κB) and SMAD signaling pathways.375 STATs are also activated by RTKs, SRC, and ABL, and STAT activation also plays a role in transformation initiated by these oncogenes.376 JAK/STAT activity is regulated by several posttranslational modifications as well as by the SOCS (suppressor of cytokine signaling) and PIAS

38 Part I: Science and Clinical Oncology

(protein inhibitor of activated STAT) proteins.375 In addition to JAK-STAT signaling, cytokine receptors can transduce messages through LCK and SYK (Src-family kinases), through BCL-2, and via PI3 kinase/AKT and Ras/Raf mediated upregulation of Fos- and Jundependent transcription.376,377 The 29-member TNF superfamily of receptors (TNFSFR1) and their corresponding 19 ligands (TNFSF) induce inflammation in addition to prosurvival and proapoptotic phenotypes.378,379 TNF ligands are TM proteins that function as membrane-integrated or cleaved, soluble trimers that bind and activate preformed, single-TM TNF receptor trimers on the cell surface.380 Conserved cysteine residues in the TNF receptor external domains dictate ligand binding, and the presence of death domains (DDs), TRAF-interacting motifs (TIMs), or neither (decoy) dictate downstream signaling. For example, on apoptotic stimuli, ligand activation of the TNF-R1 and DR3 receptors recruits the adaptor TRADD (TNFR-associated death domain) via DDs, which in turn binds to FADD (Fas-associated protein with death domain).378,379,381 FADD binds procaspase-8 and procaspase-10 via death effector domains (DEDs), and induces their cleavage to form active enzymes, which cleave caspase-3 and induce apoptosis. TRADD binding is also capable of inducing the intrinsic apoptotic cascade (mitochondrial release of ROS, cytochrome C, and Bax, which leads to caspase-9 and caspase-3 activation and apoptosis).378,382 Under proliferative stimuli, TNF-α–dependent activation of TNF-R1/TRADD and TNF-R2 converges on the recruitment of TRAF2 (TNFR-associated factor 2). TRAF2 binding sequentially recruits the RIP (receptor interacting protein), TAK1 (TGF-β activated kinase 1), and IκB kinase (IKK) trimer; this functions to degrade IκB-α (inhibitor of NF-κB-α) and in turn facilitates the activation and translocation of NF-κB.378,379 NF-κB in turn induces mediators of inflammation and cytoprotective phenotypes.383 Alternatively, TAK1 can signal to MKK3/6 (MAP kinase kinase 3/6) and to two MAP kinases, ERK (proproliferation) and p38α (to activate the transcription factor AP-1 [activator protein-1]).384 Upstream, TRAF2 can additionally trigger MEKK1 (MAP/ERK kinase kinase 1), MKK7, and JNK (c-Jun activating kinase), the recruitment of which also converges to activate AP-1 and thus regulate proliferation and survival.385–387 There is an important avenue of cross talk between the TNF-directed NF-κB and JNK pathways, the balance of which ultimately decides cell fate.388 In cancer, dysregulation of cytokine signaling promotes chronic inflammatory signals and prevents the immune system from attacking cancer cells. Unfortunately, because of their pleiotropic effects, which are often cell type and microenvironment specific, therapeutic strategies that modulate cytokine signaling have demonstrated only modest clinical activity in patients with cancer. For example, the recombinant human IFN-α2a mimetic, Roferon-A, has been shown to inhibit tumor growth in patients with melanoma and hairy cell leukemia, but such treatments have now been supplanted by more active therapies.389 Moreover, focal delivery of TNF-α to limbs affected by soft tissue sarcomas and melanomas through isolated limb perfusion techniques has been more effective than systemic use, which is toxic.390,391 Proapoptotic, anti-TRAIL therapies, such as mapatumumab, are also in clinical testing.381 Downstream components of the cytokine signaling cascades are also being explored as targets for drug development. Gain-of-function mutations in the JAK2 (hot spot V617F) and MPL genes, the latter of which encodes the thrombopoietin receptor, are common in myeloproliferative neoplasms, and the selective JAK1/2 kinase inhibitor ruxolitinib has been approved for this indication (see Table 2.1).392–394 STAT3 is frequently hyperactivated in cancer, because it is a downstream effector of both cytokine receptors and mutated and amplified tyrosine kinases.376,395 Constitutive activation of STAT5 and STAT6 plays a critical role in BCR-Abl–driven chronic myelogenous leukemia (CML), and IL-13–driven lymphomas and leukemias.376 On the basis of these findings, direct inhibitors of JAK and STAT are currently in development.374,396 Inhibition of NF-κB has been shown to induce apoptosis in leukemias and lymphomas and enhance chemotherapy and radiation

response.383 JNK1 is upregulated in hepatocellular carcinoma and prostate cancer, whereas p38α activity is either lost (in hepatocellular carcinoma) or activated in numerous cancers, and targeted inhibitors are being developed.385,397

SERINE/THREONINE RECEPTOR SIGNALING Receptor serine/threonine kinases are exemplified by the TGF-β type I and II receptors.398,399 Ligands for these receptors include the TGF-β superfamily, which comprises the TGF-β1-3 isoforms, activins, inhibins, Nodal, and Lefty, and the more distantly related bone morphogenic proteins (BMPs), growth and differentiation factors (GDFs) and müllerian inhibitory substance (MIS). TGF-β ligands regulate a diverse array of physiologic processes including growth, proliferation, survival, hormone release, and differentiation.398,400 They thus have a central role in embryonic patterning, tissue development, and morphogenesis. Signaling mediated by TGF-β, the namesake and most studied ligand of the superfamily, will be used to exemplify the general structure of the serine/threonine kinase cascades. TGF-β is ubiquitously expressed and requires a multistep maturation and secretion process to be functional and bioavailable. Initially translated as an immature proprotein, the prodomain (called latencyassociated protein [LAP]) is cleaved and noncovalently bound to the remaining mature form of TGF-β.401 Covalently bound mature, active dimers are further bound to latent TGF-β binding protein (LTBP) such that this complex is sequestered by LTBP binding to the extracellular matrix (ECM) until appropriate signals initiate matrix metalloproteinase, plasmin, or thrombin-dependent cleavage of LTBP and subsequent release of active TGF-β dimers.401,402 TGF-β dimers bind constitutively active, single TM, homodimeric TGF-β type II receptors (TβRII). Once bound by ligand, TβRII forms a complex with TGF-β type I (TβRI) receptor homodimers, thus creating a receptor heterotetramer.398,400 TβRII receptors phosphorylate and activate the intrinsic kinase activity of TβRI, which in turn phosphorylates serine residues in the C-terminal–SSXS motif of receptor-activated SMAD proteins (R-SMAD2 and R-SMAD3 for TGF-β).403 Activated R-SMAD2 and R-SMAD3 then form a heterotrimer with SMAD4, which then localizes to the nucleus, where it both binds DNA and partners with other transcription factors (i.e., Forkhead, homeobox, zinc-finger, AP-Ets, and bHLH family transcription factors) and cofactors (e.g., p300, CBP).404,405 These SMAD-containing complexes then target selective promoter elements with high affinity, leading to the induction or repression of hundreds of genes, depending on cell context. For example, TGF-β induces the expression of 4EBP1 and the cyclin-dependent kinase inhibitors INK4B and p21 and represses Myc to elicit growth inhibitory effects.406 It also activates DAPK (death-associated protein kinase), GADD45β (growth arrest and DNA damage-inducible 45β), and BIM to promote apoptosis and PDGF in smooth muscle cells to enhance proliferation, among other effects.407 TGF-β also signals through many non-SMAD effectors including Shc, which can result in enhancement of Ras/ERK signaling, and TRAF6, which activates the TAK1/MKK3&6/JNK/p38 cascades.408 TGF-β, through indirect mediators, can also activate Src, Rho, and PI3 kinase, and through pathway cross talk the Wnt, Hedgehog, and Notch cascades.409 The outcome of TGF-β–directed transcriptional responses depends on access of TGF-β to particular signaling receptors and SMAD complexes, the availability of transcription factors, and the epigenetic status of the cell.410 Alterations in TGF-β signaling are common in cancer.407,411,412 For example, mutational inactivation of TβRII is seen in colorectal cancers with microsatellite instability.413 Mutations and loss of SMAD4 expression are common in colorectal, pancreas, and head and neck cancers.414,415 Conversely, increased TGF-β expression occurs in breast, prostate, and colorectal cancers and has been associated with cancer progression and the development of metastases.407 TGF-β has also been shown to play a role in maintaining the tumor-initiating cell or cancer stem cell population in gliomas, leukemias, and breast

Intracellular Signaling  •  CHAPTER 2 39

cancer.407 It has been proposed that TGF-β signaling promotes invasion and metastasis by promoting EMT.406 Interesting to note, in pancreatic ductal adenocarcinoma, wherein loss of SMAD4 is common, TGF-β can play a novel tumor suppressor role by inducing a lethal EMT through the lineage and progenitor regulators Klf5 and Sox4.416 Intense efforts are underway to develop therapeutic strategies that inhibit the tumorigenic properties of TGF-β signaling. These include the development of selective TGFβRI inhibitors, TGF-β blocking antibodies, soluble TGF-β antisense therapies, and selective kinase inhibitors.411,417,418 The development of inhibitors of TGF-β signaling has, however, been confounded by the ability of TGF-β to both promote and suppress tumor progression in a context-specific manner.419,420

NOTCH RECEPTOR SIGNALING The mammalian Notch receptors, Notch1 to Notch4, are single-pass TM receptors that are functionally unique with regard to receptor processing and signal activation and transduction.421–424 Notch receptors are translated as immature receptors that undergo S1 cleavage by furin-like convertase during Golgi trafficking.425 This generates two subunits that are retethered at the cell membrane by noncovalent bonds in the heterodimerization domain.423,426 The extracellular domain subunit consists of ligand-binding EGF-like repeats, a heterodimer domain, three negative regulatory LIN12 and Notch repeats (LNRs) containing numerous cysteines, and a hydrophobic TM-interacting region. The other subunit contains the TM domain and the intracellular domain, which has ankyrin repeats and a RAM domain central to the Notch receptor’s unusual activity as a direct transcription factor, and a PEST motif important for the quick termination of Notch activity and ubiquitin-mediated degradation. Delta-like ligand (DLL1, DLL3, DLL4) and Jagged (JAG1 and JAG2) are the five TM-protein ligands for the Notch receptors.427 A Notch receptor on one cell binds to DLL or JAG ligand on an adjacent cell. This results in a change in Notch receptor conformation, which facilitates a series of proteolytic cleavages required for receptor activation. First, the ADAM17/TACE (a disintegrin and metalloprotease-17/ TNF-α converting enzyme) metalloprotease performs S2 cleavage of the extracellular domain.428,429 This allows subsequent S3 cleavage of the TM domain by the γ-secretase complex and release of the Notch intracellular domain (NICD).430 The active NICD translocates to the nucleus, where it binds to and converts the repressor complex CSL (CBF1, Suppressor of Hairless, Lag-1) into a transcriptional activator that recruits coactivators including the Mastermind-like family proteins and p300. NICD target genes include the HES (hairy enhancer of split) and HRT/HEY (hair-related transcription factor) transcriptional repressors, as well as cyclin D1, myc, p21, NF-κB, and the Notch receptors and ligands themselves, among others.422,426,431 Notch is also heavily involved in cross talk with other pathways, including RTKs, PI3 kinase, Ras/ERK, JAK/STAT, Wnt, Hedgehog, TGF-β/SMAD, and p53. Overall, Notch plays a role in proliferation, cell fate determination in development, and survival. Notch’s causal role in tumorigenesis was established on its discovery in 1991, when a translocation event in T-cell acute lymphoblastic leukemia or lymphoma (T-ALL) was identified that fused the T-cell receptor-β promoter/enhancer elements to Notch, generating a truncated form of Notch1 that was constitutively nuclear and active.432 Activating NOTCH1 mutations have since been found to occur in approximately 60% of T-ALL patients.433 Subsequent studies have determined that Notch and its ligands have both oncogenic and tumor suppressor properties in several hematologic and solid tumor malignancies, including melanoma, glioblastoma, and breast, lung, colorectal, and pancreatic cancers, depending on cellular context.423,426 Thus far, efforts to develop inhibitors of Notch signaling have focused primarily on inhibiting cleavage, and thus activation of Notch. Specifically, selective γ-secretase inhibitors (GSIs), such as MK-0752, PF03084014, and BMS-906024, are currently being tested in early-stage

clinical trials.422,434–436 Newer avenues of drug development include monoclonal antibodies targeting Notch receptors or ligands.437

NUCLEAR HORMONE RECEPTOR SIGNALING The nuclear hormone superfamily is composed of hormone receptors and orphan receptors (receptors for which no ligand has yet been identified). The nuclear hormone receptors are characterized structurally by a ligand binding domain, a DNA binding domain, and a hinge region that connects the ligand and DNA binding domains.438 Nuclear hormone receptors are classified into four subtypes based on ligand specificity: steroid, retinoid X receptor (RXR), monomeric or tethered orphan receptors, and dimeric orphan receptors.439 The steroid receptor ligands include estrogen, progesterone, androgen, and growth hormone. Ligand binding occurs in the cytoplasm and results in receptor homodimerization followed by nuclear translocation. Once translocated into the nucleus, the ligand-bound receptor acts as a transcription factor that modulates the expression of several downstream proteins through binding to steroid response elements, which are conserved nucleotide sequences within the regulatory regions of genes.440 In contrast to the steroid receptors, the RXR receptors form heterodimeric complexes with other partners, including the retinoic acid receptor, the thyroid hormone receptor, and vitamin D receptors. Hormone receptor blockade is a cornerstone in the treatment of estrogen receptor (ER)– and progesterone receptor–expressing breast cancers. The selective estrogen receptor modulator (SERM) tamoxifen competes with estradiol for binding to ER. Notably, tamoxifen binding results in ER dimerization, nuclear translocation, and receptor binding to estrogen response elements in the promoter regions of estradiol-target genes. It is thought that the ER/tamoxifen complex recruits transcriptional corepressors, in contrast to ER/estradiol binding, which recruits transcriptional coactivators.440,441 Although tamoxifen has antiproliferative effects in ER-expressing breast cancer cells, it causes hypertrophy and neoplastic transformation of endometrial tissue, likely as a result of cell type and context-specific recruitment of transcriptional coactivators that are differentially expressed in these tissues.440 Recently, mutations in the ESR1 gene, which encodes the ER, have been shown to be a common mechanism of acquired resistance to hormonal therapy in patients with breast cancer.442 Retrospective studies suggest that patients with ESR1-mutant breast cancer may have more durable responses to the selective estrogen receptor downregulator (SERD) fulvestrant than those treated with the aromatase inhibitor exemestane (see Table 2.1).442,443 Dihydrotestosterone is the primary ligand of the androgen receptor (AR). AR blockade by antiandrogens such as bicalutamide is a commonly used therapeutic modality for patients with locally advanced or metastatic prostate cancer. Bicalutamide competes with dihydrotestosterone for binding to cytoplasmic AR.444 The mechanism by which bicalutamide-bound AR inhibits androgen-dependent gene transcription is unclear but may involve the recruitment of transcriptional corepressors as well as histone modifications that lead to tighter chromatin binding and therefore reduced access to promoter regions by transcription factors. Enzalutamide, a nonsteroidal small-molecule antagonist of the AR that inhibits AR nuclear translocation and DNA binding is FDA approved for the treatment of patients with metastatic prostate cancer that has progressed following treatment with bicalutamide and medical castration (see Table 2.1).445 Abiraterone acetate, an irreversible inhibitor of CYP17A1 that enables intratumoral and adrenal androgen depletion, also has significant clinical activity in patients with castrateresistant prostate cancer.446

INTEGRIN RECEPTOR SIGNALING The integrin receptor family regulates cell adhesion, migration, invasion, and cell survival.447,448 Integrin receptors are heterodimeric molecules consisting of combinations of alpha and beta subunits. Each combination dictates the spectrum of ECM components to which these receptors

40 Part I: Science and Clinical Oncology

bind. Once ligated to the ECM, the receptors recruit multiple proteins to the cell membrane, including cytoskeletal molecules such as paxillin and vinculin that form focal adhesions to ECM components.449 Unlike the RTK family, integrin receptors do not possess intrinsic kinase activity but rather promote signaling by facilitating the activation of kinases such as Src or FAK.450 Integrins are also unique in that they participate in bidirectional signaling. “Inside-out” signaling occurs when intracellular adapters, of which talin-1 and kindlin-1/2/3 are the best known activators, trigger a conformational change of the cytoplasmic tails of the alpha and beta subunits, which is transduced to the extracellular component of the receptor, resulting in increased affinity for portions of the ECM.451 Conversely, “outside-in” signaling involves binding of ligand to integrins, which stimulate the activation of multiple intracellular signaling pathways.452 Collectively, the term integrin adhesome is used to describe the site at which integrins initiate contact with the ECM and other cells and recruit all of the underlying intracellular machinery (e.g., cytoskeleton, scaffolds, signaling adapters— over 200 intrinsic and transient components) to generate diverse types of adhesions (e.g., focal complexes, focal adhesions, fibrillar adhesions, podosomes, invadopodia).453 Integrins are expressed on cancer cells and have been shown to promote disease progression.447,450,454 Integrins are also present on stromal cells, including pericytes (which promote endothelial cell growth and proliferation and thus angiogenesis) and fibroblasts, where they influence the surrounding microenvironment and thus indirectly stimulate tumor growth and proliferation. For example, vascular cell adhesion molecule 1 (VCAM1) is expressed on pericytes and binds to the integrin receptor α4β1, which is found on the surface of endothelial cells, resulting in pericyte recruitment to sites of vascular maturation.455 Integrin signaling also plays a role in the activation of matrix metalloproteinase 2,456 which promotes cell invasion, and has been shown to regulate cyclin D and cyclin-dependent kinase inhibitor expression, thereby controlling cell cycle progression.457 Finally, integrin receptor activation can lead to increased secretion of growth factors, which then stimulate tumor invasion through autocrine and paracrine mechanisms.456 Tumors that express integrin receptors include melanomas, glioblastomas, and breast cancers. In melanoma, the αvβ3 and α5β1 integrin receptors promote vertical growth and metastatic spread to lymph nodes.458,459 In glioblastoma, αvβ3 and αvβ5 are expressed mainly at the edge of tumors, suggesting a role in tumor invasion.460 The expression of the α6β4 and αvβ3 integrin receptors in breast cancer is associated with higher grade and tumor size,461 the development of bone metastases,462 and decreased survival.463 To date, drug development has targeted three integrins—αIIbβ3, α4β1, and α4β7—in the context of platelet activation and blood clotting, multiple sclerosis, and inflammatory bowel disease.451 Clinical trials of monoclonal antibodies that target integrin receptors are underway in several cancer types. For example, etaracizumab, a humanized monoclonal antibody targeting αvβ3 integrin, is being tested in solid tumors and has shown activity in patients with metastatic melanoma.464 Cilengitide, an inhibitor of the αvβ3 and αvβ5 integrins, inhibited angiogenesis and tumor cell proliferation in preclinical studies; however, a phase III trial in combination with temozolomide and radiation in patients with glioblastoma did not result in improved outcomes compared with chemoradiation alone.456,465

NON–RECEPTOR TYROSINE KINASE SIGNALING SRC Signaling The SRC family of intracellular, non-RTK proteins is composed of 11 members (SRC, FYN, YES, Blk, Yrk, Frk/Rak, Fgr, Hck, Lck, Srm, and Lyn).466 They share common structural features including the so-called SRC-homology (SH) domains 1 to 4. The SH1 domain includes the kinase domain. Only SRC, FYN, and YES are expressed

ubiquitously, whereas the tissue distribution of the latter six is more restricted.467 Together, SRC family kinases have pleiotropic roles in cellular proliferation, apoptosis, differentiation, motility, adhesion, angiogenesis, and immunity.468,469 SRC is by far the most intensively studied family member and was the first gene observed to have oncogenic potential.470 Peyton Rous was awarded the Nobel Prize for a series of experiments showing that a transmissible factor was present in avian sarcomas capable of initiating tumors in recipient birds. Five decades later the viral oncogene v-Src was identified as the oncogenic factor in the Rous sarcoma virus.471–473 Bishop and Varmus later showed that v-Src was a mutant form of the cellular proto-oncogene c-Src, and Hunter and colleagues showed that its transformative capacity was dependent on its tyrosine kinase activity.14,15,474 SRC is regulated in a number of ways. First, SRC has a myristoylation site in its N-terminus that is necessary for membrane localization and that promotes its interaction with nearby membrane-bound effectors.475 The SH2 and SH3 domains facilitate protein-protein interactions and conformational changes in the protein. Inactive SRC is maintained in a closed conformation with phosphorylated Y530 (mediated by CSK, C-terminal SRC, and Csk homology kinases) interacting with the folded-over SH2 domain.476 The closed, inactive confirmation of SRC is further stabilized by proline-rich segments of the kinase domain associating with the SH3 domain.477 SRC activation requires dephosphorylation of Y530, likely by PTPα/γ/1β (protein tyrosine phosphatase α/γ/1β) or SHP1/2 (SH-containing phosphatases), which allows the kinase to assume an open conformation.466,478 Autophosphorylation of Y419 in the activation loop of the kinase domain also promotes full activity,479 whereas binding of FAK and CRK-associated substrate (CAS) to the SH2 domain induces SRC activation and links SRC signaling to the regulation of focal adhesion, actin reorganization, and migratory phenotypes.480,481 SRC is a downstream mediator of numerous receptor families including RTKs, integrin receptors, hormone receptors, cytokine receptors, and GCPRs and promotes signaling through the PI3 kinase/AKT, Ras/MAP kinase, and JAK/STAT cascades, among others.466,467,469,478,482–486 More than two decades of research have uncovered numerous SRC substrates, including p85-cortactin, p110-AFAP1, p130Cas, p125FAK, and p120-catenin.487 Although mutations in SRC are rare in human cancers, SRC is frequently activated as a consequence of other mutational events in colorectal, breast, esophageal, gastric, pancreatic, hepatocellular, ovarian, and lung cancers.466 In colorectal and hepatocellular carcinomas, SRC activation occurs in the setting of concomitant loss of CSK.488–490 Newer signaling discoveries have identified roles for SRC in promoting tissue repair after intestinal inflammatory injury, as seen in inflammatory bowel diseases and colorectal cancer, via the IL-6 cytokine coreceptor gp130 and YAP.491 Furthermore, norepinephrine-mediated, β-adrenergic/PKA activation of SRC has recently been shown to enhance tumor cell migration, invasion, and growth.492 The tyrosine kinase inhibitor dasatinib, which is used in the treatment of CML and Philadelphia chromosome–positive acute lymphoblastic leukemia (ALL), inhibits SRC family kinases, in addition to BCR-ABL, KIT, Ephrin A2 receptor, and PDGFR (see Table 2.1).493–495 Additional dual SRC/ABL and SRC selective inhibitors are approved in CML (bosutinib, ponatinib; see later section on ABL signaling) or are in clinical testing, including saracatinib, XL-228, KX2-391, and DCC2036. Most have shown limited single-agent activity and are being developed as combination therapies.469,478,484–486

ABL Signaling The ABL gene was first identified in 1980 as the oncogene responsible for driving the Abelson murine leukemia virus, and later was found to be part of the translocations found in many types of human leukemias.496 ABL1 and ABL2 isoforms have both overlapping and divergent functions. ABL is found in an autoinhibited conformation dictated by clamping of the N-terminal hydrophobic region, SH3

Intracellular Signaling  •  CHAPTER 2 41

and SH2 domains onto the C-lobe of the kinase domain.497 Intermolecular interactions with adaptors such as RIN1 or phosphorylation by SRC, among others, promote stabilization of the open, active form of ABL 23842626. The ABL tyrosine kinase is found in both the cytoplasm and the nucleus, and its function varies based on subcellular localization.498 Cytoplasmic ABL has been implicated in G1/S checkpoint regulation and interaction with actin on C-terminal binding sites,499 whereas nuclear ABL inhibits binding of the DNA repair protein Rad51 to sites of DNA damage.500,501 Activated ABL kinases are now recognized to play an important role in tumor initiation by disrupting cell polarity and by promoting invasion and metastasis by regulating invadopodia.502 In cancer, translocation of the ABL gene on chromosome 9 with the breakpoint cluster region (BCR) gene on chromosome 22 results in the expression of a BCR-ABL fusion protein.503 This translocation, the Philadelphia chromosome, is found in almost all CMLs and represents the pathognomonic molecular lesion in this disease. BCR-ABL translocations are also found in approximately one-third of acute lymphoblastic leukemias (ALLs).504,505 In addition, at least eight other fusion partners of ABL have been discovered.502 The realization that the proliferation and survival of CML cells is critically dependent on the ABL fusion protein led to the development of imatinib, an inhibitor of the ABL tyrosine kinase.505 To date, there are five FDA-approved inhibitors for CML: imatinib, dasatinib, bosutinib, nilotinib, and ponatinib (see Table 2.1).506 Moreover, imatinib and dasatinib are approved for treatment of ALL.507–511 A secondary mutation in the gatekeeper site T315I is the most common reason for acquired resistance to ABL kinase inhibitors. Combination of ATP-competitive and allosteric inhibitors such as GNF-5 may be a novel strategy to combat resistance.512

RAS/MAP KINASE PATHWAY SIGNALING First identified over 30 years ago as the oncogenes responsible for the transforming potential of the Harvey and Kirsten murine sarcoma retroviruses (Ha-MSV and Ki-MSV), RAS proteins are guanine nucleotide binding proteins.513–517 Ras proteins have intrinsic GTPase activity and cycle between inactive GDP- and active GTP-bound states. GDP/GTP exchange thus allows Ras proteins to function as binary molecular switches. In the human genome, there are three RAS genes, which encode four homologous proteins (HRas, NRas, and the alternative splice variants KRAS4A and KRAS4B) with highly conserved N-terminal and variable C-terminal regions. Following stimulation of cells by serum growth factors, cytokines, hormones, and neurotransmitters, Ras undergoes a series of C-terminal (C186AAX) posttranslational modifications that result in its localization to specific membrane microdomains.518 Membrane localization is required for the transforming properties of Ras, because mutation of Ras at C186 results in cytosolic localization and protein inactivation whereas Ras activity can be rescued by myristoylation, which promotes membrane localization.519–521 GEFs promote Ras activation by binding to GDP-bound Ras and facilitating the release of GDP and the binding of GTP. SOS1, RAS-GRF (dual specificity GEFs for RAS and RAC), and RAS-GRP (stimulated by DAG/phorbol esters and Ca+) are the mostly highly characterized RAS-GEFs.522–524 Using RTK stimulation as an example, ligand binding induces RTK dimerization and autophosphorylation of tyrosine residues in the receptor cytoplasmic tail. Phosphotyrosine docking sites recruit scaffold proteins such as SHC and permit interaction with the SH2 domains of adaptor proteins such as Grb2.525 Grb2 in turn recruits SOS1 via its SRC homology 3 (SH3) domain, thereby positioning SOS near membrane-anchored RAS (see Fig. 2.1).525–529 SOS1 in turn activates RAS via its CDC25 homology (RASGEF) domain and N-terminal RAS exchanger motif (REM or RASGEFN domain). Ras inactivation is catalyzed by GTPase-activating proteins (GAPs), which enhance the intrinsic GTPase activity of Ras proteins by 100,000-fold.530 RAS GAPs, which include p120-RASGAP,

neurofibromin (NF1), GAP1IP4BP, and CAPRI, negatively regulate RAS activity and thus function as tumor suppressors.514,518 Binding of GTP to RAS induces conformational changes in the switch I (loop 2 residues 30–38) and switch II (helix 2 and loop 4 residues 60–76) domains, which facilitate the association of RAS with regulators and downstream effectors.531,532 RAS directly interacts with over 20 effector proteins, of which the RAF kinases, PI3 kinases, and RALGDS are the best characterized (see Fig. 2.1). The canonic RAS/ RAF/MEK/ERK (classic MAP kinase) cascade is by far the most extensively characterized RAS effector pathway. This prototype of a three-tiered kinase signaling cascade exemplifies numerous RASdependent mitogen-activated protein kinase (MAPK) cascades that respond to diverse signals, including cell stress and cytokine signaling (see section on cytokines for details of the JNK and p38 pathways).533 The RAF protein family (which represent the top-tier MAPK kinase kinases [MAPKKKs], or MEK kinases [MEKKs]) is composed of three differentially expressed isoforms, A-RAF, B-RAF, and C-RAF (RAF-1).534,535 RAF, via its RAS binding domain (RBD) and cysteine rich domain (CRD), interacts with GTP-bound Ras.536–539 Binding of RAF to GTP-bound RAS results in RAF localization to the plasma membrane and its subsequent phosphorylation and activation.540 The mechanisms of RAF-1 and B-RAF phosphorylation and activation are distinct and are derived from the summation of signaling inputs from small G proteins (RAS, RAC, CDC42, RAP-1), kinases (activating inputs: SRC/PAK/PKC, inhibitory inputs: PKA/AKT/SGK), isoform homodimerization and heterodimerization, phosphatases (PP2, PP2A), scaffolding proteins (KSR, RKIP, HSP90, and so on) and cofactors (14-3-3), with phosphorylation events regulating critical aspects of RAF activation.535,541 In brief, RAS binding to RAF-1 releases 14-3-3, a negative regulator that binds to basally phosphorylated residues S259 and S261 and sequesters RAF-1 in the cytosol in a closed, inactive conformation. Liberation from 14-3-3 exposes the PKA/ AKT/SGK-dependent inhibitory phosphorylation on S259, facilitating its dephosphorylation by protein phosphatase 2A.542–546 Loss of this inhibitory phosphorylation primes RAF-1 for RAS, PAK, SRC, growth factor, and integrin-stimulated activating phosphorylations on S338, Y341, T491, and S494.547–551 A-RAF is activated similarly to RAF-1. Notably, B-RAF activation requires fewer steps owing to its constitutive phosphorylation at S445, a site analogous to S338 in RAF-1.541,552 Binding to RAS-GTP stimulates B-RAF phosphorylation at critical residues in its activation loop, T599 and S602 (analogous to T491 and S494 in RAF-1).553,554 The B-RAF isoform is the most potent activator of ERK pathway output.535,552,554 Activated Raf proteins bind and phosphorylate MEK1 and MEK2 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinases 1 and 2, or MAPKK, or MAP2K) on serines 217 and 221.555 Activated MEK, a dual-specificity threonine/tyrosine kinase, in turn phosphorylates MAPK/ERK-1/2 (mitogen-activated protein kinases/ extracellular signal-regulated kinases 1 and 2) on threonine 183 and tyrosine 185, inducing a conformational change, activation, and dissociation from MEK.556 Activated ERK then phosphorylates substrates in the cytosol (e.g., p90RSK) and in the nucleus (such as the transcription factors ELK-1, ETS-2, FOS, JUN, ATF-2, AP-1, MYC, CREB1), which promote proliferation and survival.557,558 Raf-1 has also been shown to mediate suppression of apoptosis through non–MEK-dependent interactions with ASK1, MST2, and MEKK1/NF-κB.559 RAS/RAF signaling triggers numerous regulatory mechanisms, including classic negative feedback loops, which serve to attenuate pathway output.560 The Sprouty and Sprouty-related EVH1-domaincontaining protein (Spred) proteins (encoded by the SPRY1–4 and SPRED1–4 genes, respectively) inhibit the cascade at the level of RAS and RAF activation.561–564 The dual-specificity phosphatases (MKPs/ DUSPs) dephosphorylate MAP kinases, including ERK.565,566 In addition, increased pathway activity induces ERK-dependent negative phosphorylations on B-RAF (at S151, S750, T401 and T753) and on RAF-1 (at S29, S43, S289, S296, S301 and S642) that abrogate interactions with RAS and homodimer and heterodimer formation.567,568

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RAS signaling is further regulated by RKIP (RAF kinase-inhibitory protein), which disrupts the RAF-1/MEK interaction and IMP (impedes mitogenic-signal propagation), which represses KSR-dependent scaffolding of RAF/MEK, among other functions.534,569–571 In its active, GTP-bound state, RAS alternatively binds to the p110α catalytic subunit of class I PI3 kinases,572,573 causing activation of its lipid kinase activity and thereby generating PIP3, which in turn stimulates the proproliferative and prosurvival kinases PDK1 and AKT (see section on PI3 kinase signaling for details). RAS-dependent activation of PI3 kinase can further stimulate RAC, a RHO family GTPase involved in regulation of actin and NF-κB.513,574,575 A third major class of RAS effectors is the group of GEFs for RALA and RALB, namely RALGDS, RGL, and RGL2.576–578 These RAL exchange factors stimulate phospholipase D and the CDC42/RAC-GAP RAL binding protein 1 (RALBP1) and inhibit Forkhead transcription factors to regulate transcription, vesicular trafficking, and cell cycle progression. Several additional RAS effectors have been identified. These include (1) PLCε, which generates IP3 and DAG and regulates calcium release and PKC activation; (2) T-cell lymphoma invasion and metastasis-1 (TIAM1), which facilitates actin reorganization via RAC; (3) AF6, which contributes to cytoskeletal changes; (4) RIN1, which regulates endocytosis; and (5) RASSF and NORE1, which have been shown to regulate apoptosis and cell cycle progression.513,518 Many other nondirect connections allow RAS to affect the cellular microenvironment, metabolic signaling, autophagy, inflammation, and immune responses. Overall, the complex effects of RAS activation result in a diversity of context-dependent phenotypes ranging from cell proliferation to cell death that are influenced by a wide variety of extracellular stimuli and intricately woven layers of regulation. The potent oncogenic effects of RAS signaling are highlighted by the high prevalence of mutations in the RAS genes and its proximal downstream effectors.518,579–581 RAS mutations are found in approximately 30% of all human tumors, the majority of which (85%) occur in the KRAS isoform. KRAS is frequently mutated in pancreatic cancers (58%), colorectal and biliary tree cancers (33 and 31%), and non–small cell lung adenocarcinomas (17%). Mutations in HRAS are most common in low-grade bladder cancers (11%), whereas mutation in NRAS is a frequent event in melanoma (18%) and biliary tree cancers (11%). Mutations that lock RAS in its GTP-bound state confer oncogenic potential. Point (missense) mutations in residues 12, 13, 61 (exons 2 and 3) generate a constitutively active RAS oncoprotein by abrogating intrinsic GTPase activity and inhibiting GAP binding.582 Other mutations, including those at residues 117 and 146, contribute to RAS activation by increasing GDP-to-GTP exchange.583,584 Heritable germline mutations of several RAS/MAPK pathway components have been shown to be the underlying cause of the so-called RASopathies neurofibromatosis type 1 (NF1); Noonan, Costello, and cardiofaciocutaneous syndromes; and other developmental disorders.579 More recently, deep sequencing efforts have identified recurrent somatic mutations in the RAS-GAP NF1 in glioblastoma585 and melanoma,586,587 as well as in A-RAF (hot spot at S214) in histiocytosis and lung cancer588–590 and in RAF-1 (hot spot S257).588 Mutations in BRAF are also common in human cancer and typically occur in a mutually exclusive pattern with RAS mutations. BRAF mutations have been identified in approximately 8% of all cancers, most notably in melanoma (50%–60%) and in papillary thyroid (30%–50%), biliary tree (14%), colorectal (10%), ovarian (12%), and lung cancers (3%) and hairy cell leukemia (100%).534,535,559,579,591,592 A single valine to glutamic acid substitution at residue 600 (V600E) accounts for more than 80% of all BRAF mutations and renders BRAF an active monomer in settings of low RAS activity, that is sensitive to RAF inhibition. Other nonV600E BRAF mutants have been recently identified and characterized to function as RAS-independent dimers that are insensitive to current RAF inhibitors, such as vemurafenib, that only effectively inhibit mutant monomers.593 Activating mutations in MEK1 (MAP2K1) and MEK2 (MAP2K2) are also present in approximately 1% of human tumors, more prominently in melanoma and lung cancer, and have

emerged as a mechanism of acquired resistance to RAF inhibition.594,595 Hot spots include mutations at MEK1 residues F53, K57, and P124 and at MEK2 residue F57, which is paralogous to MEK1 F53. ERK amplification and mutation, although equally rare (approximately 1% of all tumors), may also emerge as mechanisms of resistance to upstream MAPK inhibition, including alteration at the hot spot residue E322.596 Given the high prevalence of RAS pathway alterations in human cancers, significant attention has been directed toward the development of selective inhibitors of this pathway.560,580,597,598 To date, clinically effective direct inhibitors of oncogenic RAS have yet to be identified. The inability to directly target RAS has been attributed to the high affinity of the RAS-GTP interaction. Extensive efforts were thus directed toward inhibiting the posttranslation modifications required for RAS activation. Specifically, inhibitors of the enzyme farnesyltransferase, which regulates RAS localization, were tested in randomized phase III trials but were found to be inactive.599 The failure of farnesyltransferase inhibitors in KRAS-mutant tumors was predicted by the preclinical observation that geranylgeranyl modification can substitute for farnesylation in targeting KRAS and NRAS to the plasma membrane.600,601 Small molecules that irreversibly bind to the mutant cysteine in tumors with G12C K-RAS have been developed.602 Because this class of compounds is selective for the G12C mutant, they are a promising novel approach for a subset of patients with KRAS-mutant tumors. As an alternative, extensive efforts have focused on the development of selective inhibitors of key kinase effectors of RAS transformation. The selective RAF inhibitors vemurafenib and dabrafenib induce tumor regressions in most patients with BRAF V600E mutant melanoma and are FDA approved for this indication (see Table 2.1).603–612 Notably, these agents selectively inhibit RAF signaling in BRAF-mutant tumors and are thus inactive in RAS-mutant tumors.613 Several mechanisms of resistance to RAF inhibitors have emerged, including BRAF V600E splice variants or amplification; loss or mutation of NF1; parallel pathway activation of RTKs; mutation of PI3K/AKT components; mutations in NRAS, RAF1, and MEK1/2; and amplification of MITF.592,614 Sorafenib, a multikinase inhibitor of RAF, VEGFR2, and PDGFRβ, has been shown to have some clinical efficacy in ARAFmutant histiocytosis and NSCLC.588,589,615 The selective MEK inhibitor trametinib is also FDA approved for use in melanomas with BRAF mutation (see Table 2.1).605,616–619 Furthermore, the combination of a RAF and a MEK inhibitor has been shown to have greater activity than Raf inhibitor monotherapy in both preclinical models and in patients with melanoma, including the combinations of vemurafenib plus cobimetinib620 and dabrafenib plus trametinib.605,619,621,622 MEK inhibition is also emerging as a strategy to target NRAS-mutant melanoma,623 colorectal624 and thyroid cancers,625 NF1-mutant melanoma,587 GBM,626 and neuroblastoma.627 MEK inhibition in combination with immunotherapy, chemotherapy, radiation, and PI3K and CDK4/6 inhibitors is being investigated in KRAS-mutant tumors including colorectal and lung cancer (see Table 2.1).624,628 Furthermore, clinical evidence of activity of the MEK inhibitors cobimetinib, selumetinib, and trametinib has been revealed in cases and preclinical reports of MEK1-mutant histiocytosis,589 low-grade serous ovarian cancer,629 NSCLC,594 and melanoma.630 Alternative strategies for inhibiting MAP kinase signaling include HSP90 inhibitors that induce the degradation of RAF1 and mutant BRAF.631 Drug development of kinase- and dimerization-targeted ERK inhibitors (SCH772984, BVD-523, DEL-22379) is also underway and may provide a downstream alternative when upstream RAF or MEK inhibitors fail.632–635 Combinatorial approaches are also being actively pursued, given the modest activity of MEK inhibitors in patients with RAS-mutant tumors and the frequent co-occurrence of RAS and PI3 kinase pathway alterations in multiple cancer types.636–638 Given the recent success of immunotherapy in many cancers, preclinical evidence supports the triple combination of BRAF and MEK inhibitors with immunotherapy in BRAF V600E mutant melanoma, despite substantial liver toxicities described with initial trials with combined treatment of vemurafenib and ipilimumab.639 In addition, elevated

Intracellular Signaling  •  CHAPTER 2 43

antitumor immune responses in mouse models of triple negative breast cancer following combined MEK inhibition and immunotherapy suggest that hyperactivate MEK signaling may contribute to immune evasion.640 Given the prominent role of activated ERK in initiating the transcription and translation of cell cycle components such as cyclin D1, inhibitors of cell cycle kinase components have emerged as an alternative strategy to abrogate the output of MAPK signaling. CDK4, a critical serine/threonine kinase that functions in an active complex with cyclin D to phosphorylate RB and stimulate E2F release and S phase entry, is amplified in liposarcoma. Palbociclib, an inhibitor of CDK4, is FDA approved for use in postmenopausal women with ER+, HER2− metastatic breast cancer; a second drug, abemaciclib, has also show efficacy in this same patient population (see Table 2.1).641–644

PI3 KINASE/AKT/MTOR PATHWAY SIGNALING The PI3 kinase/AKT/mTOR pathway is a key regulator of growth factor–mediated proliferation and survival.645 Several extracellular growth factors stimulate PI3 kinase by binding to their cognate RTKs or GPCRs.646 Activated PI3 kinase phosphorylates the 3-OH group of the inositol ring of phosphatidylinositol, catalyzing the conversion of PIP2 to PIP3. PIP3 then binds to the pleckstrin homology domain (PH domain) of multiple proteins, facilitating their recruitment to the plasma membrane and thus regulating their function (see Fig. 2.2). PI3 kinases are grouped into class I, II, and III kinases based on their structure and substrate preferences, although class II and III kinases have been much less studied. Class I PI3 kinases are subdivided into two groups, IA and IB. Class IA comprises the PIK3CA, PIK3CB, and PIK3CD genes, which encode the catalytic p110α, p110β, and p110δ subunits, respectively. These p110 components heterodimerize with a regulatory subunit (p85), of which there are five isoforms and splice variants encoded by the PIK3R1-3 genes. Together, the p110/ p85 complex functions primarily in the generation of PIP3.647,648 The PIK3CG gene encodes the class IB p110γ isoform, which couples with p101 (PIK3R5) or p87 (PIK3R6) regulatory subunits. Notably, p110δ/γ catalytic subunits have specialized expression patterns mainly in leukocytes, whereas p110α/β are ubiquitous.649 Analogous to the activation of the Ras pathway detailed previously, the p85 regulatory subunit of PI3 kinase associates with phosphorylated tyrosine residues located on the intracellular domains of RTKs through an SH2 domain, resulting in allosteric activation of the p110 catalytic subunit. PI3 kinases can also be activated indirectly through the adaptor protein Grb2 following its association with the scaffolding protein GAB1. PI3 kinase pathway activity is negatively regulated by the tumor suppressor PTEN (phosphatase and tensin homolog), which is a dual lipid and protein phosphatase that dephosphorylates PIP3, converting it back to PIP2.650,651 The best characterized effectors of PI3 kinase are the three members of the serine/threonine kinase AKT family (AKT1, AKT2, and AKT3). Both AKT and phosphoinositide-dependent kinase 1 (PDK1) are recruited by PIP3 to the plasma membrane via their PH domain, where AKT is phosphorylated at Thr308 by PDK1, resulting in its activation.652–654 Phosphorylation at a second residue, Ser473, by mTOR complex 2 (mTORC2) further enhances AKT activity.655 Activated AKT promotes cellular proliferation, survival, and other phenotypes through activation of multiple downstream effectors. Proliferative effects are regulated through phosphorylation and inhibition of GSK3β, which phosphorylates cyclin D1 and marks it for degradation; FOXO4, a transcription factor that regulates the expression of the CDK inhibitor p27; and p21, a second CDK inhibitor that, on phosphorylation, translocates from the nucleus to the cytoplasm, where it regulates cell survival.656–659 In sum, these actions promote the expression of cyclin D1, which drives progression of cells through the cell cycle and the downregulation of cyclin-dependent kinase inhibitors that inhibit cell cycle progression.

