Ferroptosis: Mechanism and Diseases (Advances in Experimental Medicine and Biology, 1301) 3030620255, 9783030620257

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Advances in Experimental Medicine and Biology 1301

Andres F. Florez Hamed Alborzinia   Editors

Ferroptosis: Mechanism and Diseases

Advances in Experimental Medicine and Biology Volume 1301 Series Editors Wim E. Crusio, Institut de Neurosciences Cognitives et Intégratives d’Aquitaine, CNRS and University of Bordeaux UMR 5287,  Pessac Cedex, France Haidong Dong, Departments of Urology and Immunology,  Mayo Clinic, Rochester, MN, USA John D. Lambris, University of Pennsylvania, Philadelphia, Pennsylvania, USA Heinfried H. Radeke, Institute of Pharmacology & Toxicology, Clinic of the Goethe University Frankfurt Main, Frankfurt am Main, Hessen, Germany Nima Rezaei, Research Center for Immunodeficiencies, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran Ortrud Steinlein, LMU University Hospital, Institute of Human Genetics, Munich, Germany Junjie Xiao, Cardiac Regeneration and Ageing Lab, Institute of Cardiovascular Sciences, School of Life Science, Shanghai University, Shanghai, China

Advances in Experimental Medicine and Biology provides a platform for scientific contributions in the main disciplines of the biomedicine and the life sciences. This series publishes thematic volumes on contemporary research in the areas of microbiology, immunology, neurosciences, biochemistry, biomedical engineering, genetics, physiology, and cancer research. Covering emerging topics and techniques in basic and clinical science, it brings together clinicians and researchers from various fields. Advances in Experimental Medicine and Biology has been publishing exceptional works in the field for over 40 years, and is indexed in SCOPUS, Medline (PubMed), Journal Citation Reports/Science Edition, Science Citation Index Expanded (SciSearch, Web of Science), EMBASE, BIOSIS, Reaxys, EMBiology, the Chemical Abstracts Service (CAS), and Pathway Studio. 2019 Impact Factor: 2.450 5 Year Impact Factor: 2.324

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Andrés F. Florez  •  Hamed Alborzinia Editors

Ferroptosis: Mechanism and Diseases

Editors Andrés F. Florez Department of Molecular and Cellular Biology Harvard University Cambridge, MA, USA

Hamed Alborzinia Division of Stem Cells and Cancer German Cancer Research Center (DKFZ) and Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM) Heidelberg, Germany

ISSN 0065-2598     ISSN 2214-8019 (electronic) Advances in Experimental Medicine and Biology ISBN 978-3-030-62025-7    ISBN 978-3-030-62026-4 (eBook) https://doi.org/10.1007/978-3-030-62026-4 © Springer Nature Switzerland AG 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Foreword

Twenty years ago, cell death was a simpler field. Apoptosis was the only cell death process known to be regulated by a specific biochemical mechanism, and the induction of apoptosis was thought to explain the vast majority of programmed and pathological cell death in vivo. But since that time, it has become abundantly clear that, in addition to apoptosis, cells can die in response to the activation of several non-apoptotic cell death mechanisms, including necroptosis, pyroptosis, and ferroptosis. The existence of multiple distinct non-apoptotic cell death pathways raised innumerable fundamental questions and may explain the previously surprising observations that, when examined closely, inactivating the apoptotic machinery does not completely suppress cell death in development or prevent pathological cell death in vivo. Ferroptosis is an especially unique cell death mechanism in several ways. First, as the name implies, and unlike other cell death mechanisms, it is characterized by an essential role for iron – a role that still remains poorly defined. Second, unlike apoptosis and other known non-apoptotic cell death processes, ferroptosis is not defined by specific protein effectors. Rather, ferroptosis is fundamentally a metabolic process, characterized by the iron-dependent overaccumulation of toxic lipid peroxides. Third, ferroptosis is not an active process that absolutely requires the transcriptional or post-translational induction or modification of a specific death effector in response to lethal stimulation. Rather, ferroptosis has been referred to as a process of cell sabotage, or cellular imbalance, where disruption of the normally active intracellular antioxidant machinery allows for a runaway accumulation of lipid peroxides through the normal actions of the cell. This biochemical uniqueness, together with the evident importance of ferroptosis in vivo, has heightened interest on how this pathway operates and how this pathway could be turned on or off in different medical scenarios. Collectively, the chapters in this book provide a detailed introduction and overview of ferroptosis and describe recent progress in several important areas. Pouyssegur and colleagues describe how targeting system x?c may provide a new therapeutic avenue for the treatment of pancreatic cancer via the induction of ferroptosis. Venkataramani, as well as Mancias and colleagues, delves into the still mysterious role of iron in the ferroptotic mechanism, and how iron storage and recycling pathways play a critical role in governing ferroptosis sensitivity and execution. Amaral and Namasivayam discuss the emerging evidence that ferroptosis has a role to play in infectious disease, a fact that may be especially surprising given that apoptosis, necroptosis, and v

Foreword

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pyroptosis are already known players in this area. Ferroptosis was initially described in 2012 using a variety of small molecule tools to activate or inhibit this process, and as described by Rodriguez and colleagues our ability to modulate ferroptosis continues to expand in various ways. Finally, as noted above, ferroptosis is not the only type of non-apoptotic cell death to be described, and Zanetti and Weinlich describe how necroptosis represents yet another biochemically distinct pathway. As implied by this final chapter, a major question awaiting detailed elucidation is how different non-apoptotic cell death modalities contribute to complex pathological phenotypes in vivo. This book provides a solid foundation in ferroptosis and other non-apoptotic cell death pathways and establishes a solid foundation for future studies in these emerging and important areas. Department of Biology, Stanford, CA, USA August 2020

Scott J. Dixon

Contents

1 Ferroptosis: Concepts and Definitions����������������������������������������    1 Andrés F. Flórez and Hamed Alborzinia 2 Overcoming Therapeutic Challenges for Pancreatic Ductal Adenocarcinoma with xCT Inhibitors ����������������������������    7 Milica Vucetic, Boutaina Daher, Shamir Cassim, Scott Parks, and Jacques Pouyssegur 3 Iron Homeostasis and Metabolism: Two Sides of a Coin ����������   25 Vivek Venkataramani 4 The Role of NCOA4-Mediated Ferritinophagy in Ferroptosis����������������������������������������������������������������������������������   41 Naiara Santana-Codina, Ajami Gikandi, and Joseph D. Mancias 5 Emerging Role for Ferroptosis in Infectious Diseases����������������   59 Eduardo Pinheiro Amaral and Sivaranjani Namasivayam 6 Small Molecule Regulators of Ferroptosis����������������������������������   81 Sylvain Debieu, Stéphanie Solier, Ludovic Colombeau, Antoine Versini, Fabien Sindikubwabo, Alison Forrester, Sebastian Müller, Tatiana Cañeque, and Raphaël Rodriguez 7 Necroptosis, the Other Main Caspase-Independent Cell Death ��������������������������������������������������������������������������������������  123 Larissa C. Zanetti and Ricardo Weinlich Index��������������������������������������������������������������������������������������������������������  139

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About the Editors

Andrés  F.  Flórez  is currently a postdoctoral fellow in the Department of Molecular and Cellular Biology (MCB) at Harvard University. His research is focused in understanding the oncogene-based mechanisms regulating ferroptosis and to use this knowledge for designing new therapeutic strategies. He is also interested in the role of ferroptosis in controlling bacterial cell populations. Hamed Alborzinia  is a postdoctoral fellow in the Division of Stem Cells and Cancer at the German Cancer Research Center (DKFZ). He also belongs to the Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM) in Heidelberg, Germany. His main research focus is to understand the metabolic properties of cancer cells in connection to novel forms of cell death, including ferroptosis.

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Ferroptosis: Concepts and Definitions Andrés F. Flórez and Hamed Alborzinia

Abbreviations ACSL4

Acyl-CoA Synthetase Long Chain Family Member 4 FSP1 Ferroptosis Suppressor Protein 1 GPX4 Glutathione Peroxidase 4 LPCAT3 L y s o p h o s p h a t i d y l c h o l i n e Acyltran‑sferase 3 NCOA4 Nuclear Receptor Coactivator 4 NLR NOD-Like Receptor PUFAs Polyunsaturated Fatty Acids SLC7A11 Solute Carrier Family 7, Member 11 TfR1 Transferrin Receptor Protein 1

“Life had to invent death to evolve” (Dyson 1999). As a result, multicellular organisms have developed active cell death mechanisms to control their development. Limiting the number of cells spatially and temporarily while actively triggering cell death gives rises to full body programs (Tyson and Novak 2014; Elmore 2007). A. F. Flórez (*) Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA H. Alborzinia Division of Stem Cells and Cancer, German Cancer Research Center (DKFZ) and Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), Heidelberg, Germany

Apoptosis is one of the most studied forms of regulated cell death, mainly due to its role in cancer pathogenesis. It functions through signalling cascades leading to caspase activation; proteases in charge of the final destruction of the cell (Elmore 2007). This destruction is clean and contained, all components of the cell once transformed into apoptotic bodies are consumed by the immune system leaving no trace. Despite being a highly controlled and potent cell death mechanism, apoptosis is not an exclusive form of cell death and other forms have been discovered in the last decades. In the early 1990s, the mechanisms underlying autophagy were discovered in yeast (Takeshige et al. 1992), and it requires signal activation similar to apoptosis with resulting formation of autophagosomes and their delivery to lysosomes digesting big portions of cytoplasm (Gump and Thorburn 2011; Mizushima 2018; Yu et  al. 2018). Autophagy and apoptosis have helped explain how organisms maintain tissue homeostasis, and the discoveries behind led to Nobel prize winners in 2002 and 2016 (Levine and Klionsky 2017). More recently, new forms of cell death were discovered with less clear homeostatic roles. Necroptosis (Chap. 7), like apoptosis and autophagy, depends on the activation of a signalling cascade. When apoptosis is inhibited, necroptosis is triggered in response to tissue injury that leads to formation of membrane pores and ultimately cell death. Interestingly, necroptosis seems dispens-

© Springer Nature Switzerland AG 2021 A. F. Florez, H. Alborzinia (eds.), Ferroptosis: Mechanism and Diseases, Advances in Experimental Medicine and Biology 1301, https://doi.org/10.1007/978-3-030-62026-4_1

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able for embryo development (Choi et al. 2019). On the other hand pyroptosis “fiery death” (McKenzie et al. 2020) is activated in response to microbial infections and few other non-infectious stimuli. It involves the activation of proinflammatory caspases (caspase-1/4/5/11) not relevant for apoptosis, and similarly to necroptosis it appears dispensable for embryo development. NLR proteins undergo activation causing caspase proteolytic activation of gasdermins and formation of pores, causing rapid cell lysis (Bergsbaken et al. 2009; Frank and Vince 2019) (Chap. 5). In summary, apoptosis, autophagy, necroptosis and pyroptosis share a core mechanism of execution that involves a signal transduction pathway forming a proteinaceous pore that grants the scape of cytoplasmic contents causing cell lysis. In 2003, a small molecule named Erastin (Dolma et al. 2003) was found to induce a form of cell death that avoided the requirement for a signalling cascade or a protein pore at the membrane. Almost 10 years later, the mechanism for this cell death was described and coined as “ferroptosis” for its dependence on iron (Dixon et al. 2012). Ferroptosis can be thought as a more primitive form of cell death with membrane damage caused by lipid peroxidation. The lipid damage occurs by the conversion of H2O2 to highly reactive hydroxy radicals in the presence of iron as a catalyzer, a process called Fenton reaction (Gaschler and Stockwell 2017). The most important source of H2O2 is the mitochondria. It  is also  generated through activity of the cytoplasmic enzyme NADPH oxidase in conjunction with superoxide dismutases (Schieber and Chandel 2014) generating superoxide anion radical as a by-product. The mechanisms of how peroxidation of PUFAs leads to cell death is not well understood. A study using liposomes and mathematical modelling suggested (Agmon et al. 2018) that lipid peroxidation could lead to alteration of membrane curvature and generation of a lipid pore responsible for cell lysis. Recent studies have shown that iron dependent lipoxygenases can induce lipid peroxidation with labile iron mediating this reaction (Stockwell et  al. 2020) indicating that peroxidation is an active process. Iron causes lipid peroxides by reacting with H2O2

A. F. Flórez and H. Alborzinia

generating extremely reactive hydroxyl radicals, therefore it needs to be strictly regulated, usually via import, storage and extrusion (Chap. 3). The unstable, redox active Fe2+ (or labile iron) responsible for cytotoxic damage, is generated by reducing Fe3+ imported via TfR1 at the membrane (Han et al. 2020). In fact, TfR1 expression can be used as a marker of ferroptosis induction in some cell types (Feng et al. 2020). Once iron is in the cytoplasm, it is quickly stored in ferritin complexes or exported via ferroportin (Han et al. 2020). Iron release from ferritin is also regulated, and more recent evidence points to degradation of ferritin via NCOA4 as a mechanism to increase susceptibility to ferroptosis, a process called ferritinophagy (Chap. 4). Besides regulating iron metabolism, the cell carries different systems to both reduce the levels of reactive oxygen species and undo the damage caused by ROS. Of particular importance for ferroptosis are the glutathione peroxidase enzymes which help clearing peroxides while using glutathione (Dixon and Stockwell 2019). GPX4 is one the most relevant peroxidases in the context of ferroptosis, it is a selenoprotein essential for development and it has an important role in immunity and to prevent infertility and neurodegeneration (Stockwell et  al. 2020). GPX4 uses glutathione and converts it to the oxidized form (GSSG) while reducing lipid peroxides (L-OOH) to alcohol forms (L-OH). GPX4 inhibition triggers ferroptosis in carcinomas (Zou et al. 2019), prevents metastasis in melanomas (Ubellacker et al. 2020) and its currently an attractive target for drug development (Eaton et  al. 2020) and (Chap. 6). Although it is probably not the only enzyme responsible for preventing lipid peroxidation. More recently, FSP1 was demonstrated to prevent toxic peroxidation by regenerating reduced coenzyme Q10, thus working independently of glutathione. (Doll et al. 2019; Bersuker et al. 2019). Glutathione is critical for GPX4 activity, and pharmacological modulation of glutathione levels has been studied before the ferroptosis era as a therapeutic strategy to either induce cell death or protect cells tissues from oxidative injuries (Ballatori et al. 2009). GSH is synthesized from

1  Ferroptosis: Concepts and Definitions

the amino acid precursors: cysteine, glutamate and glycine, being cysteine the rate limiting step for glutathione synthesis. Cysteine is usually transported as cystine (the reduced form) via system xc- composed of two subunits, SLC7A11 and SLC3A2. Drugs such as erastin prevent cystine uptake by inhibiting this antiporter (Chap. 2). Other transporters such as SLC1A1 could serve as alternatives for cystine import (Hodgson et al. 2013) and it can be as well synthesized de novo by using methionine in a metabolic pathway named transsulfuration. Activation of this pathway might suppress ferroptosis induced by cystine deprivation (Hayano et  al. 2016). Special attention has been given to transsulfuration in the tumor context, where some tumors are more heavily dependent on transsulfuration enabling them to scape ferroptosis when cystine becomes limiting. (Zhu et al. 2019). Cancer appears as an attractive disease model to develop new ferroptosis inducers. Targeting cancer cells specifically is the most challenging and ultimate goal in oncology. In the novel Emperor of all Maladies, Siddhartha Mukherjee describes elegantly the desire for a targeted cancer therapy: Specificity refers to the ability of any medicine to discriminate between its intended target and its host. Killing a cancer cell in a test tube is not a particularly difficult task: the chemical world is packed with malevolent poisons that, even in infinitesimal quantities, can dispatch a cancer cell within minutes. The trouble lies in a finding a selective poison—a drug that will kill cancer without annihilating the patient. Systemic therapy without specificity is an indiscriminate bomb. For an anticancer poison to become a useful drug, Meyer knew, it needed to be a fantastically nimble knife: sharp enough to kill cancer yet selective enough to spare the patient. (Mukherjee 2010)