AKT-mediated antiapoptotic effects occur through phosphorylation and inhibition of Bad, which negatively regulates the antiapoptotic protein Bcl-xL; caspase-9, a proapoptotic protease; and the FOXO1 transcription factor which regulates the expression of proapoptotic genes.660–662 AKT also controls cell survival by upregulating NF-κB activity through phosphorylation and activation of Iκb kinase, which marks Iκb, an inhibitor of NF-κB, for degradation.663,664 Once released from IκB, NF-κB translocates into the nucleus, where it regulates a multitude of genes involved in cell survival. AKT also phosphorylates and activates Mdm2, an E3 ubiquitin ligase that binds to the proapoptotic tumor suppressor p53 and directs it for proteasomal degradation.665 AKT-independent effectors of PI3 kinase activation have also been identified and likely play important roles in the development and progression of some cancers. These include CDC42 and Rac1, which are involved in motility and reorganization of the cytoskeleton, and the serum glucocorticoid kinase (SGK) family of serine/threonine kinases, which promote cell survival.666,667 Many of the canonic functions of AKT with regard to cell growth and proliferation are mediated through the mTOR pathway. mTOR is a serine/threonine kinase and a member of the phosphatidylinositol kinase-related kinase family.668 mTOR is a component of two complexes, the rapamycin-sensitive mTORC1 and the rapamycininsensitive mTORC2 complexes.669 Within mTORC1, mTOR associates with Raptor (regulatory associated protein of mTOR) and mLST8, whereas the mTORC2 complex consists of mTOR, Rictor (rapamycin insensitive companion of mTOR), SIN1, and mLST8.670 In addition to its role in activating AKT as described earlier, mTORC2 also controls cytoskeletal changes through regulation of paxillin, Rho, Rac, and PKCα.671 mTORC1 activation is regulated in part by AKT phosphorylation of TSC2. TSC2 forms a heterodimeric complex with TSC1 that acts as a GTPase-activating protein (GAP) for the small GTPase Rheb, causing accumulation of inactive Rheb-GDP.672,673 TSC2 phosphorylation results in suppression of this GAP activity and subsequent activation and accumulation of Rheb-GTP, which then activates mTORC1. mTORC1 serves as a central nexus for integration of multiple extracellular signals, including oxygen and amino acid levels, growth factors, and stress. Based on these input signals, mTORC1 activity influences cellular growth, metabolism, protein synthesis, and cell cycle progression. One such example is the energy sensing mechanism of the cell comprised of LKB1 and AMPK.674 Increasing levels of AMP, a marker of decreased nutrient levels, results in AMPK phosphorylation and activation by LKB1. AMPK phosphorylates TSC2, which, as described earlier, inhibits mTORC1 activity, leading to downregulation of protein synthesis and cell growth in response to low nutrient levels.675 In part, mTORC1 activation regulates these phenotypes via phosphorylation and activation of p70S6 kinase and inhibition of 4EBP1. The former protein stimulates mRNA translation, whereas the latter inhibits translation of mRNA transcripts with a 5′ cap.676 Both mTORC1 and S6 kinase also participate in a negative feedback loop in which both proteins activate insulin receptor substrate 1 (IRS1), which results in inhibition of insulin-mediated PI3 kinase activation.677 AKT can also activate mTORC1 in a TSC2-independent manner via phosphorylation of PRAS40, a protein that interacts with mTORC1. The mechanisms responsible for PI3 kinase pathway activation in cancer are diverse and include activating mutations and amplification of PIK3CA, AKT1, AKT2, and AKT3, and mTOR; deletion or loss of PTEN expression or function; mutation in PIK3R1; loss of TSC1 or TSC2 function; RAS mutation; and dysregulated growth factor receptor and integrin activation, as outlined later.645,648,678 In human tumors, activation of PI3 kinase is frequently a direct consequence of dysregulated RTK signaling secondary to mutation, amplification, or ligand overexpression. For example, ERBB2 amplification in breast and gastric cancer results in AKT activation.27,678 Similarly, kinase domain mutations of EGFR induce constitutive AKT activation in lung cancers and glioblastomas, and AKT activity in these tumors

44 Part I: Science and Clinical Oncology

is critical for EGFR-mediating transformation.637,679 Oncogenic Ras mutations also activate PI3 kinase, and PI3 kinase activation is required for Ras-mediated tumorigenesis in genetically engineered mouse models.572,680–682 Mutations in the PIK3CA gene, which encodes the p110α catalytic subunit, are frequently observed in tumors of the colon, breast, brain, stomach, and ovary and other cancers.648,678,683 The most frequent are E542K and E545K, located in the helical domain (exon 9), and H1047R (exon 20), located in the kinase domain.684 All three mutants demonstrate increased lipid kinase activity, induce phosphorylation of AKT and its downstream effectors, and can transform chicken embryo fibroblasts.685 Exon 9 helical domain mutations block the inhibitory interaction between the p85 regulatory subunit and the p110 subunit and result in constitutive kinase activation.686 Exon 20 catalytic domain mutations result in constitutive kinase activation.678 PIK3CA mutations commonly co-occur with KRAS mutations, and ERBB2 amplification in colon and breast cancers, respectively, and expression of mutant PI3 kinase in breast cell lines as well as fibroblasts causes neoplastic transformation.687 Unlike wild-type p110α which lacks oncogenic potential, wild-type overexpression of the other three isoforms can induce transformation of cultured cells.688 PIK3R1 encodes the p85 regulatory subunit of PI3 kinase. Alterations in within this gene have also been reported in multiple cancers, including glioblastomas and colorectal, endometrial, and ovarian cancers.585,689 A recurrent PIK3R1 mutation, PIK3R1(R348∗), has been shown to have neomorphic functions resulting in activation of MEK and JNK and sensitivity to inhibitors of these effector kinases.690 Furthermore, partial loss of the PIK3R1 gene product p85α was demonstrated to increase the proportion of p110α/p85 heterodimers bound to active receptors, indicating that targeting p110α-selective inhibitors may be effective in the setting of PIK3R1 loss.691 Loss of PTEN function is the most frequently observed PI3 kinase/ AKT pathway alteration in human malignancies and is common in tumors of the prostate, breast, ovary, lung, colon, and bladder as well as melanomas and glioblastomas.678 Loss of PTEN function in tumors is mediated by a diversity of mechanisms including mutation, deletion, posttranslational modification, and promoter hypermethylation.678 Dysregulated expression of microRNAs that target the 3′-untranslated region of PTEN has also been shown to induce cell survival and cisplatin resistance.692 As AKT activation enhances proliferation and suppresses apoptosis, its activation would be predicted to have strong oncogenic function. Indeed, AKT was initially identified as a proto-oncogene in the mouse leukemia virus AKT8.693 A recurring hot spot mutation in the PH-domain of AKT1 (E17K) occurs with low frequency in breast, colorectal, bladder, endometrial, and ovarian cancers.694–696 This mutation results in constitutive localization to the plasma membrane without the need for PIP3 recruitment.684 Amplification of the AKT2 gene has been reported in ovarian and pancreatic cancers, and gain-of-function AKT3 mutations have been reported to occur in melanoma.697 Given the significant proportion of malignancies that harbor mutations in the PI3 kinase/AKT/mTOR pathway, a concerted effort is ongoing to identify selective inhibitors of various PI3 kinase pathway components. These agents can be categorized as pan-selective or selective PI3 kinase inhibitors, dual PI3 kinase/mTOR kinase inhibitors, AKT inhibitors, and mTOR inhibitors. Both isoform-specific and pan-selective inhibitors of PI3 kinase are being explored in several clinical trials across cancers. Most notably, the PI3Kδ inhibitor idelalisib is FDA approved for non-Hodgkin lymphoma and certain types of leukemia.698,699 Promising clinical responses have also been reported with α-selective PI3 kinase inhibitors in patients with several cancer types, most notably breast cancer (see Table 2.1).700–703 Although clinical activity with AKT inhibitors has been modest in patients, a pan-cancer basket trial of the ATP-competitive pan-AKT kinase inhibitor AZD5363 demonstrated significant clinical responses in patients with AKT1 E17K mutant tumors.704

Temsirolimus and everolimus, analogues of rapamycin that inhibit the mTORC1 complex, are FDA approved for use in patients with renal cell carcinomas, and everolimus is FDA approved for the treatment of patients with pancreatic neuroendocrine tumors (see Table 2.1).705–708 Everolimus also has significant clinical activity in patients with subependymal giant cell astrocytomas that arise in the setting of tuberous sclerosis, an inherited cancer-predisposition syndrome resulting from germline mutations in the TSC1 and TSC2 genes.709,710 In metastatic renal cell carcinoma, patients that benefited from treatment with everolimus more commonly harbored TSC1/2 or mTOR mutations (see Table 2.1).711 A complete response to everolimus has been reported in a patient with metastatic bladder cancer that harbored loss-of-function mutations in TSC1 and NF2, suggesting that such agents can induce significant antitumor responses in genetically selected patients.712 Additional responses to everolimus were observed in TSC1-mutant tumors but not to the degree of the complete responder, indicating that coaltered genes likely confer resistance to mTOR inhibitory therapy even in the setting of a canonic predictive biomarker of response. In sum, mTOR inhibitors are likely most effective when used in genetically defined patient populations, with the majority of patients requiring combination therapies because of the presence of coaltered genes. As an example of the latter, everolimus in combination with exemestane was FDA approved for the treatment of advanced hormone receptor– positive, HER2− breast cancer.713 Resistance to mTOR inhibition is thought to derive from multiple mechanisms, including activation of parallel signaling networks such as the MAP kinase pathway.714 Furthermore, inhibiting mTORC1 can preferentially lead to an increase in mTORC2 signaling, and, as described earlier, mTORC2 enhances AKT activation via phosphorylation of the serine 473 residue.715 As an alternative approach, potent mTOR kinase inhibitors are being developed, including RapaLink-1, and have shown significant preclinical activity in cell lines resistant to rapamycin and in in vivo models of glioblastoma.716,717

TRANSLATIONAL IMPLICATIONS As outlined earlier, mutational and epigenetic alterations induce constitutive activation of a broad array of signaling pathways in human tumors. In some instances, the growth and survival of tumor cells have been shown to be dependent on a single signaling pathway activated by a mutated oncogene or tumor suppressor, a phenomenon referred to as oncogene addiction.718 In such instances, targeted inhibitors of such pathways have demonstrated unprecedented clinical activity in molecularly defined subsets of patients (see Table 2.1). Examples include imatinib in patients with CML, erlotinib in patients with EGFR-mutant NSCLC, and vemurafenib in patients with BRAF-mutant melanoma.30,505,511,603,612,719 Despite these dramatic successes, the majority of cancer patients have yet to benefit from this approach. Potential explanations for this lack of benefit include the redundant regulation of key downstream mediators of transformation by multiple signaling pathways, the lack of specificity of the drug for the driver alteration, and intrinsic and acquired drug resistance due to second site mutations in the target gene or by other mechanisms. Progress in this field has also been delayed by the continued practice of performing clinical trials of targeted inhibitors in unselected patient populations. Recent advances in sequencing methodology have made it feasible to now prospectively sequence all patients with advanced cancer with the goal of identifying potentially “actionable” genomic alterations.720 Such prospective sequencing efforts have highlighted several challenges that have slowed the application of targeted inhibitors in cancer patients. As one example, most mutations, even in well-characterized cancer genes, are likely inert passenger mutations. Furthermore, not all activating mutations respond similarly to targeted inhibitors. For example, exon 19 EGFR deletions are sensitive to the EGFR kinase inhibitor erlotinib, whereas exon 20 insertions are intrinsically resistant.721,722 Given the large number of somatic mutations present

Intracellular Signaling  •  CHAPTER 2 45

in each human tumor, and the variable biologic and clinical significance of individual mutant alleles, there is an urgent need for clinical support tools that will aid clinicians in interpreting molecular tumor profiling and guiding treatment selection. A second challenge is that many oncogenic alterations are rare. To address this challenge, novel clinical trial designs have been formulated to test the efficacy of targeted inhibitors in patients with defined molecular events independent of the primary site of disease. Eligibility for these “basket” studies is based on the presence of a particular molecular alteration (e.g., BRAF V600E, AKT1 E17K, or an NTRK fusion) rather than site of tumor origin.268,273,704,723 Although promising results from such trials highlight the importance of tumor mutational profile in dictating drug sensitivity, lineage-specific differences also play a role in determining clinical response to inhibitors of activated signaling pathways. As an example, most BRAF V600E melanomas

respond to vemurafenib, whereas colorectal tumors with the same mutation are intrinsically resistant. Resistance in the latter results from rapid adaptation of the cancer cell resulting in activation of EGFR.724 This phenomenon of adaptive resistance, defined as a rapid reactivation of parallel signaling pathways after relief of negative feedback signals, likely abrogates the effects of selective pathway inhibitors in many cancer types.592,725,726 Because significant drug development efforts are currently focused on the development of targeted inhibitors of oncogene-activated signaling pathways, a detailed understanding of normal physiologic pathways and their dysregulation in cancer will be critical for the optimal development and clinical application of targeted kinase inhibitors. The complete reference list is available online at ExpertConsult.com.

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46 Part I: Science and Clinical Oncology 644. Beaver JA, et al. FDA approval: palbociclib for the treatment of postmenopausal patients with estrogen receptor-positive, HER2-negative metastatic breast cancer. Clin Cancer Res. 2015;21:4760–4766. 690. Cheung LW, et al. Naturally occurring neomorphic PIK3R1 mutations activate the MAPK pathway, dictating therapeutic response to MAPK pathway inhibitors. Cancer Cell. 2014;26:479–494.

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Intracellular Signaling  •  CHAPTER 2 46.e1 46.e1

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700. Davies BR, et al. Preclinical pharmacology of AZD5363, an inhibitor of AKT: pharmacodynamics, antitumor activity, and correlation of monotherapy activity with genetic background. Mol Cancer Ther. 2012;11:873–887. 701. Addie M, et  al. Discovery of 4-amino-N[(1S)-1-(4-chlorophenyl)-3-hydroxypropyl]-1(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine4-carboxamide (AZD5363), an orally bioavailable, potent inhibitor of Akt kinases. J Med Chem. 2013;56:2059–2073. 702. Davies BR, et al. Tumors with AKT1E17K mutations are rational targets for single agent or combination therapy with AKT INhibitors. Mol Cancer Ther. 2015;14:2441–2451. 703. Hyman DM, et al. Abstract B109: AZD5363, a catalytic pan-Akt inhibitor, in Akt1 E17K mutation positive advanced solid tumors. Mol Cancer Ther. 2015;14: B109. 704. Hyman DM, et al. AKT inhibition in solid tumors with AKT1 mutations. J Clin Oncol. 2017;35:2251–2259. 705. Wander SA, Hennessy BT, Slingerland JM. Nextgeneration mTOR inhibitors in clinical oncology: how pathway complexity informs therapeutic strategy. J Clin Invest. 2011;121:1231–1241. 706. Hudes G, et al. Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med. 2007;356:2271–2281. 707. Motzer RJ, et al. Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebo-controlled phase III trial. Lancet. 2008;372: 449–456. 708. Yao JC, et al. Everolimus for advanced pancreatic neuroendocrine tumors. N Engl J Med. 2011; 364:514–523. 709. Krueger DA, et al. Everolimus for subependymal giant-cell astrocytomas in tuberous sclerosis. N Engl J Med. 2010;363:1801–1811. 710. Franz DN, et al. Efficacy and safety of everolimus for subependymal giant cell astrocytomas associated with tuberous sclerosis complex (EXIST-1): a multicentre, randomised, placebo-controlled phase 3 trial. Lancet. 2013;381:125–132. 711. Kwiatkowski DJ, et al. Mutations in TSC1, TSC2, and MTOR are associated with response to rapalogs in patients with metastatic renal cell carcinoma. Clin Cancer Res. 2016;22:2445–2452. 712. Iyer G, et al. Genome sequencing identifies a basis for everolimus sensitivity. Science. 2012;338:221.

713. Baselga J, et al. Everolimus in postmenopausal hormone-receptor-positive advanced breast cancer. N Engl J Med. 2012;366:520–529. 714. Carracedo A, et al. Inhibition of mTORC1 leads to MAPK pathway activation through a PI3Kdependent feedback loop in human cancer. J Clin Invest. 2008;118:3065–3074. 715. Breuleux M, et al. Increased AKT S473 phosphorylation after mTORC1 inhibition is rictor dependent and does not predict tumor cell response to PI3K/ mTOR inhibition. Mol Cancer Ther. 2009;8: 742–753. 716. Fan Q, et al. A kinase inhibitor targeted to mTORC1 drives regression in glioblastoma. Cancer Cell. 2017;31:424–435. 717. Rodrik-Outmezguine VS, et al. Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor. Nature. 2016;534:272–276. 718. Weinstein IB, Joe A. Oncogene addiction. Cancer Res. 2008;68:3077–3080, discussion 3080. 719. Eberhard DA, et al. Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-smallcell lung cancer treated with chemotherapy alone and in combination with erlotinib. J Clin Oncol. 2005;23:5900–5909. 720. Zehir A, et al. Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients. Nat Med. 2017;23:703–713. 721. Wu JY, et al. Lung cancer with epidermal growth factor receptor exon 20 mutations is associated with poor gefitinib treatment response. Clin Cancer Res. 2008;14:4877–4882. 722. Yasuda H, Kobayashi S, Costa DB. EGFR exon 20 insertion mutations in non-small-cell lung cancer: preclinical data and clinical implications. Lancet Oncol. 2012;13:e23–e31. 723. Hyman DM, et al. Vemurafenib in multiple nonmelanoma cancers with BRAF V600 mutations. N Engl J Med. 2015;373:726–736. 724. Prahallad A, et al. Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR. Nature. 2012;483:100–103. 725. Camidge DR, Pao W, Sequist LV. Acquired resistance to TKIs in solid tumours: learning from lung cancer. Nat Rev Clin Oncol. 2014;11:473–481. 726. Holohan C, Van Schaeybroeck S, Longley DB, Johnston PG. Cancer drug resistance: an evolving paradigm. Nat Rev Cancer. 2013;13:714– 726.

46.e12 Part I: Science and Clinical Oncology

SELF-ASSESSMENT REVIEW QUESTIONS

ANSWERS

1. Next-generation sequencing has helped reveal numerous unusual genomic events, including chromosomal translocations. Which of the following genes is NOT found as oncogenic fusion partners in chromosomal re-arrangements? a. ABL b. PTEN c. PDGFRβ d. ALK e. NTRK 2. Which of the following is true of Hedgehog (SHH) signaling and targeted therapies? a. Secreted hedgehog ligands bind Smoothened receptors, which causes receptor dimerization and autophosphorylation of intracellular tyrosine residues. b. Erlotinib and gefitinib are FDA approved Smoothened receptor inhibitors. c. Sonic hedgehog (SHH) binds to the receptor Patched (PTCH), which relieves its repression of Smoothened (SMO). d. Mutations in SHH, PTCH, and SMO are found in patients with small cell lung cancer. e. Smoothened receptors couple directly to actin. 3. The Ras/MAP kinase and PI3 kinase/mTOR signaling cascades are parallel but interconnected pathways that are frequently mutated in human cancer. Which of the following has NOT been an effective approach in targeting these pathways? a. Allosteric inhibitors of MEK kinase b. Small-molecule inhibitors and kinase of mTOR c. Monoclonal antibodies that bind to EGFR d. Farnesyltransferase inhibitors of Ras e. Kinase inhibitors of mutant BRAF 4. Acquired resistance commonly occurs after treatment with targeted inhibitors. Which of the following are known mechanisms of resistance? a. Parallel pathway upregulation b. Gatekeeper mutation c. Amplification of specific RTKs d. Alternative splicing or amplification of the driver oncogene e. All of the above 5. Which of the following statements is FALSE? a. Chromosomal rearrangement of BCR and ALK to generate the Philadelphia chromosome is the driving event in CML. b. Recurrent somatic splice site alterations involving MET exon 14 (METex14) have been identified in lung cancer. c. Integrin receptors perform inside-out and outside-in signaling at the integrin adhesome. d. ESR1 mutations are a common mechanism of acquired resistance to hormonal therapy in patients with breast cancer. e. Extracellular and transmembrane FGFR3 mutations are recurrent in noninvasive and high-grade invasive bladder cancer.

1. (b) PTEN is a tumor suppressor of the AKT pathway and would not be a likely candidate fusion partner, because truncating mutations or deletions confer loss of PTEN function. All of the other genes listed have been found in patients with a variety of fusions partners. BCR-ABL, the Philadelphia chromosome, which is found in nearly all cases of chronic myelogenous leukemias (CML) and one-third of ALLs, is the classic example of an oncogenic fusion driving cancer. EML4-ALK fusions play a driving role in non–small cell lung cancer (NSCLC). COL1A1-PDGFRβ fusions occur in dermatofibrosarcoma protruberans, a rare sarcoma. TPM3-TRKA fusions were first identified in colon cancer. 2. (c)  The Smoothened receptor is a GPCR, not a receptor tyrosine kinase, and therefore it does not get activated by dimerization and transphosphorylation. Vismodegib and sonidegib are FDA-approved smoothened receptor inhibitors. Mutations in SHH, PTCH, and SMO are found in patients with inherited and sporadic basal cell carcinomas. Smoothened receptors couple to the G proteins Gαi and Gα12. 3. (d)  No direct inhibitors of Ras are available for clinical use. Farnesyltransferase inhibitors were tested but were found to be ineffective in tumors in which Ras mutations are common, likely because of the ability of geranylgeranyl modifications to substitute for farnesylation in effectively targeting Ras to the membrane. New drug development of allosteric inhibitors that specifically target KRAS G12C is underway. Trametinib is an FDA-approved allosteric inhibitor of MEK. Rapamycin and its analogues, as well as novel kinase inhibitors such as RapaLink, are efficacious mTOR inhibitors. Cetuximab and panitumumab are anti-EGFR monoclonal antibody therapies. Vemurafenib, an ATP-competitive inhibitor of BRAF, is approved for the treatment of patients with BRAF V600E mutant melanoma. 4. (e)  Several mechanisms of resistance to kinase inhibitors have emerged. For example, patients with BRAF V600E mutations that are treated with the specific RAF inhibitor vemurafenib often develop resistance mediated by BRAF V600E splice variants or amplification; loss or mutation of NF1; parallel pathway activation of RTKs; mutation of PI3K/AKT components; mutations in NRAS, RAF1 and MAP2K1/2 (MEK1/2); and amplification of MITF. 5. (a)  Translocation of the ABL gene on chromosome 9 with the breakpoint cluster region (BCR) gene on chromosome 22 results in the expression of a BCR-ABL fusion protein, called the Philadelphia chromosome. This event is the pathognomonic molecular lesion in almost all chronic myelogenous leukemias (CML). It is also found in approximately one-third of acute lymphoblastic leukemias (ALLs). The ABL tyrosine kinase inhibitor imatinib, as well as four other ABL inhibitors, are FDA approved in CML; imatinib and dasatinib are approved for ALL.

Cellular Microenvironment and Metastases

3 

Erinn B. Rankin and Amato J. Giaccia

S UMMARY

OF

K EY

P OI NT S

• Metastases are responsible for more than 90% of all cancer-related deaths. • Gene dysregulation, the tumor microenvironment, and host cells drive the metastatic spread of tumor cells. • Metastasis can be subdivided into invasion and migration from the primary tumor; intravasation into the vasculature; dissemination and survival in the circulation; extravasation from the vasculature; survival and metabolic adaptation in

the distant tissue; dormancy; and reactivation and proliferation in the new tissue microenvironment. • Colonization of metastatic tumor cells requires the ability to metabolically adapt, develop angiogenesis, overcome dormancy, and proliferate in a foreign tissue. • The formation of a premetastatic niche is essential for the growth of extravasating metastatic tumor cells. • Organ specificity of tumor metastases is determined both by

One of the most important challenges in clinical oncology is the prevention and treatment of metastatic disease. With advances in surgical techniques and conventional and targeted therapies, localized disease is effectively managed in the clinic. However, metastatic disease is the primary cause of cancer-related deaths. Tumor metastasis involves tumor cell invasion and migration from the primary tumor, intravasation into the vasculature, dissemination and survival in the circulation, extravasation into distant tissues, survival and metabolic adaptation in the distant tissue, dormancy, reactivation, and overt colonization to form a new macroscopic tumor at a distant site.1 This process is highly inefficient; it has been estimated that less than 0.01% of tumor cells that enter the circulation develop into metastases. Despite this inefficiency, metastases are responsible for more than 90% of all cancer-related deaths. Understanding the biology and vulnerabilities of metastatic tumor cells is of critical importance to improve overall survival rates in cancer patients. With little evidence to support mutations in “metastasis genes” as drivers of metastasis, current data suggest that microenvironmental factors as well as epigenetic changes may play key roles in metastasis. This chapter describes the cellular and molecular traits driving tumor metastasis and addresses how the tumor microenvironment influences this process. Most important, it discusses how this knowledge can be translated into current and future cancer therapies.

TUMOR MICROENVIRONMENT AND METASTASIS Although cellular intrinsic traits acquired by tumor cells are required for successful metastatic colonization, cellular and molecular factors within the tumor microenvironment significantly contribute to metastatic progression. The tumor microenvironment contains a number of cell types that promote tumor progression, including cancer stem cells, angiogenic vascular cells, infiltrating immune cells, and cancerassociated fibroblasts (CAFs).2 In addition, extracellular factors within

blood flow and by tissue-specific factors. • Primary tumors possess stem cells that can recapitulate the tumor from a single cell, and a subset of these cancer stem cells may inherently possess altered gene expression changes with increased metastatic potential. • Antimetastatic therapy will likely require the targeted inhibition of many pathways that control proliferation, invasion, angiogenesis, and immune evasion.

the tumor microenvironment such as hypoxia and extracellular vesicles promote metastatic phenotypes within these cell types.

Cancer Stem Cells The traditional view of tumors with a relatively homogeneous population of tumorigenic cells has been significantly revised with the isolation and characterization of cancer stem cells. Stem cells are primal cells that retain the ability to renew themselves through cell division and can differentiate into a wide range of specialized cell types. Cells with stem cell properties have been identified in a variety of solid tumors including colon, breast, head and neck, and pancreatic tumors, glioblastomas, medulloblastomas, and melanoma.3 This rare subpopulation of tumor cells exhibits enhanced tumor-initiating potential: the ability to self-renew and differentiate into multiple cell types. The origin of cancer stem cells remains unclear and may differ among tumor types. It has been hypothesized that cancer stem cells arise from either transformed resident stem and/or progenitor cells, or may represent a dedifferentiated epithelial tumor cell. In intestinal cancer, mouse modeling studies have identified intestinal stem cells as the cells of origin. For example, intestinal stem cell–specific deletion of adenomatous polyposis coli (APC) leads to rapid cellular transformation with uncontrolled cellular growth and solid tumor formation. In contrast, deletion of APC in short-lived transit-amplifying cells was not sufficient to induce long-term tumor growth.4 Stem cell properties can also be acquired by tumors cells through epithelialmesenchymal transition (EMT). Induction of EMT in immortalized human mammary epithelial cells was sufficient to induce the expression of stem cell markers, enhance self-renewal, and increase the number of tumor-initiating cells.5 Although the molecular mechanisms that drive the cancer stem cell phenotype in tumor cells remains largely unknown, a study showed that the transcription factors Slug and Sox9 drive EMT and the cancer stem cell phenotype in breast cancer 47

48 Part I: Science and Clinical Oncology

Invasion Intravasation Extravasation

s, VEGF, CCLI8, E MMP GF

Endothelial

Macrophage

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Endothelial cells play an active role in tumor cell intravasation, extravasation, and dormancy (Fig. 3.1). Studies have shown that metabolic reprogramming of tumor endothelial cells toward a glycolytic phenotype contributes to the early stages of metastasis by promoting an abnormal tumor vasculature, tumor intravasation, and dissemination. Cantelmo and colleagues performed mRNA sequencing analysis of tumor-associated endothelial cells and discovered that tumor endothelial cells expressed a hyperglycolytic signature compared with normal endothelial cells.12 The glycolytic phenotype within tumor-associated endothelial cells could be inhibited through genetic and pharmacologic inhibition of the glycolytic enzyme 6-phosphofructo-2-kinase/fructose2,6-bisphosphatase-3 (PFKFB3). Moreover, PFKFB3 inhibition in endothelial cells was sufficient to normalize the tumor vasculature and significantly reduce tumor cell intravasation and metastasis.12 There are multiple mechanisms by which PFKFB3 inhibition in endothelial cells may have impaired metastatic dissemination, including enhancing the integrity of the endothelial cell barrier, improving vessel maturation and pericyte coverage, and inhibiting cancer cell adhesion molecules. These findings suggest that targeting glucose metabolism in tumor endothelial cells may offer therapeutic benefit for anticancer therapy.

ava

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OSTN F, P -CS GM I2, on CL asi on Inv asati niche CX av tic Intr tasta me

Endothelial Cells

Intr

Pre

Tumor angiogenesis facilitates the hematogenous spread of metastatic tumors. The aberrant production of proangiogenic factors by tumor cells results in malformed and irregular tumor blood vessels that often contain breaks in their lining that facilitate tumor cell intravasation and dissemination. Endothelial cells and pericytes are important structural and functional components of the tumor and distant tissue vasculature, and both have been considered as potential targets in metastatic therapy.11

io sat n ava atio Intr avas cy n tr Ex orma D

IL-3 3, C D2 48

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, B3 KF PF

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Invasion Intravasation Premetastatic nic he

cells.6 Future studies are eagerly awaited to further delineate the intracellular signaling pathways driving the cancer stem cell phenotype in vivo. The role of cancer stem cells in multistage tumor progression, particularly with respect to metastasis, is poorly defined. Given that metastasis is an infrequent event that is achieved by only a small portion of cancer cells that reach distant sites, it has been hypothesized that cancer stem cells may be responsible for metastatic disease. Evidence to support this hypothesis has been generated in models of breast and pancreatic cancer. In the MMTV-PyMT model of breast cancer, Malanchi and colleagues observed that only the CD90+ CD24+ population of cancer stem cells isolated from primary breast tumors contained cells with the ability to form metastases in the lung when introduced into the tail vein.7 Furthermore, this study identified the extracellular matrix protein, periostin, produced by fibroblasts and stromal tumor cells, as a critical factor to maintain the cancer stem cell phenotype and metastatic potential in these cells.7 In human breast cancers, single-cell analysis of early-stage metastatic cells demonstrated that these samples express distinct stemlike, EMT, prosurvival, and dormancy-associated gene expression signatures compared with metastatic cells from high-burden tissues.8 Moreover, transplanted stemlike metastatic cells from low-burden tissues exhibited tumor-initiating capacity and were able to differentiate into luminal-like cancer cells.8 Similarly in pancreatic cancer, lineage-tracing studies showed that circulating pancreatic tumor cells exhibit EMT and cancer stem cell properties, and initiate tumor formation. A critical role for inflammation to maintain the ability of cancer stem cells to metastasize was observed in this study.9 In addition, the extracellular protein tenascin C (TNC), expressed by stem cell niches and cancer cells, was also found to promote stem cell signaling and lung metastases in breast cancer cells.10 Collectively, these data strongly implicate the tumor microenvironment in promoting the cancer stem cell phenotype and the initiation of tumor metastasis.

Figure 3.1  •  Multiple cellular components of the tumor microenvironment

(TME) contribute to metastasis. Key cell types and mechanisms of action are shown.

Adhesion to the endothelium is an initial step in extravasation that is followed by transendothelial cell migration.13 Tumor cell adhesion to the endothelium requires the expression of cognate ligands and receptors by cancer cells and endothelial cells.13 A variety of ligandreceptor interactions have been shown to contribute to tumor extravasation, including interactions with selectins, integrins, cadherins, CD44, and immunoglobulin (Ig) superfamily receptors (for a review, see Reymond and colleagues13). For example, endothelial cell P- and E-selectins bind to tumor cells through cell-cell adhesion molecules such as integrins and CD44.14–19 Studies have suggested a role for the immunosuppressive cytokine interleukin (IL)-35 in facilitating cancer cell adhesion to endothelial cells. IL-35 was found to be highly expressed by human pancreatic cancer cells in which it promoted ICAM1 expression to facilitate endothelial cell adhesion and transendothelial migration via an ICAM1-fibrogen-ICAM1 bridge.20 Finally, endothelial cells within in the perivascular niche of the bone marrow have been implicated in promoting tumor cell dormancy. Similar to hematopoietic stem cells, dormant cancer cells have been found to be localized within perivascular niches of the bone marrow microenvironment.21 Within stable microvasculature niches, endothelial cells promote tumor cell quiescence through the production of thrombospondin-1 (TSP1)21. In contrast, endothelial cells isolated from sprouting neovasculature promote tumor proliferation through the production of tumor-promoting factors including transforming growth factor–β1 (TGF-β1) and periostin from sprouting endothelial cell tips.21

Pericytes Similar to endothelial cells, pericytes are thought to play a protumorigenic role by supporting blood vessel maturation and function (see Fig. 3.1). Pericytes promote endothelial cell survival and stabilize the tumor vasculature.22 In addition, pericytes can directly facilitate distant metastasis by promoting tumor cell intravasation through the upregulation of the transmembrane receptor endosialin (CD248).23 Studies have demonstrated that pericytes can also promote tumor cell intravasation and metastasis through the IL-33–dependent recruitment of macrophages.24 Therefore it has been proposed that targeting pericytes alone or in combination with endothelial cells may be an effective strategy in the treatment of cancer.24,25 However, clinical data indicate

Cellular Microenvironment and Metastases  •  CHAPTER 3 49

that low pericyte coverage is associated with metastasis and poor prognosis in a number of cancers.25 A recent study demonstrated that depletion of pericytes suppressed tumor growth and enhanced metastasis of murine models of breast cancer.25 Enhanced metastasis was associated with increased levels of tumor hypoxia, EMT, and Met receptor activation.25 These findings indicate that pericyte coverage may be important to stabilize the tumor vasculature and prevent the hypoxic selection of aggressive tumor cells (see later discussion of hypoxia and metastasis). These findings raise concerns when considering antipericyte treatments in cancer therapy, especially in the setting of metastasis.

Immune Cells Most solid tumors contain immune infiltrates derived from both the myeloid and lymphoid lineages that can have both positive and negative effects on metastasis.2 This section focuses on immune cells that stimulate metastatic progression.

Macrophages Studies have linked a unique subset of macrophages, termed tumorassociated macrophages (TAMs), to tumor progression and metastasis. Clinically, the presence of excessive macrophages is associated with poor prognosis in the majority of patients with solid tumors including breast, prostate, cervix, bladder, endometrial, and kidney cancer.26–28 TAMs express a variety of growth factors and cytokines that promote tumor angiogenesis, invasion, intravasation, metastasis, and immune suppression (see Fig. 3.1)28,29. For example, macrophages within the primary tumor contribute to tumor cell invasion and extravasation by releasing a variety of proteases including serine, cysteine, and metalloproteases that remodel the extracellular matrix to enhance tumor cell invasion and migration (for a review, see Cho and colleagues2). Perivascular macrophages are thought to play a particularly important role in tumor cell intravasation by promoting vascular permeability. In preclinical models of breast cancer, genetic ablation of vascular endothelial growth factor (VEGF) from macrophages was sufficient to reduce transient vascular permeability, leading to reduced numbers of circulating tumor cells.30 Moreover, macrophages can promote tumor cell invasion through the release of chemokines and cytokines including CCL18 and epidermal growth factor (EGF; for a review, see Ruffell and colleagues28). Many questions remain regarding the factors that control the biologic activities of TAMs within the tumor microenvironment. TAMs localize to regions of hypoxia, and thus it has been hypothesized that the hypoxic microenvironment may influence the unique characteristics and gene expression profiles of TAMs. Indeed, TAMs express high levels of the hypoxia inducible transcription factor (HIF) HIF-2, which has been found to be an independent prognostic factor of outcome.31 When exposed to hypoxia, macrophages upregulate the expression of mitogenic and proangiogenic cytokines.32 Experimentally, deletion of myeloid HIF-2 reduced TAM infiltration into tumors and impeded tumor proliferation and growth.32 Thus, targeting TAMs and CD11b cells may be a viable mechanism for antimetastatic therapies.