Ferroptosis inducers could become that nimble knife to eliminate cancer cells. The metabolic imbalances required to activate ferroptosis in cancer cells can be summarized into the so-called hallmarks of ferroptosis (Dixon and Stockwell 2019), in allusion to the hallmarks of cancer (Hanahan and Weinberg 2011). Activating these hallmarks could tilt the balance towards ferroptosis in cancer cells while sparing normal cells. On

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one hand iron, specifically the labile pool, could undergo imbalances via higher import, observed in several cancers through increased expression of TfR1 (Manz et al. 2016), or release from ferritin via ferritinophagy thus increasing sensitivity to ferroptosis. The PUFAs, substrates for peroxidation, are regulated at the level of unsaturation and tightly controlled via ACSL4 (Doll et  al. 2017) and LPCAT3 (Dixon et al. 2015). The last hallmark, ROS counteracting mechanisms, depend on glutathione biosynthesis via cystine import or transsulfuration, and selenium which is essential for proper GPX4 functioning. Activating these three hallmarks pharmacologically could open new therapeutic opportunities for cancer treatment, even for those whose treatments options are limited such as pancreatic cancer (Chap. 2). The mechanisms behind the hallmarks of ferroptosis are illustrated in Fig. 1.1. We hope this book serve as a guide to navigate the field of ferroptosis in the context of human diseases and the potential of this pathway to become a new source of therapies spanning a wide range of pathologies. Conflict of Interest  The authors declare no potential conflict of interest.

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Fig. 1.1  Hallmarks of ferroptosis (explained in the text). Cys cysteine, Cysta cystathionine, Hcy homocysteine, SAH S-adenosyl-l-homocysteine, SAM S-adenosyl-l-­ methionine, NAA neutral aminoacids, PUFA polyunsaturated fatty acid, PL phospholipid, 2-KG, 2-ketoglutaric acid, TfR1 transferrin receptor 1, GCL glutamate cysteine ligase, GLS2 glutaminase 2, GSS glutathi-

one synthetase, GSR glutathione reductase, CTH cystathionine gamma-lyase, CBS cystathionine-β-­ synthase, FSP1 ferroptosis suppressor protein, ACSL4 acyl-CoA synthetase long chain family member 4, LPCAT3 lysophosphatidylcholine acyltransferase 3, GPX4 glutathione peroxidase 4

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Overcoming Therapeutic Challenges for Pancreatic Ductal Adenocarcinoma with xCT Inhibitors Milica Vucetic, Boutaina Daher, Shamir Cassim, Scott Parks, and Jacques Pouyssegur

Abbreviations GPx4 Glutathione Peroxidase 4 GSH Glutathione FSP1 Ferroptosis Suppressor Protein 1 TFR1 Transferrin Receptor 1 EMT Epithelial-to-Mesenchymal Transition CSC Cancer Stem Cell 5-FU 5-Fluorouracil BSO Buthionine Sulfoximine CAFs Cancer-Associated Fibrob‑lasts CD98/4F2hc Chaperon Heavy-Chain Subu‑nit of the Transport System xc− ceCT contrast-enhanced Computed Tomography COXs Cyclooxygenases

M. Vucetic · B. Daher · S. Cassim Department of Medical Biology, Centre Scientifique de Monaco (CSM), MC, Monaco S. Parks Trev and Joyce Deeley Research Centre, BC Cancer, Victoria, BC, Canada Genome British Columbia Proteomics Centre, University of Victoria, Victoria, BC, Canada J. Pouyssegur (*) Department of Medical Biology, Centre Scientifique de Monaco (CSM), MC, Monaco University Côte d’Azur, (IRCAN), CNRS, INSERM, Centre A. Lacassagne, Nice, France e-mail: [email protected]

CTLA-4

Cytotoxic T-Lymphocyte-­ Associated Protein 4 CYP450s Cytochrome p450s CySSCy Oxidized form of Cysteine (Cystine) DFO Deferoxalmine DMT1 Divalent Metal Transporter 1 ER Endoplasmic Reticulum 18 FDG F-fluorodeoxyglucose FSPG (4S)-4-(3-18F-Fluoropropyl)L-Glutamate H2O2 or ROOH Hydrogen or Organic perox‑ides INF-γ Interferon-γ LOH Lipid alcohols LOOH Lipid peroxides LOXs Lipoxygenases MET Mesenchymal-to-Epithelial Transition mTORC1 Mechanistic Target of Rapa‑mycin Complex 1 NAC N-Acetyl-Cysteine PanIN lesion Non-Invasive Intraepithelial Neoplasia PD1 Programmed Associate Pro‑tein 1 PD-1L PD1 Ligand PDAC Pancreatic Ductal Adenocarcinoma PE Phosphatidylethanolamine

© Springer Nature Switzerland AG 2021 A. F. Florez, H. Alborzinia (eds.), Ferroptosis: Mechanism and Diseases, Advances in Experimental Medicine and Biology 1301, https://doi.org/10.1007/978-3-030-62026-4_2

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PEG-PLGA NP

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Polyethylene Glycol-­­ or chemotherapy with radiotherapy, used in neoP o l y ( L a c t i c -­c o -­G l y c o l i c adjuvant and/or adjuvant settings; however, the Acid) Nanoparticle outcomes of patients with pancreatic cancer have PPAR-γ Peroxisome Proliferator-­ been changed only modestly (Garrido-Laguna Activated Receptor-γ and Hidalgo 2015) (Table 2.1). Nowadays, single RSL3 RAS-selective Lethal 3 agent  – gemcitabine, which was shown to be SHH Sonic Hedgehog superior to 5-FU in 1997 (Burris et al. 1997), is TGF-β Tumour Growth Factor-β the standard-of-care treatment for the patients xCT Light Chain of the Transport with metastatic PDAC. Modest improvement in System xc− survival has been recently achieved with the β-ME β-Mercaptoethanol combination of gemcitabine and nab-paclitaxel, as well as with FOLFIRINOX (folinic acid, 5-FU, irinotecan, and oxaliplatin) (Von Hoff et  al. 2013; Conroy et  al. 2011). Clearly new 2.1 Pancreatic Cancer treatment strategies are urgently needed for both resectable and nonresectable PDAC. Therapeutic Challenges Couple features of the PDAC add up and make Pancreatic cancer, 90% of which makes pancre- progress in development of adequate treatment atic ductal adenocarcinoma (PDAC), represents rather slow and difficult, and consequently, result one of the most aggressive and deadliest malig- in dismal prognosis for the PDAC patients. These nancies with a 5-year survival rate of approxi- include: (1) early stages of pancreatic cancer are mately 5% (Kalser and Ellenberg 1985). It has usually asymptomatic making early diagnosis been estimated that PDAC will surpass breast, extremely difficult especially without any effecprostate and colorectal cancer by 2030, and tive screening tests for this type of cancer, (2) become second leading cause of cancer-related preclinical data suggest that pancreatic cancer is deaths in the USA (Rahib et  al. 2014). Despite systemic disease even at its inception suggesting great efforts of the scientists and clinicians dur- that systemic treatment is required even in the ing past decades to improve diagnostic strategies, case of localized disease (Rhim et al. 2012), (3) surgical procedures and chemotherapeutic regi- unique desmoplastic response of pancreatic stromens, the overall prognosis of PDAC patients mal compartment interferes with drug delivery remains alarmingly poor. The statistic reflects from one side (Hwang et al. 2008) but from the wide range of the cause; however, still one of the other it provides the tumour-restraining effect main problems regarding PDAC is lack of the (Rhim et al. 2014; Lee et al. 2014), (4) low antiadequate treatment and/or resistance to the con- genicity of pancreatic cancer might explain why ventional chemotherapeutic approaches. success of immune therapy has not been transAlthough surgery resection represents the lated to its treatment (Winograd et  al. 2015). only therapy offering long-term survival for Highly promising therapeutic approach in patients with PDAC, 5-year survival rate even difficult-­to-treat tumour types is identification of with these patients remains very low (15–20%) leading and actionable oncogenic driver, such as (Oettle et  al. 2007) suggesting a need for (neo) BRAF V600E mutation in melanoma (Chapman adjuvant chemo(radio)therapy. In 1985, the et  al. 2011). However, activating KRAS mutaresults of the GITSG trial showed beneficial tion, found in >90% cases of PDAC (Almoguera effect of adjuvant 5-fluorouracil [5-FU] chemo- et  al. 1988), is traditionally considered undrugtherapy plus radiotherapy followed by 2-year gable due to extremely high affinity of RAS pro5-FU treatment in comparison with observation teins for GTP. To circumvent this problem, efforts only (Kalser and Ellenberg 1985). Since that have been made to target downstream compotime, plenty of clinical trials had been undertaken nents of the RAS pathway, such as RAF or MEK with different combination of chemotherapeutics protein kinases. Unfortunately, clinical trials with