Neutrophils Neutrophils represent 50% to 75% of the total circulating leukocytes in blood. They play an important role in inflammation and are early responders to pathogens including bacteria, fungi, and viruses. Neutrophils mediate direct antimicrobial activities through the release of enzymes and toxic factors, the generation of reactive oxygen species, and the release of nuclear material into extracellular traps called neutrophil extracellular traps (NETs).33 Although neutrophils can inhibit the metastatic seeding of disseminated tumor cells under some conditions, accumulating evidence suggests a prometastatic role for neutrophils (see Fig. 3.1).34–37 Study findings have suggested that that metastatic cancer cells promote the formation of NETs through the release of G-CSF.37 NETs may promote metastasis through multiple mechanisms including (1) trapping

circulating tumor cells within DNA meshes at distant tissues, (2) promoting vascular permeability and the premetastatic niche, and (3) stimulating tumor cell invasion and proliferation at distant tissue sites.36–38 Important to note, digesting NETs with DNase I is sufficient to reduce metastasis in multiple metastatic tumor models, suggesting that therapeutic targeting of NETs may prevent metastasis.36,37

Platelets Platelets are the second most abundant cell type in the blood and play an important role in hemostasis, thrombosis, inflammation, and vascular biology.39 Platelets are among the first cell types that interact with cancer cells in the circulation.20 Tumor cells release platelet coagulation factors such as thrombin to promote platelet aggregation around circulating tumor cells. Indeed, increased blood coagulation is often observed in cancer patients as a result of elevated levels of thromboplastin, procoagulant A, and phosphatidylserine produced by tumor cells.40,41 Platelets facilitate metastasis through multiple mechanisms (see Fig. 3.1). Platelets are thought to form a physical barrier around disseminated cancer cells, protecting them from shear stress and lysis mediated by natural killer cells.42 In addition, platelets can facilitate tumor cell extravasation by enhancing tumor cell adhesion to endothelial cells and by promoting endothelial cell permeability.43–46 Finally, platelets have been shown to directly stimulate cancer cell EMT and invasion through the release of TGF-β.47 Therapeutic inhibition of platelet binding to cancer cells through the disruption of α6β1 integrin/ADAM9 receptor may be an effective therapeutic strategy to target platelet-mediated metastasis in vivo.48

Fibroblasts and Mesenchymal Stem Cells Accumulating evidence supports a critical role for CAFs in tumor progression and metastasis (see Fig. 3.1). Clinically it was observed that epigenetic changes and mutations commonly found in cancer cells, such as p53 and PTEN mutations, can also be found in cancerassociated stroma.49,50 These studies provided early evidence to suggest that alterations in the stroma may contribute to tumor progression. Gene expression profiling studies of CAFs in human specimens and murine tumor models revealed an “activated” proinflammatory gene signature.51 Among the cytokines produced by CAFs, the chemokine CXCL12 is of particular interest, given its role in driving tumor cell migration and recruitment of endothelial progenitor cells expressing the CXCR4 receptor. Consistent with these data, an important role for CAFs in promoting tumor invasion has been observed in several murine models of cancer.51 In breast cancer, Hu and colleagues identified a role for CAFs in the transition of mammary ductal carcinoma in situ (DCIS) to invasive carcinoma. Coinjection of fibroblasts with DCIS cells resulted in invasive carcinomas, whereas injection of DCIS cells alone was only sufficient to induce mammary DCIS.52 CAFs promote tumor cell invasion by establishing invasion-permissive tracks in the extracellular matrix through the secretion of factors that induce matrix remodeling.51,53 CAFs can also support tumor cell survival and migration through the release of extracellular vesicles carrying annexin A6/LDL receptor–related protein/thrombospondin 1 (ANXA6/LRP1/ TSP1) complexes.54 In addition to promotion of tumor invasion, supportive roles for CAFs in the maintenance of cancer stem cell signaling and the establishment of the premetastatic niche have been found.51 Further characterization of the distinct subsets of CAFs relevant to metastasis in various tumor types is needed. In addition, further elucidation of the interplay between tumors and the stroma is warranted and may reveal novel strategies in the treatment of metastatic disease. Studies have suggested an important role for a subset of CAFs, mesenchymal stem cells (MSCs), in tumor progression and metastasis. Although MSCs are commonly isolated in CAF preparations, MSCs are functionally distinct in that they are able to undergo multipotent differentiation. MSCs have been identified as important components of the tumor stroma that promote the progression of ovarian, colon, and pancreatic cancers.55–57 Cancer-associated MSCs have recently

50 Part I: Science and Clinical Oncology

been shown to promote pancreatic cancer cell proliferation, invasion, and transendothelial cell migration through the production of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF).55 These findings suggest that inhibition of tumorMSC cross talk may be a therapeutic strategy for the treatment of pancreatic cancer.

Extracellular Vesicles Extracellular vesicles are emerging as key factors that promote metastasis. Studies have shown that tumor and stromal cells secrete small vesicles that contain a variety of bioactive molecules such as proteins, lipids, RNA, and DNA that can promote intercellular signaling and tumor progression. Extracellular vesicles can be derived from either the endosome (exosome) or the plasma membrane (microvesicle) to mediate intercellular signaling with tumor and stromal cells within the local and distant microenvironments. Clinically, extracellular vesicles and their cargo have been associated with tumor progression. The concentrations of exosomes have been found to be increased in the peripheral blood of patients with ovarian, breast, and pancreatic cancers compared with healthy control patients (for a review, see Becker and colleagues58). Specific nucleic acid and protein expression signatures within exosomes have also been associated with tumor progression and metastasis. For example, exosomal miR-141 expression in the serum of prostate cancer patients is significantly higher in metastatic prostate patients compared with patients with localized disease.59 An exosomal protein signature containing the melanoma-specific protein tyrosinase-related protein-2 (TYRP2), very late antigen 4 (VLA-4), HSP-70, and the MET oncoprotein has been associated with metastasis in melanoma patients.60 In pancreatic cancer, the level of circulating glypican-1–positive exosomes correlates with poor patient survival.61 Functionally, extracellular vesicles have been shown to play an important role in the tumor microenvironment and promote metastasis through a variety of mechanisms including tumor immune suppression, invasion, angiogenesis, and the premetastatic niche (for a review, see Kalluri and colleagues62). For example, melanoma cells promote immune evasion through the release of FasL-bearing microvesicles that trigger Fas-dependent apoptosis of lymphoid cells.63 Tumor-derived exosomes can also directly promote the immunosuppressive phenotype of myeloid-derived suppressor cells through the activation of STAT3 signaling in these cells.64 Tumor-derived exosomes promote an invasive phenotype by activating stromal cells to produce matrix metallo­ proteinases (MMPs) such as MMP1.65 In addition, tumor-derived extracellular vesicles can directly promote the invasive capacity of nonmetastatic cells at local and distant sites by transferring mRNAs involved in migration and metastasis.66 Conversely, stromal microvesicles have also been shown to promote tumor cell invasion and metastasis. Fibroblast-secreted exosomes promote breast cancer cell migration through the activation of planar cell polarity (PCP) signaling.67 Astrocyte-mediated transfer of exosomal PTEN-targeting microRNAs leads to PTEN loss in brain metastatic tumor cells that can support metastatic outgrowth through mechanisms involving the increased expression of the chemokine CCL2 and adaptive metastatic outgrowth.68 Exosome-mediated transfer of miR-105 by breast cancer cells promotes vascular leakiness and metastasis by disrupting endothelial cell tight junctions.69 Tumor-derived exosomes may also play a key role in establishing a premetastatic niche at distant sites by activating signaling within organ-specific cells to recruit bone marrow–derived macrophages, activate Src phosphorylation and proinflammatory S100 gene expression, and increase nutrient availability.70–72 The factors that regulate extracellular vesicle formation and content in cancer remain poorly understood. Ostrowski and colleagues used an RNA interference screen to identify factors important in exosome secretion in cancer cells. These studies revealed an important role for the Rab GTPases Rab27a and Rab27b and the Rab27 effectors Slp4 and Slac2b in exosome secretion.73 Studies have implicated hypoxia as an important microenvironmental factor that can influence both

exosome content and shedding. The shedding of exosomes from tumor cells is increased under hypoxic conditions. In breast cancer cells, hypoxia promotes microvesicle shedding through the HIF-dependent regulation of the small GTPase RAB22A, a protein that is localized to budding microvesicles and promotes metastasis.74 Hypoxia also plays an important role in regulating exosome content and function. Clinically, the levels of hypoxia-regulated proteins in plasma exosomes are significantly higher in glioblastoma multiforme (GBM) patients compared with healthy control patients.75 Moreover, exosomes derived from hypoxic GBM cells are potent inducers of angiogenesis compared with exosomes isolated from normoxic GBM cells.75 Given that extracellular vesicles can be detected in patient biologic fluids and contribute to cancer progression, there is great interest in use of exosomes as diagnostic biomarkers and therapeutic targets in cancer.

Hypoxia Hypoxia is a potent microenvironmental factor driving metastatic tumor progression. Clinically, hypoxia is associated with metastasis and poor survival in variety of cancer patients.76–79 Hypoxia selects for cells with low apoptotic potential and increases genomic instability, allowing for rapid mutational adaptations.80–82 In addition, hypoxia directly increases the expression of genes involved in glycolysis, angiogenesis, cell survival, invasion, immune suppression, the cancer stem cell phenotype, and metastasis. All of these changes allow cells to adapt to oxygen-deprived conditions and permit cells to escape these conditions by establishing new blood supplies or by physically moving from an oxygen-poor environment to an oxygen-rich environment. The primary molecular mediators of hypoxic signaling are HIF-1 and HIF-2. In the presence of oxygen, the alpha subunits of HIF-1 and HIF-2 are rapidly degraded through the cooperative actions of prolyl hydroxylase (PHD) enzymes PHD1, PHD2, and PHD3, and the E3 ubiquitin ligase substrate recognition component VHL.83,84 Under hypoxia, HIF-1 and HIF-2 are stabilized and activate the expression of genes containing hypoxia response elements (HREs).76 Over 200 genes that allow cells to survive and adapt to low oxygen tensions are activated in response to HIF-1 and HIF-2. Consistent with the association between hypoxia and metastasis, HIF-1 and HIF-2 are highly expressed in primary tumors and metastases. Immunohistochemical analysis of human cancer specimens indicates that the majority of primary tumors and metastases have increased HIF-1 and/or HIF-2 protein compared with the normal adjacent tissue.76–78 Moreover, increased HIF expression is often associated with increased patient mortality.85 Experimentally, overexpression of HIF in tumor cells promotes metastasis,86 whereas inactivation of HIF significantly decreases the metastatic potential of metastatic tumor cells.87–89 These clinical and experimental findings indicate an important role for HIF in metastatic tumor progression. HIFs influence multiple steps within the metastatic cascade (Fig. 3.2). HIF-1 and HIF-2 activate the early stages of metastasis in the primary tumor by promoting EMT, the cancer stem cell phenotype, invasion, and migration (for a review, see Rankin and Giaccia79). HIFs promote invasion and migration through multiple mechanisms. First, HIF regulates the E- to N-cadherin switch during EMT phenotype through the direct activation of E-cadherin repressors including Twist1, Zeb1/2, Snail.86,90,91 HIF signaling has also been shown to indirectly promote EMT through the activation of Notch, TGF-β, integrin-linked kinase (ILK), and the receptor tyrosine kinase AXL (for a review, see Rankin and Giaccia79). Second, HIF directly upregulates the expression of multiple factors driving cellular invasion and migration. Most notably, HIF drives the expression of the extracellular matrix protein LOX.92 Increased LOX expression correlates with decreased distant metastasis-free survival and overall survival in patients with breast and head and neck cancer.92 In addition, LOX activation promotes the invasive and metastatic potential of breast cancer cells. Erler and colleagues demonstrated that genetic and pharmacologic inhibition

Cellular Microenvironment and Metastases  •  CHAPTER 3 51

Invasion/migration

Intravasation/ extravasation

Premetastatic niche

Distant growth

BMDC Fibroblast

SNAIL TWIST ZEB1/2 MMPs LOX CTGF CCR5 PGF

AXL CDCP1 ZEB1/2 MET PLOD1/2 AKAP12 RAB222 PLUAR

LOX LOXL2 LOXL4 SDF-1 VEGF CXCL12 CCL2 EXOSOMES

ANGPTL44 L1CAM VEGF UPAR MMPS SDF-1/CXCR4 VCAM1 ICAM1

HK1/2 VEGF LDHA ANGPT1/2 PDK1 PDFG CKB PIGF ENO1 PAI-1 ALDHA TIE-2 GLUT-1/3 PGK1

Figure 3.2  •  Hypoxia inducible transcription factor (HIF) signaling regulates multiple steps within the metastatic cascade. The key steps of the metastatic cascade and target genes by which HIF signaling regulates these processes are shown.

of LOX in metastatic breast cancer is sufficient to prevent hypoxiainduced cell invasion and metastasis. These findings indicate that LOX is a promising therapeutic target for the treatment of metastatic disease. HIF signaling also facilitates tumor cell intravasation and extravasation from the vasculature. HIF activity in tumor cells results in the release of factors that modulate endothelial-endothelial cell and endothelial-tumor cell interactions. The upregulation of angiopoietinlike 4 (ANGPTL4) by HIF promotes tumor cell motility and intravasation of tumor cells through blood vessels.88 Simultaneously, HIF strengthens tumor cell–endothelial cell interactions through the activation of L1 cell adhesion molecule (L1CAM).88 Another mechanism by which HIF promotes tumor cell intravasation and extravasation is through the activation of genes that control vascular permeability. Hypoxic induction of VEGF, angiopoietin 2, MMPs, and UPAR cooperatively act to destabilize the vascular wall and allow for tumor cell entry.93 Third, HIF activity in the primary tumor is involved in the formation of the metastatic niche. As mentioned earlier, priming the premetastatic site for the recruitment and survival of metastatic tumor cells is a critical step in successful metastatic colonization. In breast cancer cells, HIF activity results in the upregulation and release of LOX and LOX-like proteins (LOXL2 and LOXL4). These proteins recruit bone marrow–derived cells (BMDCs) into the lung and prime the lung for metastatic colonization.87,92,94 BMDCs produce chemokines that recruit tumor cells and stimulate blood vessel invasion.95–98 Studies have also demonstrated a role for LOX in the establishment of the premetastatic niche in the bone wherein tumor-secreted LOX is both necessary and sufficient to induce osteolytic bone lesions and cortical bone loss in mice before the arrival of tumor cells.99 Another mechanism by which HIF signaling controls the directional migration of metastatic tumor cell sites is through the upregulation of SDF1/CXCR4 signaling.100,101 Stromal cells within target tissue sites produce stromal derived factor-1 (SDF1), which recruits cancer cells expressing the receptor CXCR4.29 Although these studies have indicated an important cellular intrinsic role for hypoxia and HIF signaling in the primary tumor, future studies are eagerly awaited to elucidate the role of HIF signaling in tumor support cells. Finally, HIF promotes the late stages of metastasis at a distant site by stimulating angiogenesis. As in the primary tumor, angiogenesis is a critical step for metastatic tumor growth. VEGF-A is a proangiogenic factor produced by tumor cells that stimulates the recruitment and

proliferation of endothelial cells and supports pericytes.29 VEGF-A is a well-established HIF target and is significantly induced by HIF signaling in both primary tumors and metastases.102 In summary, HIF affects multiple aspects of tumor metastasis, indicating that therapeutic targeting of HIF may be an effective strategy to selectively inhibit multiple aspects of metastasis.

PATTERNS OF METASTASIS Seed and Soil Hypothesis The patterns of colonization cannot solely be explained by the circulatory, lymphatic, and transcelomic routes described earlier. The propensity for certain tumors to seed in particular organs was first discussed as the “seed and soil” theory by Paget in 1889.103 For example, prostate cancer often metastasizes to the bones, colon cancer has a tendency to metastasize to the liver, and the primary site for ovarian metastasis is the omentum.104 Colonization is an extremely inefficient process that is heavily dependent on the interactions between “seeding” tumor cells and the “soil” microenvironment of the secondary site. Many factors including formation of a premetastatic niche and interactions between circulating tumor cells and the distant microenvironment determine patterns of colonization.

Premetastatic Niche Over the past decade, studies have convincingly shown that formation of a premetastatic niche is essential for the growth of extravasating metastatic tumor cells.98 Soluble factors and BMDCs are recruited to the distant site before the arrival of tumor cells to establish a permissive environment for malignant colonization. The mechanisms by which the premetastatic niche is formed remain largely unknown. However, recent studies have shown that factors secreted by the primary tumor are directly involved in establishing a permissive ECM environment and in recruiting BMDCs to the distant site. BMDCs expressing VEGFR1, c-kit, CD133, and CD134 have been detected at distant sites and increase angiogenesis at the premetastatic sites. Targeted inhibition of VEGFR1 prevented niche formation and subsequent metastatic progression. This tissue preconditioning may thus represent a key step that could be targeted therapeutically, although studies with anti-VEGF therapy have failed to show significant benefit in preventing metastatic growth for long periods of time. The role of

52 Part I: Science and Clinical Oncology

hematopoietic progenitor cells and other BMDCs in tumor progression is reviewed by Kaplan and colleagues.105 An additional function of the premetastatic niche is to guide metastases to specific organs. Kaplan and coworkers demonstrated that injection of secreted factors collected from cancer cells that metastasize to multiple organs could permit cancer cells that only metastasize to the lung when grown as subcutaneous tumors in mice to display widespread metastasis through governing BMDC accumulation.98 Elevated fibronectin expression by fibroblasts and fibroblast-like cells resident at premetastatic sites seems to be a critical factor in the development of the premetastatic niche. The key tumor-secreted factors that determine metastatic sites and mediate premetastatic niche formation have yet to be identified, although a role for tumor necrosis factor–α (TNF-α), TGF-β, and VEGF-α pathways has been demonstrated.106 MMPs may also play an important role in this process. For example, VEGFR1 signaling has been shown to be required for premetastatic induction of MMP-9 expression in endothelial cells and macrophages of the lungs by distant primary tumors.107 This is thought to make the lung microenvironment more compliant for invasion of metastasizing cells. This concept is supported by the finding that pericyte recruitment and angiogenesis are not observed in tumor-bearing mice with MMP-9 knockout bone marrow cells.108 Furthermore, stromalderived MMP-2 and MMP-9 have also been shown to contribute to establishment and growth of metastases.109 Thus, whereas there is evidence that MMPs play multiple roles in metastases, clinical trials with MMP inhibitors have failed to show significant efficacy. In large part, this has been due to unexpected normal tissue toxicities and conflicting roles in metastases.

Organ Specificity The organ distribution of metastases from a primary tumor is not random. Minn and colleagues used bioluminescence imaging to reveal patterns of metastasis formation by human breast cancer cells in mice.110 They also showed that individual cells from the pleural effusion of a breast cancer patient showed distinct patterns of organspecific metastasis.111 Single-cell progenies derived from this population

37+U3 7XPRUFHOO

demonstrated different abilities to metastasize to the bone, lung, or adrenal medulla. These studies indicated that there are particular requirements for circulating tumor cells to colonize specific organs. The factors contributing to tissue specific colonization include cellular intrinsic factors within the disseminated tumor cells and specific factors within the tissue microenvironment. Some of the key cellular intrinsic molecules determining organ-specific metastasis have been identified and are briefly discussed in the following section. In addition, we are beginning to unravel the mechanisms by which infiltrating tumor cells “educate” the normal tissue stroma at distant sites to support metastatic growth. Elucidation of the complex signaling networks that exist between tumor cells and their tissue microenvironment offers new opportunities for targeted therapy against cancer.

Metastases to the Bone There are two types of bone metastases: osteoblastic and osteolytic.112 Osteoblastic metastases are observed in patients with advanced prostate cancer. Both the differentiation of osteoblastic precursors and the activity of osteoblast cells are stimulated by tumor and microenvironmental signals such as bone morphogenetic protein (BMP), fibroblast growth factor receptors (FGFRs), and insulin-like growth factor 1 receptor (IGF1R).113 Runx-2 is a key transcription factor that regulates the differentiation of osteoblasts and osteoblastic precursor cells, and represents a potential new target for inhibiting osteoblastic metastases by preventing osteoblastic precursor differentiation.114 In contrast, osteolytic metastases are observed in patients with breast cancer or multiple myeloma, and in these patients interactions between tumor cells and the bone microenvironment result in bone resorption and metastatic growth as a result of the unique interplay between osteoblasts and osteoclasts (Fig. 3.3).112,115 Parathyroid hormone–related peptide (PTHrP) secreted by the tumor cells stimulates osteoblasts to produce receptor activator of nuclear factor–κB (RANK) ligand (RANKL). Consequently, bone-resorbing osteoclast cells are activated by RANKL when it binds to the RANK receptor. Activated osteoclasts upregulate MMPs, which degrade the bone matrix–releasing growth factors such as TGF-β, IGFs, platelet-derived growth factor (PDGF), fibroblast

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Figure 3.3  •  The vicious cycle of osteoclastic bone metastasis. Interactions between the tumor cells and the bone microenvironment create a vicious cycle

of osteolytic metastatic lesion development. Parathyroid hormone–related peptide (PTHrP), secreted by the tumor cells, stimulates osteoblasts to produce receptor activator of nuclear factor–κB (RANK) ligand (RANKL). Bone-resorbing osteoclast cells are activated by RANKL when it binds to the RANK receptor. The activated osteoclasts upregulate matrix metalloproteinases (MMPs) that degrade the bone matrix–releasing growth factors such as transforming growth factor–β (TGF-β), insulin-like growth factors (IGFs), platelet-derived growth factor (PDGF), fibroblast growth factors (FGFs), and bone morphogenetic proteins (BMPs). These factors stimulate tumor cells to release PTHrP, thus completing the vicious cycle. (Modified from Steeg PS. Tumor metastasis: mechanistic insights and clinical challenges. Nat Med. 2006;12:895–904.)

Cellular Microenvironment and Metastases  •  CHAPTER 3 53

growth factors (FGFs), and BMP.112,116,117 These factors stimulate tumor cells to release PTHrP, thus restarting this pathway of bone resorption. Gene profiling has identified other important mediators of osteoclastic bone metastases including CXCR4, MMP-1, CTGF, and osteopontin.118 Tumor cells additionally induce osteoclast formation by overexpressing ILs such as IL-8 and IL-11, and by downregulating macrophage colony-stimulating factor.119,120 All of these factors represent new targets for metastases, although the importance of each factor in osteoclastic bone metastases requires further clarification.

Metastases to the Brain Brain metastases are most commonly observed in patients with breast cancer, lung cancer, and melanoma. Vascular access to the brain is strictly regulated by the blood-brain barrier, an endothelial layer surrounding the brain, connected by tight junctions and further lined by a basement membrane, pericytes, and astrocytes.121 Macromolecules are not usually able to traverse the blood-brain barrier, and it remains unclear how tumor cells are able to penetrate the blood-brain barrier. However, once the tumor cells are within the brain parenchyma, glial cells permit establishment and growth of metastases by secreting chemokines, cytokines, and growth factors.122 Other neurotransmitter hormones in the brain such as norepinephrine have also been reported to increase tumor cell motility and metastatic spread.123 Little is known about the key factors that determine colonization of the brain, mostly because there is a lack of good in vivo models of brain metastasis. Overexpression of Stat3 increases melanoma metastasis to the brain and increases invasion of the melanoma cells and angiogenesis, although the pathways modulated by Stat3 signaling require elucidation.124 The dependence of brain metastases on VEGF has been demonstrated experimentally in animals through inhibition studies in which VEGF neutralization reduced brain metastases.125,126 In general, patients with brain metastases have an extremely poor prognosis. It is of concern that there has been an increase in the incidence of brain metastases in patients whose systemic disease is well controlled.127–129 For example, patients with breast tumors that overexpress Her2 and who are treated with Her2 targeting trastuzumab (see later discussion) have an incidence of brain metastases twice that of breast cancer patients who are treated with other agents.128 This is thought to be because the brain offers a sanctuary for metastatic breast tumor cells when systemic disease is being controlled.130 The development of drugs that can cross the blood-brain barrier and target brain metastases is of paramount importance in the development of new targeted therapies to tackle this problem. Currently, the best treatment for oligometastases to the brain is radiosurgery.

Metastases to the Lung Pulmonary metastases are frequently observed in patients with sarcoma, breast cancer, melanoma, gastrointestinal cancer, and kidney cancer. Because cardiac output from the pulmonary artery circulates through the lungs, a high incidence of pulmonary metastases in cancer patients can be expected on the basis of blood flow alone. Metastases therefore often initiate in pulmonary arterioles and later traverse the basement membrane into the lung parenchyma. TGF-β and NF-κB facilitate this process in breast cancer, as does osteopontin in hepatocellular cancer, and ezrin in osteosarcoma and breast cancer.131–136 In vivo studies have identified a gene expression signature for lung metastasis including several membrane-localized and secreted proteins that has been validated in breast cancer patients.110 Interesting to note, this group of genes was able to induce lung metastasis when expressed together but not individually, suggesting essential cooperation between proteins. Increased expression of antiapoptotic proteins such as Bcl-2 and Bcl-xL is also observed in lung metastases, facilitating survival and resulting in resistance to therapy.137–141 Adding to the complexity of signaling interactions needed for successful metastatic colonization in the lung, studies have revealed

that interactions between breast cancer stem cells and the lung stroma are necessary for metastatic colonization. The ECM components TNC and periostin (POSTN) have recently been shown to be essential factors required for tumor initiating cells to form metastases in the lung.7,10,142 Malanchi and colleagues showed that tumor-initiating cells induce POSTN expression in lung myofibroblasts to initiate colonization and maintain their stem cell phenotype.7 Similarly, Oskarsson and colleagues demonstrated a role for the extracellular protein TNC in supporting the breast cancer cell stem cell phenotype and metastases in the lung.10 Interesting to note, previous studies showed that TNC and POSTN form complexes together with collagen type I and fibronectin in the ECM. It was shown that POSTN promotes the incorporation of TNC into the ECM to strengthen the ECM architecture, suggesting that these factors may act through similar pathways in promoting cancer stem cell metastasis.143 In support of this notion, maintenance of the cancer stem cell phenotype by TNC and POSTN was mediated at least in part through the induction of WNT signaling. These studies suggest that targeting signaling pathways mediated through the ECM may be an effective strategy against cancer stem cell–driven metastasis.

Metastases to the Liver Liver metastases are observed in patients with breast, lung, and pancreatic cancers. However, liver metastases are most commonly found in patients with metastatic colorectal cancer, because the liver is the first capillary bed encountered by the metastasizing cells. The circulatory system of the liver, in particular the liver sinusoids, does not have a barrier limiting macromolecule flux, and it is well perfused and highly permeable, permitting metastasizing cancer cells to establish themselves and grow. Thus, tumor cell invasion and survival are key determinants in metastatic colonization of the liver.144 Tumor cell survival within the liver is dependent on the cells’ ability to metabolically adapt to the local tissue microenvironment.145 The liver is characterized by regions of hypoxia and highly glycolytic hepatocytes.146 Preclinical studies have revealed that disseminated colon cancer cells experience acute hypoxia and competition for glycolytic substrates early after hepatic dissemination.147 To survive, metastatic colorectal cells release the enzyme creatine kinase brain-type (CKB) into the extracellular space.147 CKB controls the amount of rapidly mobilized high-energy phosphates by catalyzing the transfer of a high-energy phosphate group from adenosine triphosphate (ATP) to the metabolite creatine, producing phosphocreatine.148 Under hypoxia, tumor cells import phosphocreatine and use it as a source of high-energy phosphate that can be transferred to adenosine diphosphate (ADP) to generate ATP.148 These findings suggest that hepatic hypoxia is a barrier to disseminated tumor cell survival and that disseminated tumor cells metabolically adapt to overcome metabolic stress associated with hypoxia. Consistent with these findings, Dupuy and colleagues observed that disseminated breast cancer cells in the liver are dependent on glycolysis for survival. Glycolytic reprogramming in metastatic breast cancer cells was mediated by the HIF target gene pyruvate dehydrogenase kinase (PDK1).149 Under hypoxic conditions, PDK1 upregulation represses mitochondrial function by reducing pyruvate entry into the TCA cycle and promoting the conversion of pyruvate to lactate, thereby supporting glycolysis through the regeneration of regenerate NAD.150 Together, these findings highlight the importance of metabolic plasticity in promoting tumor cell survival within foreign tissue microenvironments.

CLINICAL RELEVANCE AND APPLICATIONS The future of cancer therapy lies in the ability to effectively prevent and treat metastatic disease. Metastases are largely resistant to current cytotoxic therapies and as a result are responsible for more than 90% of all cancer-related deaths. Over the past decade, many of the cellular and molecular constituents that drive metastatic tumor progression

54 Part I: Science and Clinical Oncology

have been defined. Based on these findings, a number of therapeutic agents targeting key components of the metastatic cascade have been developed and tested in preclinical as well as clinical settings. Many of the initial targeted therapies developed for tumor metastasis inhibit factors driving tumor cell invasion and migration. Preclinical studies have demonstrated that agents that inhibit MMPs, the receptor tyrosine kinase AXL, miR-10b, fascin, and exosomes are effective in blocking the initiation and/or progression of metastatic tumors in mice (see reviews by Valastyan and Weinberg151 and Rankin and colleagues152). Of all of these agents, MMP inhibitors have been the most tested in clinical trials. Disappointingly, these inhibitors failed to increase survival in patients with advanced cancer and were associated with adverse side effects.153 Important lessons regarding clinical trial design were learned from these trials. Because MMP inhibitors and other inhibitors of invasion are likely to function at the early stages of metastasis, it is important to thoughtfully identify patient populations that may benefit most from these types of therapies. In addition, it is likely that the combination of targeted agents controlling distinct aspects of metastasis may prove to be the most effective when targeting metastatic cancer. An emerging strategy in the treatment of metastasis involves the therapeutic targeting of cellular and molecular constituents within the tumor microenvironment. As mentioned earlier, hypoxia and HIF signaling are critical drivers of both the tumorigenic and metastatic phenotypes. HIF and hypoxia-induced proteins represent therapeutic targets that have the potential to be tumor specific because these proteins are highly elevated in both the primary tumor and metastases in comparison with normal tissue.154 In addition, targeting HIF is an attractive strategy for the treatment of metastatic disease because HIF controls multiple aspects of metastasis including EMT, invasion, migration, metastatic niche formation, and metastatic tumor growth. In this regard, a number of small-molecule HIF inhibitors including digoxin and acriflavine have been identified and have shown success in preclinical studies by preventing lung metastasis in mice bearing primary breast tumors.155 Although the efficacy of these inhibitors in treating established metastatic disease remains unknown, these compounds do inhibit metastatic xenograft growth after tumor implantation.88 In addition to targeting HIF directly, a number of HIF-induced proteins have been targeted for metastatic therapy. One very promising target is LOX, a hypoxia-induced secreted protein involved in multiple stages of metastasis.92 LOX contributes to tumor cell invasion by cross-linking collagens in the ECM, which stimulates integrin-mediated cell-matrix adhesion and activation of FAK, and in addition provides a route (“highway”) by which tumor cells may travel. Furthermore, LOX is involved in the formation and maintenance of the metastatic niche, allowing metastatic dissemination and growth. Targeting secreted LOX through antibody or small-molecule inhibition significantly reduced formation and growth of metastases to the lung, liver, bone, and brain, in several preclinical studies. Another promising hypoxia-induced target is CTGF.156 CTGF is an extracellular matrix

protein that exerts a number of prometastatic activities including migration, invasion, angiogenesis, and anoikis.157 Both genetic and therapeutic inhibition of CTGF have shown efficacy in primary and metastatic tumor growth inhibition of preclinical models of pancreatic cancer.158,159 The results of clinical trials targeting LOX and CTGF are eagerly awaited. Among the therapies targeting the cellular components of the tumor microenvironment, those that target endothelial cells are the most advanced and have shown success within the clinic.29 Targeting VEGF signaling on vascular and lymphatic endothelial cells has shown clinical benefits in patients with metastatic cancer.160 A variety of VEGF inhibitors have been approved by the US Food and Drug Administration (FDA) for the treatment of metastasis. The humanized anti-VEGF-A monoclonal antibody bevacizumab has been approved as a first-line therapy in combination with 5-fluorouracil for metastatic colon cancer. In addition, in patients with metastatic non–small cell lung cancer, bevacizumab increased survival in combination with chemotherapy.161 In addition to biologic therapies, small-molecule VEGF receptor inhibitors have also been developed for the treatment of metastasis. Both sorafenib and sunitinib have been approved by the FDA for the treatment of metastatic renal cancer. Phase III clinical trials demonstrated that sorafenib monotherapy resulted in a significant increase in progression-free survival in this patient population. The results of future clinical trials targeting additional key cellular components of the tumor microenvironment, such as TAMs, are eagerly awaited.

CONCLUSION Metastatic disease, not the primary tumor, kills the majority of cancer patients. For such an important determinant of long-term survival, progress in understanding the crucial genes and pathways that drive metastatic progression has been slow. The reasons for this slow progress have been numerous, including inadequate animal models that reflect the metastatic process in humans, failure to identify genes that specifically affect metastatic tumor growth, the complexity of host and metastatic tumor interactions, and premature clinical trials focusing on “attractive” gene targets. Recent studies have elucidated many of the cellular and molecular factors driving metastatic progression. Based on the findings of these studies, new opportunities to target metastatic disease have been identified. The future of metastatic therapy looks very promising, in large part because we understand the mistakes of the past and are using multiple genomic and proteomic approaches to target what has for so long seemed to be an intractable problem. It is only when we are able to attack the problem of metastases that we will make significant inroads in our war against cancer. The complete reference list is available online at ExpertConsult.com.

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105. Kaplan RN, Psaila B, Lyden D. Bone marrow cells in the ‘pre-metastatic niche’: within bone and beyond. Cancer Metastasis Rev. 2006;25(4):521–529. 106. Hiratsuka S, Watanabe A, Aburatani H, Maru Y. Tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis. Nat Cell Biol. 2006;8(12):1369–1375. 107. Hiratsuka S, Nakamura K, Iwai S, et al. MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis. Cancer Cell. 2002;2(4):289–300. 108. Chantrain CF, Shimada H, Jodele S, et al. Stromal matrix metalloproteinase-9 regulates the vascular architecture in neuroblastoma by promoting pericyte recruitment. Cancer Res. 2004;64(5):1675– 1686. 109. Masson V, de la Ballina LR, Munaut C, et al. Contribution of host MMP-2 and MMP-9 to promote tumor vascularization and invasion of malignant keratinocytes. FASEB J. 2005;19(2):234–236. 110. Minn AJ, Gupta GP, Siegel PM, et al. Genes that mediate breast cancer metastasis to lung. Nature. 2005;436(7050):518–524. 111. Minn AJ, Kang Y, Serganova I, et al. Distinct organ-specific metastatic potential of individual breast cancer cells and primary tumors. J Clin Invest. 2005;115(1):44–55. 112. Mundy GR. Metastasis to bone: causes, consequences and therapeutic opportunities. Nat Rev Cancer. 2002;2(8):584–593. 113. Logothetis CJ, Lin SH. Osteoblasts in prostate cancer metastasis to bone. Nat Rev Cancer. 2005;5(1):21–28. 114. Harada S, Rodan GA. Control of osteoblast function and regulation of bone mass. Nature. 2003;423(6937):349–355. 115. Kozlow W, Guise TA. Breast cancer metastasis to bone: mechanisms of osteolysis and implications for therapy. J Mammary Gland Biol Neoplasia. 2005;10(2):169–180. 116. Kang Y, He W, Tulley S, et al. Breast cancer bone metastasis mediated by the Smad tumor suppressor pathway. Proc Natl Acad Sci USA. 2005;102(39):13909–13914. 117. Yin JJ, Selander K, Chirgwin JM, et al. TGF-beta signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development. J Clin Invest. 1999;103(2):197–206. 118. Kang Y, Siegel PM, Shu W, et al. A multigenic program mediating breast cancer metastasis to bone. Cancer Cell. 2003;3(6):537–549. 119. Morgan H, Tumber A, Hill PA. Breast cancer cells induce osteoclast formation by stimulating host IL-11 production and downregulating granulocyte/ macrophage colony-stimulating factor. Int J Cancer. 2004;109(5):653–660. 120. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. 2003;423(6937):337–342. 121. Abbott NJ, Ronnback L, Hansson E. Astrocyteendothelial interactions at the blood-brain barrier. Nat Rev Neurosci. 2006;7(1):41–53. 122. Lassman AB, DeAngelis LM. Brain metastases. Neurol Clin. 2003;21(1):1–23, vii. 123. Entschladen F, Drell TL, Lang K, Joseph J, Zaenker KS. Neurotransmitters and chemokines regulate tumor cell migration: potential for a new pharmacological approach to inhibit invasion and metastasis development. Curr Pharm Des. 2005;11(3):403–411. 124. Xie TX, Huang FJ, Aldape KD, et al. Activation of stat3 in human melanoma promotes brain metastasis. Cancer Res. 2006;66(6):3188–3196. 125. Kim LS, Huang S, Lu W, Lev DC, Price JE. Vascular endothelial growth factor expression promotes the growth of breast cancer brain metastases in nude mice. Clin Exp Metastasis. 2004;21(2):107–118. 126. Yano S, Shinohara H, Herbst RS, et al. Expression of vascular endothelial growth factor is necessary but not sufficient for production and growth of brain metastasis. Cancer Res. 2000;60(17):4959–4967.

Cellular Microenvironment and Metastases  •  CHAPTER 3 55.e3 55.e3 127. Omuro AM, Kris MG, Miller VA, et al. High incidence of disease recurrence in the brain and leptomeninges in patients with nonsmall cell lung carcinoma after response to gefitinib. Cancer. 2005; 103(11):2344–2348. 128. Clayton AJ, Danson S, Jolly S, et al. Incidence of cerebral metastases in patients treated with trastuzumab for metastatic breast cancer. Br J Cancer. 2004;91(4): 639–643. 129. Bendell JC, Domchek SM, Burstein HJ, et al. Central nervous system metastases in women who receive trastuzumab-based therapy for metastatic breast carcinoma. Cancer. 2003;97(12):2972–2977. 130. Steeg PS. Tumor metastasis: mechanistic insights and clinical challenges. Nat Med. 2006;12(8):895–904. 131. Yu Q, Stamenkovic I. Transforming growth factorbeta facilitates breast carcinoma metastasis by promoting tumor cell survival. Clin Exp Metastasis. 2004;21(3):235–242. 132. Siegel PM, Shu W, Cardiff RD, Muller WJ, Massague J. Transforming growth factor beta signaling impairs Neu-induced mammary tumorigenesis while promoting pulmonary metastasis. Proc Natl Acad Sci USA. 2003;100(14):8430–8435. 133. Luo JL, Maeda S, Hsu LC, Yagita H, Karin M. Inhibition of NF-kappaB in cancer cells converts inflammation-induced tumor growth mediated by TNFalpha to TRAIL-mediated tumor regression. Cancer Cell. 2004;6(3):297–305. 134. Ye QH, Qin LX, Forgues M, et al. Predicting hepatitis B virus-positive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning. Nat Med. 2003;9(4):416–423. 135. Khanna C, Wan X, Bose S, et al. The membranecytoskeleton linker ezrin is necessary for osteosarcoma metastasis. Nat Med. 2004;10(2):182–186. 136. Sarrio D, Rodriguez-Pinilla SM, Dotor A, Calero F, Hardisson D, Palacios J. Abnormal ezrin localization is associated with clinicopathological features in invasive breast carcinomas. Breast Cancer Res Treat. 2006;98(1):71–79. 137. Ladeda V, Adam AP, Puricelli L, Bal de Kier Joffe E. Apoptotic cell death in mammary adenocarcinoma

cells is prevented by soluble factors present in the target organ of metastasis. Breast Cancer Res Treat. 2001;69(1):39–51. 138. Martin SS, Ridgeway AG, Pinkas J, et al. A cytoskeleton-based functional genetic screen identifies Bcl-xL as an enhancer of metastasis, but not primary tumor growth. Oncogene. 2004;23(26):4641–4645. 139. Pinkas J, Martin SS, Leder P. Bcl-2-mediated cell survival promotes metastasis of EpH4 beta­ MEKDD mammary epithelial cells. Mol Cancer Res. 2004;2(10):551–556. 140. Inbal B, Cohen O, Polak-Charcon S, et al. DAP kinase links the control of apoptosis to metastasis. Nature. 1997;390(6656):180–184. 141. Wong CW, Lee A, Shientag L, et al. Apoptosis: an early event in metastatic inefficiency. Cancer Res. 2001;61(1):333–338. 142. O’Connell JT, Sugimoto H, Cooke VG, et al. VEGF-A and tenascin-C produced by S100A4+ stromal cells are important for metastatic colonization. Proc Natl Acad Sci USA. 2011;108(38):16002–16007. 143. Kii I, Nishiyama T, Li M, et al. Incorporation of tenascin-C into the extracellular matrix by periostin underlies an extracellular meshwork architecture. J Biol Chem. 2010;285(3):2028–2039. 144. Chambers AF, Groom AC, MacDonald IC. Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer. 2002;2(8):563–572. 145. Lehuede C, Dupuy F, Rabinovitch R, Jones RG, Siegel PM. Metabolic plasticity as a determinant of tumor growth and metastasis. Cancer Res. 2016;76(18):5201–5208. 146. Jungermann K. Metabolic zonation of liver parenchyma. Semin Liver Dis. 1988;8(4):329–341. 147. Loo JM, Scherl A, Nguyen A, et al. Extracellular metabolic energetics can promote cancer progression. Cell. 2015;160(3):393–406. 148. Wyss M, Kaddurah-Daouk R. Creatine and creatinine metabolism. Physiol Rev. 2000;80(3):1107–1213. 149. Dupuy F, Tabaries S, Andrzejewski S, et al. PDK1dependent metabolic reprogramming dictates metastatic potential in breast cancer. Cell Metab. 2015;22(4):577–589.

150. Papandreou I, Cairns RA, Fontana L, Lim AL, Denko NC. HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. Cell Metab. 2006;3(3):187–197. 151. Valastyan S, Weinberg RA. Tumor metastasis: molecular insights and evolving paradigms. Cell. 2011;147(2):275–292. 152. Rankin EB, Fuh KC, Taylor TE, et al. AXL is an essential factor and therapeutic target for metastatic ovarian cancer. Cancer Res. 2010;70(19):7570–7579. 153. Coussens LM, Fingleton B, Matrisian LM. Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science. 2002;295(5564):2387– 2392. 154. Harris AL. Hypoxia—a key regulatory factor in tumour growth. Nat Rev Cancer. 2002;2(1):38–47. 155. Wong CC, Zhang H, Gilkes DM, et al. Inhibitors of hypoxia-inducible factor 1 block breast cancer metastatic niche formation and lung metastasis. J Mol Med. 2012;90(7):803–815. 156. Higgins DF, Biju MP, Akai Y, Wutz A, Johnson RS, Haase VH. Hypoxic induction of Ctgf is directly mediated by Hif-1. Am J Physiol Renal Physiol. 2004;287(6):F1223–F1232. 157. Chu CY, Chang CC, Prakash E, Kuo ML. Connective tissue growth factor (CTGF) and cancer progression. J Biomed Sci. 2008;15(6):675–685. 158. Dornhofer N, Spong S, Bennewith K, et al. Connective tissue growth factor-specific monoclonal antibody therapy inhibits pancreatic tumor growth and metastasis. Cancer Res. 2006;66(11):5816– 5827. 159. Aikawa T, Gunn J, Spong SM, Klaus SJ, Korc M. Connective tissue growth factor-specific antibody attenuates tumor growth, metastasis, and angiogenesis in an orthotopic mouse model of pancreatic cancer. Mol Cancer Ther. 2006;5(5):1108– 1116. 160. Sleeman J, Schmid A, Thiele W. Tumor lymphatics. Semin Cancer Biol. 2009;19(5):285–297. 161. Jain RK. Antiangiogenesis strategies revisited: from starving tumors to alleviating hypoxia. Cancer Cell. 2014;26(5):605–622.

4 

Control of the Cell Cycle Marcos Malumbres

S UMMARY

OF

K EY

P OI N T S

• Most cells in postnatal tissues are quiescent. Exceptions include abundant cells of the hematopoietic system, skin, and gastrointestinal mucosa, as well as other minor progenitor populations in other tissues. • Many quiescent cells can reenter into the cell cycle with the appropriate stimuli, and the control of this process is essential for tissue homeostasis. • The key challenges for proliferating cells are to make an accurate copy of the 3 billion bases of DNA (S phase) and to segregate the duplicated chromosomes equally into daughter cells (mitosis). • Progression through the cell cycle is dependent on both extrinsic and

intrinsic factors, such as growth factor or cytokine exposure, cell-tocell contact, and metabolic constraints. • The internal cell cycle machinery is controlled largely by oscillating levels of cyclin proteins and by modulation of cyclin-dependent kinase (Cdk) activity. One way in which growth factors regulate cell cycle progression is by affecting the levels of the D-type cyclins, Cdk activity, and the function of the retinoblastoma protein. • Cell cycle checkpoints are surveillance mechanisms that link the rate of cell cycle transitions to the timely and accurate completion of prior dependent events; p53 is a checkpoint protein that induces cell

Most cells in the adult body are quiescent—that is, they are biochemically and functionally active but do not divide to generate daughter cells. However, specific populations retain the ability to proliferate throughout the adult life span, which is essential for proper tissue homeostasis. For example, cells of the hematopoietic compartment and the gut have a high rate of turnover, and active proliferation is essential for the maintenance of these tissues. On average, about 2 trillion cell divisions occur in an adult human every 24 hours (about 25 million per second). The decision about whether to proliferate is tightly regulated.1,2 It is influenced by a variety of exogenous signals, including nutrients and growth factors, as well as inhibitory factors, and the interaction of the cell with its neighbors and with the underlying extracellular matrix. Each of these factors stimulates intracellular signaling pathways that can either promote or suppress proliferation. The cell integrates all of these signals, and if the balance is favorable, the cell will initiate a series of processes, collectively known as the cell cycle, that lead to cell division into two daughter cells. During the past four decades, extensive effort has been placed on unraveling the basic molecular events that control the cell division cycle. Studies in a variety of organisms have identified evolutionarily conserved machinery that regulates eukaryotic cell cycle transitions through the action of key enzymes, including cyclin-dependent kinases (Cdks) and other kinases.3 It is essential that proliferating cells copy their genomes and segregate them to the daughter cells with high fidelity. Eukaryotic cells therefore have evolved a series of surveillance pathways, termed cell cycle checkpoints, that monitor for potential problems during the cell cycle process.4 Human cells are continuously 56

cycle arrest, senescence, or death in response to cellular stress. • Checkpoints minimize replication and segregation of damaged DNA, or the abnormal segregation of chromosomes to daughter cells, thus protecting cells against genome instability. • Disruption of cell cycle controls is a hallmark of all malignant cells. Frequent tumor-associated alterations include aberrations in growth factor signaling pathways, dysregulation of the core cell cycle machinery, and/or disruption of cell cycle checkpoint controls. • Because cell cycle control is disrupted in virtually all tumor types, the cell cycle machinery provides multiple therapeutic opportunities.

exposed to external agents (e.g., reactive chemicals and ultraviolet light) and to internal agents (e.g., byproducts of normal intracellular metabolism, such as reactive oxygen intermediates) that can induce DNA damage. A major goal of specific cell cycle checkpoints is to detect DNA damage and activate cell cycle arrest and DNA repair mechanisms, thereby maintaining genomic integrity. Anything that disrupts proper cell cycle progression can lead to either the reduction or the expansion of a particular cell population. It is now clear that such changes are a hallmark of tumor cells, which carry mutations that impair signaling pathways that suppress proliferation and/or activate pathways that promote proliferation. In addition, most (if not all) human tumor cells have mutations within key components of both the cell cycle machinery and checkpoint pathways.1,5,6 This characteristic has important clinical implications, because the presence of these defects can modulate cellular sensitivity to chemotherapeutic regimens that induce DNA damage or mitotic catastrophe. This chapter focuses on the mechanics of the cell cycle and checkpoint signaling pathways and discusses how this knowledge can lead to the efficient use of current anticancer therapies and to the development of novel agents.

CELL DIVISION CYCLE Overview of the Cell Cycle Machinery Cell division proceeds through a well-defined series of stages (Fig. 4.1). First, the cell moves from the nonproliferative, quiescent

Control of the Cell Cycle  •  CHAPTER 4 57 G1/S

Intra-S

G2/M

DNA replication & repair Cell cycle entry G1

G0

Microtubule nucleation spindle

Centrosome duplication and separation

G1

S

SAC

Cytokinesis

Chromosome condensation

Chromosome segregation

M

M

G2

Cyclin D Cyclin E

Cyclin A

Cyclin B

Restriction point

Figure 4.1  •  The cell division cycle. One round of cell division requires high-fidelity duplication of deoxyribonucleic acid during the S phase of the cell

cycle and proper segregation of duplicated chromosomes during mitosis, or the M phase. Before and after the S phase and M phase, the cell transits through “gap” phases, termed G1 and G2. Quiescent (G0) cells require the appropriate mitogenic stimuli for reentry into the cell cycle by inducing D-type cyclins. These stimuli are required up to a specific moment, known as the restriction point, at which time the cell cycle becomes independent of the mitogenic signals. The cellular processes required for transition through the different cell cycle phases are controlled by the action of regulatory pathways and mostly are driven by the expression of specific cyclins and the activation of cyclin-dependent kinases. These transitions are monitored by signaling pathways known as the cell cycle checkpoints (represented by blue columns) that function to arrest the cell cycle at different phases (G1/S, intra–S phase, G2/M) in the presence of DNA damage. In addition, the spindle assembly checkpoint (SAC) delays the exit from mitosis in the presence of defective chromosome alignment by inhibiting the degradation of cyclin B1 and the subsequent inactivation of cyclin-dependent kinases.

(also known as G0) state into the first gap phase, or G1, in which the cell essentially is readying itself for the cell division process. This process involves a dramatic upregulation of both transcriptional and translational programs, not only to yield the proteins required to regulate cell division but also to essentially double the complement of macromolecules so that one cell can give rise to two cells without a loss of cell size. Not surprisingly, this process takes a significant amount of time (from 8 to 30 hours in cultured cells) and energy.7 Studies with cultured cells show that mitogenic growth factors are essential for continued passage through the G1 phase. Specifically, if growth factors are withdrawn at any point during this phase, the cell will not divide. However, as it nears the end of the G1 phase, the cell passes through a key transition point, called the restriction point, whereupon it becomes growth factor independent and is fully committed to undergoing cell division.1 The cell then enters the DNA synthesis phase, or S phase, in which each of the chromosomes is replicated once and only once. This is followed by a second gap phase, called G2, which lasts 3 to 5 hours, and the cell then initiates mitosis, or the M phase, a rapid phase (lasting about 1 hour) in which the chromosomes are segregated. On completion of mitosis, the daughter cells can enter quiescence or initiate a second round of cell division, depending on the milieu. Progression throughout the different phases of the cell cycle depends on the activity of key molecules that drive transcription, translation, or the structural changes required for cell division (Table 4.1). A large number of these changes are modulated by protein phosphorylation and dephosphorylation, but other molecular processes such as SUMOylation, acetylation, or ubiquitin-dependent protein degradation are crucial for ordered cell cycle progression. Many of these cell cycle regulators have been involved in tumor development or may be attractive targets for cancer therapy and will be introduced in the following sections.

Cyclin-Dependent Kinases and Their Regulators The Cdks constitute a large subfamily of highly conserved Ser/Thr kinases that are defined by their dependence on a regulatory subunit, called a cyclin.8–11 The first identified human Cdk, called Cdk1

(originally cdc2), was cloned by virtue of its ability to complement a mutant cdc2 yeast strain.12 Subsequent studies identified additional human Cdks and determined that they regulate distinct cell cycle stages; for example, Cdk4 and Cdk6 regulate cell cycle entry, whereas Cdk2 may have specific roles during the G1-to-S transition and S phase. Cdk1 is essential in the control of G2 and mitosis and also may play additional roles in earlier stages. The human genome encodes about 15 additional Cdks, although the functional relevance of many of them is still unknown.3,8–11 The activity of these kinases is controlled by multiple regulatory mechanisms. Cdks act in association with a cyclin subunit that binds to the conserved PSTAIRE helix within the kinase.13 Cyclin binding causes a reorientation of residues within the active sites that is essential for kinase activity.8,13 The associated cyclin also determines the substrate specificity of the resulting cyclin/Cdk complex. The cyclins are quite divergent, especially in their N-terminal sequences, but they all share a highly conserved 100–amino acid sequence, called the cyclin box, that mediates Cdk binding and activation.9 As their name implies, cyclins originally were identified as proteins whose expression was restricted to a particular stage of the cell cycle14 because of cell cycle–dependent regulation of both cyclin gene transcription and protein degradation. The human genome encodes more than 25 cyclin-like proteins, yet only four distinct subclasses—D-, E-, A-, and B-type cyclins—are thought to play key roles in cell cycle regulation (see Fig. 4.1).9 Each of these classes has a few paralogs (e.g., cyclin D1, D2, and D3; cyclin E1 and E2; cyclin A1 and A2; and cyclin B1, B2, and B3). The relative roles of these paralogs are not completely clear in most cases. Although some functional redundancy may exist, published evidence suggests differences in regulation, expression pattern, and substrate specificity.9,15 The activation of cyclin/Cdk complexes requires considerable posttranslational regulation.8,9,16 First, kinase activation is dependent on phosphorylation of a threonine residue that is adjacent to the active site (Thr160 in Cdk2). This phosphorylation is catalyzed by a kinase, called Cdk-activating kinase (CAK).17,18 In mammalian cells, phosphorylation occurs after cyclin binding. Although it appears that

58 Part I: Science and Clinical Oncology

Table 4.1  Representative Molecules Involved in Cell Cycle Regulation Protein Family/ Complex KINASES Cyclin-dependent kinases Wee1/Myt1 Aurora-A holoenzyme Chromosome passenger complex (CPC) Polo-like kinases NIMA-related kinases Haspin Mastl

Representative Members

Function

Heterodimeric complexes formed of a cyclin (A, B, D, and E types) and a Cdk (Cdk1, Cdk2, Cdk4, Cdk6); Cdk7 functions as a Cdk-activating kinase Wee1, Myt1 Aurora-A and its nonkinase activator, Tpx2 Aurora-B (Aurora-C?), Incenp, survivin, borealin

Phosphorylation of multiple proteins to drive progression throughout the different phases of the cell cycle

Plk1–Plk5 Nek1–Nek11 Haspin Mastl

Centrosome function, chromosome segregation, and cytokinesis Centrosome function and mitosis Phosphorylates histone H3 to recruit the CPC Inhibition of PP2A phosphatases

p16INK4a, p15INK4b, p18INK4c, p19INK4d p21Cip1, p27Kip1, p57Kip2

Inhibition of Cdk4 and Cdk6 during G1 progression Cdk inhibition and other roles in transcription or the cytoskeleton

Inactivation of Cdks Spindle dynamics, chromosome segregation, and cytokinesis Chromosome segregation

CDK INHIBITORS INK4 proteins Cip/Kip inhibitors

TRANSCRIPTIONAL CONTROL Retinoblastoma family E2F transcription factors

pRB, p107, p130 E2F1–E2F8

Repression of the transcription of genes required for the cell cycle Transcription of genes encoding S-phase and mitotic regulators

Cdc14 Cdc25 PP1

Cdc14a, Cdc14b Cdc25a, Cdc25b, and Cdc25c Multiple complexes with different regulatory subunits

Control of transcription and cell cycle progression Cdk activation and cell cycle progression Protein dephosphorylation

PP2A

Multiple complexes with different regulatory subunits

Protein dephosphorylation; major Cdk-counteracting phosphatase

E3 ubiquitin ligase formed of Rbx1, Cul1, Skp1, and an F-box protein (e.g., Skp2 or βTrCP) E3 ubiquitin ligase composed for multiple subunits including Cdc20 or Cdh1 as coactivator molecules

Targets multiple cell cycle regulators (e.g., p27Kip1 or cyclin E) for ubiquitin-dependent degradation during interphase Targets multiple cell cycle regulators for ubiquitin-dependent degradation during mitosis (cyclin B, securin) and G1 (e.g., Aurora-A, Plk1, or Tpx2)

More than 600 proteins including Eg5, CenpE, MCAK Smc family (1–4), Rad21, Pds5, and SA (Stag) proteins, among others

Microtubule-based motor proteins that hydrolyze ATP to generate energy for movement along microtubule fibers Structure and regulation of DNA

PHOSPHATASES

UBIQUITIN LIGASES SCF APC/C

OTHER FUNCTIONS Kinesins Cohesins and condensins Kinetochore proteins

Mitotic checkpoint complex (MCC)

Linking the chromosomes to microtubules and regulation of More than 100 proteins including the CENP (centromere-binding proteins) family, and the Knl1, microtubule dynamics Mis12, Ndc80, and Dam1 complexes, among many others Mad2, Bub3, BubR1, and Cdc20 Inhibit the APC/C until complete bipolar attachment of chromosomes to the mitotic spindle

APC/C, Anaphase-promoting complex/cyclosome; ATP, adenosine triphosphate; Cdk, cyclin-dependent kinase; CPC, chromosomal passenger complex; NIMA, never in mitosis, gene A; SCF, Skp1–Cullin1–F-box.

at least two mammalian CAKs exist, the major CAK is a trimolecular complex composed of Cdk7, cyclin H, and Mat1. The Cdk7/cyclinH/ Mat1 complex also is required for the control of basal transcription via regulation of RNA polymerase II function.11,18 Second, when it is first formed, the cyclin/Cdk complex frequently is subject to inhibitory phosphorylation of Thr14 and Tyr15 residues within the Cdk’s active site by the Wee1 (Tyr15) and Myt1 (Thr14 and Tyr15) kinases.9,19

Activation of the cyclin/Cdk complex is then dependent on the action of a dual-specificity phosphatase called Cdc25. Mammalian cells have three different Cdc25 proteins, called Cdc25a, Cdc25b, and Cdc25c, which show some specificity for different cyclin/Cdk complexes and cell cycle stages.20 Cdks are modulated by a series of Cdk inhibitors (CKIs) that play a key role in restricting the activity of the cyclin/Cdk complexes in

Control of the Cell Cycle  •  CHAPTER 4 59

response to either external signals or internal stresses.1,21 The CKIs can be divided into two distinct families based on their biological properties. The first CKI family is named INK4, based on their roles as INhibitors of Cdk4. The INK4 family has four members called p16INK4a, p15INK4b, p18INK4c, and p19INK4d (encoded by the CDKN2A-D genes in humans). These INK4 proteins specifically prevent the binding of cyclins to monomeric Cdk4 and Cdk6 but do not inhibit other Cdks.13,21 The second CKI family is named Cip/Kip and includes three members: p21Cip1 (also called p21Waf1), p27Kip1, and p57Kip2 (encoded by the CDKN1A-C genes in humans).21 These Cip/Kip proteins have two major activities. First, they do not bind to monomeric Cdks but associate with and inhibit the activity of cyclin/Cdk complexes already formed. Second, Cip/Kip proteins may promote the assembly of cyclin D/Cdk4/6 complexes without dramatically perturbing its kinase activity.21,22 This activity is modulated by phosphorylation of Cip/Kip proteins by Src, Jak2, and Akt kinases,23–26 directly linking Cdk regulation with the activity of these upstream mitogenic pathways. In addition to regulating the cell cycle, Cip/Kip proteins play important roles in apoptosis, transcriptional regulation, cell fate determination, cell migration, and cytoskeletal dynamics.27,28

Retinoblastoma Proteins and E2F Transcription Factors The retinoblastoma protein (pRB) was originally identified by virtue of its association with hereditary retinoblastoma.29 It behaves as a classic tumor suppressor: affected persons inherit a germline mutation within one allele of the pRB-encoding gene, RB1, and loss of heterozygosity is seen in all of the tumors. Subsequent studies showed that the transforming ability of small DNA tumor viruses, including human papillomavirus, adenovirus, and simian virus, was dependent on the ability of virally encoded oncoproteins (E7, E1A, and SV40, respectively) to bind and inhibit pRB.30–32 Moreover, the RB1 gene is inactivated in approximately one-third of all sporadic human tumors. pRB and the pRB-related proteins p107 and p130, collectively known as the pocket proteins, are transcriptional repressors whose major function is to inhibit the expression of cell cycle–related proteins (see Table 4.1).33 This suppressive activity is largely dependent on the ability to prevent cell cycle entry through inhibition of the E2F transcription factors.33,34 The E2F proteins regulate the cell cycle– dependent transcription of numerous targets, including core components of the cell cycle control (e.g., cyclin E and cyclin A) and DNA replication (e.g., Cdc6, Cdt1, and the Mcm proteins) machineries.33–35 pRB

Rbx1

regulates E2F through two distinct mechanisms. First, its association with E2F is sufficient to block the transcriptional activity of E2F. Second, the pRB/E2F complex can recruit histone deacetylases to the promoters of E2F-responsive genes and thereby actively repress their transcription. Cell cycle entry requires the phosphorylation of pRB by cyclin/Cdk complexes and the consequent dissociation of pRB from E2F.1,8,33 Studies have identified eight E2F genes that encode nine different E2F proteins.34,35 Pocket proteins can regulate a subset of these factors: E2F1, E2F2, E2F3a, E2F3b, E2F4, and E2F5. These E2F proteins associate with a dimerization partner, called DP, and the resulting complexes function primarily as either activators (E2F1, E2F2, and E2F3a) or repressors (E2F4 and E2F5) of transcription under the direction of the pocket proteins. Observations have suggested that several of these factors may act either as positive or negative regulators of transcription, depending on the cell type or the differentiation state.34,36–38 Most classic E2F target genes are regulated by the coordinated action of these repressor and activator E2Fs.

Ubiquitin-Dependent Protein Degradation The original observation that cyclin levels are tightly regulated during the cell cycle implies that these proteins are regulated not only at the transcriptional level but also at the protein level. It is now evident that ubiquitin-mediated protein degradation is a major regulatory mechanism to ensure ordered transition through the different phases of the cell division cycle. Ubiquitylation depends on an enzymatic cascade, in which ubiquitin ligases recruit specific substrates for modification. About 600 ubiquitin ligases are encoded by the human genome. Among them, the Skp1–Cullin1–F-box (SCF) and the anaphase-promoting complex/cyclosome (APC/C) are known for driving the degradation of cell cycle regulators to accomplish irreversible cell cycle transitions.39–41 SCF has three core components: a RING finger protein, called Rbx1, which recruits the E2-ubiquitin conjugate; a cullin (Cul1); and Skp1 (Fig. 4.2). Skp1 acts to recruit a family of proteins, called F-box proteins, which determine the target specificity of the SCF complex. Once SCF binds its substrate, it transfers a ubiquitin molecule to lysine residues within the target protein to create a polyubiquitin chain, which targets the substrate to the proteasome for degradation.42 The APC/C is a much larger complex, but it also contains a RING finger protein, called Apc11, to recruit the E2-ubiquitin conjugate,

Apc11

E2

Ubiquitin

Ubiquitin Substrate P

Cul1

E2

Apc2

Substrate P Activator (Cdc20 or Cdh1)

F-box protein

Skp2 SCF ubiquitin ligase

APC/C ubiquitin ligase

Figure 4.2  •  Ubiquitin ligases. The Skp1–Cullin1–F-box (SCF) and anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligases play a key role in

enabling forward passage through key cell cycle transitions. These ligases are both large complexes that include three core components: a scaffolding protein called a cullin in SCF, a protein that recruits the E2 and its associated ubiquitin molecule, and a specificity factor (called the F-box protein in SCF and the activator in APC/C) that recruits the substrate. SCF and APC/C catalyze polyubiquitination of their substrates, which acts as a signal for substrate degradation by the 26S proteasome. SCF has numerous substrates whose degradation promotes passage through the early stages of the cell cycle, including the cyclindependent kinase inhibitor p27Kip1, cyclin E, E2F-1, and Cdt1. APC/C is essential for mitotic progression (by promoting degradation of securin and the mitotic cyclins) and for cell cycle exit (by promoting degradation of multiple cell cycle regulators).

60 Part I: Science and Clinical Oncology

and a core cullin subunit (Apc2). In addition, APC/C is activated by a cofactor that, in a manner comparable with that of the F-box proteins of SCF, establishes substrate specificity (see Fig. 4.2).39 Cdc20 is the mitotic cofactor of APC/C, and it targets several cell cycle regulators (A- and B-type cyclins, Nek2, and securin) during mitotic entry and the metaphase-to-anaphase transition. Cdh1 (also known as FZR1 in mammals) replaces Cdc20 during mitotic exit and is the cofactor responsible for the elimination of many cell cycle regulators in the G0 or G1 phase to prevent unscheduled DNA replication.43 Recent studies have provided new insights into the intricate relationship between ubiquitylation and the cell division apparatus, including new roles for atypical ubiquitin chains, new mechanisms of regulation, and extensive cross talk between ubiquitylation enzymes.44–49

Mitotic Spindle and Mitotic Kinases and Kinesins In addition to the central role of Cdks in the cell cycle, many other kinases play critical roles in cell cycle progression.3 Aurora and Polo kinases were first identified in genetic studies in flies as a result of their essential role in mitotic progression.50–52 Each of these families of kinases is represented by a single member in yeast, whereas three Aurora kinases (Aurora-A, Aurora-B, and Aurora-C) and five Polo-like kinases (Plk1 through Plk5) exist in humans (see Table 4.1).52,53 Aurora-A participates in several processes required for building a bipolar spindle, including centrosome separation and microtubule dynamics. Aurora-A is activated by Tpx2, and the Aurora-A–Tpx2 holoenzyme may have critical implications in tumor development.51,52,54 Aurora-B, on the other hand, is part of the chromosome passenger complex (CPC) that localizes to the kinetochores from prophase to metaphase and to the central spindle and midbody in cytokinesis. Other components of the CPC include INCENP, survivin, and borealin, and this complex regulates proper microtubule-kinetochore attachment and promotes biorientation during mitosis.51 Aurora-C may play similar roles to Aurora-B, although it is mostly expressed in germ cells and may play a specific role in meiosis and during the first embryonic cycles.55–57 Plk1 functions as a major regulator of centrosome maturation, mitotic entry, and cytokinesis, whereas Plk4 is a critical regulator of centriole duplication.50,58–60 The other members of the family, Plk2, Plk3, and Plk5, are mostly involved in stress responses during interphase or in neuron biology.3,53 A different group of additional cell cycle kinases with potential interest in tumor biology is the never in mitosis, gene A (NIMA)–related kinase family (Nek1 through Nek11). Four of these proteins, Nek2, Nek6, Nek7, and Nek9, are involved in cell cycle progression, whereas all other family members are likely to play critical roles in cilia and centrioles.3,61–63 Nek2, the closest relative to the Aspergillus NIMA kinase, localizes to the centrosome and plays a role in establishing the bipolar spindle by initiating the separation of centrosomes and contributing to microtubule organization at the G2/M transition by phosphorylating several centrosomal substrates. Nek2 also may play additional roles in chromosome condensation and the mitotic checkpoint. Nek9, Nek6, and Nek7 function in a kinase cascade that participates in centrosome separation and the formation and/or maintenance of the mitotic spindle.61,63 A structurally different kinase, Haspin, is involved in the phosphorylation of histone H3 on threonine 3 (H3T3).64,65 This kinase is activated by Cdk1 and Plk1,66 and phosphorylation of H3T3 is required for proper Aurora-B localization and activity and coupling of mitotic chromosome structure with transcription.64,65,67 The activity of all these kinases is functionally linked to many other proteins associated with chromosomes, microtubules, or different organelles, whose activity is essential for the structural changes associated with cell cycle progression and chromosome segregation.68 Among them, microtubule-associated proteins such as kinesin motor proteins determine the dynamic behavior of the mitotic spindle required for chromosome movement during mitosis.69,70 During mitosis, microtubules associate with chromosomes through a large protein assembly known as the kinetochore.71 The position of kinetochores in the

centromeric region of chromosomes is determined by a complex epigenetic, DNA sequence–independent mechanism.72–74 Kinetochores regulate the proper bipolar attachment between microtubules and chromosomes in order to distribute the replicated genome from a mother cell to its daughters.71,75 Given the relevance of the kinetochore in genome integrity, the composition of the kinetochore and the identification of various physical and functional modules within its substructure is under deep investigation.76,77

Cell Cycle Phosphatases Cdc14 phosphatases are the major Cdk-counteracting proteins in yeast. Although two family members exist in mammals, their relevance in the cell cycle is not well understood.78,79 In eukaryotes, two major complexes, PP1 and PP2A, account for more than 90% of protein phosphatase activity. In fact, these enzymes correspond to hundreds of phosphatase complexes assembled from a few catalytic subunits (PP1α, PP1β/δ, and PP1γ1/2 for PP1, and the Cα and Cβ isoforms for PP2A) and a diverse array of regulatory subunits.80 Recent evidence suggests that these protein families cooperate in the dephosphorylation of most cell cycle kinase targets, including the retinoblastoma family or mitotic phosphoproteins.78,81–83 PP1 and PP2A are major phosphatases responsible for pRB dephosphorylation during mitotic exit, although the relative roles of these complexes or the particular holoenzymes involved are not clear.81,82 Similarly, both PP1 and PP2A are required for dephosphorylation of hundreds of mitotic proteins that are phosphorylated by Cdk1, as well as the other mitotic kinases.83,84 Thus it has been suggested that the cell cycle ultimately is regulated by the dynamic equilibrium between Cdks (and partially by the other mitotic kinases) and PP1/PP2A activity. In the absence of Cdk activity, the balance tilts in favor of the phosphatases. When Cdks are activated, phosphatase activity is overtaken. Cdk1 is able to directly inhibit PP1 by direct phosphorylation of the catalytic subunit. The Cdk-dependent inhibition of PP2A, on the other hand, is not direct; rather, it is mediated by a new kinase known as Greatwall in flies and Xenopus or Mastl in mammals.85–87 Cdk1 phosphorylates and activates Mastl, which in turn phosphorylates Arpp-19 and Ensa, two highly related proteins that function as inhibitors of a particular PP2A holoenzyme encompassing a regulatory subunit of the B55 family.85–89 Reactivation of PP1 and PP2A phosphatases is a mandatory step for the exit from mitosis and the transition to interphase.90–92

Entry Into the Cell Cycle Because most adult cells are quiescent, the mechanisms that determine their quiescent state and their reentry into the cell cycle with the appropriate stimuli are key determinants of tissue homeostasis. In quiescent cells, several DP/E2F complexes associate with the promoters of E2F-responsive genes and recruit pRB-family members, along with their associated histone deacetylases, to actively repress their transcription.93,94 This repression machinery therefore prevents the expression of proteins required for DNA synthesis and chromosome segregation. In addition, CKIs normally are expressed in quiescent cells, preventing the activation of Cdks.21 D-type cyclins are present at very low levels in most quiescent cells, in large part because they are phosphorylated by an abundant kinase called Gsk3β and then exported to the cytoplasm for degradation.95 The transcription of D-type cyclins is induced in response to a wide variety of mitogenic stimuli.96,97 In addition, Gsk3β is inhibited by mitogens, thus preventing the degradation of these cyclins. D-type cyclins then associate with Cdk4 and Cdk6, and the resulting complexes phosphorylate pRB proteins, partially inactivating their transcriptional suppressor function (Fig. 4.3).98–101 pRB inactivation causes the release of its associated DP-E2Fs. Repressive E2Fs such as E2F4 and E2F5 dissociate from the DNA, and the free E2F complexes—DP-E2F1, DP-E2F2, and DP-E2F3—now occupy the promoters and activate their transcription. DP-E2F targets include many genes encoding

Control of the Cell Cycle  •  CHAPTER 4 61

Inhibitory growth factors

Mitogens

P

Cip/Kip INK4

P

pRb

pRb

P

P

E2F 1,2,3

Cdk4/6 CycD

p107 p130

CycE

p107 p130

P

P

P

E2F 4,5

Cdk2

HDAC

DNA synthesis and mitotic regulators

E2F 1,2,3

E2F 4,5

P

G0 /G1

G1/S

Transcriptional repression

Transcriptional activation

Figure 4.3  •  Entry into the cell cycle. The pocket proteins—pRB, p107, and p130—regulate a subset of the E2F family of proteins among many other transcription factors. The pocket proteins bind to these E2Fs during the G0/early G1 phases, block their transcriptional activity, and recruit histone deacetylase (HDAC), which comprises repressive complexes. Mitogenic signaling leads to the induction of D-type cyclins and the activation of interphase cyclin-dependent kinase (Cdk) complexes, which phosphorylate the pocket proteins and release their associated E2Fs. This process allows the activating E2Fs to induce the transcription of genes required for DNA synthesis and mitosis.

essential proteins required for DNA synthesis as well as mitosis.33–35 Through the control of pRB proteins, cyclin D-Cdk4/6 activity is a central mediator of the reentry of cells into the cell cycle. Not surprisingly, Cdk4/6 activity is frequently hyperactivated in cancer cells, as described later. Recent data suggest that Cdk6 may have kinaseindependent, transcriptional functions, although the exact mechanisms behind these functions and their relevance in tumor development deserve further investigation.102,103 Cyclin E is itself an E2F-responsive gene, and this regulation creates a strong feed-forward loop. Cyclin E binds to Cdk2, and this complex may promote further pRB inactivation.104,105 Cyclin E–Cdk2 also phosphorylates the CKI p27Kip1 on Thr187.106,107 This action creates a high-affinity binding site for the SCF ubiquitin ligase bound to the F-box protein Skp2, leading to p27Kip1 degradation during the G1/S transition.108 Finally, cyclin E-Cdk2 phosphorylates itself on multiple sites, creating a recognition site for SCF-Fbw7/Cdc4 and thereby ensuring its own destruction.109,110 The fact that lack of Cdk2 in the mouse does not result in defective mitotic cycles suggests that the activity of this protein overlaps with other Cdks, with Cdk1 being the best candidate.111–113 Indeed, Cdk1 is able to bind interphase cyclins such as cyclin D and cyclin E, and it is sufficient for G1/S transition, at least in the absence of other interphase Cdks.114–115 Cdk2 is however an essential role in meiosis, although the molecular basis for this requirement is not fully understood.112,116

DNA Replication The DNA replication machinery is optimized to ensure that the genome is copied once—and only once—in each cell cycle.117 This optimization is achieved through a two-step process that first establishes a prereplication complex (pre-RC) at each origin of replication, a process that is frequently referred to as origin licensing, and subsequently transforms pre-RCs into the preinitiation complex (pre-IC) that activates DNA replication (Fig. 4.4). These two steps occur at distinct stages of the cell cycle to ensure that origins are licensed only once per cell cycle and rereplication cannot occur. Pre-RC formation takes place in the initial steps of the cell cycle. The first event in this process is the recruitment of the multiprotein complex called the origin recognition

MCM

MCM

Cdt1

Cdc6 ORC

G1

MCM

Cdc6

MCM

Cdt1

S

DDK + Cdk dependent

ORC

C

t 1 d

Figure 4.4  •  Origin licensing and firing. The origin replication complex (ORC) associates with replication origins. During the G1 phase, Cdc6 and Cdt1 are loaded on chromatin, and they in turn load the mini chromosome maintenance (MCM) complex on chromatin, at which point licensing is considered complete, and the multiprotein complex is called the prereplication complex (pre-RC). Once cells pass the G1-to-S transition, this complex is activated to form the preinitiation complex (pre-IC), and DNA replication is initiated. Activation requires both cyclin-dependent kinase (Cdk) and Ddf4-dependent kinase (DDK) activity. It results in recruitment of numerous proteins and activation of the MCM complex, which unwinds the DNA. Subsequently, core components of the replication machinery, including DNA polymerase α and DNA polymerase ε, are recruited to initiation sites. The transition from pre-RC to pre-IC results in inhibition of Cdt1 by ubiquitinmediated degradation and geminin binding. Origin licensing cannot occur again until activation of anaphase-promoting complex/cyclosome at the end of mitosis allows accumulation of Cdt1.