2  Overcoming Therapeutic Challenges for Pancreatic Ductal Adenocarcinoma with xCT Inhibitors

9

Table 2.1  List of successful (resulted in new FDA-approved treatment regiments) and on-going (under review) stage II/III clinical trials in pancreatic ductal adenocarcinoma Regimen Observation 5-fluorouracil/radiotherapy 5-fluoruracil Gemcitabine Gemcitabine Gemcitabine/erlotinib Gemcitabine FOLFIRINOX Gemcitabine Gemcitabine/nab-paclitaxel Gemcitabine gemcitabine/rigosertib (ON 1910. Na) 5-fluorouracil/folinic acid Onivyde (irinotecan) 5-fluorouracil/folinic acid/onivyde Gemcitabine/nab-paclitaxel Gemcitabine/nab-paclitaxel/ anti-PDL1/anti-CTLA4 Gemcitabine Gemcitabine/TH-302 Gemcitabine/nab-paclitaxel Gemcitabine/nab-paclitaxel/ ibrutinib Gemcitabine Gemcitabine/bevacizumab 5-fluorouracil 5-fluorouracil/cis-platin

Number of patients

Median overall survival (months)

Progression-free survival (months)

43

Not reported

342

11 20 4.41 5.65 5.91 6.24 6.8

0.92 2.33 3.55 3.75 3.3

861

11.1 6.7

6.4 3.7

8.5 6.4 6.1

5.5 3.4 3.4

4.2 4.9 6.1 NA

1.5 2.7 3.1 NA

Wang-Gillam et al. (2016) NCT01494506 Data not published NCT02879318

430

7.6 8.9 10.78 vs 9.69

3.7 5.5 6.01 vs 5.32

Data not published NCT01746979 Data not published NCT02436668

590

NA

NA

200

NA

NA

Data under review No data given Data under review No data given

126 569

160

417

180

693

Author(s) (year) Clinical trial ID Kalser and Ellenberg (1985) Burris et al. (1997) Moore et al. (2007) NCT00026338 Conroy et al. (2011) NCT00113658 Von Hoff et al. (2013) NCT00844649 O’Neil et al. (2015) NCT01360853

NA – Not Applicable

sorafenib (FDA-approved RAF inhibitor) and trametinib (FDA-approved MEK inhibitor) did not show any benefits in patients with advanced PDAC (Infante et al. 2013; Cascinu et al. 2014). Elegant work from Stockwell’s group at Columbia University and collaborators in the period from 2003–2008 identified compounds activating iron-dependent, non-apoptotic cell death in oncogenic-RAS-harboring cancer cells (Yang and Stockwell 2008; Yagoda et  al. 2007; Dolma et  al. 2003) by using synthetic lethal screening strategy (Hartwell et al. 1997). In 2012, the term ferroptosis was coined to describe this form of cell death induced by the small molecule erastin (Dixon et  al. 2012), which irreversibly

inhibits the uptake of oxidized form of cysteine (cystine, CySSCy) from extracellular space via xCT transporter (Sato et al. 2018). This erastin-­ mediated cystine import inhibition lead consequently to glutathione depletion, followed by inactivation of the glutathione peroxidase 4 (GPx4). GPx4 is a unique Se-cysteine enzyme that converts toxic lipid hydroperoxides (LOOH) into non-toxic lipid alcohols (LOH) (Ursini et al. 1982). GPx4 inactivation results in overwhelming LOOH accumulation, and ultimately, cell death (depicted in Fig.  2.1). In this review, we summarize literature data dealing with ferroptosis induction in PDAC, point out the current problems in the treatment (late diagnosis, early

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Fig. 2.1  Ferroptosis overview (explained in the text). cystine, CySSCy; cysteine, CySH; glutathione, GSH; glutathione peroxidase 4, GPx4; hydrogen/organic peroxides, H2O2/ROOH; polyunsaturated fatty acids, PL-PUFAs; phosphatidyl-ethanolamine, PE; lipid peroxides, PL-PUFA-OOH; transferrin receptor 1, TFR1; diva-

lent metal transporter 1, DMT1; cyclooxygenases, COXs; cytochrome p450s, CYP450s; lipoxygenases, LOXs; lipid alcohols, PL-PUFA-OH; buthionine sulfoximine, BSO; RAS-selective lethal 3, RSL3; N-acetyl-cysteine, NAC; β-mercaptoethanol, β-ME; deferoxamine, DFO; ferroprosis suppressor protein 1, FSP1

metastatic potential, low antigenicity, desmoplastic response of stromal compartment) that could be overcame with ferroptosis inducers, but also emphasize possible unwanted resistant mechanisms which has yet to be investigated in more details.

et al. 2013). One of the main roles of CySH in the cell, besides its role in constitution of the amino acid backbone of the proteins, is synthesis of glutathione (GSH). GSH is the most important non-­ enzymatic antioxidant component composed of cysteine, glutamic acid and glycine. It serves as co-substrate for many antioxidant enzymes, including glutathione peroxidase 4 (GPx4) (Brigelius-Flohe and Maiorino 2013). In the context of ferroptosis, the GPx4 plays irreplaceable role as neutralizer of oxidative damage in the membrane compartments of the cell. In the presence of ‘labile’ Fe2+ ions (Fenton reaction), oxidants such as hydrogen or organic peroxides (H2O2 or ROOH) attack membrane polyunsaturated fatty acids, such as phosphatidylethanolamine (PE), converting them to highly toxic LOOH. Due to its high redox potency, the level of iron in the cell is kept under tight regulation. Extracellular Fe3+, bound to transport protein

2.2

Ferroptosis and PDAC

Basic mechanisms underlying ferroptosis have been schematically depicted in the Fig  2.1. Oxidised form of cysteine (cystine, CySSCy) is the dominant form of amino acid cysteine (CySH) in vivo and, in most cases, the only form of this amino acid in in vitro conditions (Brigham et al. 1960; Droge and Kinscherf 2008; Bannai et  al. 1989). Under basal conditions, cancer cells take up CySSCy via xCT transporter, reduce it and use it for many different purposes (Lewerenz