62 Part I: Science and Clinical Oncology

complex to the origin DNA.118 The origin recognition complex recruits additional proteins including Cdc6, Cdt1, and finally the mini chromosome maintenance (MCM) complex, a helicase that is required to unwind the DNA strands to form the pre-RC. Once cells enter S phase, the transformation of the pre-RC to the pre-IC requires the activity of two kinases: a Cdk and the Ddf4-dependent kinase, which is composed of the Dbf4 regulatory subunit and the Cdc7 kinase.119,120 The action of these kinases allows numerous additional proteins to associate with the pre-RC and form the pre-IC.121 Assembly of the pre-IC is thought to trigger DNA unwinding by the MCM complex, recruitment of the DNA polymerases, and initiation of the replication process, frequently called “origin firing.” The transformation of the pre-RC to the pre-IC can occur at different time points in S phase, depending on whether the origin fires early or late.117 The system can tolerate this heterogeneity because the pre-RC is disassembled after firing and cannot reform until the subsequent cell cycle. This process occurs through several mechanisms. The MCM complex travels with the replication fork in its role as the DNA helicase. Some evidence also indicates that phosphorylation of Orc1 reduces its ability to bind to origins. Finally, and most important, Cdt1 is prevented from participating in pre-RC formation outside of the G1 phase in two distinct ways. First, Cdt1 is marked for destruction by ubiquitination.122 This process is mediated by SCF-Skp2 and particularly by an E4 ubiquitin ligase that includes Rbx1 (to recruit the E2-ubiquitin), a cullin (Cul4), Ddb1, and Dtl/Cdt2 (the substrate specificity factor).123–125 Important to note, this Cul4-Ddb1-Dtl/Cdt2 complex functions independently of Cdt1 phosphorylation. Instead, Cdt1 is targeted only when proliferative cell nuclear antigen is present on the DNA, which occurs primarily as a consequence of the initiation of DNA replication.126 Second, cells possess a protein called geminin that sequesters Cdt1 and prevents it from participating in pre-RC formation. Geminin is present specifically in S-, G2-, and early M-phase cells. The APC/C ubiquitinates geminin and thereby triggers its destruction during mitotic exit. This action creates a window between late mitosis and the end of G1 phase (when APC/C-Cdh1 is inactivated) in which geminin is absent, and therefore Cdt1 is free to participate in pre-RC formation.127 The A-type cyclins are first transcribed late during the G1 phase under the control of the E2F transcription factors in a similar manner to that of cyclin E. Cyclin A associates with both Cdk2 and Cdk1 and acts both during S-phase and G2/M.128,129 At the start of S phase, cyclin A–Cdk2 enters the nucleus and is specifically localized at nuclear replication foci, where it is thought to be actively involved in the firing of replication origins.130 As was described previously, cyclin A–Cdk2 also is required to phosphorylate E2F-1 and mediate its degradation, which is required to prevent E2F1 from triggering apoptosis.131,132 Given the redundancy between different cyclin and Cdk family members, the specific role of cyclin A in DNA synthesis is unclear. Studies in mouse models identified a partially overlapping role between E- and A-type cyclins in the control of DNA synthesis in a cell-type specific manner.133 Fibroblasts lacking both cyclin A1 and cyclin A2 are able to proceed to S phase, and this is abrogated if E-type cyclins are deleted. However, A-type cyclins are essential in hematopoietic stem cells, suggesting cell-type differences probably caused by the levels of expression of the encoding genes. Although these observations suggest overlapping functions for E- and A-type cyclins in the activation of Cdks, a new kinase-independent role as an RNA-binding protein has been recently proposed for cyclin A2.134 Cyclin A2 binds directly to the 3′ UTR of Mre11 mRNA in a Cdk-independent manner to promote its translation. Mre11 is a component of the MRN complex, composed of Mre11, Rad50, and Nbs1, which plays a central role in DSB repair and replication fork restart.135 Depletion of cyclin A2 results in a reduction in Mre11 levels and defective repair of replication errors. Thus cyclin A2 participates in DNA synthesis in a Cdkdependent manner while reinforcing stabilization of the key machinery responsible for repairing DNA lesions caused by replication errors.134

Mitosis The mitotic machinery is optimized to ensure that the replicated chromosomes are faithfully segregated to the daughter cells. This segregation is achieved through the use of a specialized microtubulebased structure, the mitotic spindle, on which the original chromosomes and their newly replicated copies, called sister chromatids, align and then are partitioned to opposite poles of the cell. The mitotic spindle is a highly dynamic structure that is maintained by many protein families, including motor molecules and other microtubule-associated proteins.69,70,136,137 The appropriate side-by-side alignment of the sister chromatids, termed biorientation, is facilitated by the physical tethering of the sister chromatids to one another. This process, called cohesion, actually occurs in S phase in a manner that is coordinated with the replication process.138–140 Cohesin is mediated by four proteins that together make up the cohesin complex. Two of these proteins, Smc1 and Smc3, have a long coiled structure with a dimerization domain at one end that allows them to heterodimerize to form a V-like structure. Important to note, the remaining ends of Smc1 and Smc3 can associate with each another to form a functional adenosine triphosphate (ATP) domain. This domain acts in an ATP-dependent manner to recruit two additional proteins, Scc1 and Scc3, which form a closed-ring structure that most likely encircles the chromosomes.141–143 Cohesin loading onto chromosomes, catalyzed by a separate complex called kollerin, is thought to be mediated by the entry of DNA into cohesin rings, whereas dissociation, catalyzed by Wapl and several other cohesin subunits, is mediated by the subsequent exit of DNA.144 Increasing evidence indicates that cohesin participates in other cellular processes that involve DNA looping such as transcriptional regulation. Interesting to note, mutations in genes encoding cohesin subunits and other regulators of the complex have been identified in several tumor types.145 The related family of chromosomal proteins, condensins (condensin I and condensin II), are formed from a conserved pair of Smc proteins (Smc2 and Smc4) and distinct sets of non-SMC regulatory subunits.146 Condensins participate in a diverse array of chromosomal functions including chromosomal organization in interphase or the assembly of mitotic chromosomes.

Mitotic Entry In addition to its role in DNA replication as discussed earlier, cyclin A/ Cdk complexes are critical mediators of the changes that occur during the G2 phase in preparation for mitosis.128 Here they are thought to play a key role in initiating the condensation of chromatin and also might participate in the activation of the cyclin B/Cdk1 complexes. Data have suggested that cyclin A2 is also crucial for loading of kinesins to microtubules, thus contributing to the formation of a functional spindle.134 Cyclin A2 is destroyed at nuclear envelop breakdown in an APC/C-Cdc20–dependent manner.147 A-type cyclins are then substituted with B-type cyclins. Cyclin B1 protein accumulates steadily through the G2 phase and associates mainly with Cdk1, although it also may associate with Cdk2.129 The resulting cyclin B1/Cdk1 complex is mostly sequestered in the cytoplasm, and it is retained in an inactive form throughout the G2 phase via the inhibitory phosphorylation of Thr14 and Tyr15 in Cdk1’s active site by the Myt1 and Wee1 kinases.148–150 Activation of cyclin B1–Cdk1 occurs in a highly synchronous manner during G2-prophase transition (see Fig. 4.1).151 This activation is mediated by two changes. First, the activities of Myt1 and Wee1 are downregulated at the transition between the G2 and M phases. Second, a dramatic increase in the activity of the Cdc25 phosphatases occurs that relieves the inhibitory phosphorylation of Thr14 and Tyr15. These activity changes are triggered by the phosphorylation of Myt1, Wee1, Cdc25a, and Cdc25c. Three different kinases are thought to contribute to this phosphorylation: Polo-like kinase (Plk1), cyclin A–Cdk1, and cyclin B1–Cdk1 itself. The involvement of cyclin B1–Cdk1 creates a powerful feed-forward loop; once a small amount of cyclin B1–Cdk1 is activated, it simultaneously

Control of the Cell Cycle  •  CHAPTER 4 63

inactivates its own inhibitors and activates its activators, enabling a rapid transformation of the entire cyclin B1–Cdk1 pool from the inactive state to the active state. Once active, cyclin B1–Cdk1 phosphorylates components of the centrosomes and initiates a process called centrosome separation, in which the centrosomes move to opposing poles of the nascent spindle, an event essential for formation of the mitotic spindle.68,152 The activation of mitotic kinases leads to dramatic changes in DNA structure. Largely on the basis of these morphologic changes, mitosis is divided into five different stages—prophase, prometaphase, metaphase, anaphase, and telophase (Fig. 4.5)—before separation of the daughter cells or cytokinesis.

Prophase Prophase is characterized by condensation of sister chromatids— essentially packaging into a more compact chromatin structure. This process involves condensins I and II, which require phosphorylation by mitotic Cdks.145 During prophase, the nuclear envelope is still intact; consequently, differences in subcellular localization of the condensin and Cdk complexes allow only condensin II and cyclin A-Cdk1 (nuclear), and not condensin I and cyclin B-Cdk1 (cytoplasmic), to initiate condensation. In a parallel process called resolution, the sister chromatids are untangled via the action of topoisomerase II.141,142 Resolution requires removal of the chromosome arm cohesin through phosphorylation of Scc3 by Plk1 and histone H3 by the Aurora-B kinase. Important to note, the cohesin complex at the centromere is somehow protected from this modification by a protein called shugosin (Sgo).153–155 The second major event in prophase is the translocation of the cytoplasmic cyclin B1–Cdk1 to the nucleus, where it phosphorylates components of the nuclear envelope and triggers its breakdown,68,156 which defines the transition from prophase to prometaphase. To prevent the activity of phosphatases induced by the mixture of cytoplasmic and nuclear compartments, Mastl (which is mostly nuclear in prophase) is exported to the cytoplasm, thus inhibiting the Cdk-counteracting phosphatase PP2A-B55.68,157 Cdk1 phosphorylates more than 100 proteins and participates in such activities as chromosome condensation, nuclear envelope breakdown, modifications in the Golgi apparatus, and formation and dynamics of the mitotic spindle.9,68 Cdk1-deficient mouse embryos arrest during the first embryonic divisions, indicating that this kinase is absolutely required for mitosis and cannot be compensated by other mammalian Cdks.115,158

Prometaphase During prometaphase, the condensation process is accelerated because condensin I and cyclin B-Cdk1 now have access to the DNA. The sister chromatids become attached to spindle microtubules through the kinetochore, which is assembled onto centromeric DNA.71,72,74–76 Microtubules nucleated from the centrosomes attach to the kinetochore through a process called search and capture, in which individual microtubules grow and shrink until they contact and bind the kinteochore.73 Typically, one sister chromatid of the pair attaches first, and this attachment is further stabilized through the recruitment of additional microtubules from the same pole of the mitotic spindle to create a kinetochore fiber—that is, highly bundled microtubules bound to the kinetochore. The sister chromatids oscillate in the cell until the second sister chromatid is captured by microtubules emanating from the other pole. These oscillations continue until all of the chromosomes are properly aligned on the metaphase plate.

Metaphase Metaphase is defined as the point at which all of the chromosome pairs are fully condensed, attached to the mitotic spindle, and aligned at the center—termed the metaphase plate. The pulling of the kinetochore fibers toward the poles creates tension through the cohesin complex at the kinetochores, which indicates that the sister chromatids have achieved appropriate biorientation. The cell constantly monitors

the attachments of microtubules to the chromosomes, and possibly the tension that is generated by microtubules on the kinetochores ensures that the sister chromatids are properly aligned at the metaphase plate.159,160 This process is the basis of one of several cell cycle checkpoints, called the mitotic spindle assembly checkpoint (SAC), which is described in more detail in the following sections.

Anaphase Anaphase is characterized by the segregation of the chromosomes.161 This event is controlled by the mitotic ubiquitin ligase APC/C-Cdc20. APC/C-Cdc20 ubiquitinates, and thereby triggers the degradation of, cyclin B1 and a protein called securin.39 Both securin and cyclin B1/ Cdk1 complexes are able to bind and inhibit a protease called separase.162,163 APC/C-Cdc20 activity results in the degradation of cyclin B and securin and the subsequent separase activation. Once released, separase cleaves the Scc1 component of the cohesin complex, which opens the cohesin ring, unlinking the sister chromatids and allowing them to be pulled to opposite poles (see Fig. 4.5). The spindle poles then move farther apart to ensure that the chromosomes are fully segregated. The separase-dependent cleavage of Scc1 also is essential to link segregation of chromatids with the separation of centrioles during mitotic exit.163 Cyclin B degradation results in the parallel inhibition of Cdk1 activity, thereby releasing the inhibitory mechanism that limit PP1 and PP2A activity during the earlier phases of mitosis.84,90,92 The reactivation of these phosphatases results in the massive dephosphorylation of mitotic phosphoproteins and results in the disassembly of the mitotic spindle, chromosome decondensation, and the reformation of the nuclear envelope.161 During anaphase, Cdh1, which is inhibited by Cdk-dependent phosphorylation during mitosis, is dephosphorylated and replaces Cdc20 as the main APC/C activator.39 APC/C-Cdh1 is responsible for the degradation of multiple cell cycle regulators, including Cdc20. APC/C-Cdh1 also activates the ubiquitination and degradation of geminin, allowing accumulation of Cdt1 for origin relicensing in the subsequent G1 phase, and the mitotic cyclins, allowing loss of Cdk kinase activity. Loss of Cdh1 does not result in major abnormalities during mitotic exit but results in earlier entry into the following S phase because of increased Cdk activity and DNA damage.164,165 Cdh1 therefore is required to prevent unscheduled entry into S phase and genomic instability.43

Telophase The reactivation of phosphatases during mitotic exit leads to the dismantling of the mitotic spindle, leaving two discrete sets of chromosomes with each nascent daughter cell.161 The elimination of mitotic phosphoresidues by these phosphatases also results in DNA decondensation, and the nuclear envelope reforms around the segregated chromosomes to create two new nuclei, an event that defines telophase.166

Cytokinesis Finally, the cell undergoes cytokinesis, or cytoplasmic division. This process involves formation of a structure containing actin and myosin, called the contractile ring, on the inner face of the cell membrane. The position of the contractile ring is carefully controlled. In cultured cells, the ring typically begins to form in anaphase, and its position is established by the position of the metaphase plate. As the membrane grows, the contractile ring contracts steadily to form a constriction, termed the “cleavage furrow,” which ultimately separates the two nuclei and forms the two daughter cells.167,168 This process partially depends on several mitotic kinases such as Aurora-B and Plk1, which are located at the midbody.51,52,58,169 Plk1 controls the activity of RhoA guanosine triphosphatases, which are major regulators of the actomyosin ring required for abscission.58,170 Aurora-B also ensures that abscission does not occur in the presence of DNA bridges to avoid DNA damage in a process known as the abscission checkpoint.168,171,172

64 Part I: Science and Clinical Oncology

Interphase

Cyclin A/B-Cdk1 activity Chromatin begins to condense Centrosomes move to poles and mitotic spindle starts to form

NE breakdown

Chromosomes attach to microtubules of spindle

Prophase

Prometaphase

Chromosomes align at metaphase plate

Sister chromatids separate Chromatin expands Cytoplasm divides

Metaphase

APC

Cytokinesis Anaphase

Telophase

Figure 4.5  •  Key stages of mitosis. As the parent cell enters prophase, the chromosomes begin to condense, and multiple proteins associate to form the

kinetochores. The centrosomes segregate to the poles to begin formation of the mitotic spindle. Nuclear envelope (NE) breakdown denotes the start of prometaphase. In this phase, the sister chromatids continue to condense, and they attach to spindle microtubules via their kinetochores. During metaphase, the sister chromatids align at the metaphase plate and eventually achieve appropriate biorientation. At the onset of anaphase, the sister chromatids separate and move toward the poles of the spindle. During telophase, the two daughter nuclei are reformed and the daughter cells are finally separated by cytokinesis. APC/C, Anaphase-promoting complex/cyclosome.

Control of the Cell Cycle  •  CHAPTER 4 65

CELL CYCLE CHECKPOINTS

G1/S Checkpoint

At key transitions during eukaryotic cell cycle progression, signaling pathways monitor the successful completion of events in one phase of the cell cycle before proceeding to the next phase. These regulatory pathways are commonly referred to as cell cycle checkpoints.4 In a broader context, cell cycle checkpoints are signal transduction pathways that link the rate of cell cycle phase transitions to the timely and accurate completion of prior dependent events. Checkpoint surveillance functions are not confined to monitoring normal cell cycle progression; they also are activated by both external and internal stress signals. The checkpoint pathways include sensor proteins that detect these lesions and simultaneously trigger two processes: they recruit additional effector complexes to correct the problems and activate signaling pathways that induce a temporary cell cycle arrest. In certain situations, which are determined by the cell type and the degree of damage, the checkpoint pathways eventually can induce permanent cell cycle arrest (a process called senescence) or apoptosis. To minimize the possibility of errors, checkpoints exist at the four different phases of the cell cycle: G1 (to prevent entry into S phase), intra S, G2 (to prevent mitosis), and M (to avoid mitotic exit with abnormal segregation of chromosomes) (see Fig. 4.1). In general, these checkpoints monitor the status and structure of DNA during cell cycle progression. In particular, cells scan the chromatin for partially replicated DNA, as well as DNA strand breaks and other DNA lesions that can result from both extrinsic (e.g., chemicals, ionizing or ultraviolet radiation) and intrinsic (e.g., byproducts of intracellular metabolism) DNA-damaging agents. In addition, checkpoints also monitor the proper structure of chromosomes and their biorientation to ensure equal distribution between the two daughter cells.

The molecular pathway that determines cell cycle entry with the appropriate mitogenic stimuli (previously described) is not considered a cell cycle checkpoint, strictly speaking. However, several of its components also are used by a checkpoint that monitors DNA alterations before replication. In G1 cells, double-stranded DNA breaks (DSBs) are the most common and most deleterious type of DNA damage. The central components of the DNA damage response (DDR) are two members of the phosphoinositide 3-kinase–related kinase family: ataxia telangiectasia mutated (ATM) and ATM- and rad3-related (ATR).173 ATM originally was identified by virtue of its mutation in a hereditary syndrome, ataxia-telangiectasia, which is associated with radiation hypersensitivity and cancer predisposition.174 ATR also is associated with a hereditary syndrome called Seckel syndrome.175 Early studies suggested that ATM and ATR played distinct roles in the response to DSBs (ATM) versus replicative defects and single-stranded breaks (ATR). However, we now know that the regulation is more complex; considerable cross talk occurs between ATM and ATR, and they share many mediators and effectors, but the precise composition and role of the DDR complexes vary depending on both the type of damage and the stage of the cell cycle (Fig. 4.6).176 These DSBs are recognized by the multifunctional Mre11-Rad50Nbs1 (MRN) complex.135,173 This complex recruits ATM to the site of damage. The active ATM then recruits proteins to modify the chromatin at the region of the break and activate repair and signaling. As a first step in this process, ATM phosphorylates histone H2AX to form γH2AX, which helps hold the damaged ends together and acts as a binding platform for additional factors, including Mdc1, 53BP1, and Brca1, as well as more MRN and ATM. In contrast to the S and G2 response, no recruitment of ATR to DSB in G1 cells occurs, and

G1

G2/M

Intra S

Replicationassociated error

DNA damage

DNA damage

DSB

DSB ssDNA

MRN

RPA

MRN

ATM

ATR

ATM

γH2AX

γH2AX

ATM

Mediators repair machinery Chk2P

γH2AX

ATR

Mediators repair machinery Chk2P

Chk1P

Mediators repair machinery Chk1P

Chk2P

Figure 4.6  •  Ataxia telangiectasia mutated/ATM- and rad3-related (ATM/ATR) signaling is activated by DNA damage and replication stress. The cell

constantly monitors the chromatin for lesions, using complex signal transduction pathways that center on the ATM and ATR kinases. The precise mechanism of response varies according to the type of DNA damage and the cell cycle stage. Double-stranded breaks (DSBs) are the most deleterious form of DNA damage. DSBs are recognized by the Mre11-Rad50-Nbs1 (MRN) complex that consists of Mre11, Rad50, and Nbs1. This complex recruits ATM to the site of damage. ATM phosphorylates histone H2AX to form γH2AX, which creates a binding platform for additional proteins that propagate the DNA damage response and activate repair. For S and G2 phase cells, but not G1 phase cells, ATR also is recruited to the damage site. ATR and/or ATM signal to their effector kinases (Chk1 and Chk2, respectively) to influence cell cycle progression as described in Fig. 4.7. Errors in DNA replication also can activate the DNA damage response machinery through the presence of single-stranded DNA (ssDNA), which is a hallmark of the replication fork. The ssDNA is coated with replication protein A (RPA) and bound by ATR. Active ATR then recruits the DNA damage and repair machinery, including ATM, leading to the sequential activation of Chk1 and then Chk2.

66 Part I: Science and Clinical Oncology

G1, Intra S, G2/M

Intra S

G1/S

DNA damage

Replicative stress

Oncogenic stress

Chk1

P

Chk2

and/or P

Chk1

P

Chk2

p14ARF

and P

Hdm2

Phosphorylation of all three Cdc25 proteins Cdc25a

Cdc25b Cdc25c

P p53 P P

Ubiquitination Degradation

P

Bound and inhibited by 14-3-3

p53

Oligomerizes to form active transcription factor

Cdk activation

p21Cip1

Pro-apoptotic genes

Cdk activity

Figure 4.7  •  DNA damage, replicative stress, and oncogenic stress induce cell cycle arrest. DNA damage and replication stress lead to the rapid phosphorylation

and activation of the Chk1 and/or Chk2 kinases. These kinases enforce cell cycle arrest through two mechanisms. Chk1 and Chk2 both phosphorylate the Cdc25 phosphatases, which triggers their ubiquitination and degradation (Cdc25a) or binding and inhibition by 14-3-3 (Cdc25b and Cdc25c), thereby preventing activation of cyclin/Cdk kinase complexes. Chk1 and Chk2 also phosphorylate p53 and prevent it from being targeted by Hdm2 for ubiquitin-mediated degradation. As a result, p53 accumulates and activates transcription of p21Cip1, inhibiting Cdk2 and Cdk1 kinase complexes, or proapoptotic genes. Oncogenic stress also leads to cell cycle arrest by activating replicative stress and/or inducing transcription or the p14ARF tumor suppressor and suppressing Hdm2-mediated inhibition of p53. Cdk, Cyclin-dependent kinase.

thus ATM is solely responsible for checkpoint activation. The recruitment of additional ATM amplifies the signal, and ATM acts via phosphorylation and activation of the effector kinase Chk2.177,178 Chk2 influences the G1 cell cycle arrest via two mechanisms (Fig. 4.7). First, it phosphorylates all three members of the Cdc25 family. Phospho-Cdc25a is ubiquitinated by SCF-TrCPβ and degraded, whereas phospho-Cdc25b and phospho-Cdc25c are bound and sequestered by a cytoplasmic protein called 14-3-3.20,179 This process is a rapid response that can take effect within minutes after DNA damage, and it has a widespread effect on cell cycle progression by preventing activation of Cdks. Second, Chk2 phosphorylates p53, a critical regulator of cell cycle checkpoints.180 In normal, nonstressed cells, p53 protein is maintained at low steady state levels because it has a very short half-life. This half-life is a result of rapid ubiquitination of p53 by Hdm2 (the human ortholog of murine Mdm2 protein) and its consequent degradation.181,182 The importance of Mdm2 for maintenance of appropriate p53 levels in vivo is highlighted by the fact that absence of Mdm2 in knockout mice results in early embryonic lethality that is rescued by a dual knockout of Mdm2 and p53.183,184 Phosphorylation of p53 by Chk2 is sufficient to prevent its association with Hdm2/ Mdm2,183 which leads to an accumulation of p53, which functions as a transcriptional activator; p53 induces expression of many genes involved in cell cycle arrest, including the CKI p21Cip1.185,186 This p53-mediated arrest takes longer to develop than does the Cdc25 response (because it requires transcription and protein synthesis), but it appears to be much more robust. Moreover, in addition to inducing cell cycle arrest, p53 has the capacity to induce apoptosis through the transcriptional activation of proapoptotic regulators (e.g., the BH3-only proteins Puma and Noxa).187

Important to note, p53 also is activated by other stress signals (see Fig. 4.7). In particular, it is now well established that numerous oncogenes trigger a stress response (called oncogene-induced stress) that leads to the activation of p53.188,189 The emerging view is that this process occurs through two distinct mechanisms. First, oncogene activation is thought to yield replicative stress that activates p53 via activation of Chk kinases and phosphorylation of Hdm2/Mdm2 as just described.190,191 Second, many oncogenes activate transcription of cell cycle inhibitors such as p16INK4a, p15INK4a, p21CIP1, or p19ARF in a p53-independent manner.188,192–195 The protein p19ARF is encoded by the INK4A/ARF (CDKN2A) locus, and it actually shares two coding exons, which are read in alternate reading frames (hence the name ARF) with the p16INKa tumor suppressor.194 The ARF protein product, called p14ARF in humans and p19ARF in mice, binds to Hdm2/ Mdm2 and prevents it from regulating p53.196–199 As with the DDR, this mechanism frees p53 to activate the transcription or proarrest or proapoptotic targets. As an additional DDR in G1 cells, genotoxic agents also inhibit origin licensing by way of an ATM/ATR-independent process that is achieved through regulation of Cdt1.200 As described previously, Cdt1 is required for pre-RC formation. In an undamaged cell, Cdt1 is available during the G1 phase but is inhibited after origin firing by degradation (mediated by the SCF-Skp2 and Cul4-Ddb1-Dtl/Cdt2 ubiquitin ligases) and geminin binding. As a key feature of this regulatory system, Cdt1 is completely resistant to Cul4-Ddb1-Dtl/Cdt2 in the G1 phase. However, DNA damage allows the Cul4-Ddb1-Dtl/ Cdt2 complex to ubiquitinate Cdt1 and induce its degradation. Important to note, the degradation of Cdt1 is extremely rapid, occurring within minutes of the DNA damage. Both Cdt1 and Cdt2 are

Control of the Cell Cycle  •  CHAPTER 4 67

phosphorylated in response to DNA damage, which results in the ubiquitination of Cdt1. This process depends on the activity of the p97 AAA+-ATPase and its cofactor Ufd1, a complex required for the extraction of ubiquinated proteins from the chromatin.124,201,202 As a result, origin licensing is completely blocked until the damage is repaired and Cdt1 is resynthesized.

Intra–S Phase Checkpoint One of the major goals of cell cycle checkpoints is to prevent the deleterious consequences of replicating damaged DNA. Therefore S-phase cells must respond virtually instantaneously to DNA damage to halt initiation of new replication forks throughout the S phase.203 The most deleterious type of damage is DSBs. DSBs can occur through the action of DNA-damaging agents (from either extrinsic or intrinsic sources) or as a consequence of the replication process itself—for example, if the replication fork passes through nicked DNA or if replication stalls at sites of DNA damage.204 The cell senses the damage in different ways, depending on whether the lesion is associated with replication. Ultimately, both ATM and ATR are recruited to the site of damage, but the order of binding is different.176,203 Replication-linked DSBs are distinguished by the presence of single-stranded DNA (ssDNA), a hallmark of the replication process. The ssDNA is coated by replication protein A (RPA) and bound by ATR and its regulator subunit ATRIP, even during the normal replication process. In response to DNA damage, the ATR kinase is activated, and it then recruits a variety of complexes that mediate both repair and checkpoint activation, including ATM. In contrast, nonreplication-associated DSBs initially recruit and activate ATM through the MRN-dependent process described previously for the G1/S checkpoint. However, in S-phase cells, DSB resection causes the formation of ssDNA (through the action of the MRN endonuclease), which is then bound by RPA and ATR/ATRIP.203 Thus in S-phase cells, ATR and ATM jointly orchestrate the DDR. ATR contributes to the checkpoint response in a similar manner to ATM: it activates Chk1, which also can phosphorylate the Cdc25 proteins and p53.205–208

G2 Checkpoint Whereas the G1/S and intra–S phase checkpoints prevent cells from unfaithful replication, the G2 checkpoint is required to prevent the passage of DNA lesions to the two daughter cells during mitosis.173,209,210 DSBs are detected exactly as described previously for the S-phase nonreplication-associated DSBs. Similarly, the ATR/Chk1 and ATM/ Chk2 pathways enforce arrest through inhibition of G2 and mitotic Cdk complexes via the rapid removal of the Cdc25 phosphates and the p53-dependent induction of p21CIP1 to inhibit mitotic Cdk complexes (cyclin A/B in combination with Cdk1/2 kinases).208,210,211 Ubiquitin ligases also are involved in this process. SCF-βTrCP regulates the levels of Cdc25, Claspin, and Wee1, whereas APC/C-Cdh1 is critical for the elimination of Plk1, a kinase essential for checkpoint recovery.211,212

Spindle Assembly Checkpoint The SAC acts to ensure that appropriate partitioning of the chromosomes occurs during mitosis.159,160 The concept that chromosome segregation is prevented until all condensed sister chromatid pairs are aligned at the metaphase plate with the appropriate biorientation has already been introduced. This process actually is controlled by a signaling network that constitutes the SAC (Fig. 4.8). The core components of this checkpoint—called Mad1, Mad2, BubR1, Bub1, and Bub3 in humans—originally were identified through screens in yeast for “mitotic arrest deficient” (MAD) and “budding uninhibited by benzimidazole” (BUB) mutants.159 Other components of the checkpoint include the Mps1 kinase and the three subunits of the Rod, Zwich, and ZW10 (RZZ) complex. During prometaphase, these proteins

localize to the outer kinetochore and, in the absence of biorientation, prevent the Cdc20 activator from binding to the APC/C. The kinetochore is made of approximately 30 scaffold proteins that link chromatin and the mitotic spindle.71,72,75–77 Additional regulatory elements include sensors of microtubule-kinetochore attachment and a complex signaling pathway that modulates APC/C-Cdc20 activity. Lack of tension or lack of attachment at the kinetochore results in stable Mad1/Mad2 complexes that convert an inactive open-Mad2 conformation into a closed-Mad2 conformation that is able to bind to Cdc20.159,213,214 The Mad2-Cdc20 association triggers the recruitment of BubR1-Bub3 into an APC/C-inhibitory complex (the mitotic checkpoint complex [MCC]). Because the closed-Mad2 signal is diffusible, a single unattached kinetochore is sufficient to form these complexes and inhibit APC/C-Cdc20 activity. As a result, separase is inhibited by the high levels of securin and cyclin B/Cdk1 complexes, being unable to cleave the centromeric cohesin (see Fig. 4.8). Once all chromosomes are bipolarly attached to the mitotic spindle, the SAC is satisfied and the Mad1/Mad2 complex is removed from the kinetochores.71,77 How the checkpoint signaling is inhibited currently is unclear. Cdc20 is now released from the MCC complex and activates the APC/C, leading to the rapid ubiquitination and degradation of cyclin B and securin. Inactivation of these two proteins results in two major processes. First, lack of securin and cyclin B results in the activation of separase, a caspase-like protease that cleaves cohesin, the molecule that holds sister chromatids together at the centromere. Second, inhibition of Cdk1, due to the lack of cyclin B, leads to the activation of mitotic phosphatases such as PP1 and PP2A, triggering mitotic exit as described earlier.161

CELL CYCLE DEREGULATION IN HUMAN CANCERS The central relevance of cell cycle regulation in cancer is underscored by the finding that virtually all human tumors carry mutations in (1) the basic cell cycle machinery that controls entry into the cell cycle and (2) checkpoint regulators such as the p53 pathway (Table 4.2) and by the observation that most human tumors display aberrant chromosome numbers.1,5,6,173,215 Together, the pRB and p53 pathways are critical gatekeepers of cell cycle progression and stress response, and their function has crucial implications in the maintenance of genome integrity at the level of both structural and numeric aberrations in chromosomes.216,217

Unscheduled Cell Cycle Entry in Cancer The alterations in the cell cycle machinery that occur most frequently include loss or mutation of the pRB tumor suppressor; overexpression of cyclins, Cdks, and Cdc25 phosphatases; and loss of expression of CKIs.1 Mutations that affect the pRB pathway have been identified in most human cancers.1,29,217 The RB1 gene originally was identified by virtue of its mutation in both familial and sporadic retinoblastoma, but it is defective in many other tumor types, especially osteosarcoma and lung cancer. In tumors that lack RB1 mutations, alterations in other components of the signaling pathways that regulate pRB frequently are found, including cyclin overexpression or loss of CKIs. Nearly 50% of invasive breast cancers have elevated cyclin D expression compared with surrounding normal breast epithelium, and in transgenic mice with overexpression of human cyclin D1 or cyclin E in mammary gland cells, mammary adenocarcinomas develop.218–220 Similarly, Cdk4 and Cdk6 gene amplification occurs in breast cancers, sarcomas, gliomas, and melanomas, and specific translocations lead to cyclin D or Cdk6 overexpression in hematopoietic disorders.1,221 Modifications of CKIs that act upstream of pRB activity also commonly are found in human tumors. The CKI p27Kip1 often is aberrantly expressed in human breast cancer, and reduced p27Kip1 protein levels are correlated with more aggressive breast tumors.222–224 Likewise, decreased expression

68 Part I: Science and Clinical Oncology

Prometaphase

No attachment

Pole

Metaphase

“Wait” Unattached kinetochore Low Mad2

Cohesion Spindle checkpoint proteins

Cdc20

High Mad2

Attachment

Anaphase

Securin ubiquitination + degradation

Active APC/C

Inactive APC/C

Securin Active separase

Inactive Separase

Cohesion (by Scc1 cleavage)

Figure 4.8  •  The spindle assembly checkpoint (SAC). Improper chromosome alignment on the mitotic spindle, disruption of microtubule dynamics, or

unattached kinetochores maintains the SAC in an active state. Checkpoint signaling is mediated by the Bub1, Bub3, BubR1, and Mad2 proteins, which localize to kinetochores. These core spindle checkpoint regulators prevent Cdc20 from activating the anaphase-promoting complex/cyclosome (APC/C) and therefore protect securin and cyclin B, two major APC/C-Cdc20 targets, from ubiquitin-mediated degradation. As a result, securin and cyclin B1-Cdk1 remain bound to separase, which prevents cleavage of Scc1 and loss of centromeric cohesin. The “wait” signal is inhibited at the end of the metaphase by the appropriate biorientation of the sister chromatids at the metaphase plate. The sensing mechanism involves detecting attachment or tension at the kinetochores that is created by the pulling of the spindle fibers toward the poles. Mad2 then dissociates from the attached kinetochore, which allows Cdc20 to activate APC/C, targeting securin and cyclin B for degradation. This process leads to the inactivation of Cdk1 and the activation of separase, triggering sister chromatid segregation and mitotic exit.

of the CKI p57Kip2 is found in human bladder cancers. Although p21Cip1 is not commonly mutated in human cancer, its expression is strongly reduced in multiple tumors as a consequence of defective p53 signaling.225 The CKI most frequently affected appears to be p16INK4a; it was identified as a tumor suppressor that is associated with familial melanoma, and it is inactivated by point mutation, deletion, and/or promoter methylation in approximately 30% of all human tumors.221,226 In contrast, point mutations in p15INK4b, p18INK4c, and p19INK4d are rare, but promoter methylation and reduced protein expression of some of these inhibitors have been seen in a variety of tumor types.1,221,227–229 Germline mutations in p16INK4a predispose individuals to melanoma, whereas deletion of p15INK4b and p16INK4a is linked to the pathogenesis of lymphomas, mesotheliomas, and pancreatic cancers.1,221 Although point mutations in Cdks are not common in human tumors, a specific mutation of Cdk4 (R24C) that prevents its inhibition by INK4 proteins has been found in persons with familial melanoma.230 Knockin mice harboring this mutation show not only an increased susceptibility to melanoma but also the development of multiple tumor types, indicating the relevance of this regulatory circuit in human tumors.231,232 Both Cdc25a and Cdc25b phosphatases also are overexpressed in more than 30% of primary breast tumors, 40% to 60% of non–small cell lung cancers, 50% of head and neck tumors, and a significant fraction of non-Hodgkin lymphomas.20,233,234 All these events can result in increased activation of Cdks, defective pRB signaling, unscheduled cell proliferation, and override of checkpoint arrest.

Mutations in p53 and Checkpoint Regulators The most frequently altered cell cycle checkpoint signaling molecule is the p53 tumor suppressor. The importance of p53-dependent signaling in tumor suppression is underscored by the frequency of mutation (~50%) in sporadic tumors235 and the finding that germline mutations of p53 result in Li-Fraumeni syndrome, a highly penetrant familial cancer syndrome that is associated with significantly increased rates of brain tumors, breast cancers, and sarcomas.236 In human tumors that lack p53 gene mutation, p53 function may be disrupted by alterations in cellular proteins that modulate the levels, localization, and biochemical activity of p53. For example, in some tumors with wild-type p53 alleles, Mdm2 gene amplification occurs, resulting in Mdm2 protein overexpression and subsequent p53 inactivation.237 In human papillomavirus–induced cervical carcinoma, p53 typically is not mutated; however, the human papillomavirus E6 protein binds p53 and targets it for degradation, abrogating p53-dependent signaling.238 Proteins that reside upstream of p53 (including ATM and Chk2) also are targeted for mutation in human tumors, and their discovery and analysis have greatly deepened our insight into DDR signaling pathways. ATM mutations occur in ataxia-telangiectasia, a disorder in which patients have increased sensitivity to radiation and an elevated incidence of leukemias, lymphomas, and breast cancer.174,239 ATM-null mice exhibit growth retardation, neurologic dysfunction, infertility, defective lymphocyte maturation, and sensitivity to ionizing

Control of the Cell Cycle  •  CHAPTER 4 69

Table 4.2  Alteration of Cell Cycle Regulators in Human Tumorsa Protein (Gene)

Tumors Associated With Mutations or Altered Expression

Hereditary Syndromes Associated With Germline Mutations

ATM Aurora-A (AURKA) Bub1 BubR1 (BUB1B)

Breast carcinomas, lymphomas, leukemias Wide array of tumors Colon, lung, and pancreatic cancer Wilms tumor, rhabdomyosarcoma, and acute leukemia

Brca1/2 Cdc25a Cdc25b Cdk4 Cdk6 Chk1 Chk2 Cyclin D1 (CCND1)

Breast and ovarian carcinoma Carcinomas of the breast, lung, head, and neck and lymphoma Carcinomas of the breast, lung, head, and neck and lymphoma Wide array of cancers Wide array of cancers Colorectal and endometrial carcinomas Carcinomas of the breast, lung, colon, urogenital tract, and testis Wide array of cancers

Ataxia-telangiectasia NR NR Mosaic variegated aneuploidy and premature chromatid separation syndrome Familial breast and ovarian cancer NR NR Familial melanoma NR NR Li-Fraumeni syndrome NR

Cyclin D2 (CCND2)

Lymphoma and carcinomas of the colon, testis, and ovary

NR

Cyclin D3 (CCND3)

Lymphoma, pancreatic carcinoma

NR

Cyclin E (CCNE1/2)

Wide array of cancers

NR

Mdm2 (HDM2) Mps1 (TTK) Mre11 Nbs1 p15INK4b (CDKN2B) p16INK4a (CDKN2A) p27Kip1 (CDKN1B) p53 (TP53) p57Kip2 (CDKN1C) p130 (RBL2) pRB (RB1) SA1 (STAG2) Tpx2

Soft tissue tumors, osteosarcomas, esophageal carcinomas Lung, gastric and bladder cancer Lymphoma Lymphomas, leukemias Wide array of cancers Wide array of cancers Wide array of cancers Wide array of cancers Bladder carcinomas Wide array of cancers Wide array of cancers Bladder, colorectal, glioblastoma, melanoma and Ewing’s sarcoma Lung, bone, and pancreatic cancer

NR NR Ataxia-telangiectasia–like disorder Nijmegen breakage syndrome NR Familial melanoma NR Li-Fraumeni syndrome NR NR Familial retinoblastoma NR NR

a

Only genetic alterations or defects that are present in more than 10% of primary tumors are represented. NR, Not reported.

radiation.240,241 The DNA double-strand break repair gene Mre11 also is mutated in persons with an ataxia-telangiectasia–like disorder.242 Mutations of Chk2 and Chk1 also arise in human cancers. Chk2 mutations have been reported in several cancers, including lung cancer, whereas Chk1 mutations have been observed in human colon and endometrial cancers.243,244 In addition, heterozygous alteration of Chk2 occurs in a subset of persons with Li-Fraumeni syndrome who lack p53 gene mutations.245 These findings support the theory that in human tumors in which p53 is intact, the function of this signaling pathway might be disrupted by alterations in cellular proteins that modulate the levels or activity of p53. In addition, the breast cancer susceptibility tumor suppressors Brca1 and Brca2 are known to participate in the DDR and repair.246 Similarly, Fanconi anemia proteins, which originally were identified by virtue of their association with a recessive development disorder called Fanconi anemia, which is associated with increased cancer predisposition (particularly acute myeloid leukemia), also function in the DDR.246 Mutations in the Nbs1 gene, a component of the MRN complex that sensors damage, are responsible

for Nijmegen breakage syndrome, a rare autosomal-recessive disorder characterized by chromosome instability, hypersensitivity to ionizing radiation, and high susceptibility to the development of tumors.135,247

Aneuploidy and Chromosomal Instability Abnormal chromosome numbers, called aneuploidy, is a frequent feature of cancer cells.6,215,248 In addition, many cancer cells also display chromosomal instability or an aberrantly high change in the karyotype. Chromosomal instability may lead to aneuploidy, but not all aneuploidy cells display chromosomal instability, because many cancer cells exhibit stable aneuploidy karyotypes. Aneuploidy has a high rate of frequency (more than 75%) in human tumors, and about one-quarter of the genome is affected by whole-arm or whole-chromosome number alterations.249 Yet it is not clear whether aneuploidy is a cause or consequence of cancer, and the relative level of chromosomal instability may have either oncogenic or tumor suppressor properties during tumor development.6,250–252

70 Part I: Science and Clinical Oncology

In vitro estimates suggest that normal cells missegregate a chromosome every hundred cell divisions.215 This rate is dramatically increased in cells that display chromosomal instability, which missegregate a chromosome in every one to three cell divisions in vitro.253 These defects occur for multiple reasons, including aberrant centrosome numbers, abnormal microtubule-kinetochore attachments, and imperfect SAC. In particular, there has been considerable speculation that disruption of the SAC could occur during tumor progression.252,254,255 Notably, inactivating mutations in BUB1 have been identified in human colon carcinoma cell lines, which are known to have a high degree of aneuploidy.256 These mutations facilitate the transformation of cells that lack the breast cancer susceptibility gene BRCA2.257 The gene encoding BubR1, BUB1B, is also mutated in persons with mosaic variegated aneuploidy and the premature chromatid separation syndrome. Both BUB1 and BUB1B also are silenced by promoter hypermethylation in specific tumors.258 Additional mutations and epigenetic alterations have been found in the SAC components Mad1 and Mad2.258 Most of these alterations are loss-of-function mutations, suggesting that the SAC is not functional in a variety of tumors. Moreover, haploinsufficiency of Mad2 has been shown to cause elevated rates of lung tumor development in mice.259 Interesting to note, overexpression of Mad2 also results in increased tumor susceptibility and frequent relapses of chromosomally unstable tumors.260 In fact, Mad2 is also frequently overexpressed in human tumors. Several other mitotic regulators such as separase, securin, condensins, Cdc20, or Aurora kinases, as well as the SAC component Mps1, are included in the overexpression signature that marks chromosomally unstable cancers.258,261 All these data, together with the recent evidence gathered in mouse models, suggest that small aberrations, either overexpression or downregulation, in the levels of mitotic and SAC regulators may contribute to aneuploidy and/or chromosomal instability in human tumors.6,255 Because functional disruption of SAC proteins results in the abrogation of the checkpoint and a failure to arrest in mitosis in the presence of microtubule poisons such as taxol and nocodazole, these aberrations also may play a role in the response to mitotic poisons currently used in the clinic.262 Multiple cell cycle aberrations, including pRB or p53 inactivation, lead to aberrant levels of mitotic regulators resulting in “oncogeneinduced mitotic stress.”216 The consequences of this aberration in cancer cell proliferation are possibly limited by additional alterations in other mitotic regulators. For instance, the cancer-associated upregulation of the SAC component Mad2 can be balanced by upregulation of its target Cdc20, thus maintaining the balance between SAC and APC/C activity required for genomic stability.216 In the same line, mutations in the APC/C component Cdc27 may slow down APC/C activity, thereby facilitating the correction of chromosome segregation errors in the presence of the weak SAC frequently found in tumor cells.263 Tumor cells also accumulate mutations in other genes whose deregulation may induce chromosomal instability, such as cohesin subunits and their regulators. However, whether chromosome missegregation is the major contribution of cohesin mutations to cancer progression is not clear at present.146 Chromosome missegregation not only results in whole-chromosome aneuploidy but also may generate chromosome breaks and rearrangements. A possible mechanism for DNA damage caused by chromosome missegregation is based on the defective DNA replication of the micronuclei present in cells affected by aneuploidy.264 These micronuclei undergo asynchronous DNA replication, resulting in DNA damage and pulverization of chromosomes. This process may provide, at least partially, an explanation for “chromothripsis,” a situation in which chromosomes undergo massive local DNA breakage and rearrangements.264,265 In addition, chromosomes that missegregate may be damaged during cytokinesis, resulting in DNA double-strand breaks that can lead to unbalanced translocations in the daughter cells.266 All these processes provide additional mechanisms by which defective chromosome segregation may induce genomic aberrations during tumor development.