2  Overcoming Therapeutic Challenges for Pancreatic Ductal Adenocarcinoma with xCT Inhibitors

transferrin, is taken up by cells via transferrin receptor 1 (TFR1), transported into endosome where it undergoes reduction to Fe2+ by metalloreductases (Knutson 2017). Divalent metal transporter 1 (DMT1) mediates the transport of Fe2+ from the endosome into the cytoplasmic labile iron pool, where most of it is ligated by heme, bound in FeS clusters, or stored in the iron storage protein ferritin (See Chap. 3). However, small amount of free and catalytically active Fe2+ ions is present in the cytoplasm coming either directly from endosomes or from autophagic degradation of ferritin in the process known as ferritinophagy (See Chap. 4) (Hou et al. 2016). In addition to being formed through non-­ specific propagation of radicals, oxidized lipids can also be synthesized in an enzymatically-­ regulated manner by cyclooxygenases (COX), cytochrome p450s (CYP450s), and lipoxygenases (LOX). Under basal conditions, the level of LOOH is controlled by the action of GPx4 enzyme. This Se-protein uses reducing power of GSH to covert toxic LOOH into non-toxic LOH, protecting membrane and cellular integrity (Ursini et al. 1982). However, blocking CySSCy import via xCT (erastin) (Dolma et  al. 2003), synthesis of GSH (buthionine sulfoximine, BSO) or GPx4 activity (RAS-selective lethal 3, RSL3) (Yang et  al. 2014), antioxidant balance within the cell is disturbed and accumulation of lipid peroxide leads to the loss of membrane assembly, composition, structure, dynamics, and finally cell death (ferroptosis). This type of the cell death is classified under ‘regulated’ forms of cell death as some compounds can specifically prevent it. N-acetyl-cysteine (NAC) serves as alternative donor of cysteine to the cell in the conditions where xCT is blocked (Daher et  al. 2019). Also, β-mercaptoethanol (β-ME) shifts balance toward reduced form of cysteine in the extracellular space (Ishii et al. 1981a; Ishii et al. 1981b), which then can enter the cells bypassing xCT.  Vitamin E (tocopherol α) is hydrophobic antioxidant that can approach to the membrane compartment and stop chain-formation of LOOH (Kagan et al. 2017), while deferoxamine (DFO) chelate ‘labile’ metal irons including Fe2+ (Dixon et al. 2012).

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In the initial report from 2003, Stockwell’s group identified genotype-selective antitumour agent that becomes lethal to tumour cells only in the presence of RAS oncogenes and they named it erastin, for ‘eradicator of RAS and small T (ST) oncoproteins-expressing cells’ (Dolma et al. 2003). In this initial work, erastin has been discovered as HRAS-selective lethal compounds; however, later studies showed that the cells carrying mutated KRAS oncogene, including pancreatic cancer cell lines, show similar sensitivity toward erastin (Yang and Stockwell 2008; Yagoda et  al. 2007). In the same report, system xc− has been identified as the key target of erastin action. System xc− is a Na+-independent and Cl−dependent exchanger of the anionic forms of cystine and glutamate. It is a disulfide-linked heterodimer composed of the transporter light-­ chain (xCT, encoded by SLC7A11 gene) and a chaperone heavy-chain subunit (CD98hc aka 4F2hc, encoded by SLC3A2 gene) (Lewerenz et  al. 2013). Controlling cystine uptake, xCT directly regulates the intracellular cysteine pool (conditionally-essential, proteinogenic amino acid), but also glutathione (GSH)  – the major, evolutionary conserved, non-enzymatic antioxidant component (Bannai and Tateishi 1986; Sasaki et  al. 2002). The importance of xCT-­ mediated cystine import for cancer cells growth, redox balance, and survival in particular in in vitro conditions, has been recognized for a long time (Bannai 1986; Combs and DeNicola 2019; Bannai and Tateishi 1986; Vucetic et  al. 2017); however, the exact nature of cystine deprivation-­ induced cell death has remained elusive until recent contextualization of ferroptosis. About the same time when erastin was described as RAS-­ specific lethal compound, Lo and colleagues showed that pancreatic cancer cell lines rely on the system xc−-dependent cystine uptake for growth, as well as for development of gemcitabine resistance (Lo et  al. 2008). A recent study discovered connection between RAS-­ mutated genotype and xCT-mediated cystine uptake. Namely, it has been revealed that the ETS-1 transcription factor downstream of the RAS-RAF-MEK-ERK signalling cascade directly activates the xCT promoter in synergy

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with the ATF4 transcription factor (Lim et  al. 2019). Our group using CRISPR-Cas9 genetic approach showed that genetic invalidation of xCT transporter leads, not only to growth arrest, but inevitably to ferroptosis of PDAC cells in vitro, unless alternative donor of cysteine (such as N-acetyl-cysteine) has been added (Daher et  al. 2019). In accordance with the data of Lo and colleagues who showed correlation between gemcitabine resistance and expression profile of xCT, we demonstrated that erastin-mediated xCT inhibition potentiates cytotoxic effects of both gemcitabine and cis-platin in pancreatic cancer cells. This synergistic effect of xCT inhibition and chemotherapy has also been described in the case of cis-platin resistant ovarian cancer cells (Sato et al. 2018).

2.2.1 xCT-Based Ferroptosis Inducers as Systemic Therapeutics for PDAC Data suggesting PDAC dissemination as very early event (will be discussed in more details later in the chapter) point out the need for systemic therapy even for localized form of the disease. Although GPx4, as a gatekeeper of ferroptosis sounds like an obvious target for this, mounting amount of findings challenge use of GPx4 inhibitors in systemic manner. Pharmacological and genetic inhibition of the GPx4 seems to inevitably lead to ferroptosis in both cancerous and non-cancerous cell types. Also, genetic invalidation of GPx4 in mice is not compatible with life (Friedmann Angeli et  al. 2014). Fortunately, xCT-based ferroptotic inducers can be much better alternative, unless a quantitative window that distinguishes the sensitivity toward GPx4 inhibition between cancer and normal tissues is identified. Additionally, recent discoveries of (1) the alternative, GSH/ GPx4-indeendent ferroptosis suppressor pathway, that involves ubiquinol (Coenzyme Q10) and its regenerating protein (now known as ferroptosis suppressor 1  – FSP1) (Bersuker et  al. 2019; Doll et  al. 2019); (2) as well as deubiquitinases-­ dependent mechanism of the

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resistance to GSH collapse (Harris et  al. 2019), all suggest that this part of the ferroptosis-­ regulating axis might be dispensable in cancer. On the other side, resistant mechanism to xCT-­ inhibition to date still has not been reported. Even more, recent study points out additional roles of xCT-dependent cysteine availability, which are important for the process of ferroptosis but independent of GSH biosynthesis. Namely, by using mass spectrometry analysis of radiolabeled cysteine Badgley and coworkers showed that cysteine, besides incorporation into GSH, is incorporated into the Coenzyme-A via pantothenate pathway. Coenzyme-A, by its side is involved in lipid metabolism and ubiquinol biosynthesis, suggesting its involvement into the GSH-independent mechanism of ferroptosis (Badgley et al. 2020). Great advantage of targeting xCT transporter for PDAC treatment is its rather restricted expression in vivo, with brain, spinal cord, and lymphoid organs as the primary tissues with constitutive xCT expression (Kim et  al. 2001). xCT mRNA and protein were also found to be present in the pancreas (Kim et al. 2001; Lo et al. 2008). On the other side, upregulated expression of xCT has been detected virtually in almost all tumour types, including pancreatic cancer tissues (Lo et  al. 2008). Furthermore, xCT knockout mice are viable, healthy in appearance and fertile, although oxidative shift of the plasma cystine/cysteine redox balance, characteristic of aging (Jones et al. 2002; Hack et al. 1998), cancer patients and people infected with HIV or smokers (Hack et  al. 1997, 1998; Moriarty et al. 2003), was observed (Sato et  al. 2005). These results suggest that potential systemic administration of specific xCT inhibitor could be used without any or with manageable side effects. Unfortunately, somewhat discouraging results came from clinical trial on glioma with xCT inhibitor – sulfasalazine, which failed due to the lack of response and severe side effects (Robe et al. 2006, 2009). Possible explanation for this outcome lies in the fact that preclinical studies on already FDA-­approved drugs used in different contexts, such as sulfasalazine (initially approved for use for Crohn’s disease and rheumatoid arthritis) are rather underestimated.

2  Overcoming Therapeutic Challenges for Pancreatic Ductal Adenocarcinoma with xCT Inhibitors

Although sulfasalazine has been recognized as inhibitor of xCT (Gout et al. 2001), our study univocally showed that it lacks specificity (Daher et al. 2019). On the other side, erastin, although not devoid of side effects at high concentrations (32), seems to be much more specific toward xCT (Daher et al. 2019; Sato et al. 2018; Dixon et al. 2012), and recently a great progress has been made with novel formulation of the drug (such as imidazole keton erastin) and delivery systems (such as polyethylene glycol-poly(lactic-co-glycolic acid) nanoparticule, PEG-PLGA NP), which showed promising results in diffuse large B cell lymphoma xenograft model (Zhang et al. 2019). However, it still remains to be examined if this or other new formulations of erastin will show consistent effects in the preclinical models before they get selected for clinical trials. It is worth noting here that some other compounds proved to induce ferroptosis in PDAC in in vitro settings. These include: natural products, such as biologically active alkaloid largely existing in the long pepper – piperlongumine, and cotylenin A, diterpenoid produced by plant-pathogenic fungi; as well as anti-malarial  – artesunate (Yamaguchi et al. 2018; Eling et al. 2015). In each case, ferroptotic effects of the molecules were restricted toward PDAC cells (lipid peroxidation, cell death) and reverted by ferroptosis inhibitors such as ferrostatin-1.

2.2.2 x  CT-Based Early Detection of Metastasized PDAC As mentioned previously, one of the main problems in PDAC treatment are asymptomatic early stages of pancreatic cancer, and consequently, late diagnosis of the disease. This issue is of particular importance as, according to elegant work from Rhim and colleagues (Rhim et al. 2012), it has been demonstrated that pancreatic epithelial cells have the capacity to seed the liver unexpectedly early, at the point when primary tumour still cannot be detected in the pancreas by rigorous histologic analysis. These early metastatic lesions are very difficult to detect by existing techniques (Sperti et al. 1997; Cheng et al. 2019).

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Newly developed positron emission tomography (PET) tracer (4S)-4-(3-18F-Fluoropropyl)-L-­ glutamate (FSPG), designed to detect xCT activity (Baek et al. 2012; Koglin et al. 2011) has been used for imaging brain, breast, lung and liver cancers with modest success (Baek et  al. 2012, 2013; Kavanaugh et al. 2016; Mittra et al. 2016; Mosci et al. 2016). On the other side, FSPG PET has been shown to be effective in preclinical and clinical settings for detection of PDAC metastasis in liver that are undetectable by contrast-­enhanced computed tomography (ceCT) or even 18F-fluorodeoxyglucose (FDG) (Cheng et  al. 2019). A possible explanation for these promising results comes from the fact that xCT expression is detectable in normal pancreas and increased in pancreatic cancer (Lo et  al. 2008), but not in organs which are initial sites for PDAC metastasis, such as liver (Bassi et al. 2001; Kim et al. 2001). Additional benefit from FSPG may come from indirect measurement of cancer response to chemotherapy, as increased xCT expression/activity correlates with increased resistance of PDAC to chemotherapy (Lo et  al. 2008). Further studies with more robust cohorts are required to prove these rather encouraging results.

2.3

 etabolic Status Dictates M Sensitivity Toward Ferroptosis

Approximately 70% of patients who undergo resection of small pancreatic tumours with R0 surgical margins, and no sign of metastasis (based on the available techniques), experience hepatic recurrence of the disease within 10 months postoperatively (Neoptolemos et  al. 2004). This clearly supports the hypothesis by which the seeding of distant organs represents an early event of PDAC development, sometimes even before primary tumours can be detected by standard histological examination (Rhim et al. 2012). Indeed, metastatic PDAC has been documented in the cohort of patients with chronic pancreatitis, where histological analysis of pancreas revealed only non-invasive intraepithelial neoplasia

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(PanIN lesions) (Sakorafas and Sarr 2003). High metastatic potential of cancer cells is tightly associated with epithelial-to-mesenchymal transition (EMT), which allows cancer cells to detach from the tissue of origin, acquire invasive properties and seed distant tissue/organs, where inverse process (MET) and settlement takes place. Although the role of EMT in cancer biology has been proven many times in vitro, its relevance in  vivo remained controversial until recently when epithelial linage tracing in a mouse model allowed final proof for EMT preceding invasion and dissemination of carcinoma cells (Rhim et al. 2012). Highly mesenchymal phenotype, not only allows invasion and dissemination of cancer cells, but also provides elevated resistance to several therapeutic regimens (Shibue and Weinberg 2017), and tumour- initiating state sometimes termed the cancer stem cell (CSC) state (Mani et al. 2008; Morel et al. 2008). Due to high selective pressure of EMT, the success rate of metastasis is extraordinarily low from one side (Gupta et al. 2005); but from another a high-­ mesenchymal state of human cancer cells is associated with acquired resistance to many different insults, including chemotherapy (Shibue and Weinberg 2017). That is why recent report of Viswanathan and colleagues came as a surprise. Namely, the authors showed that therapy-resistant high-­mesenchymal state of cancer cells are extremely sensitive to ferroptosis inducers and depend on xCT-GSH-GPx4-axis that protects against it (Viswanathan et al. 2017). These data pointed out specifically ZEB1-driven mesenchymal phenotype as the most sensitive toward GPx4 inhibition (Viswanathan et  al. 2017). Interestingly, it has been shown that ZEB1 plays a key role in the formation of precursor lesions, invasion and dissemination in pancreatic cancer model (KPC model) (Krebs et  al. 2017). Accordingly, results from our group showed similar differences in sensitivity between two pancreatic cell lines: MiaPaCa-2 being highly mesenchymal and sensitive even at low concentration of erastin (0.5 μM) and Capan-2 being epithelial and less sensitive to this ferroptosis

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inducer (Daher et al. 2019). However, induction of EMT in Capan-2 by tumour growth factor-β (TGF-β) induced increased expression of ZEB1, strongly decreased expression of epithelial marker E-cadherin and set sensitivity of these cells to the same level as in MiaPaCa-2 (Daher et al. 2019). Molecular mechanisms underlying high sensitivity of ZEB1-driven mesenchymal states toward ferroptosis are still underexamined. It is proposed that this might involve direct transcriptional regulation of peroxisome proliferator-activated receptor gamma (PPARγ, master regulator of lipid metabolism) by ZEB1. PPARγ signalling could stimulates uptake, accumulation, mobilization of lipids, and consequently EMT-associated remodelling of plasma membrane, a key compartment affected by oxidative insults during ferroptosis (Viswanathan et  al. 2017). However, although direct evidence for this association are still lacking, activation of mechanistic target of rapamycin complex 1 (mTORC1) upon TGF-β treatment (Daher et al. 2019) might suggest that changes in lipid metabolism during EMT can be achieved through mTORC1 as it plays a central role in lipid metabolism (for further reading see (Ricoult and Manning 2013)). On the other side, prevention of lipid peroxide accumulation in xCT-KO mesenchymal cells observed upon treatment with protein synthesis inhibitor  – cycloheximide (Daher et  al. 2019) suggests that general metabolic remodelling upon EMT, including both lipid and protein synthesis, plays role in high sensitivity toward ferroptosis. Two observations from our group go in favour of “high sensitivity of highly-mesenchymal cell” hypothesis, which we would modify to “high sensitivity of highly-metabolically active/proliferative cells”: (1) epithelial cell phenotype susceptibility to ferroptosis should not be excluded neither neglected (Daher et  al. 2019), and (2) observation from in vitro clonal growth of epithelial Capan-2 cells (Fig. 2.2). The effects of erastin are preferentially and readily visible on the edges of the clones, which are generally seen as more proliferative, while interior of such dense, ‘proliferative silent’ clones were more “resistant”