THERAPEUTIC MANIPULATION OF CELL CYCLE CONTROLS Research during the past several decades has shown that alterations in cell cycle machinery and checkpoint signaling lead to tumorigenesis. These findings have important implications for the optimization of current therapeutic regimens and for the selection of novel cell cycle targets for the future development of anticancer agents. A leading goal in cancer research is to identify compounds that will target key cell cycle controls in a tumor-specific manner.

Targeting Cyclin-Dependent Kinase Activity Considerable debate has occurred about whether inhibition of Cdk activity is a rational strategy for anticancer therapies.267 Cdk activity frequently is elevated in human tumors, but it also is required to maintain specific cell populations in adults (e.g., the hematopoietic compartment and gut) that are essential for viability.3 Thus the key issue is whether tumor cells may require different Cdks for proliferation or whether sufficient difference in the Cdk activity exists to create a therapeutic window. During the last few years, the analysis of Cdk and cyclin mouse models has yielded considerable insight into this question but also has raised additional questions.268 These mouse models show that the cell cycle machinery is extremely robust; it adapts easily to the loss of Cdks or cyclins by using other Cdks or cyclins to substitute for the missing activity. For example, cells are able to proliferate without specific interphase Cdks because novel cyclin/Cdk complexes can be formed that allow the cell cycle to progress.111–115 This ability raises the possibility that tumor cells will develop resistance to Cdk-inhibitory drugs rapidly simply by adapting their cell cycle machinery. On the positive side, studies in cell lines and mouse models clearly show that tumors can be more dependent on Cdk activity, or at least a specific Cdk activity, than are normal tissues. In parallel to these biologic studies, numerous small-molecule inhibitors have been developed with different specificity profiles against Cdks (Table 4.3).268–270 Which Cdk should be targeted in each tumor type? Pioneering studies in the mouse showed that loss of D-type Cdk had little or no effect on the development and maintenance of many tissues but can greatly suppress the development of certain tumor types, depending on the tissue and the identity of the initiating oncogenic lesions.271–274 Mammary gland tumor proliferation dramatically depends on cyclin D1/Cdk4 complexes, whereas the absence of these molecules does not alter normal mammary gland development. In a pioneering study in mouse models, cyclin D1–Cdk4 was shown to be required for Her2 (ErbB2)- or HRas-driven tumors, whereas it was dispensable for Myc- or Wnt-induced neoplasias, suggesting that the efficacy of CKIs may have a strong dependence on the genetic background of target tumors.272,275,276 Similarly, Cdk4 is required for KRAS-mutant lung tumors, but it seems dispensable for similar lung tumors with wild-type KRAS alleles.274 On the other hand, cyclin D3/Cdk6 complexes are critical for Notch1-mutant T-cell leukemias.277 These studies and parallel efforts in human cancer cell lines led to multiple clinical trials to test the effect of specific Cdk4/6 inhibitors in a wide spectrum of tumors.278 The success of some of these trials resulted in the rapid approval of palbociclib for the treatment of hormone-positive breast cancer, and other Cdk4/6 inhibitors such as abemaciclib and ribociclib have showed significant efficacy in advanced clinical trials in multiple tumor subtypes.279–281

Targeting DNA Damage Response Proteins In the past decade, there has been a growing appreciation that many tumor cells carry mutations that disrupt their DDR. This characteristic is a major factor in establishing the resistance of tumors to chemotherapeutic agents, many of which work by causing DNA damage and triggering apoptosis through induction of DNA damage pathways.

Control of the Cell Cycle  •  CHAPTER 4 71

Table 4.3  Cell Cycle Regulators With Therapeutic Interest Target Representative Small-Molecule Inhibitors MITOTIC SPINDLE

Preclinical or Clinical Data

Tubulin

Stabilizing: taxanes, eleutherobins, epothilones, laulimalide, sarcodictyins, and discodermolide; polymerization inhibitors: vinca-alkaloids, cryptophycins, halichondrins, estramustine ARRY-520, AZD4877, MK-0731, SB-715992 (ispinesib), SB-743921

In clinical use for solid and hematopoietic tumors

ATR

AZD6738, VX-970, ATR-101

Aurora kinases Cdks (1, 2, 4, 6)

AT9283, CYC116, GSK1070916A, MLN8237, PF-03814735, VX-680 AG-024322, EM-1421, LEE-011 (ribociclib), LY2835219 (abemaciclib), P276-00, PD-0332991 (palbociclib) LY2606368, UCN01, CCT245737 BI2536, BI6727 (volasertib), GSK461364, NMS-1286937, TKM-080301

Clinical trials in monotherapy or combination with radiotherapy in solid tumors Clinical trials in solid tumors and hematopoietic malignancies Clinical trials in solid tumors and leukemias; Cdk4/6 inhibitors approved for hormone-positive breast cancer Clinical trials in combination with DNA damaging agents Clinical trials in solid tumors and hematopoietic malignancies

Eg5 (KSP)

Clinical trials in advanced solid tumors, lymphoma, and taxane-resistant cancer

KINASES

Chk1 Plk1 Mps1 (TTK)

NMS-P153, BAY1161909, NTRC 0066-0

Clinical trials in metastatic solid tumors

PHOSPHATASES Cdc25

ARQ-501, IRC 083864

Preclinical studies and clinical trials in advanced solid tumors

ABT-888, AZD-2281 (olaparib), AG014699, BMN-673, CEP 9722, MK-4827 (niraparib)

Clinical trials in solid tumors and hematopoietic malignancies; olaparib and rucaparib are approved for treating ovarian cancer

DNA REPAIR PARP

UBIQUITIN LIGASES APC/C

TAME, APCIN

Preclinical studies

APC/C, Anaphase-promoting complex/cyclosome; Cdk, cyclin-dependent kinase; ATR, ATM- and rad3-related; PARP, poly (ADP-ribose) polymerase.

Therefore considerable attention has focused on designing cancer treatments that would be effective in cells with an impaired DDR. Because it is difficult to restore the function of mutant or missing proteins, the prevailing strategy is to identify drugs that would synergize with the defective DDR to selectively kill the tumor cells and not the normal cells. For example, inhibitors of poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) selectively kill cells that lack either Brca1 or Brca2.282,283 The rationale for this action is that these proteins provide two alternative repair mechanisms in response to DNA damage: homologous recombination (Brca1 and Brca2) and base excision repair (PARP). Therefore loss of one but not both of these pathways can be tolerated. As a second example, inhibition of Chk1 sensitizes p53 mutant cells to DNA damage.284 Because p53 is mutated in approximately half of all human tumors and the absence of p53 is a major predictor of poor response to classic chemotherapeutic agents, considerable efforts are being made to develop small-molecular inhibitors of Chk1. Although the initial model proposed that this effect was due to the simultaneous abrogation of the G2 (Chk1) and G1 (p53) checkpoints, new evidence suggests that the toxicity of Chk1 inhibitors may be due to the generation of replicative stress (RS) in cells, with the less restrictive S-phase entry due to the lack of p53.285 Chk1 inhibitors therefore might synergize with other mutations that promote a promiscuous S-phase entry. A similar rationale applies to other molecules that target the RS response, such as inhibitors of the Chk1activating kinase ATR. Indeed, ATR inhibitors display a selective effect in Myc-driven tumors that display high levels of RS as a consequence of the overexpression of Myc.286 This strategy can be generalized to multiple oncogenic alterations that result in cell cycle defects, causing an increased reliance on ATR checkpoint activity, as recently shown for different tumor types.287–291

Targeting the Mitotic Spindle Microtubule poisons, such as taxol and vinblastine, kill cancer cells at least partially by exploiting their effects on the mitotic spindle.292 Taxanes, such as docetaxel or paclitaxel, are microtubule-stabilizing drugs widely used to treat breast and ovarian tumors, non–small cell lung cancer, and Kaposi sarcoma. Vinca alkaloids, such as vinblastine or vincristine, are microtubule-destabilizing compounds and have shown clinical efficacy against a broad range of hematologic malignancies. Both classes of drugs bind tubulin and inhibit microtubule dynamics, impairing the formation of a functional spindle. The SAC senses lack of proper attachment of kinetochores and arrests cells in prometaphase in the presence of these compounds. Thus inhibition of spindle dynamics results in abnormal chromosome segregation that frequently results in either aneuploidy or cell death. The low mitotic index in human tumors suggests that microtubule poisons may have therapeutic potential in a mitotic-independent manner although this deserves further exploration.293 About 30 microtubule poisons are currently in clinical development. The success of these molecules in the clinic led to the search for additional compounds that target specific regulators of microtubule dynamics rather than tubulin itself. In a pioneer screening, monastrol was identified as an inhibitor of the kinesin Eg5, a protein required for centrosome separation and the formation of a bipolar spindle.294 New drugs have been characterized that result in similar defects by inhibiting CenpE, another kinesin with critical roles in microtubuleto-kinetochore attachment.295–297 Inhibition of Aurora-A or Plk1 also may be considered to be an antispindle strategy because these kinases, although involved in other mitotic processes, are essential for centrosome maturation and separation and the formation of a bipolar spindle.52,295

72 Part I: Science and Clinical Oncology

In general, the inhibition of all these molecules results in an SACdependent arrest that impairs proliferation or viability of targeted cells. The Plk1 inhibitor volasertib has shown considerable promise in clinical studies, having reached phase III trials and been granted the breakthrough therapy designation for its effects in acute myeloid leukemia.298

Targeting Mitotic Entry and Exit As indicated in the earlier sections of this chapter, multiple enzymatic activities are required for mitosis. Cdk1 is the major engine in this process, and its activity is essential for mitosis. However, it has not been considered as a major target because its inhibition may have strong undesired effects.270 Yet some evidence suggests that it may be an interesting target in specific situations. Cdk1 is specifically required in cells transformed with Myc but not in cells transformed by other oncogenes. Inhibition of Cdk1 rapidly downregulates survivin expression, a protein required to avoid apoptosis in the presence of an excess of Myc. Cdk1 inhibition therefore results in Myc-dependent apoptosis and regression of Myc-dependent lymphoma and hepatoblastoma tumors.299 Cdk1 also is implicated in DNA repair by homologous recombination, and its inhibition results in impaired Brca1 activity. Cdk1 inhibition synergizes with PARP inhibitors, and partial inhibition of Cdk1 therefore may sensitize Brca1/2-proficient cancer cells to inhibit PARP, suggesting specific applications of Cdk1-targeting compounds in cancer cells.300 Cdk1 activators such as Cdc25 phosphatases are additional targets whose inhibition results in impaired Cdk activity and cell cycle arrest.20 The therapeutic usefulness of inhibition of additional mitotic kinases such as Mastl, Nek proteins, or Haspin, among others, is currently undergoing preclinical evaluation.3,295,301 Inhibiting mitotic entry or progression or preventing spindle dynamics may result in different outcomes, including cell death or the exit from the cell cycle without chromosome segregation.295 In fact, a rapid exit from mitosis is a major resistance mechanism that generates viable cells in the presence of mitotic poisons. This finding led to the evaluation of mitotic exit pathways as new targets for therapy.302 In fact, genetic ablation of APC/C-Cdc20 completely prevents mitotic exit, and these mutant cells arrest in mitosis until they die.90 This action results in complete tumor repression in mouse models, and a first generation of APC/C inhibitors is currently available to test this strategy in preclinical studies (see Table 4.3).90,303,304

Targeting the Spindle Assembly Checkpoint and Aneuploidy Similarly to what was discussed for the DNA damage checkpoint, a different concept is provided by the use of SAC inhibitors (checkpoint abrogation) to increase instability in cancer cells. Checkpoint kinases such as Mps1 (also known as TTK) and Aurora-B (not considered to be a “core” checkpoint protein but involved in an error-correction mechanism) are required for the proper bipolar spindle attachment of chromosomes.3 Inhibition of these kinases results in rapid exit from mitosis without chromosome segregation, generating tetraploid or aneuploid cells. Important to note, inhibition of these molecules

prevents mitotic arrest in the presence of microtubule poisons and therefore generates aberrant cells.52,305,306 It has been shown that the reduction in these checkpoint proteins makes tumor cells more sensitive than untransformed cells to low doses of spindle poisons.307 The efficacy of several Aurora or Mps1 small-molecule inhibitors currently is being tested in preclinical or clinical assays (see Table 4.3).52,295,306,308 Aneuploidy is a hallmark of cancer and is now considered as a highly attractive therapeutic target.215,309 Most tumor cells are aneuploid, whereas this abnormality is infrequent (although this has not been precisely established) in wild-type cells. This specificity is especially interesting given the problems that most therapeutic strategies that target mitosis have in specifically inhibiting tumor cells. Aneuploid cells display specific defects in cell cycle kinetics, growth rate, metabolism, and the response to specific stresses.310,311 These defects can be exploited by targeting specific cellular pathways that protect cells from the deleterious effects of aneuploidy. For instance, inhibiting the proteotoxic and metabolic stress pathways specifically reduces the viability of aneuploidy cells because of the unbalanced load of proteins in these cells.312 As previously described, targeting the mitotic checkpoint may increase the levels of aneuploidy in the tumor cells, which opens the opportunity to combine different types of antimitotic strategies to further induce chromosomal instability and aneuploidy and inhibit the pathways that cancer cells use to tolerate this abnormal state.309

SUMMARY During the past several decades, investigators have uncovered a wealth of information about the proteins that control the division of human cells. A key finding is that deregulation of the cell cycle machinery and/ or its checkpoints is a universal alteration in human cancer.1,2 Because the basic machinery that controls the cell cycle is similar in all cell types, the hope is that common strategies will be developed against a wide variety of cancers. Even though several of the currently used anticancer therapies target nonselective and non–mechanism-based targets, their effectiveness, albeit limited in many cases, is likely due to the fact that they ultimately target cell cycle regulatory or DDR signaling pathways, the status of which is different in normal cells versus tumor cells. Identification of all the components of the cellular machinery that control the cell cycle both positively and negatively is vital to the continued development of anticancer agents that can preferentially eliminate cancer cells and minimize the toxicity to normal tissues. The complexity of the human genome makes this task difficult because many members of the major protein families that regulate the cell cycle have not been studied thus far. In addition, our knowledge of the in vivo relevance of these proteins in different tissues is limited. It is obvious from the mouse models studied that the efficacy of inhibiting specific cell cycle targets depends on the tumor type and the oncogenic environment in each specific tumor. As our understanding of cell cycle regulation and checkpoint signaling improves, the goal is to use this knowledge in the design of mechanism-based therapeutics that will bring anticancer therapy to a new level. There can be little doubt of the value of targeting the cell cycle in drug discovery. The complete reference list is available online at ExpertConsult.com.

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3. Malumbres M. Physiological relevance of cell cycle kinases. Physiol Rev. 2011;91:973–1007. 4. Hartwell LH, Weinert TA. Checkpoints: controls that ensure the order of cell cycle events. Science. 1989;246:629–634.

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Control of the Cell Cycle  •  CHAPTER 4 73.e1 73.e1

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290. Morgado-Palacin I, Day A, Murga M, Lafarga V, Anton ME, Tubbs A, et al. Targeting the kinase activities of ATR and ATM exhibits antitumoral activity in mouse models of MLL-rearranged AML. Sci Signal. 2016;9:ra91. 291. Karnitz LM, Zou L. Molecular pathways: targeting ATR in cancer therapy. Clin Cancer Res. 2015;21:4780–4785. 292. Dumontet C, Jordan MA. Microtubule-binding agents: a dynamic field of cancer therapeutics. Nat Rev Drug Discov. 2010;9:790–803. 293. Komlodi-Pasztor E, Sackett D, Wilkerson J, Fojo T. Mitosis is not a key target of microtubule agents in patient tumors. Nat Rev Clin Oncol. 2011;8:244–250. 294. Mayer TU, Kapoor TM, Haggarty SJ, et al. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science. 1999;286:971–974. 295. Manchado E, Guillamot M, Malumbres M. Killing cells by targeting mitosis. Cell Death Differ. 2012;19:369–377. 296. Wood KW, Chua P, Sutton D, Jackson JR. Centromere-associated protein E: a motor that puts the brakes on the mitotic checkpoint. Clin Cancer Res. 2008;14:7588–7592. 297. Wood KW, Lad L, Luo L, et al. Antitumor activity of an allosteric inhibitor of centromereassociated protein-E. Proc Natl Acad Sci USA. 2010;107:5839–5844. 298. Gjertsen BT, Schöffski P. Discovery and development of the Polo-like kinase inhibitor volasertib in cancer therapy. Leukemia. 2015;29:11–19. 299. Goga A, Yang D, Tward AD, et al. Inhibition of CDK1 as a potential therapy for tumors overexpressing MYC. Nat Med. 2007;13:820–827. 300. Johnson N, Li YC, Walton ZE, et al. Compromised CDK1 activity sensitizes BRCA-proficient cancers to PARP inhibition. Nat Med. 2011;17:875– 882.

301. Higgins JM. Haspin: a newly discovered regulator of mitotic chromosome behavior. Chromosoma. 2010;119:137–147. 302. Huang HC, Shi J, Orth JD, Mitchison TJ. Evidence that mitotic exit is a better cancer therapeutic target than spindle assembly. Cancer Cell. 2009;16:347–358. 303. Zeng X, Sigoillot F, Gaur S, et al. Pharmacologic inhibition of the anaphase-promoting complex induces a spindle checkpoint-dependent mitotic arrest in the absence of spindle damage. Cancer Cell. 2010;18:382–395. 304. Sackton KL, Dimova N, Zeng X, Tian W, Zhang M, Sackton TB, et al. Synergistic blockade of mitotic exit by two chemical inhibitors of the APC/C. Nature. 2014;514:646–649. 305. Liu X, Winey M. The MPS1 family of protein kinases. Annu Rev Biochem. 2012;81:561–585. 306. Dominguez-Brauer C, Thu KL, Mason JM, Blaser H, Bray MR, Mak TW. Targeting mitosis in cancer: emerging strategies. Mol Cell. 2015;60:524–536. 307. Janssen A, Kops GJ, Medema RH. Elevating the frequency of chromosome mis-segregation as a strategy to kill tumor cells. Proc Natl Acad Sci USA. 2009;106:19108–19113. 308. Koch A, Maia A, Janssen A, Medema RH. Molecular basis underlying resistance to Mps1/TTK inhibitors. Oncogene. 2016;35:2518–2528. 309. Manchado E, Malumbres M. Targeting aneuploidy for cancer therapy. Cell. 2011;144:465–466. 310. Williams BR, Prabhu VR, Hunter KE, et al. Aneuploidy affects proliferation and spontaneous immortalization in mammalian cells. Science. 2008;322:703–709. 311. Sheltzer JM, Amon A. The aneuploidy paradox: costs and benefits of an incorrect karyotype. Trends Genet. 2011;27:446–453. 312. Tang YC, Williams BR, Siegel JJ, Amon A. Identification of aneuploidy-selective antiproliferation compounds. Cell. 2011;144:499–512.

5 

Pathophysiology of Cancer Cell Death Lorenzo Galluzzi, Andreas Linkermann, Oliver Kepp, and Guido Kroemer

S UMMARY

OF

K EY

P OI N T S

• Regulated cell death mainly occurs via extrinsic apoptosis, intrinsic apoptosis, necroptosis, and mitochondrial permeability transition (MPT)–driven regulated necrosis. Autophagy operates as a bona fide cell death mechanism in a few, mostly developmental, settings.

• Oncogenesis results from multiple molecular alterations, one of which frequently impairs the ability of cancer cells to die in response to exogenous or endogenous signals. • Several oncoproteins and oncosuppressor proteins regulate, either directly or in an indirect

Perhaps biased by their focus on the cell’s vital functions, biologists have disregarded the existence of programmed cell death (PCD; see later for a definition) for a long time. Sporadic observations of PCD were made throughout the 19th century by scientists such as Carl Vogt, August Weismann, Ludwig Stieda, Élie Metchnikoff, Walther Flemming, Sigmund Mayer, and John Beard.1 The concept of PCD was first theorized in the 1960s, thanks to the work of Sir Richard Lockshin.2 In 1972 John Kerr, Alastair Currie, and Andrew Wyllie introduced the term apoptosis (from Greek apo, “from, off, without,” and ptosis, “falling”) to describe one type of cell death that manifests with peculiar morphologic traits.3 At that time and for the subsequent 30 years, apoptosis was thought to be the only form of PCD, an oversimplistic notion that has been invalidated only in this century.4,5 In the middle of the first decade of the century, it indeed became clear that other subroutines of cell death, notably necrosis, can occur in a regulated fashion and account for some instances of PCD.6–8 Regulated cell death (RCD) constitutes a conserved mechanism whose usefulness trespasses evolutionistic barriers.9,10 For instance, in unicellular organisms that grow in colonies (such as yeast), the controlled demise of old and damaged cells increases the probability that fit individuals will survive adverse environmental conditions, and hence will perpetuate their genes.11 Conversely, in metazoans (including humans), PCD is critical for embryonic and postembryonic development, as well as for the maintenance of adult tissue homeostasis.12 In line with this notion, defects in the molecular mechanisms that mediate cell death contribute to the development of a wide array of human diseases. On one hand, the excessive demise of postmitotic cells decisively contributes to the pathogenesis of diseases encompassing ischemia (of the heart, brain, and kidney) and neurodegeneration. On the other hand, insufficient rates of cell death have been associated with autoimmune disorders and cancer.13 The first hint that the molecular machinery for cell death is involved in oncogenesis came in the mid-1980s, when it was found that a translocation between chromosomes 14 and 18 (t14;18), which is common in lymphoma patients, leads to the overexpression of the protein BCL2.14 Subsequent work clarified that BCL2 promotes lymphomagenesis by inhibiting the programmed demise of excessive B cells rather than by stimulating their proliferation.15 This was the first demonstration that disabled cell death constitutes one of the 74

fashion, the molecular machinery for apoptotic or necrotic cell death. • Targeting deregulated cell death signaling pathways in cancer constitutes a clinical reality and underlies promising approaches for the development of novel anticancer regimens.

hallmarks of cancer, irrespective of the histologic origin of malignant cells.16 During the subsequent two decades, it rapidly became clear that defects in the signaling pathways leading to cell death not only contribute to oncogenesis and tumor progression, but also determine, at least in some instances, the resistance of neoplastic cells to chemotherapy and radiotherapy.17 Our understanding of cell death is constantly advancing, and this knowledge has already been translated into multiple therapeutic successes. Nevertheless, future investigations will have to provide deeper insights into the cancer-associated defects of cell death, in all its forms.

FUNDAMENTAL SCIENCE: MECHANISMS OF CELL DEATH In cell death research, the attribute “programmed” is used to highlight the implication of one particular instance of cell death in developmental programs (and hence its physiologic relevance), whereas the adjective “regulated” is employed to stress the notion that one particular instance of cell death can be inhibited by specific pharmacologic or genetic interventions (and hence is mediated by genetically encoded molecular mechanisms).4 Thus all instances of PCD are regulated by definition, but not vice versa. Additional recommendations on how to define specific cell death subroutines based on morphologic5 or biochemical4 parameters have been formulated in the past few years, and will be respected throughout this chapter. The Nomenclature Committee on Cell Death (NCCD) has expressed doubts regarding the causal implication of specific biochemical processes in RCD.18 Indeed, inhibiting the mechanisms that until now were considered to be the cause of RCD (in all its instances) generally delays, but does not prevent, cell death (at least in mammalian organisms).18 This has far-reaching conceptual and therapeutic implications, especially for the development of cytoprotective clinical interventions.18 According to currently accepted models, there are two main types of cell death: apoptosis and necrosis.19–23 Additional cell death subroutines with very specific biochemical traits have been described, although they often constitute particular cases of apoptosis and necrosis.4,24,25 Macroautophagy (hereafter referred to as autophagy) has also been implicated as a potential mechanism of cell death, a notion that remains highly debated (see later).26–28

Pathophysiology of Cancer Cell Death  •  CHAPTER 5 75

Apoptosis For a long time, instances of cell death have been catalogued as apoptotic based on purely morphologic manifestations, including cytoplasmic and nuclear shrinkage (pyknosis), nuclear breakdown (karyorrhexis), and plasma membrane blebbing.5,29 This morphologic definition, reflecting the original observations by Kerr, Currie, and Wyllie,3 has been abandoned in favor of a biochemical one.4 Thus, the term apoptosis is now used to define an instance of RCD that is precipitated by the activation of caspase 3 (CASP3), implying that it can be delayed by chemical CASP3 inhibitors as well as by specific genetic interventions that inactivate CASP3.18 Apoptosis can be triggered by the activation of either of two distinct but not mutually exclusive signaling pathways. Thus, whereas extrinsic apoptosis is initiated by an extracellular stimulus acting on plasma membrane receptors, intrinsic apoptosis follows the permeabilization of mitochondrial membranes driven by an intracellular stimulus.4 Of note, the enzymatic activity of CASP3, caspase 6 (CASP6), and caspase 7 (CASP7)—which are cumulatively known as executioner caspases—is responsible for multiple (but not all) classic morphologic and biochemical manifestations of apoptosis, including karyorrhexis, internucleosomal DNA degradation, and the exposure of the phospholipid phosphatidylserine on the outer surface of the plasma membrane.30 Extrinsic apoptosis is frequently elicited by the ligand-induced activation of plasma membrane proteins of the death receptor family, such as Fas cell surface death receptor (FAS) and the tumor necrosis factor (TNF) superfamily member 1A (TNFRSF1A, best known as TNFR1).31 Alternatively, apoptotic signals can be transduced by the so-called dependence receptors (such as Patched 1, PTCH1) when the extracellular concentration of trophic factors, such as Sonic Hedgehog (SHH), falls below a critical threshold level. This means that, at odds with death receptors, dependence receptors deliver proapoptotic signals in the absence rather than in the presence of their ligands.32 Death receptors undergo spontaneous trimerization owing to the so-called preligand assembly domain (PLAD).33 Ligand binding stabilizes this configuration and hence allows for the recruitment of several proteins at the cytoplasmic tails of death receptors, including (but in some instances not limited to) pro-caspase 8 (pro-CASP8), receptor interacting serine/threonine kinase 1 (RIPK1, also known as RIP1), FAS-associated protein with a death domain (FADD), and cellular inhibitor of apoptosis proteins (cIAP1 and cIAP2, which are E3 ubiquitin ligases that also inhibit apoptosis owing to their ability to interfere with caspase activation).34 This multiprotein platform is known as death-inducing signaling complex (DISC) and stimulates the conversion of pro-CASP8 into caspase 8 (CASP8), which proteolitically activates CASP3 and hence initiates the degradative cascade that precipitates extrinsic apoptosis.35 Of note, the components of the signaling pathways that emanate from specific death receptors exhibit some degree of variation, yet all converge (in apoptosis-permissive conditions; see later) to the activation of CASP8. The molecular mechanisms that link dependence receptors to CASP3 activation are not precisely understood, yet appear to involve the adaptor protein DRAL and CASP9 (Fig. 5.1).36 Intrinsic apoptosis (also known as mitochondrial apoptosis) can be activated by a plethora of stimuli, including intracellular damage (to virtually any of the subcellular compartments) and oncogenic stress (see later). Cells are equipped with a heterogeneous set of intracellular sensors that respond to microenvironmental perturbations by activating signaling pathways for the reestablishment of homeostasis and the repair of damage, and then, if damage is irreparable, by igniting intrinsic apoptosis.37 The central step in this cascade is regulated by the integrity of mitochondrial structure and function. Indeed, if proapoptotic signals are predominant, mitochondrial membranes get permeabilized, resulting in the abrupt cessation of adenosine triphosphate (ATP) synthesis and other metabolic functions, as well as in the spillage of several proteins that are normally confined in the mitochondrial intermembrane space.37 These proteins include cytochrome c, somatic (CYCS, a semisoluble

component of the mitochondrial respiratory chain), diablo IAP-binding mitochondrial protein (DIABLO), and HtrA serine peptidase 2 (HTRA2).38–40 Once in the cytosol, CYCS—together with dATP and the adaptor protein apoptotic peptidase activating factor 1 (APAF1)— drives the assembly of the apoptosome, a supramolecular platform for the activation of CASP9.40 DIABLO and HTRA2 also stimulate caspase activation, although indirectly, by sequestering and/or degrading cytosolic caspase inhibitors of the IAP family.38,39 Other mitochondrial proteins with cytotoxic potential are released in the cytosol during the course of intrinsic apoptosis, including apoptosis inducing factor, mitochondria associated 1 (AIFM1, which normally contributes to the stability and function of respiratory complex I) and endonuclease G (ENDOG).41,42 Although both AIFM1 and ENDOG can translocate to the nucleus and mediate large-scale DNA degradation independently of caspases,42,43 their implication in intrinsic apoptosis has been dismissed.44,45 According to current models, mitochondrial outer membrane permeabilization (MOMP) is initiated at the mitochondrial outer membrane (OM) by the pore-forming activity of proapoptotic multidomain proteins of the Bcl-2 family.19 These proteins, such as BCL2 associated X, apoptosis regulator (BAX), and BCL2 antagonist/ killer 1 (BAK1), contain several Bcl-2 homology (BH) domains as well as a transmembrane domain that allow for their constitutive or inducible insertion into the OM. MOMP execution by BAK1 and BAX is regulated by other members of the same protein family. In particular, antiapoptotic proteins such as BCL2, apoptosis regulator (BCL2), BCL2-like 1 (BCL2L1, best known as BCL-xL), and MCL1, BCL2 family apoptosis regulator (MCL1) inhibit MOMP by binding to BAX and BAK1 and hence by maintaining them in an inactive conformation.46 Conversely, the so-called BH3-only proteins (small members of the Bcl-2 family that often contain only the BH3 domain) can promote the pore-forming activity of BAK1 and BAX by two different mechanisms. Thus, BH3-only proteins can either stimulate the conformational activation of BAX and BAK1 in a direct fashion or competitively displace BAX, BAK1 or other BH3-only proteins from inhibitory interactions with BCL2, BCL-xL and MCL1. BH3-only proteins can be regulated at the transcriptional level as well as by rapid posttranslational modifications (e.g., phosphorylation, proteolytic processing), de facto constituting sensors of intracellular stress that directly impinge on the regulation of intrinsic apoptosis.47 Of note, the signaling pathways leading to extrinsic and intrinsic apoptosis exhibit some degree of cross talk. Thus, whereas in some cells including lymphocytes (type I cells) the activation of death receptors leads to pro-CASP3 processing and apoptosis without any mitochondrial involvement,48 in other cells such as hepatocytes (type II cells), CASP8 not only activates CASP3 but also mediates the proteolytic cleavage of the BH3-only protein BH3 interacting domain death agonist (BID), generating a MOMP-inducing fragment.49 Thus, in type II cells, MOMP functions as an amplifier of apoptotic signaling by eliciting the CASP9-mediated activation of CASP3. Interesting to note, whether cells respond to death receptor ligation in a type I- or type II-like manner depends on the expression levels of the cIAP-like protein X-linked inhibitor of apoptosis (XIAP).50 The cross talk between extrinsic and intrinsic apoptosis is pathophysiologically relevant in vivo, as demonstrated by the fact that the hepatocytes of Bid−/− mice are partially protected from FAS-induced apoptosis (see Fig. 5.1).51

Necrosis Classically, necrosis was defined as an instance of cell death lacking the peculiar morphological manifestations of apoptosis as well as the accumulation of cytoplasmic vacuoles that characterize autophagic cells. Somehow, this was in line with the belief that necrosis would always proceed in an unregulated fashion and would only terminate accidental instances of cell death.5 In the 1990s, Vandenabeele’s and Schulze-Osthoff ’s groups demonstrated that engagement of FAS does not always lead to cell death via extrinsic apoptosis.52–54 This observation

76 Part I: Science and Clinical Oncology

Figure 5.1  •  Apoptosis. Extrinsic apoptosis is often ignited by the ligation of death receptors such as FAS. This allows for the stabilization of receptor

trimers and for recruitment at their intracellular tails of a multiprotein complex known as death-inducing signaling complex (DISC). Within the DISC, pro-CASP8 undergoes spatial-proximity induced autoactivation, hence becoming able to cleave multiple substrates including BID and pro-CASP3. As an alternative, extrinsic apoptosis can be initiated by so-called “dependence receptors,” such as PTCH1, when the concentrations of their ligands (e.g., SHH) falls below a specific threshold. In this case, the activation of CASP3 proceeds via a molecular mechanism involving CASP9 and the adaptor proteins DRAL and TUCAN. Intrinsic apoptosis is activated in response to intracellular stress conditions (e.g., DNA damage) and involves a central step of mitochondrial regulation. Thus, if proapoptotic signals (often relayed by BH3-only proteins) predominate over antiapoptotic ones, mitochondrial membranes lose their structural integrity because of the pore-forming activity of Bcl-2 family members such as BAK1 and BAX. Consequent mitochondrial outer membrane permeabilization (MOMP) allows for the release of mitochondrial proteins into the cytosol, including direct activators of caspases, such as CYCS, as well as proteins that indirectly facilitate caspase activation, such as DIABLO and HTRA2. Cytosolic CYCS directs the assembly of an APAF1-containing, dATP-dependent supramolecular platform that catalyzes CASP9 activation, hence initiating a proteolytic cascade that culminates in CASP3 (CASP6 and CASP7) activation. Extrinsic apoptosis and intrinsic apoptosis exhibit some degree of cross talk. Indeed, in some cell types, CASP8 can convert the BH3-only protein BID into a MOMP-promoting fragment, further accelerating caspase activation and apoptosis. FADD, FAS-associated protein with a death domain; FASLG, FAS ligand; IAPs, inhibitor of apoptosis proteins; tBID, truncated BID.

instilled in some researchers the suspicion that, similar to apoptosis, necrosis also might be orchestrated by a refined molecular machinery; this ignited an intense wave of research that has not yet come to an end.6 Important to note, regulated necrosis occurs during mammalian development, in particular at the bone growth plate (i.e., the zone of the bone that controls its length), as well as during adult tissue homeostasis, for instance in the lower regions of intestinal crypts.55,56 Moreover, multiple instances of necrotic RCD have been involved in the pathophysiology of diseases including viral infection, neurodegeneration, ischemia, and others.8,22,57

Necroptosis The best-characterized pathway of regulated necrosis, which is known as necroptosis, can be elicited by the ligation of death receptors in

conditions in which CASP8 is inhibited (either by pharmacologic or by genetic interventions). In this context, DISC-bound RIPK1 does not get degraded by CASP8 as it occurs during extrinsic apoptosis, but recruits and functionally interacts with its homolog receptor interacting serine/threonine kinase 3 (RIPK3) and mixed-lineage kinase like (MLKL), generating the so-called necrosome.58–60 Although PGAM family member 5, mitochondrial serine/threonine protein phosphatase (PGAM5) has been suggested to contribute to necroptosis downstream of RIPK1 and RIPK3 by virtue of its capacity to promote mitochondrial fragmentation,61,62 such a possibility has now been discarded.63,64 Rather, it is now clear that phosphorylated MLKL can form oligomers that relocalize to the inner leaflet of the plasma membrane and compromise its stability, hence precipitating RCD.65–71 Whether phosphorylated MLKL is sufficient to permeabilize the plasma membrane or whether

Pathophysiology of Cancer Cell Death  •  CHAPTER 5 77

additional factors are required for necroptosis execution remains a matter of debate. RIPK3 activation is tonically inhibited by a heterodimer composed of CASP8 and the long isoform of CASP8 and FADD-like apoptosis regulator (CFLAR), best known as FLIPL, but the underlying molecular mechanisms remain to be elucidated.72 Moreover, RIPK3 oligomerization and consequent phosphorylation of MLKL is controlled by the RIP homotypic interacting motif (RHIM) of RIPK1, the absence of which results in spontaneous necroptosis.73,74 Important to note, stimuli other than death receptor ligands (e.g., alkylating DNA damage, viral infection) trigger bona fide RIPK3- and MLKL-dependent necroptosis (Fig. 5.2).75,76

Mitochondrial Permeability Transition–Driven Regulated Necrosis In conditions of intense oxidative stress and/or in the presence of high cytosolic Ca2+ concentrations, mitochondria experience an abrupt increase in the permeability to ions and small solutes of the mitochondrial inner membrane (IM), a process known as mitochondrial permeability transition (MPT).77,78 MPT results in virtually immediate

dissipation of the mitochondrial transmembrane potential (Δψm), rapid cessation of all Δψm-dependent mitochondrial activities (including ATP synthesis and protein import), and osmotic breakdown of the organelle.77,78 MPT has been ascribed to the activity of a multiprotein protein complex that is assembled at the juxtaposition sites between the OM and the IM, the so-called permeability transition pore complex (PTPC).79 The main components of the PTPC, including members of the voltage-dependent anion channel (VDAC) family (located in the OM) and the adenine nucleotide translocase (ANT) family (located in the IM), as well as peptidylprolyl isomerase F (PPIF, a protein of the mitochondrial matrix best known as CYPD), normally mediate physiologic functions. For instance, ANT catalyzes the exchange of adenosine diphosphate (ADP) and ATP between the cytosol and the mitochondrial matrix. In response to cytosolic Ca2+ overload oxidative stress, the PTPC has been proposed to assume a high-conductance conformation leading to rapid mitochondrial breakdown.37 So far, mouse knockout experiments failed to attribute to specific components of the PTPC a critical role in the regulation of MPT, perhaps because of the existence of multiple and (at least partially) redundant isoforms of these proteins.80–82 One notable exception is represented by CYPD, whose absence has been shown to limit pathologic cell death in multiple circumstances, in vitro and in vivo.83,84 Thus MPT-driven necrosis can be defined biochemically as a form of RCD that can be retarded by pharmacologic or genetic inhibition of CYPD.18 Recent findings suggest that the c subunit of the F1FO ATPase (which in humans is encoded by ATP5G1, ATP5G2, and ATP5G3) may constitute the long-sought pore-forming component of the PTPC,85 but robust genetic evidence in support of this hypothesis is missing (Fig. 5.3).77,78

Other Forms of Regulated Cell Death Several other specific instances of apoptotic and necrotic RCD have been described throughout the past two decades, most of which eventually impinge on the core molecular machinery of apoptosis (CASP3 activation), necroptosis (RIPK3 and MLKL activation), or MPT-driven regulated necrosis (CYPD activation).23 Of all such specific instances, we find of particular relevance (because of their peculiar mechanistic aspects and pathophysiologic implications) ferroptosis, pyroptosis, and parthanatos.

Figure 5.2  •  Necroptosis. Necroptosis can be triggered by the ligation of TNFR1 when caspases (notably CASP8) are inactive (for instance, owing to the presence of the pan-caspase inhibitor Z-VAD-fmk). In these conditions, RIPK1 is deubiquitinated and engages in physical and functional interactions with its homolog RIPK3 as well as with MLKL in the context of a supramolecular entity called necrosome. Phosphorylated MLKL forms oligomers that precipitate necroptosis by relocalizing at the inner leaflet of the plasma membrane and favoring its irreversible permeabilization. CYLD, CYLD lysine 63 deubiquitinase; FADD, FAS-associated protein with a death domain; CFLARL, CASP8 and FADD like apoptosis regulator, long isoform; Ub, ubiquitin, TNF, tumor necrosis factor; TRADD, TNFRSF1A associated via death domain.

Figure 5.3  •  MPT-driven regulated necrosis. In response to oxidative stress

or cytosolic Ca2+ overload, the so-called permeability transition pore complex (PTPC) assumes a high-conductance conformation that allows for the unregulated entry of solutes and water into the mitochondrial matrix. This CYPD-dependent process causes a structural and functional breakdown of the mitochondrial network that rapidly seals the cell fate. Δψm, Mitochondrial transmembrane potential; MPT, mitochondrial permeability transition; IM, inner mitochondrial membrane; OM, outer mitochondrial membrane; RN, regulated necrosis.

78 Part I: Science and Clinical Oncology

Ferroptosis Ferroptosis is an iron-dependent form of RCD that involves the depletion of intracellular antioxidants including reduced glutathione (GSH), and consequent lethal lipid peroxidation.86–88 Ferroptosis is tonically inhibited by glutathione peroxidase 4 (GPX4), a major gatekeeper of intracellular redox balance.89,90 Moreover, various molecules cumulatively known as ferrostatins have been shown to retard ferroptosis in vitro and in vivo, presumably as they inhibit lipid peroxidation by lipoxygenases.86,91 Ferroptosis appears to be particularly relevant for the synchronized demise of renal tubules in the context of acute kidney injury.92,93

Pyroptosis For a long time, pyroptosis was defined as a cell death modality associated with the secretion of interleukin (IL)-1B and IL-18,94 two key mediators of systemic inflammation.95 Thus, pyroptosis was thought to originate from, or at least to be associated with, the activation of so-called inflammatory caspases, that is, caspase 1 (CASP1), caspase 4 (CASP4), caspase 5 (CASP5), or mouse caspase 11 (Casp11, the murine orthologue of human CASP4 and CASP5).96 Study findings have demonstrated that inflammatory caspases proteolytically cleave gasdermin D (GSDMD) to generate an N-terminal fragment that precipitates pyroptosis by forming pores in the plasma membrane on interaction with phosphoinositides.97–101 Thus pyroptosis should now be defined as a GSDMD-dependent instance of RCD.102

Parthanatos Alkylating DNA damage activates repair mechanisms involving the NAD+-dependent enzyme poly (ADP-ribose) polymerase 1 (PARP1).103 Catalytically active PARP1 recruits multiple components of the DNA repair machinery, hence facilitating the recovery of genetic homeostasis.103 However, in the context of irreparable alkylating DNA damage, PARP1 hyperactivation has lethal consequences for the cell because (1) it favors a nonrecoverable NAD+ depletion that culminates in irreversible bioenergetic crisis, and (2) it promotes the selective release of AIFM1 from mitochondria, thereby unleashing its concealed nucleolytic activity.104,105 Parthanatos can be defined as a PARP1dependent form of RCD.

Autophagy Autophagy entails the engulfment of intracellular structures (including organelles, protein aggregates, and portions of cytoplasm) by doublemembraned vacuoles called autophagosomes.106 Autophagosomes normally fuse with lysosomes, leading to the degradation of their content by lysosomal hydrolases. Baseline levels of autophagy contribute to the maintenance of intracellular homeostasis by ensuring the removal of old and damaged (and hence potentially dangerous) organelles, notably mitochondria.107,108 In addition, autophagy is upregulated in response to a wide array of stressful conditions, including nutrient deprivation, hypoxia, and the presence of xenobiotics, including multiple anticancer agents.109 For a while, it was thought that the continuative activation of autophagy would eventually lead to the exhaustion of cellular resources and cell death. Such an “autophagic cell death” was defined morphologically by an extensive vacuolization of the cytoplasm, representative of an elevated number of autophagosomes and autolysosomes (the organelles that are generated by the autophagosomal-lysosomal fusion).5,26 However, the association between the accumulation of autophagosomes and cell death has rarely if ever been proved to be causal, in particular in settings of stress-induced cancer cell death. Indeed, the inhibition of autophagy by pharmacologic or genetic means often accelerates (rather than inhibits) cell death, suggesting that autophagy constitutes a stress response mechanism that attempts (but fails) to avoid the cellular demise.110 These results cast doubts on the appropriateness of the term autophagic cell death, which

inadvertently suggests a cause-effect relationship between these two phenomena.26 Thus far, autophagy has been demonstrated to mediate cell death in several developmental scenarios, notably during the metamorphosis of insects.111,112 Moreover, at least in some instances, autophagy appears to contribute to the execution of human cancer cells that succumb to specific experimental cell death inducers in vitro.113 These observations de facto justify the use of the expression autophagic cell death under highly selected circumstances.4 Defects of the autophagic machinery have been associated with a plethora of human pathophysiologic conditions, including accelerated aging, neurodegeneration, and cancer.114,115 However, this appears to be more strictly related to the role that autophagy exerts in the regulation of intracellular homeostasis rather than as a bona fide cell death mechanism28,107 and hence is not discussed here in further detail.

FUNDAMENTAL SCIENCE: CELL DEATH AND CANCER According to classic models, single molecular alterations are per se unable to fully transform normal cells into highly aggressive cancer cells. Rather, oncogenesis seems to proceed along with a progressive increase in genetic instability and with the accumulation of several molecular defects. Often, if not always, one of these alterations consists in the interruption of the signaling cascades that ensure the homeostatic death of continuously proliferating cells.116 As they evolve, premalignant and malignant cells are indeed subjected to elevated levels of stress, in part as a result of the overactivation of cell-intrinsic oncogenic signaling pathways (so-called oncogenic stress), and in part due to microenvironmental conditions, which are often characterized by hypoxia and nutrient shortage (especially in rapidly proliferating neoplasms).117 Thus, in virtually all scenarios, carcinogenesis requires the (at least partial) suppression of cell death signaling pathways. This can result from loss-of-function mutations in proteins that transduce lethal signals or execute cell death (as many oncosuppressor proteins), but also from gain-of-function alterations in molecules that normally deliver pro-survival signals (as several oncoproteins). Defects in the molecular pathways that regulate cell death also are instrumental to tumors when it comes to resistance to chemotherapy and radiotherapy.17 Important to note, intrinsic alterations are generally insufficient for a healthy cell to form a clinically manifest aggressive neoplasm, owing to the existence of a systemic layer of control on cellular homeostasis mediated by the immune system (so-called “cancer immunosurveillance”).118 For the sake of simplicity, however, the cell-extrinsic regulation of oncogenesis and tumor progression is not discussed in further detail here.

Oncogenes and Cell Death Regulation Oncogenes—that is, genes that stimulate malignant transformation— were originally identified in tumorigenic viruses and then were shown to exist as inactive variants (or proto-oncogenes), also in the human genome.119 Proto-oncogenes including MYC and NRAS are involved in the regulation of mitogenic signals and hence play a critical role in the control of tissue homeostasis. Per se, proto-oncogenes are not tumorigenic and must acquire gain-of-function alterations to become so. These alterations can be as gross as chromosomal translocations that bring proto-oncogene coding sequences in the proximity of strong transcriptional regulators (as in the case of MYC, which is often rearranged in lymphoma patients), or as specific as point mutations that render proto-oncogene products constitutively active (as in the case of NRAS, which is affected by hyperactivating mutations in 20% to 25% of all cancers).120,121 By transducing strong and persistent mitogenic signals, constitutively active MYC and NRAS, in addition to other oncoproteins such as epidermal growth factor receptor (EGFR, which is often hyperactivated in lung and colon cancer) and Abelson tyrosine kinase (ABL, which is fused to BCR on the t(9;22) translocation in chronic myelogenous leukemia), facilitate the

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escape of malignant precursors from the growth control that usually guarantees tissue homeostasis. As mentioned earlier, however, one single alteration of this kind is insufficient to convert normal cells into their malignant counterparts. The hyperactivation of mitogenic pathways causes consistent functional alterations and drives cells into a state of constant stress featuring significant metabolic rewiring and increased generation of reactive oxygen species. Because oncogenic stress would normally lead to cell death, only premalignant cells in which the cell death signaling pathways are blocked can progress toward tumorigenesis.116 Premalignant cells resist oncogenic stress by activating one (or more) of several pathways that (1) directly counteract the execution of cell death (because of the deregulation of additional proto-oncogenes) or (2) facilitate the management of intracellular stress (because of the elicitation of per se nononcogenic stress response pathways). In general, this leads to two distinct phenomena of dependence, commonly referred to as “oncogene addiction” and “non–oncogene addiction.”117 In the former scenario, not only the tumorigenic phenotype but also the survival of cancer cells becomes dependent on the hyperactivation of one or more proto-oncogenes. This has provided a rationale for the use of multiple oncogene-targeted agents in anticancer therapy, some of which (such as the BCR-ABL–targeting compound imatinib) have impressive rates of therapeutic responses.116 In the latter scenario, cancer cells become dependent on the activation of per se nononcogenic mechanisms of intracellular stress management, such as those centered around molecular chaperones of the heat-shock protein (HSP) family.117 Major examples of prosurvival systems that often get hyperactivated throughout (and contribute to) oncogenesis are provided by antiapoptotic Bcl-2 family members, the nuclear factor–κB (NF-κB) pathway, and AKT1. Antiapoptotic Bcl-2 family members have been shown to regulate cell death by multiple mechanisms, including the sequestration of

their proapoptotic counterparts into inactive complexes, in addition to Ca2+-related and metabolic effects (Fig. 5.4).46,122,123 After the discovery that the overexpression of BCL2 as a result of the t(14;18) translocation underlies multiple instances of lymphomagenesis,14 preclinical and clinical evidence has accumulated to demonstrate that not only BCL2 but also its antiapoptotic homologues BCL-xL and MCL1 exert bona fide oncogenic functions.124 The overexpression of BCL2 shortens the latency period required for transgenic mice overexpressing MYC in B cells to develop lymphomas.15 Along similar lines, both BCL-xL and MCL1 have been shown to cooperate with MYC in murine models of hematopoietic malignant transformation.125,126 However, consistent with the notion of multistep oncogenesis described earlier, mice engineered for the overexpression of BCL2, BCL-xL, or MCL1 in hematopoietic cells exhibit perturbed myelopoieis or lymphopoieis but are only slightly more prone to the development of age-related lymphomas than their normal counterparts.125,127,128 Of note, the protein expression levels of BCL2, BCL-xL, and MCL1 are elevated in a wide range of human tumors.129 NF-κB is a transcription factor that participates in the cellular response to a wide array of conditions, including (but not limited to) cytokine stimulation, infection, and oxidative stress. Under normal circumstances, NF-κB subunits such as p65RELA and p50NFKB1 are held in check in the cytoplasm by inhibitory interactions with IκBα. In the presence of NF-κB–inducing conditions, however, IκBα is phosphorylated by the so-called IκB kinase (IKK, a multiprotein complex including two catalytic subunits [IKKα and IKKβ] and one regulatory component [IKKγ/NEMO]), resulting in its proteosomal degradation and in the nuclear translocation of NF-κB.130 NF-κB homodimers and heterodimers control the expression of more than 150 target genes, including genes that code for mitogens, other transcription factors (including MYC), inhibitors of extrinsic apoptosis (e.g., the so-called FLIPs), IAPs, and BCL2 and BCL-xL (Fig. 5.5).131

Figure 5.4  •  Bcl-2 family proteins. Human Bcl-2 family proteins. Official gene names (within brackets) and MW are reported. BH, Bcl-2 homology domain; TM, transmembrane domain.

80 Part I: Science and Clinical Oncology

Figure 5.5  •  The nuclear factor–κB (NF-κB) canonic pathway. In the

canonic activation pathway (a “noncanonic” pathway exists but is not described here for the sake of simplicity), the IκB kinase (IKK) complex (comprising one regulatory subunit, known as IKKγ or NEMO, and two catalytic subunits, IKKα and IKKβ) responds to specific signals (including the ligation of death receptors) or nonspecific stress by phosphorylating IκB, thereby targeting it for proteosomal degradation. The degradation of IκB unmasks a nuclear localization signal that allows preformed NF-κB homodimers and heterodimers to enter the nucleus and bind DNA. In the nucleus, NF-κB dimers control the expression of genes involved in several distinct pathophysiologic processes, including innate and adaptive immunity, inflammation, proliferation, and cell death and survival. P, Phosphate; Ub, ubiquitin.

Thus, the activation of NF-κB orchestrates the response of cells to stress while delivering a strong prosurvival (and in some cases mitogenic) signal. Multiple lines of evidence demonstrate that the NF-κB system exerts bona fide oncogenic functions. First, REL genes are homologues of the avian reticuloendotheliosis virus v-rel oncogene, which is strictly required for virus-induced malignant transformation.132,133 Second, the human leukemia/lymphoma virus type I (HTLV1) can transform target cells via a mechanism that depends, at least in part, on the activation of cellular IKK by the viral protein Tax.134 Finally, several components of the NF-κB pathway get overexpressed or hyperactivated during malignant transformation. For instance, REL genes are amplified or affected by gain-of-function mutations in a constituent proportion of B-cell lymphomas,135 and the IκB-related protein BCL3 (which stimulates the nuclear translocation and activity of NF-κB dimers)136 is involved in a chromosomal rearrangement, t(14;19), that is found in some hematologic malignancies.137 Of note, the NF-κB system appears to play a critical role in the handling of oncogenic stress, as demonstrated by the fact that multiple oncoproteins including RAS and BCR-ABL stimulate NF-κB nuclear translocation and transcriptional activity.138,139 One of the hallmarks of cancer cells as originally formulated by Hanahan and Weinberg in 2000 consists of their ability to circumvent the absence of extracellular growth factors and de facto emancipate from homeostatic tissue regulation.140 Several growth factors, including IL-2, platelet-derived growth factor (PDGF), and insulin-like growth factor 1 (IGF1), promote proliferation and survival via transmembrane receptors that activate phosphoinositide-3-kinases (PI3-Ks, also known as phosphatidylinositol-3 kinases). Active PI3-K generates plasma membrane-bound phosphatidylinositol-3,4,5-triphosphate, in turn stimulating the activation of the serine/threonine kinase AKT1 (also known as protein kinase B [PKB]). By phosphorylating multiple

Figure 5.6  •  AKT1 signaling. Several growth factor receptors are

coupled to phosphoinositide-3-kinase (PI3-K), catalyzing the conversion of membrane-bound phosphatidylinositol-4,5-diphosphate (PIP2) into phosphatidylinositol-3,4,5-triphosphate (PIP3). By binding to a pleckstrin homology (PH) domain, PIP3 recruits AKT1 at the intracellular leaflet of the plasma membrane, hence allowing for its phosphorylation-dependent activation by phosphoinositide-dependent kinase 1 (PDK1). Active AKT1 phosphorylates multiple substrates, thereby transducing multipronged prosurvival signals. For instance, AKT1 inhibits the BH3-only protein BAD by facilitating its sequestration by 14-3-3 proteins, stimulates the degradation of the oncosuppressive transcription factor p53, promotes glucose uptake (hence favoring the maintenance of bioenergetic such as FKHRL1. Moreover, AKT1 indirectly activates the mechanistic target of rapamycin (MTOR), hence stimulating cell growth and proliferation while inhibiting autophagy. By catalyzing the dephosphorylation of PIP3, phosphatase and tensin homolog (PTEN) functionally antagonizes PI3-K, de facto inhibiting AKT1 activation. P, Phosphate.

substrates, AKT1 transduces prosurvival signals via distinct mechanisms. Among others, AKT1 inhibits the BH3-only protein BCL2-associated agonist of cell death (BAD) by facilitating its sequestration by chaperones of the 14-3-3 protein family, stimulates the degradation of the oncosuppressor tumor protein p53 (TP53, best known as p53; see later), promotes glucose uptake and hence favors the maintenance of bioenergetic homeostasis, and blocks the nuclear translocation of proapoptotic Forkhead transcription factors such as Forkhead box O3 (FOXO3).141 Moreover, AKT1 indirectly activates mechanistic target of rapamycin (MTOR), thus stimulating cell growth and proliferation while inhibiting autophagy (Fig. 5.6).142 Several preclinical and clinical studies have demonstrated the importance of AKT1 in tumorigenesis. Transgenic mice expressing constitutively active Akt1 under the control of a T cell–restricted promoter develop lymphoma early in life.143 Amplification of AKT1 or gain-of-function AKT1 mutations have been detected in several neoplasms, including (but not limited to) breast and gastric cancers. In addition, loss-of-function of the oncosuppressor PTEN (the phosphatase that directly antagonizes PI3-K and hence inhibits AKT1, see later) is common across a wide array of tumors.144 Of note, the inhibition of MTOR limits tumor formation in Pten+/− mice,145 demonstrating that AKT1-driven oncogenesis relies, at least in part, on MTOR hyperactivation. This is particularly interesting in view of the fact the MTOR inhibition stimulates autophagy,

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which is considered as a bona fide oncosuppressor mechanism during early tumorigenesis.146

Oncosuppressors and Cell Death Regulation The existence of tumor suppressor genes (also known as oncosuppressor genes)—that is, genes whose products actively counteract tumorigenesis —was first suspected in the 1960s, when Harris and colleagues observed that highly malignant Ehrlich ascites cells lose the ability to form tumors on fusion with virtually nontumorigenic cells.147 The molecular characterization of the first tumor suppressor gene, however, lagged until the 1980s, when the cDNA encoding the protein product of the RB1 gene (a negative regulator of cell cycle progression involved in the development of pediatric retinoblastoma) was isolated and sequenced.148 Since then, several other oncosuppressor proteins have been identified, encompassing negative regulators of mitogenic signals, such as PTEN; proapoptotic Bcl-2 family members such as BAX; and proteins that couple the management of cellular stress with cell death induction, such as ATM and p53. Oncosuppressor genes are inactivated during oncogenesis not only with loss-of-function mutations and deletions, but also after epigenetic gene silencing. Thus, promoter hypermethylation at CpG islands or histone deacetylation can turn off oncosuppressor gene expression, owing to local chromatin remodeling and impaired access by essential transcription factors.149 These instances are exemplified by the loss of CASP8 expression in pediatric neuroblastomas or by the loss of p14ARF (an upstream activator of p53; see later) expression in multiple tumor types.150,151 Irrespective of the underlying mechanisms, both alleles of one tumor suppressor gene must be inactivated for the establishment of tumor-permissive conditions. In this context, germline mutations in oncosuppressor genes are associated with familial tumor syndromes (as one copy of the gene is lost in the whole organism), whereas somatic mutations are frequently detected in sporadic tumors (in this case, both alleles have been inactivated during tumorigenesis).152 As mentioned earlier, the protein product of PTEN (phosphatase and tensin homolog) functions as a phosphatase, catalyzing the conversion of phosphatidylinositol-3,4,5-triphosphate into phosphatidylinositol4,5-diphosphate and de facto antagonizing the enzymatic activity of PI3-Ks and negatively regulating AKT1 (see Fig. 5.6).153 PTEN was discovered in the search for a tumor suppressor on chromosome 10 that is often lost in glioblastoma and breast, endometrial, and prostate cancer.154 Subsequent preclinical and clinical observations supported the notion that PTEN loss of function sustains tumorigenesis in many different organs.155,156 The oncosuppressive functions of PTEN have been shown to be particularly sensitive to gene dosage, even beyond the classic concept of haploinsufficiency.157 Of note, the frequency of PTEN inactivation in human tumors is exceeded only by that of p53 (see later).144 Pro-apoptotic BCL-2 family members, in particular BAX and BAK1, exert crucial oncosuppressive functions as they precipitate RCD in response to adverse conditions, including oncogenic stress (see Fig. 5.4). Results from multiple murine models support the notion that BAX and BAK1 operate in vivo to counteract oncogenesis independent of p53. For instance, murine cells expressing adenoviral E1A (a proliferative factor) and dominant negative p53 are unable to form tumors in mice unless Bax and Bak1 are lost.158 In addition, loss-of-function mutations in BAX and BAK1 have been detected in many hematopoietic and solid malignancies.129,159 By acting as stress sensors that activate intrinsic apoptosis, BH3-only proteins could also exert, at least theoretically, prominent oncosuppressive functions. This said, evidence implicating them as bona fide tumor suppressors is scant, and only a few articles report their loss in cancer patients.160 The reasons underlying this apparent discrepancy have not yet been clarified, but perhaps are linked to the fact that whereas BAX and BAK1 operate at the convergence of multiple lethal signaling pathways, single BH3-only proteins are involved in highly specific signaling cascades and perhaps are relatively redundant among themselves.

p53 was originally identified in 1979 by two methodologically distinct approaches: as a cellular protein coimmunoprecipitating with the large-T antigen in SV40-transformed cells,161,162 and as the target of antibodies isolated from the serum of immunodeficient mice bearing human tumors.163,164 Since then, p53 has been subjected to an intense wave of investigation, leading to the discovery that p53 regulates an ever-growing list of cellular processes.165 Historically, the first function ascribed to p53 was that of a stress-responsive transcription factor. In physiologic conditions, the intracellular levels of p53 are regulated by MDM2, an E3 ubiquitin ligase that polyubiquitinates p53 and hence targets it to proteosomal degradation.166 In response to a variety of adverse conditions, including DNA damage and oncogenic stress, p53 escapes MDM2-mediated degradation and accumulates in the cytoplasm. This can be due to posttranslational modifications of p53 that reduce its affinity for MDM2 or to the expression of MDM2 inhibitors such as p14ARF.167 Irrespective of the underlying molecular mechanisms, stabilized p53 assembles into transcriptionally proficient tetramers that can regulate the expression of distinct sets of genes, leading to highly diverse functional outcomes.168 Among the most common p53-regulated responses are reversible cell cycle arrest, senescence, and cell death. Cell death is accomplished via the upregulation of multiple genes involved in the execution of apoptosis, including APAF1, BAX, FAS, and the BH3-only protein BCL2 binding component 3 (BBC3, best known as PUMA),168–171 as well as via transcriptionindependent mechanisms whereby cytoplasmic p53 stimulates MOMP by interacting with both proapoptotic and antiapoptotic members of the Bcl-2 family or MPT by interacting with CYPD (Fig. 5.7).172–176 The inactivation of p53, be it mutational, due to the overexpression or overactivation of p53 negative regulators such as MDM2, or resulting from the inactivation of upstream p53-activating factors such as p14ARF, is the most common molecular alteration in human cancer, affecting more than 50% of neoplasms, all confounded. In addition, the p53 status has been shown to influence prognosis and/or therapeutic outcome in multiple oncologic settings, including (but not limited to) breast, lung, and colorectal cancer.177 These clinical data reflect a huge number of preclinical observations demonstrating that p53 exerts bona fide oncosuppression in vivo.178 Of note, recent research has focused on physiologic aspects of the p53 biology, unveiling a critical role for baseline p53 levels in the maintenance of bioenergetic, redox, and genomic homeostasis.179–182 Thus, p53 appears to exert oncosuppressive functions both as it regulates intracellular homeostasis, thereby preventing malignant transformation, and as it orchestrates the elimination of potentially tumorigenic cells.

CLINICAL RELEVANCE AND APPLICATIONS As introduced earlier, genetic, epigenetic, and functional alterations of the mechanisms that underlie RCD (1) are highly prevalent in human cancer, (2) are often required for oncogenesis and/or tumor progression, and (3) underlie many instances of cancer chemoresistance and radioresistance. The clinical relevance of these alterations is therefore dual. On one hand, the characterization of the genotype and functional phenotype of specific neoplasms can provide prognostic and/or predictive information. This allows for the stratification of patients into precise risk groups and—at least in some instances—for the implementation of personalized therapeutic regimens.183 On the other hand, a great number of anticancer agents that specifically target alterations in cell death–regulating signaling pathways have been developed, and some of them have successfully entered clinical routines. At odds with conventional chemotherapeutics, which frequently kill tumor cells because of their elevated proliferative potential, targeted anticancer agents act on cancer cell–specific defects and therefore are generally associated with a reduced incidence and severity of side effects.184 These agents include compounds that interrupt oncogene or nononcogene addiction as well as strategies that attempt to reconstitute the lost function of tumor suppressors. In most cases, these approaches lead to the apoptotic or necrotic demise of cancer cells, although in

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The list of successful approaches that target deregulated cell death signaling in cancer cells is continuously growing and encompasses, among others, the proteosomal inhibitor bortezomib, which blocks NF-κB activation and is approved by the US Food and Drug Administration (FDA) for the treatment of multiple myeloma and mantle cell lymphoma189; so-called BH3 mimetics, that is, small molecules that inhibit antiapoptotic members of the Bcl-2 protein family, such as venetoclax (an orally available agent licensed by the FDA for the treatment of chronic lymphocytic leukemia)190; rapamycin, an MTOR inhibitor that is approved by the FDA for transplant rejection and is now being evaluated in combination therapies against multiple neoplasms191; and p53-reconstituting strategies, aimed at either reintroducing wild-type p53 in p53-deficient cancer cells (by gene therapy) or at blocking MDM2 in p53-proficient cells (by pharmacologic inhibitors).192,193 Of note, a recombinant adenovirus engineered to express wild-type p53 (known as gendicine) is the first gene therapy product approved for clinical use in humans.194 In addition to targeted approaches, such as the aforementioned, promising results have been obtained with epigenetic regulators such as suberoylanilide hydroxamic acid (SAHA, also known as vorinostat), a histone deacetylase inhibitor that is currently approved by the FDA for the treatment of cutaneous T-cell lymphoma,195 and azacitidine or decitabine, methyltransferase inhibitors that are currently used for the therapy of myelodysplastic syndromes.196

WHAT THE FUTURE HOLDS

Figure 5.7  •  The p53 system. In physiologic settings, p53 levels are maintained under control by MDM2, an E3 ubiquitin ligase that, on polyubiquitination, targets it to proteosomal destruction. In response to multiple stress stimuli (e.g., DNA damage and oncogene activation), p53 is subjected to posttranslational modifications that reduce its affinity for MDM2. Alternatively, stressful conditions result in the upregulation of MDM2 inhibitors such as p14ARF. In both cases, the cytoplasmic levels of p53 rise, allowing p53 to assemble into tetramers that enter the nucleus and regulate a plethora of transcriptional responses. Furthermore, a pool of monoubiquitinated p53 persists into the cytoplasm, where it can facilitate apoptosis by directly activating the proapoptotic protein BAX and/or by inhibiting antiapoptotic members of the Bcl-2 family, or it can promote mitochondrial permeability transition (MPT)-driven regulating necrosis by interacting with CYPD upon entering mitochondria. Ac, Acetyl; Me, methyl; Ne, Nedd8; P, phosphate; Su, SUMO; Ub, ubiquitin.

some cases the therapeutic benefit results from the activation of cellular senescence (an irreversible arrest in cell cycle progression).185 Cell death mechanisms have also attracted attention for the development of strategies for chemosensitization and radiosensitization. In this case, restoring the proficiency of cancer cells to undergo stress-induced cell death is not therapeutic per se but restores the tumor sensitivity to conventional chemotherapeutics, which is often lost during tumor progression.186 Of note, the success of targeted anticancer agents appears to depend, at least in part, on the activation of immune effector mechanisms,187 which has been demonstrated in multiple settings, in vitro and in vivo, including models of pure oncogene addiction.188

Finely manipulating cell death mechanisms in cancer, a strategy that 20 years ago appeared as a distant and hardly reachable goal, now constitutes a reality with crucial clinical implications. Future investigations will have to provide further insights into the molecular cascades that underlie cell death in both physiologic (homeostatic cell death) and pathologic (in response to oncogenic stress) scenarios, and will have to unravel the mechanisms through which these are disabled during oncogenesis and tumor progression. This will be instrumental not only for the discovery of novel drug targets and hence for the development of new anticancer agents, but also for the precise identification of patients who may respond to particular therapeutic regimens. Furthermore, strategies for the therapeutic induction of nonapoptotic RCD modes, including necroptosis and MPT-driven necrosis, or oncosuppressive signaling networks that operate in multiple ways, such as mitotic catastrophe,179 should be elaborated. These approaches will be particularly relevant for the treatment of chemoresistant and radioresistant cancers, which nearly always exhibit alterations in the molecular machinery that initiates or executes apoptotic cell death. Finally, it will be indispensable to understand how distinct cell death subroutines cross talk (i.e., to which extent they can substitute for each other in scenarios in which one is specifically disabled by tumorigenic alterations) and to what extent the immune system contributes to the therapeutic efficacy of currently used anticancer regimens. In this context, the induction of immunogenic cell death, which converts dying tumor cells into a vaccine that is capable of eliciting a tumor-specific immune response,197–199 may constitute a particularly interesting therapeutic goal. The complete reference list is available online at ExpertConsult.com.

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Pathophysiology of Cancer Cell Death  •  CHAPTER 5 83.e1 83.e1

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83.e4 Part I: Science and Clinical Oncology

SELF-ASSESSMENT REVIEW QUESTIONS

ANSWERS

1. Which one of the following molecular alterations may be common among lymphoma patients? a. A translocation between chromosome 14 and chromosome 19, resulting in the overexpression of BAX b. A loss-of-function point mutation in AKT1 c. An amplification in the chromosomal region 12q13-14, encompassing MDM2 2. The detection of a point mutation in TP53 that negatively affects p53-regulated gene transactivation would prompt for: a. The administration of a hypothetical FDA-approved MDM2 inhibitor b. The administration of DNA damaging agents c. The use of gendicine 3. Which one of the following may constitute the basis for the development of novel anticancer regimens? a. The screening of a combinatorial chemical library for the identification of MLKL activators b. The discovery of a new inhibitor of caspases c. The identification of a new inhibitor of BAX 4. Which one of the following statements is best supported by experimental evidence? a. Death receptors and dependence receptors activate the same signaling pathways. b. Death receptors and dependence receptors lead to CASP3 activation. c. Death receptors and dependence receptors operate in similar settings.

1. (c)  BAX overexpression is expected to counteract oncogenesis, similar to inactivating mutations in AKT1. Conversely, the amplification of MDM2 is relatively common among tumors, leading to the functional inactivation of the p53 system. 2. (c)  Increasing the levels of mutant p53 by employing MDM2 inhibitors is expected to be useless in the presence of inactivating TP53 mutations. Along similar lines, the use of DNA-damaging agents would presumably yield poor results, because these compounds most often function by activating p53. Conversely, gendicine would lead to the restoration of a functional p53 system, perhaps exerting direct anticancer effects or rendering cancer cells responsive to chemotherapy. 3. (a)  Inhibition of BAX or caspases appears to be therapeutically useful in conditions featuring excessive cell death, such as ischemia or neurodegeneration. Conversely, novel activators of MLKL that would selectively trigger the necrotic demise of cancer cells might be very promising, because cancer cells are often intrinsically resistant to the therapeutic induction of apoptosis. 4. (b)  Although both death receptors and dependence receptors trigger extrinsic apoptosis, not only do they respond to different conditions, but also they engage distinct signaling cascades. Still, both death receptors and dependence receptors eventually lead to the proteolytic activation of CASP3.

6 

Cancer Immunology Diane Tseng, Liora Schultz, Drew Pardoll, and Crystal Mackall

S UMMARY

OF

K EY

P OI N T S

• Cancer is characterized by genetic and epigenetic instability leading to both unique and sometimes common mutations and “ectopic” overexpression of genes not normally expressed in the tissue of origin. Cancer-specific proteins arising as a result of genetic mutations appear to provide the most potent antigens visible to the T-cell arm of the immune system. Because the number of cancer-specific antigens expressed varies greatly among individual patients and among cancer histologies, cancer immunogenicity varies substantially. • As cancers develop, they are sculpted by “immune pressure” to eliminate antigens, or diminish the degree to which antigenic peptides are processed or presented to the

immune system, through a process termed editing. Multiple cells and molecular interactions in the tumor microenvironment also inhibit antitumor immune responses, including regulatory T cells and myeloid-derived suppressor cells. Tumor-induced immunosuppression is also mediated by signaling CTLA-4 and PD-1, inhibitory receptors on T cells. • Inhibition of CTLA-4 and/or PD-1 signaling on T cells enhances antitumor immunity by unleashing naturally induced adaptive antitumor immune responses that have undergone active suppression. Clinical trials using antibodies that block CTLA-4 have demonstrated impressive effects in melanoma, and clinical trials using antibodies that block PD-1 signaling have

OVERVIEW At least two features of the immune system make it a unique therapeutic tool against cancer. First, the diversity of receptors in the adaptive immune system (T-cell receptor [TCR] for T cells and antibodies made by B cells) offers unparalleled capacity for target specificity, far greater than any synthetic drug library. Second, diverse cell-killing weaponry from both the innate immune system and cytotoxic T cells offers the potential to kill any cell, once appropriately recognized. Central to the concept of successful cancer immunotherapy are the dual tenets that tumor cells express an antigenic target distinct from their normal cellular counterparts and that the immune system is capable of recognizing these antigenic differences. The notion that the immune system can be used as an anticancer therapy emerged from experiments in animal models of carcinogeninduced cancer. It was demonstrated that a number of experimentally induced tumors could be rejected on transplantation into syngeneic immunocompetent animals.1–4 Extensive studies by Prehn on the phenomenon of tumor rejection suggested that the most potent tumor rejection antigens were unique to the individual tumor.5 These studies led to the hypothesis that the immune system may be harnessed to eliminate cancer cells while sparing normal tissue. This chapter reviews the biology of tumor–immune system interactions and discusses how scientific insights from immunology have translated into novel strategies for harnessing the immune response to treat cancer, highlighting recent 84

demonstrated impressive effects in many cancers, including melanoma, non–small cell lung cancer, renal cell carcinoma, bladder carcinoma, head and neck cancer, Hodgkin lymphoma, Merkel cell carcinoma, and others. • Synthetic biology can be used to engineer immunotherapies toward antigens differentially expressed on cancer versus normal tissues, and such therapeutics do not require inherent tumor immunogenicity to be effective. Examples of such therapeutics are monoclonal antibodies, bispecific antibodies, and T cells engineered to express chimeric antigen receptors. T cells expressing a chimeric antigen receptor targeting CD19 have demonstrated impressive effects against B-cell malignancies.

developments in T-cell checkpoint-blocking antibodies, adoptive therapies with engineered T cells, and tumor vaccines.

THE ANTIGENIC PROFILE THAT DISTINGUISHES TUMORS FROM NORMAL TISSUES Genetic instability, a hallmark of cancer, is a primary generator of tumor-specific antigens. On average, cancers express between 50 and 1000 missense mutations in coding regions, roughly 20% of which create neoantigenic peptides presented by at least one of the individual’s human leukocyte antigen (HLA) alleles and thus recognized by T cells.6–14 In addition, deletions, amplifications, and chromosomal rearrangements can result in new genetic sequences resulting from juxtaposition of coding sequences not normally contiguous in untransformed cells. The vast majority of these mutations occur in intracellular proteins, and therefore the “neoantigens” they encode would not be readily targeted by antibodies. However, the major histocompatibility complex (MHC) presentation system for T-cell recognition renders peptides derived from all cellular proteins available on the cell surface as peptide MHC complexes capable of being recognized by T cells. In accordance with the original findings of Prehn,5 the vast majority of tumor-specific antigens derived from genetic mutations are unique to individual tumors. As a result, antigen-specific immunotherapies

Cancer Immunology  •  CHAPTER 6 85

Immune surveillance Normal cell

Genetic alterations Transformation progression

Tumor cell x x

x x

Resistance mechanisms Tolerance induction

ELIMINATION SURVIVAL SURVIVAL

Figure 6.1  •  The balance among immune surveillance, resistance, and tolerance. Transformation of normal cells to cancer cells involves the creation

of neoantigens as a result of mutation as well as upregulation of self-antigens. Successful immune surveillance of tumors based on recognition of these tumor-specific antigens would lead to tumor elimination at early stages. Clinically relevant tumor survival and progression require that tumors develop resistance mechanisms that inhibit tumor-specific immune responses to kill tumor cells. Alternatively, if the tumor develops mechanisms to induce immune tolerance to its antigens, antitumor effector responses do not develop. Finally, tumors could respond to immune pressure from T cells by eliminating mutations that are efficiently presented by the tumor’s major histocompatibility complex, a process termed immune editing. Evidence is accumulating that tumors actively develop immune resistance mechanisms as well as immune tolerance mechanisms in order to survive despite displaying antigens capable of recognition by the immune system. Evidence for immune editing has been demonstrated in experimental carcinogen-induced tumors in mice but not yet in human cancers.

targeted at truly tumor-specific antigens are most commonly patient specific. However, some tumor-specific mutations are shared (e.g., KRAS codon 12 G->A in colon and pancreatic cancers; BRAF V600E found in melanomas; p53 codon 249 G->T mutation found in hepatocellular carcinomas)15–18 and could serve as potential immunotherapy targets. Tumors also manifest global alterations in DNA methylation and chromatin structure resulting in dramatic shifts in gene expression.19 Tumors often overexpress hundreds of genes relative to their normal counterparts, including genes that are normally completely silent in their normal cellular counterparts. Overexpressed genes in tumor cells are attractive targets for both antibody- and cell-based immunotherapies, because overexpressed genes are shared among many tumors of a given tissue origin or sometimes multiple tumor types. For example, mesothelin, which is targeted by T cells from vaccinated pancreatic cancer patients,20 is highly expressed in virtually all pancreatic cancers, mesotheliomas, and most ovarian cancers.21,22 Whereas mesothelin is expressed at low to moderate levels in the pleural mesothelium, it is not expressed at all in normal pancreatic or ovarian ductal epithelial cells. Another example of tumor-selective expression of epigenetically altered genes is the so-called cancer-testis antigens.23 These genes are expressed almost exclusively in germ cells of the testis and ovaries, and some appear to encode proteins associated with meiosis.24–26 Many cancer-testis antigens are recognized by T cells from nonvaccinated and vaccinated cancer patients.23 A final category of tumor antigen consists of tissue-specific differentiation antigens shared by tumors of similar histologic origin. Commonly generated melanoma-reactive T cells from melanoma patients recognize melanocyte antigens,27 including tyrosinase, a melanocyte-specific protein required for melanin synthesis.28,29 Although tissue-specific antigens are not truly tumor specific, they are nonetheless potentially effective targets when the tissue is dispensable (e.g., prostate cancer or melanoma).