2  Overcoming Therapeutic Challenges for Pancreatic Ductal Adenocarcinoma with xCT Inhibitors

Fig. 2.2  Contact inhibition of proliferation decreases sensitivity toward ferroptosis. Cell-to-cell contact activates the Hippo signaling cascade through NF2 tumour suppressor (also know as Merlin) that induces the MST1/MST2 complex to phosphorylate the LATS1/LATS2 complex that leads to the phosphorylation dependent inhibition of the transcriptional activity of Yes-associated protein 1 (YAP1) (Wu et al. 2019). Thus, in the epithelial cells with

(Fig.  2.2). More solid proof for this ­observation-­based hypothesis is a recent study showing that epithelial cells through E-cadherin suppress ferroptosis by activating the intracellular NF2 and Hippo signalling (Wu et  al. 2019). Activation of NF2-Hippo pathway leads to phosphorylation, nuclear exclusion and inactivation of the pro-­oncogenic transcription co-activator YAP, ultimately resulting in suppression of cell division and proliferation (Harvey et  al. 2013) (Fig.  2.2). Interference with NF2-Hippo axis allows activation of the pro-oncogenic YAP and upregulation of several ferroptosis modulators, thereby restoring sensitivity to this type of cell death (Wu et  al. 2019). Taking all these results together, it is possible to speculate that the ferroptosis inducers might be most efficient in the non-contact mesenchymal cells (metastatic cells), while in the epithelial, densely-packed tumour parts their effects weaken, although it is still not negligible. It is worth noting here that highly sensitive to ferroptosis are also drug-­ tolerable and apparently “quiescent” persister cells (Hangauer et al. 2017). Is NF2-Hippo axis is implicated in “quiescence” of these cells, remains yet to be examined.

15

high expression of E-cadherin activate Hippo pathway, which ultimately results in phosphorylation and nuclear exclusion of YAP transcriptional factor. This leads to suppressed proliferation and metabolic activity of the tumour cells, and consequently decreased sensitivity to ferroptosis induction. Contact-dependent resistance to ferroptosis is visible in the highly epithelial Capan-2 cells upon treatment with 1 μM erastin (micrograph on the right side)

2.4

Promoting Ferroptosis and Antigenicity in PDAC

Great promises in treatment of the different malignancies nowadays hold T-cell stimulating immunotherapies, such as immune checkpoint blockage. Checkpoint signals are part of the complex balance by which the activity of the T cells is regulated (Finn 2012). Accumulating literature data show that cancer cells can use the inhibitory signals from checkpoint molecules in order to suppress host immune response. The working hypothesis in the field starts with this premise and exploits the re-activation of the antitumour immune response by targeting (inhibiting) checkpoint molecules (for further reading see also (Wei et al. 2018)). However, in spite of spectacular clinical success, this type of therapy is still in its infancy that is reflected through low response rate (app 30%) even in sensitive tumour types (Brahmer et  al. 2012; Hodi et  al. 2010; Topalian et al. 2012). Two common checkpoint targets are: programmed associate protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-­ 4). Unfortunately, clinical

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study with monoclonal antibodies against PD-1 or PD-1 ligand (PD-1L) failed to show any response in pancreatic cancer patients, although PD-1L expression has been associated with poor prognosis in such patients (Nomi et  al. 2007). Similarly, a phase II study in 27 patients with advanced PDAC with ipilimumab, monoclonal antibody against CTLA-4, showed a delayed response in one patient only. The main reason for the immunotherapy ineffectiveness in patients with pancreatic cancer is still elusive. However, one possible explanation may be low immunogenic nature of pancreatic cancer cells (Winograd et al. 2015). The immunogenicity of tumour cells, which is prerequisite for recognition by the immune system (effector T cells) is, at least in part, reflected by the mutation profile. Thus, melanoma and lung cancers, which have approximately two times more somatic mutations in comparison with pancreatic, colon and breast cancers, should theoretically be more antigenic and thus more suitable for immunotherapy (Vogelstein et al. 2013). Interestingly, genetic invalidation of xCT in pancreatic and colon cancer cell lines enhanced the efficacy of CTLA-4 blockage in a mouse xenograft model (Arensman et al. 2019). These data go in favour of the mentioned hypothesis, as it has been proven that xCT inhibition induces ROS accumulation and intracellular cysteine depletion, both of which favour unfolded protein response, i.e. endoplasmic reticulum (ER) stress (Dixon et al. 2014). ER stress mediates the mitoxantron-induced exposure of calreticulin on the surface of the cells; a process that primes immune cells to attack tumours and is recognized as immunogenic cell death (Obeid et al. 2007; Panaretakis et al. 2009). The findings in the same study showed that systemic depletion of host xCT had no effect on T-cell proliferation in vivo, as well as on the generation of primary and memory anti-tumour immune responses (Arensman et al. 2019). These data are of great importance as previous reports showed that the proliferation and activation of human T cells in vitro are dependent of xCT-­ mediated cystine uptake (Levring et  al. 2015; Garg et al. 2011), suggesting that systemic inhibition of this transporter may have deleterious

effects on the immune response. However, although xCT is indeed required for T cell proliferation in vitro based on Arensman and colleagues’ data, in vivo, xCT seems dispensable for normal lymphoid and myeloid development (74). xCT−/− mice used in the study were viable, fertile and appeared healthy, as in the report of Bannai’s group from 2005 (Sato et al. 2005). Two important conclusions can be drown here: (1) systemic inhibition of xCT should be feasible without disturbing host (anti-tumour) immune response, (2) results obtained in vitro should be taken with caution, especially in the case of xCT-inhibition/ invalidation, and this issue will be covered in more detail in the following section.

2.5

 ow Stromal Compartment H Influences Sensitivity to Ferroptosis

One of the most important features of pancreatic cancer is the presence of a very dense and reactive stromal compartment, which includes extensive deposition of extracellular matrix components, low vascularization, as well as recruitment and activation of cancer-associated fibroblasts (CAFs) (Sinha and Leach 2016; Kleeff et al. 2016). This so-called “desmoplastic response” is in constant communication with malignant cells through Sonic hedgehog (SHH) and CXC-motive chemokine receptors, allowing them to survive and thrive even in nutrient depleted conditions (Pickup et  al. 2014). Additionally, remodelling of extracellular matrix increases tissue stiffness, which from one side interferes with drug delivery, and from the other induces signalling pathways that promote progression and metastasis through mechanical pressure (Humphrey et  al. 2014; Kalluri 2016). Remodelling of the extracellular matrix in distant organ before seeding of metastatic cells occurs, i.e. formation of so-called pre-metastatic niches, has been suggested as a facilitating step for cancer cell invasion and growth in liver or lung (Hoffmann et al. 2008; Nukui et al. 2000; Tseng et  al. 2005). Preclinical models revealed that interference with the hedgehog pathway