IMMUNE SURVEILLANCE HYPOTHESIS OF CANCER The immune surveillance hypothesis, first conceived nearly a half century ago,30,31 proposed that a fundamental role of the immune system is to survey the body for tumors as it does for infection with pathogens, recognizing and eliminating them based on their expression of tumor-associated antigens (TAAs). In animal models, carcinogeninduced tumors can be divided into those that grow progressively (termed progressor tumors) and those that are rejected after an initial period of growth (termed regressor tumors).2,3 The phenomenon of regressor tumors was explained based on ongoing immune surveillance of cancer. A corollary to the original immune surveillance hypothesis is that progressor tumors in animals (presumed to represent clinically

progressing cancers in humans) fail to be eliminated because they develop active mechanisms of either immune escape or resistance (Fig. 6.1). Genetically manipulated mice have provided clear evidence that the immune system can eliminate both carcinogen-induced and spontaneously arising cancers.32–34 When profoundly immunodeficient mice were treated with carcinogens or crossed onto a cancer-prone p53 knockout, the incidence of cancers was modestly but significantly increased relative to nonimmunodeficient counterparts when observed over an extended period (greater than 1 year). Further, tumors that arise in immunodeficient animals behave as regressor tumors when transplanted into immunocompetent animals. These findings are consistent with a model wherein tumors that arise in immunodeficient animals would have been eliminated had they arisen in immunocompetent animals. Epidemiologic studies of patients with heritable immunodeficiencies have also confirmed a significantly increased risk of certain cancers that are distinct from the epithelial cancers commonly observed in normal immunocompetent adults.35–37 Although many cancers observed in immunodeficient individuals are associated with viral infections, (e.g., Epstein-Barr virus [EBV]–associated lymphomas,38 Kaposi sarcoma–associated herpesvirus [KSHV]–associated Kaposi sarcoma,39 and human papillomavirus [HPV])–associated cervical cancer40,41), immunodeficient individuals also demonstrate an increased incidence of non–pathogen-associated cancers, particularly melanoma.42

IMMUNE HALLMARKS OF CANCER: AVOIDING IMMUNE DESTRUCTION AND TUMOR-PROMOTING INFLAMMATION Avoiding Immune Destruction Evidence from murine and human tumors demonstrates the capacity for tumors to induce T-cell tolerance to their antigens.43–46 Tolerance induction among tumor antigen–specific T cells can be an active process involving direct antigen recognition or can be associated with failure of antigen recognition by T cells—that is, the immune system “ignores” the tumor.47,48 However, tumors do not uniformly tolerize T cells. Ohashi and colleagues observed that lymphocytic choriomeningitis virus (LCMV) GP33–specific TCR transgenic CD8 T cells adoptively transferred into mice expressing pancreatic islet cell tumors that express GP3349 manifested evidence for CD8 T-cell activation as a result of antigen cross-presentation in the draining lymph nodes. Despite the activation of tumor-specific T cells, the tumors grew progressively, indicating that the degree of immune activation induced by tumor growth was insufficient to ultimately eliminate the tumors. These results suggest that in some cases T-cell responses are induced

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to developing tumors, but if the level of immune activation ultimately does not “keep up” with tumor progression, the ultimate result is tumor outgrowth. In the case of the LCMV GP33-specific TCR transgenic mice, because neither anergic nor deletional tolerance was observed, animals treated with the dendritic cell (DC) stimulatory anti-CD40 antibody demonstrated significant slowing of tumor growth. Thus depending on the mechanism of immune escape at play, agents that affect the overall activation state of either antigen-presenting cells or T cells could be used to shift the balance between tumor immune evasion and tumor immune recognition. T cells retrieved from patients with cancer tend either to be of low affinity for their cognate antigen or to recognize antigens that bind poorly to their presenting HLA (human MHC) molecule, resulting in inefficient recognition by T cells. Furthermore, tumors commonly acquire defects in MHC class I expression to avoid immune destruction. MHC class I proteins display small peptide antigens on the surface of cells and are key for immune recognition by CD8 T cells. For example, activated HRAS leads to reduced levels of messenger RNA (mRNA) transcripts of the transporter associated with antigen processing (TAP), a key protein in the antigen-processing pathway.50 Repression of proteins involved in the antigen presentation pathway are linked to overexpressed epidermal growth factor receptor (EGFR) or HPV E7 oncoprotein.51,52 MHC class I downregulation can also result from MHC gene mutations, defects in other genes in the antigen presentation

pathway such as ERAP1 and ERAP2, and epigenetic regulation of TAP genes.53–55

Tumor-Promoting Inflammation in the Tumor Microenvironment The tumor microenvironment is replete with suppressive mechanisms that dampen antitumor immune immunity (Fig. 6.2). The inhibitory cells, molecules, and signaling pathways found in the tumor microenvironment are not unique to tumors, but rather compose an array of physiologic mechanisms evolved to regulate immune responses to self-antigens and to downmodulate immune and inflammatory responses to foreign antigens so that collateral tissue damage is limited. Tumors co-opt and upregulate mechanisms for resistance to immune attack, and much activity in the field of immuno-oncology is focused on delineating these pathways and developing therapeutics capable of reversing the effects of these mediators.

Regulatory T Cells and Cancer Regulatory T cells (Tregs) maintain tolerance to self-antigens, downregulate immune responses to pathogens,56,57 and induce tolerance to tumor antigens.58,59 CD4+ Tregs are characterized by expression of Foxp3, a central master regulatory transcription factor.60,61 Other Tregs produce inhibitory cytokines such as interleukin (IL)-10 and

Maturation ↑STAT3

Inhibitory cytokines (VEGF, IL-10, TGF-β)

Tumor

DC/MΦ

PD-L1 PD-L1/2

IDO TDO

↑STAT3

B7-H3 B7-H4

LAG-3

PD-1

CD4 T cell

Adenosine

Treg A2aR

PD-L1

IL-10, TGF-β

PD-L1

↑STAT3 MDSC

IDO

IFNγ

PD-1

PD-L2

LAG-3 Antitumor CD8 killer cell Tim-3 BTLA

Figure 6.2  •  The immune microenvironment of the tumor is generally inhibitory to antitumor immune responses. Activation of oncogenic pathways such as STAT3 in the tumor leads to a cascade of molecular and cellular processes in the tumor microenvironment that block the killing function of innate immune effectors such as natural killer (NK) cells and granulocytes and block dendritic cell (DC) maturation and activation/effector function of Th1 T cells and CD8 cytotoxic T cells. This inhibitory microenvironment is organized by both cytokines, such as interleukin (IL)-10, IL-6, and transforming growth factor–β (TGF-β), and multiple cell membrane coinhibitory molecules such as PD-L1, PD-L2, B7-H3, and B7-H4 that interact with their cognate checkpoint receptors expressed at high levels on T cells in the microenvironment. Myeloid-derived suppressor cells (MDSC) produce nitric oxide (NO) that inhibits T cells, and both tumor cells and myeloid cells produce tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO), which deplete tryptophan. Regulatory T cells (Treg) also accumulate in the tumor microenvironment, further blunting antitumor T-cell responses via production of inhibitory cytokines. Tumors also produce adenosine as a byproduct of cell death, which inhibits immune responses and enhances local Treg generation and suppressive function via interaction with the adenosine A2a receptor. BTLA, B- and T-lymphocyte attenuator; IFN-γ, interferon-γ; VEGF, vascular endothelial growth factor.

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transforming growth factor–β (TGF-β). In addition, a recently described IL-12 family “hybrid” cytokine, IL-35, consisting of the alpha subunit of IL-12 and the beta subunit of IL-27, is produced by Tregs and suppresses antitumor immunity.62 Numerous murine studies have demonstrated that Tregs expand in animals with cancer and limit the potency of antitumor immune responses.63 It is now appreciated that treatment with low-dose cyclophosphamide is a relatively simple and reasonably effective way to temporarily eliminate cycling Tregs.64–67 In addition, antibodies neutralizing IL-35 limited tumor growth in multiple preclinical mouse models, but their use alone was not sufficient to eliminate tumors.62 As new cell membrane molecules that define Tregs are identified, the capacity to block regulatory T-cell activity with antibodies to these molecules presents new opportunities for immunotherapeutic strategies to break tolerance to tumor antigens. Evidence for a role of Tregs in suppressing antitumor immunity in humans includes an extensive study correlating Treg number in resected ovarian cancers with clinical outcome. Patients with greater numbers of CD4+CD25hiFoxp3+ cells had a worse outcome.58 A number of clinical trials have been performed using a toxin-conjugated IL-2 reagent (denileukin diftitox) that would bind CD25 and selectively kill Tregs. Clinical efficacy of this agent was described in phase III studies of recurrent or refractory cutaneous T-cell lymphoma, and the drug was approved by the US Food and Drug Administration (FDA) for this indication, but the agent is currently not available owing to the manufacturer’s decision to prioritize development of a newer improved formulation of the drug.68

Immature Myeloid Cells and Tumor-Associated Macrophages Immature myeloid cells (iMCs)69,70 are a heterogeneous class of cells that include myeloid-derived suppressor cells (MDSCs)71–74 as well as tumor-associated macrophages (TAMs). In mice, iMCs and MDSCs are characterized by coexpression of CD11b (considered a macrophage marker) and Gr1 (considered a granulocyte marker) while expressing low or no MHC class II or the CD86 costimulatory molecule. In humans, MDSCs are most commonly described as CD33+ but lack MHC class II expression, as well as markers of mature macrophages, DCs, or granulocytes. A number of molecular species produced by tumors tend to drive iMC and MDSC accumulation, including IL-6, colony stimulating factor 1 (CSF-1), IL-10, and gangliosides. IL-6 and IL-10 are potent inducers of STAT3 signaling, which has been shown to be important in iMC and MDSC persistence and activity. Macrophages have been divided into M1 type (Th1/interferon [IFN] instructed) and M2 type (Th2/IL-4 and IL-13 instructed). M1 macrophages are characterized by expression of genes encoding the nitric oxide (NO)–producing iNOS-2 enzyme and the cytokine IL-12, which amplifies Th1 responses. M1 macrophage activation is thought to be one of the mediators of Th1-orchestrated antitumor immunity, whereas M2 macrophages are thought to be tumor promoting. Macrophage infiltration into tumors has been largely associated with poor patient prognosis in multiple tumor types. Therapeutic strategies for targeting TAMs include inhibition of macrophage recruitment to the tumor or promotion of their antitumor properties or effector function. Strategies for preventing macrophage accumulation in tumors include disruption of the CCL2/CCR2 axis in phase I clinical trials in patients with advanced-phase solid cancers.75,76 The CSF-1/CSF-1R pathway is also being evaluated as a therapeutic strategy to target tumor TAMs, and agents targeting these pathways have been shown to reduce TAM accumulation in tumors or repolarize them into an antitumor phenotype in early-phase clinical trials.77 Strategies to promote phagocytosis and antigen presentation by TAMs are very promising—for example, by disrupting the CD47-SIRPalpha axis, which has been shown to be effective in preclinical models and is being tested in ongoing clinical trials.78–80

Immature Dendritic Cells DCs found within the tumor microenvironment typically have an immature, unactivated phenotype characterized by low levels of proinflammatory cytokine production, CD86, and surface MHC class

II expression (Fig. 6.3). Immature DCs can present antigens to T cells, but without costimulatory molecules, induce T-cell tolerance,81,82 whereas in the context of microbial ligands or endogenous “danger signals,” DCs are activated to present antigen together with costimulatory signals that result in an effector T-cell phenotype. A major inhibitory signaling pathway induced in tumor infiltrating DC is the STAT3 pathway, which, when activated, strongly antagonizes Toll-like receptor (TLR) and CD40-mediated DC activation. Tumor-derived factors such as IL-10, IL-6, and vascular endothelial growth factor (VEGF) (in part induced by STAT3 signaling in the tumor cell) can induce STAT3 activation in DCs, and such mechanisms shift tumorspecific T cells from a state of activation to tolerance (Fig. 6.4). Numerous cancer immunotherapies seek to induce activation and maturation of DCs, such as engaging CD40.

Immune Inhibitory Molecules Expressed in the Tumor Microenvironment A large number of immune inhibitory molecules are expressed in the microenvironment of tumors, spanning enzymes that metabolize certain amino acids on which lymphocytes are highly dependent, enzymes that produce immune-inhibitory products, cytokines that inhibit antitumor immune responses by acting on immune effector cells to either inhibit their tumor-lytic activity, and membrane-bound ligands that bind to inhibitory receptors on lymphocytes (so-called checkpoints). Myeloid cells in the tumor microenvironment express a number of enzymes whose metabolic activity ultimately results in inhibition of T-cell responses. These include production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). NO production by iMCs and MDSCs as a result of arginase and inducible nitric oxide synthase (iNOS) activity has been well documented, and inhibition of this pathway with a number of drugs can mitigate the inhibitory effects of iMCs and MDSCs. ROS, including H2O2, have been reported to block T-cell function associated with the downmodulation of the zeta (ζ) chain of the TCR signaling complex,83 a phenomenon well recognized in T cells from cancer patients and associated with generalized T-cell unresponsiveness. Another mediator of T-cell unresponsiveness associated with cancer is the production of indoleamine 2,3-dioxygenase (IDO).84 IDO appears to be produced by DCs either within tumors or in tumor-draining lymph nodes. It is interesting to note that IDO in DCs has been reported to be induced via backward signaling by B7-1/2 on ligation with CTLA-4.85,86 The major IDO-producing DC is thought to be either a plasmacytoid dendritic cell (PDC) or a PDC-related cell that is B220+87; however, IDO has been subsequently shown to be expressed by multiple cell types in the immune microenvironment, including tumor cells themselves.88 IDO appears to inhibit T-cell responses through catabolism of tryptophan. Activated T cells are highly dependent on tryptophan and are therefore sensitive to tryptophan depletion. Thus Munn and Mellor have proposed a bystander mechanism, whereby DCs in the local environment deplete tryptophan via IDO upregulation, thereby inducing metabolic apoptosis in locally activated T cells.84 IDO has two isoforms, IDO-1 and IDO-2, encoded by distinct genes. The role of IDO-2 in human cancer is still unclear; a major IDO-2 polymorphism in humans encodes an inactive enzyme. Epacadostat is an oral inhibitor of IDO-1 and is being tested in combination with pembrolizumab (anti-PD-1) in patients with advanced melanoma, which has demonstrated promising results in phase I/II clinical trials.89

Transforming Growth Factor–β: A Major Inhibitory Cytokine in the Tumor Microenvironment TGF-β is a major inhibitory cytokine implicated in blunting antitumor immune responses. TGF-β is produced by a variety of cell types, including tumor cells, and has pleiotropic physiologic effects. For most normal epithelial cells, TGF-β is a potent inhibitor of cell proliferation, causing cell cycle arrest in the G1 stage.90 In many cancer cells,

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GM-CSF IL-4, FLT-3L Bone marrow progenitor

Dendritic cell progenitor Antigen uptake/Processing Microbial infection danger signals

No danger signals

Exogenous LPS CpG

Endogenous TNF- CD40L

Activated DC

Tolerizing DC

Moderate MHC II Costimulatory molecules High MHC II Costimulatory molecules

Figure 6.3  •  A dendritic cell (DC) can either activate adaptive immunity or tolerize T cells depending on its state of maturation. DC progenitors develop from hematopoietic (bone marrow–derived) progenitors under the influence of various cytokines, particularly granulocyte-macrophage colony-stimulating factor (GM-CSF). Under circumstances of microbial infection, specific pathogen-associated molecular patterns (PAMPs) engage pattern recognition receptors (PRRs), as well as endogenous proinflammatory cytokines and “danger”-associated molecules (DAMPs), inducing DC maturation. PAMPs include unmethylated CpG tracks of DNA characteristic of DNA viruses and bacteria; uncapped RNA, characteristic of RNA viruses; flagellin, the major protein component of bacterial flagella; and lipopolysaccharide (LPS), a major bacterial cell wall component. DC maturation leads to upregulation of costimulatory molecules, major histocompatibility complex (MHC), and certain cytokines, as well as decreased expression of coinhibitory molecules, allowing them to efficiently activate antigen-specific T cells to effector cells (right side of figure). In the absence of these danger signals, DCs follow a default pathway (left side of figure) in which they become tolerizing DCs that present antigen to T cells in the absence of costimulatory signals and with an excess of coinhibitory signals. This represents a steady state pathway for continuous presentation of self-antigens. The consequence is that these T cells are turned off (anergy) or deleted, inducing tolerance. IL-4, Interleukin-4; TNF-α, tumor necrosis factor–α.

LN Activated DCs

Activation of tumorspecific T cells

Danger

A Inhibition of danger signals

Danger

LN Tolerizing DCs

Anergy/ Deletion of tumorspecific T cells

B Figure 6.4  •  Inhibition of dendritic cell (DC) activation in the tumor microenvironment can shift tumor-specific immune responses from activation

to tolerance. Based on the scenario presented in Fig. 6.3, if a tumor is able to produce factors that inhibit local DCs from becoming activated in response to the endogenous danger signals associated with tissue invasion, it could shift tumor-specific T cells from a state of activation (A) to one of tumor-specific tolerance (B). LN, Lymph node.

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however, mutations in the TGF-β pathway confer resistance to cell cycle inhibition, allowing uncontrolled proliferation, and promote tissue invasion through induction of matrix metalloproteinases. In vivo, TGF-β directly stimulates angiogenesis; this stimulation can be blocked by anti-TGF-β antibodies.91 Elevated serum TGF-β levels are associated with poor prognosis in a number of malignancies, including prostate cancer,92 lung cancer,93 gastric cancer,94 and bladder cancer.92 Several inhibitors of the TGF-β pathway have been tested in phase I clinical trials, including fresolimumab, a human anti-TGF-β1/2/3 monoclonal antibody (mAb), and TβM1, a human anti-TGFβ1 mAb. These molecules were shown to be safe, but data on efficacy have been limited or not yet reported.95,96

COINHIBITORY LIGANDS AND RECEPTORS THAT DOWNMODULATE TUMOR IMMUNITY Major molecules successfully targeted in clinical cancer immunotherapy are the growing class of ligand-receptor pairs, commonly referred to as immune checkpoints. In considering the mechanism(s) of action of inhibitors of various checkpoints, it is critical to appreciate the diversity of immune functions that they regulate. For example, the two immune checkpoint receptors that were first targeted in the context of clinical cancer immunotherapy, CTLA-4 (CD152) and PD-1 (CD279), regulate immune responses at very different levels and by very different mechanisms (Fig. 6.5). The clinical activity of blocking antibodies for each of these receptors implies that antitumor immunity can be enhanced at multiple levels and that combinatorial strategies can be intelligently designed, guided by mechanistic considerations and preclinical models. This section focuses particular attention on the CTLA-4 and PD-1 pathways because they were the two checkpoints whose inhibition has revolutionized clinical cancer immunotherapy.

B7-1/2

CD28 +

APC

CTLA-4 Checkpoint: A Global Regulator of T-Cell Activation CTLA-4 is expressed exclusively on T cells, where it primarily regulates the amplitude of the early stages of T-cell activation, by counteracting the activity of the T-cell costimulatory receptor CD28.97–99 CD28 does not affect T-cell activation unless the TCR is first engaged by cognate antigen. Once antigen recognition occurs, CD28 signaling strongly amplifies the TCR signal to activate T cells. CD28 and CTLA-4 share identical ligands: CD80 (B7-1) and CD86 (B7-2).100–104 Because CTLA-4 has higher affinity for both ligands, its expression on the surface of T cells dampens the activation of T cells both by outcompeting CD28 in binding CD80 and CD86 and by actively delivering inhibitory signals to the T cell.105–110 The specific signaling pathways by which CTLA-4 blocks T-cell activation are still under investigation, although a number of studies have suggested that activation of the phosphatases SHP2 and PP2A are important in counteracting kinase signals induced by TCR and CD28.98 However, CTLA-4 also confers “signaling-independent” T-cell inhibition through sequestration of CD80 and CD86 from CD28 engagement, as well as active removal from the antigen-presenting cell surface.111 The central role of CTLA-4 in maintaining T-cell activation in check is dramatically demonstrated by the systemic immune hyperactivation phenotype of CTLA-4 knockout mice.112,113 Even though CTLA-4 is expressed by activated CD8 killer T cells, the major physiologic role of CTLA-4 appears to be through distinct effects on the two major subsets of CD4 T cells: downmodulation of helper T-cell activity and enhancement of regulatory T-cell suppressive activity.97,114,115 CTLA-4 blockade results in a broad enhancement of immune responses dependent on helper T cells, and conversely, CTLA-4 engagement on Tregs enhances their suppressive function. CTLA-4

T cell still in secondary lymphoid tissue

B7-1/2

CD28 +

APC

Signal 1

Signal 1 – CTLA-4 Antigen-experienced T cell

Activation of naïve or resting T cells

B7-1/2

CD28 +

DC

Traffic to periphery

Tissue or tumor

Signal 1

Signal 1 – PD-L1

Activation of naïve or resting T cells

Inflammation

PD-1 Antigen-experienced T cell

Figure 6.5  •  CTLA-4 and PD-1 checkpoints act to regulate different elements of the T-cell response. Naïve T cells and resting T cells express little

CTLA-4 or PD-1 on their surface. On initial T-cell activation via triggering of the T-cell receptor (TCR) by cognate peptide/major histocompatibility complex (MHC) complexes together with engagement of CD28 by B7-1 and/or B7-2, CTLA-4, which is stored preformed in intracellular vesicles, rapidly migrates to the cell surface while the T cell is still engaged with its antigen-presenting cell (APC; in general, a dendritic cell), usually in the secondary lymphoid tissue. The greater the TCR stimulus, the more CTLA-4 is expressed on the surface. Inhibitory interaction of CTLA-4 with B7-1 and B7-2 results in a counterregulatory signal that downmodulates the ultimate amplitude of T-cell activation (top row). In contrast, the PD-1 checkpoint primarily operates in the periphery (bottom row). PD-1 is induced more slowly than CTLA-4 when T cells become activated. Inflammatory signals, such as interferon-γ, at sites of effector T-cell function (in the tissue or in tumors) induce PD-L1 (and PD-L2) expression, which downmodulates tissue immune responses, thereby protecting tissues from collateral damage.

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is a target gene of the transcription factor Foxp3,116,117 the expression of which determines the Treg lineage,118,119 and Tregs therefore express CTLA-4 constitutively. Although the mechanism by which CTLA-4 enhances the inhibitory function of Tregs is not known, Treg-specific CTLA-4 knockout or blockade significantly inhibits their ability to regulate both autoimmunity and antitumor immunity.114,115 Thus in considering the mechanism of action for CTLA-4 blockade, both enhancement of effector CD4 T-cell activity and inhibition of Tregdependent immune suppression are likely important factors.

PD-1 Checkpoint: A Pathway That Functions Within the Tumor Microenvironment In contrast to CTLA-4, the major role of PD-1 is to limit the activity of T cells in the peripheral tissues at the time of an inflammatory response to infection and to limit autoimmunity (see Fig. 6.5).120–126 This translates to a major immune resistance mechanism within the tumor microenvironment.127–129 PD-1 expression is induced when T cells become activated.121 When engaged by one of its ligands, PD-1 inhibits kinases involved in T-cell activation via the phosphatase SHP2.120 Because PD-1 engagement inhibits the TCR stop signal, this pathway may also modify the duration of T-cell/APC or T-cell/ target cell contact.130 PD-1 is expressed on a large proportion of tumor-infiltrating lymphocytes (TILs) from many different tumor types.131,132 Similar to CTLA-4, PD-1 is highly expressed on Tregs, where it may enhance their proliferation in the presence of ligand.133 Because many tumors are highly infiltrated with Tregs, PD-1 pathway blockade may also enhance antitumor responses by diminishing the number and/or suppressive activity of intratumoral Tregs. Increased PD-1 expression on CD8 TILs may reflect an anergic or exhausted state, as has been suggested by decreased cytokine production by PD-1+ versus PD-1− TILs from melanomas.131 PD-1 is also expressed on other non–T lymphocyte subsets, including B cells, natural killer (NK) cells, and macrophages.134,135 Thus whereas PD-1 blockade is typically viewed as enhancing the activity of effector T cells in tissues and in the tumor microenvironment, it likely also enhances NK activity in tumors and tissues and may also enhance antibody production either indirectly or through direct effects on PD-1+ B cells.136 PD-1 has also been shown to be a marker of dysfunctional macrophages and monocytes.137 The two ligands for PD-1 are B7-H1/PD-L1 (CD274) and B7-DC/ PD-L2 (CD273).120,138–140 These B7 family members share 37% sequence homology and arose via gene duplication, positioning them within 100 kb of each other in the genome.140 An unexpected molecular interaction between PD-L1 and CD80 was discovered,141 whereby CD80 expressed on T cells (and possibly antigen-presenting cells) can potentially behave as a receptor rather than a ligand, delivering inhibitory signals when engaged by B7-H1142,143; the relevance of this interaction in tumor immune resistance has not yet been determined. PD-L1 has also been proposed to function as a receptor to transmit survival signals to cancer cells and T cells, although the downstream pathways mediating this function are not understood.144,145 Understanding the role of these various interactions in given cancer settings is highly relevant for selection of both antibodies and recombinant ligands for use in the clinic. Just as PD-1 is highly expressed on TILs from many cancers, PD-1 ligands are commonly upregulated on many different human tumors.128,146 On solid tumors, the major PD-1 ligand to be expressed is B7-H1/PD-L1.128,147,148 In addition to tumor cells, B7-H1/PD-L1 is commonly expressed on myeloid cells in the tumor microenvironment such as macrophages and DCs.149–151 Although most of the analyses of PD-1 ligand expression has focused on B7-H1/PD-L1, B7-DC/PD-L2 has also been reported to be upregulated on a number of tumors. B7-DC/PD-L2 is highly upregulated on certain B-cell lymphomas such as primary mediastinal, follicular cell B-cell lymphoma and Hodgkin disease.152 Upregulation in these lymphomas is commonly associated with gene amplification or rearrangement

to the CIITA locus, which is highly transcriptionally active in B-cell lymphomas.153 Two general mechanisms for regulation of B7-H1/PD-L1 expression have emerged: innate (tumor cell intrinsic) and adaptive (tumor cell extrinsic) (Fig. 6.6). In the innate (tumor cell intrinsic) mechanism, oncogenes drive expression of PD-L1. For some tumors such as glioblastoma, deletion or silencing of phosphatase and tensin homolog (PTEN) has been shown to drive PD-L1 expression, implicating the PI3K-AKT pathway.154 In addition, constitutive anaplastic lymphoma kinase (ALK) signaling, observed in certain lymphomas and occasionally in lung cancer, has been reported to drive PD-L1/B7-H1 expression via STAT3 signaling.155 In Hodgkin disease, genomic gains in chromosome 9p24 are frequently observed, a region containing the oncogene JAK2, as well as PD-L1 and PD-L2. PD-L1 expression in tumors can also be driven by IFNs, which illustrates an adaptive mechanism (tumor cell extrinsic) for its regulation. This hypothesis is supported by work from studies in melanoma demonstrating a high correlation between cell surface PD-L1/B7-H1 expression on tumor cells and lymphocytic infiltration and intratumoral IFN-γ expression. This correlation was seen not only among tumors but within individual B7-H1/PD-L1+ tumors at the regional level, in which regions of lymphocyte infiltration were exactly the regions where B7-H1/PD-L1 was expressed on both tumor cells and infiltrating leukocytes.129

Additional Checkpoints Participate in Tumor Immune Resistance and Tolerance Successful clinical outcomes of CTLA-4 and PD-1 pathway targeting have garnered interest in a number of additional checkpoints (Fig. 6.7). In addition to defined lymphocyte inhibitory receptors, a number of B7 family inhibitory ligands—in particular B7-H3 (CD276) and B7-H4—do not yet have defined receptors, but murine knockout experiments support an inhibitory role for both these molecules,156 and they are upregulated on tumor cells or tumor-infiltrating cells.157 Lymphocyte activation gene-3 (Lag-3), T cell immunoglobulin-3 (Tim-3), and adenosine A2a receptor (A2aR) are also associated with inhibition of lymphocyte activity. Antibody targeting of these receptors, either alone or in combination with a second immune checkpoint blocker, has been shown to enhance antitumor immunity in animal models of cancer. Because many tumors express multiple inhibitory ligands and TILs express multiple inhibitory receptors, dual or triple blockade of immune checkpoints could further augment antitumor reactivity. Human blocking antibodies specific for a number of these “secondgeneration” inhibitory receptors are under development. Lag-3 was first discovered as a CD4 homologue expressed on activated CD4 T cells, CD8 T cells, and a subset of NK cells.158 Lag-3 has been shown to play a role in enhancing Treg function,159,160 and subsequently to inhibit CD8 effector function.161 Lag-3 binds to MHCII, which is upregulated on some epithelial cancers (typically in response to IFN-γ) but is also expressed on tumor-infiltrating macrophages and DCs. Another Lag-3 ligand that has been recently proposed is LSECtin, a member of the DC-SIGN family.162 Lag-3 is one of a number of immune checkpoint receptors coordinately upregulated on both Tregs and anergic T cells, and simultaneous blockade can result in enhanced reversal of this anergic state relative to blockade of either receptor. In particular, PD-1 and Lag-3 are commonly coexpressed on anergic or exhausted T cells.163,164 Dual blockade of Lag-3 and PD-1 could synergize to reverse exhaustion among tumor-specific CD8 T cells. Phase I/IIb clinical trials are underway examining the safety and efficacy of anti-Lag-3 antibodies alone or in combination with nivolumab for the treatment of B-cell malignancies or solid tumors. Tim-3, first discovered to be expressed on IFN-γ expressing CD4 cells and CD8 T cells, binds to the ligand galectin-9, which is upregulated in a number of cancer types such as breast cancer. Additional ligands for Tim-3 have also been identified, including CEACAM1, HMGB1, and phosphatidyl serine.165–167 In preclinical models, Tim-3 is often coexpressed with PD-1 on a subset of T cells

Cancer Immunology  •  CHAPTER 6 91

Innate (tumor cell intrinsic) resistance MHC-pep TCR

Oncogenic pathway (STAT3, Akt)

Constitutive tumor signaling induces PD-L1 on tumor cells PD-L1 PD-1 T Cell

Tumor Adaptive resistance T cell-induced PD-L1 upregulation

T Cell Tumor

T Cell Tumor

IFN-γ

Figure 6.6  •  Two mechanisms for PD-L1 induction on tumors: innate and adaptive. PD-L1 can be constitutively expressed on tumors as a consequence of oncogene-driven transcriptional activation. Alternatively, PD-L1 can be induced in an adaptive fashion when there are the right inflammatory cytokines in the tumor microenvironment consequent to an ongoing immune response to the tumor. This mechanism of tumor resistance to immune attack is co-opted from physiologic PD-L1 expression for tissue protection in the setting of antimicrobial immune responses. Innate and adaptive mechanisms of PD-L1 expression on tumors can coexist. The adaptive resistance mechanism implies that PD-L1 expression is a “marker” of endogenous antitumor immunity. IFN-γ, Interferon-γ; MHC, major histocompatibility complex; TCR, T-cell receptor.

that are highly dysfunctional,168 and blocking Tim-3 with anti-Tim-3 antibodies has been shown to enhance antitumor immunity.157 Coordinate blockade of PD-1 and Tim3 in preclinical models was reported to enhance antitumor responses and tumor rejection under circumstances in which only modest effects from blockade of each individual molecule were observed.169–171 It is interesting to note that Tim-3 has been shown to be upregulated after anti-PD-1 therapy, both in preclinical mouse models of lung cancer and in patients, supporting upregulated Tim-3 as a mechanism of resistance to anti-PD-1 therapy.172 Anti-Tim-3 antibodies are currently being studied in phase I clinical trials for patients with advanced solid tumors. A2aR binds adenosine and inhibits T-cell responses, in part by driving CD4 T cells to express Foxp3 and develop into Tregs.173 Knockout of this receptor results in enhanced and sometimes pathologic inflammatory responses to infection. This receptor is particularly relevant in tumor immunity because the rate of cell death in tumors from cell turnover is high and dying cells release adenosine. In addition, Tregs express high levels of the exoenzymes CD39, which converts extracellular adenosine triphosphate (ATP) to adenosine monophosphate (AMP), and CD73, which converts AMP to adenosine.174 A2aR engagement by adenosine to drive T cells to become Tregs can produce a self-amplifying loop within the tumor, as illustrated by the evidence that tumors grow more slowly in A2aR knockout mice and tumor vaccines are more effective against established tumors in these mice.175 A2aR can be inhibited either by antibodies that block adenosine binding or by adenosine analogues, some of which are fairly specific for A2aR. Currently, an oral small-molecule inhibitor of A2aR CPI-444 is being tested in advanced cancers as monotherapy and in combination with atezolizumab in phase I clinical trials (ClinicalTrials.gov ID: NCT02655822). Another oral small molecule inhibitor of A2aR, PBF-509, is being tested in phase I clinical trials for patients with

non–small cell lung cancer as monotherapy and in combination with the PD-1 antibody PDR001 (ClinicalTrial.gov ID: NCT02403193).

IMPLICATIONS FOR CANCER IMMUNOTHERAPY Recent successes in clinical immunotherapy of cancer have emerged directly from a basic understanding of molecular and cellular immunology applied to the cancer setting. Recent advances have been made in three major areas of cellular cancer immunotherapy that seek to use T cells as the antitumor weapon: checkpoint blockade, cellular therapy, and cancer vaccination.

Checkpoint Blockade Therapeutic strategies targeting the CTLA-4 and PD-1 immune checkpoints have changed paradigms in cancer therapy by emphasizing the importance of targeting the immune system rather than the tumor and offering the possibility of cure in some patients. CTLA-4 was the first immune checkpoint receptor to be clinically targeted in human metastatic melanoma.176 Antibodies targeting PD-1 or its ligand PD-L1 have subsequently revolutionized treatment of a variety of human cancers, including lung cancer, melanoma, kidney and bladder cancer, Hodgkin disease, head and neck cancer, mismatch-repair deficient cancers, and Merkel cell cancers.177–183 Encouraged by murine studies demonstrating antitumor effects of CTLA-4 blockade, human anti-CTLA-4 antibodies were developed and one antibody, ipilimumab, was shown to produce a roughly 10% objective response rate in melanoma according to standard RECIST (Response Evaluation Criteria in Solid Tumors) clinical criteria.184 Anti-CTLA-4 produced significant immune-related toxicity in 25%

92 Part I: Science and Clinical Oncology

Antigen-presenting cell or tumor cell

T Cell

CD28 B7.1/B7.2

+

CTLA-4

–

? PD-L1/PD/L2

+

PD-1

ICOS-L

–

ICOS

B7-H3

?

B7-H4

?

+ – –

LIGHT +

HVEM

BTLA

MHC/pep

TCR

– Signal 1

LAG-3 4-1BBL OX40L LIGHT PS/galectin9 – +

CD200R CD40

–

4-1BB

+

OX40

+

LIGHT-R Tim-1/ Tim-3

+ –

CD200 CD40L

Cytokines (IL-1, IL-6, IL-10, IL-12, IL-18)

Figure 6.7  •  Multiple costimulatory and coinhibitory ligand-receptor interactions ultimately determine the amplitude of T-cell activation and the potency of effector T-cell responses in tissue and tumor. B7 family ligands and CD28 family receptors are shown in purple and TNF/TNFR family ligand-receptor pairs and shown in blue. There are additional inhibitory ligand-receptor pairs that do not fit into either of these families. Some of the receptors for B7 family members are not yet discovered. Although TNF/TNFR interactions are usually one-on-one pairs, B7 family ligands often interact with multiple receptors. HVEM is a TNFR family member; in addition to its interaction with the TNF family member LIGHT, it also interacts with the inhibitory receptor BTLA, which is a member of the CD28 family. Additional signals of activation or inhibition are contributed by cytokines. BTLA, B- and T-lymphocyte attenuator; HVEM, herpesvirus entry mediator; ICOS, inducible T-Cell COStimulator; IL, interleukin; MHC, major histocompatibility complex; PS, phosphatidylserine; TCR, T-cell receptor; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor.

to 30% of patients, commonly involving the skin, liver, or colon. Subsequent clinical development of ipilimumab culminated in two successful phase III trials in patients with advanced melanoma and FDA approval in 2011.185,186 Since these studies, ipilimumab has also been shown to prolong survival in patients with stage III melanoma in the adjuvant setting following surgery.187 In addition, ipilimumab is being investigated in the neoadjuvant setting in the context of regionally advanced but surgically operable melanoma.188 In the case of PD-1, the basic discoveries that this pathway downmodulated immune responses in the tissue, together with upregulation of PD-1 ligands in human tumors, motivated clinical testing in the mid-2000s. Despite the fact that most of the patients in the initial phase I trial had end-stage disease, a number of dramatic clinical responses were observed in multiple cancer types. This led to larger clinical trials of both anti-PD-1 and anti-PD-L1, culminating

in the demonstration of major clinical efficacy in multiple tumor types.189,190 The durability of the responses, even after cessation of treatment, far exceeds that of chemotherapy or tyrosine kinase inhibitors. These findings reinforce the notion that a “reeducated” immune system not only has memory, but also can adapt to maneuvers by the tumor to develop resistance. Just as predicted from the mouse knockout studies, toxicity from anti-PD-1 or anti-PD-L1 therapy is lower than from anti-CTLA-4 therapy and also of different distribution than anti-CTLA-4 therapy (colitis is most common with anti-CTLA-4 therapy, and pneumonitis is more common with anti-PD-1). A number of interesting clinical insights have emerged from the clinical experience with checkpoint inhibitors, which call for novel approaches to monitoring the safety and efficacy of these drugs. The ipilimumab experience illustrated that the kinetics of clinical responses to anti-CTLA-4 (i.e., tumor shrinkage) tends to be slower than

Cancer Immunology  •  CHAPTER 6 93

chemotherapy or tyrosine kinase inhibitors, and in some patients tumor regression is proceeded by apparent progression as assessed with computed tomography (CT) or magnetic resonance imaging (MRI) scans. This finding suggests a mechanism of action in which ultimate tumor regression is preceded by immune activation and tumor infiltration of activated lymphocytes. In addition, although formal regressions are induced in a relatively small proportion of patients and complete responses as assessed with CT or MRI are rare (