2  Overcoming Therapeutic Challenges for Pancreatic Ductal Adenocarcinoma with xCT Inhibitors

enhanced gemcitabine delivery to the tumour and prolonged survival in mice (Yauch et  al. 2008; Olive et al. 2009). Also, intravenous injection of recombinant human hyaluronidase, an enzyme involved in degradation of extracellular matrix component  – hyaluronic acid, was associated with increased tumour vascularization and enhanced doxorubicin delivery in PDAC-derived neoplastic tissues in the KPC mouse model (Provenzano et  al. 2012). Unfortunately, these results could not be replicated in clinical settings, although the hyaluronidase-modified trial still holds the promise (JI 2012, June 4; Medicine 2014). The inconsistency between preclinical and clinical data is still unclear and many different factors could influence it, including recently suggested tumour-restraining effects of the stroma (Rhim et al. 2014). Some newly developed ferroptosis inducers seem to be highly potent in overcoming the problem of drug delivery to different cancers. Composite nanomaterial referred to as Cornell dots (C′ dots) (~6  nm surface-functionalized poly(ethylene glycol)-coated (PEGylated) near-­ infrared (NIR) fluorescent silica nanoparticles with diameters controllable down to the sub-­ 10 nm range) act as molecular ferroptosis inducers (FINs) via, according to the model, increased delivery of iron into cells. For the moment, ferroptosis induction by C′ dots has been confirmed in renal carcinoma and fibrosarcoma xenograft models (Kim et al. 2016), and if it will work in the case of pancreatic cancer, remains to be determined. The question that arises is – can stromal compartment influence sensitivity of tumour cells to ferroptosis? An interesting study of Wang and co-­ workers showed that T cells and fibroblasts, as major cellular components of tumour microenvironment, play crucial role in platinum-based chemoresistance in ovarian cancer through modulation of the GSH/cysteine release (Wang et al. 2016). Authors showed that ovarian cancer cells, although expressing xCT transporter, depend on the release of the cysteine from fibroblasts upon cis-platin treatment. According to the hypothesis, cysteine disulfide is taken up by fibroblasts via xCT transporter, reduced, and then

17

exported (as such or in the form of GSH tripeptide) into extracellular space where cancer cells can use it, these results might explained the in vivo tumour growth of highly in vitro sensitive xCT−/− pancreatic and colon cancer cells observed by our group and others (Daher et  al. 2019; Arensman et al. 2019). Namely, xCT−/− tumours show delayed growth when compared with their wild type counterparts in mice xenograft models; however, they still preserve the capacity to form tumours (Daher et  al. 2019; Arensman et  al. 2019). Evenmore, our recent study clearly showed that cystine-cysteine shuttle between xCT-/- and xCT-proficient cells of different origin prevents ferroptotic phenotype of the former even in in vitro conditions (Meira et al. 2021). These results clearly s­ uggests that some of the phenotypic characteristics observed in the culture with xCT inhibition or knockout, should be taken with caution, as the culturing media under 21% of oxygen contains almost exclusively oxidized form of cysteine, which in  vivo is not the case (Brigham et al. 1960; Droge and Kinscherf 2008). This is something that has been largely neglected in the literature, although it has been recognized 30 years ago by Bannai and colleagues (Bannai et al. 1989). The importance of cysteine and its transporters in  vivo, especially in conditions of xCT inhibition still remains to be elucidated. Although cysteine in  vivo might be provided from the blood, yet stromal fibroblasts are also good candidates, especially as xCT−/− tumours isolated from mice show high content of infiltrated fibroblasts (Daher et  al. 2019) (Fig.  2.3). More information about cysteine source in vivo could be provided by systemic inhibition of xCT in future studies. As previously introduced, not only fibroblasts but also stromal T cells can modulate cysteine/ GSH level in cancer cells through the action of interferon-γ (INF-γ). According to very recent findings, INF-γ secreted by effector CD8+ T cells inhibits xCT gene expression through JAK-­ STAT1 signalling in fibroblasts and/or tumour cells. In this way, cystine uptake is suppressed, as well as cysteine shuttle between fibroblasts and tumour cells, which consequently lead to accumulation of lipid peroxides and ferroptosis (Wang

18

M. Vucetic et al.

Fig. 2.3  CySSCy-CySH-GSH interplay between cancer cells, fibroblasts and T cells in the tumour mass. In in vivo systems extracellular cysteine is present predominantly in the oxidized form (CySSCy), which is taken up by cancer cells via xCT transporter. However, the effect of the reduced cysteine form (CySH), present in living organism, is largely neglected. CySH can come from the blood, but also from surrounding stromal cells. Data show that cancer associated fibroblasts can also provide source of CySH to the cancer cells, either directly or in the form of GSH, which is broken down to amino acid constituents in the extracellular space by the action of gamma-glutamyl

transferase (GGT). On the other side, it seems that the presence of immune cells, more precisely T cells in the tumour mass, has negative effect on this cancer cell-­ fibroblast collaboration. Interferon-γ secreted by T cells via its receptor activates signaling that ultimately leads to suppression of xCT expression, which from one side disable both tumour cells and fibroblasts to use CySSCy, and from another down-regulates capacity of fibroblasts to provide CySH for tumour cells (Wang et al. 2016, 2019). Similar effect should be observed with the inhibitors of xCT, such as erastin

et  al. 2016, 2019). This has been proven in a mouse model where checkpoint inhibitors and complete enzymatic depletion of cyst(e)ine by cysteinase were combined (Wang et  al. 2019). Accordingly, xCT gene expression in patients with cancer was shown to be negatively associ-

ated to the CD8+ T cell response signature, INF-γ and patient outcome (Wang et  al. 2019). Altogether these findings suggest that combinatory effects of immune therapy and ferroptotic inducers have great potential for cancer treatment.

2  Overcoming Therapeutic Challenges for Pancreatic Ductal Adenocarcinoma with xCT Inhibitors

2.6

Concluding Remarks

Dismal prognosis of pancreatic cancer patients is the consequence of unique set of processes underlying PDAC development and progression, as well as chemoresistance to conventionally used therapeutics. The most alarming problems in the PDAC treatment are: late diagnosis, early disseminating potential of pancreatic cancer cells and desmoplasmic response of compact stromal compartment. Recent contextualization of novel type of cell death shed new light to possible alternative strategy for PDAC treatment. Stockwell’s group in 2012 coined term “ferroptosis” to describe iron-dependent cell death triggered by inhibition of cystine uptake by xCT transporter and mediated by inactivation of indispensible Se-cystein enzyme  – GPx4, and accumulation of membrane lipid peroxides. Mounting evidence support the hypothesis that ferroptosis inducers, especially inhibitors of xCT transporter, can be used to treat PDAC, considering high specificity and great efficacy of these molecules toward mesenchymal and cancer cells resistant to chemotherapy. According to very recent findings, xCT inhibition had been placed in the context of immunotherapy, as its inhibition probably increases antigenicity of pancreatic cancer cells and significantly improves efficacy of checkpoint inhibitors. Finally, xCT activity-based tracers seem to hold promise for use as early detection markers of PDAC metastasis in the organs that naturally do not express it, such as liver. Although many avenues had been opened for ferroptosis inducers to be used in the treatment of highly aggressive PDAC, there are some important issues that remain to be addressed. One of the most important and yet almost completely underestimated aspects is the import of reduced cysteine. Although xCT-dependent cystine transport has been proven to definitely be a prerequisite for cancer cell growth and survival in vitro, extrapolation of these results to in vivo can lead to inaccurate conclusions. The effects of reduced form of cysteine, which is present in vivo and almost not at all in culturing conditions, still have not been investigated. Discrepancies in the phe-

19

notype of xCT knockout in cancer and T cells between in vitro vs in vivo conditions suggests that monomeric form of cysteine might be a cause of potential resistant mechanisms to xCT-­ based drugs. Clearly, future investigations should illuminate the various cyst(e)ine import/export systems to improve our therapeutic interventions in various forms of cancers. Acknowledgments  This work was supported by the government of Monaco, including PhD thesis (BD) and post-­ doctoral (MV) fellowships, and by ‘Le Groupement des Entreprises Monégasques dans la Lutte contre le cancer’ (GEMLUC) including post-doctoral fellowships (MV and SC). Conflict of Interest  The authors declare no potential conflict of interest.

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2  Overcoming Therapeutic Challenges for Pancreatic Ductal Adenocarcinoma with xCT Inhibitors genic cell death. EMBO J 28(5):578–590. https://doi. org/10.1038/emboj.2009.1 Pickup MW, Mouw JK, Weaver VM (2014) The extracellular matrix modulates the hallmarks of cancer. EMBO Rep 15(12):1243–1253. https://doi.org/10.15252/ embr.201439246 Provenzano PP, Cuevas C, Chang AE, Goel VK, Von Hoff DD, Hingorani SR (2012) Enzymatic targeting of the stroma ablates physical barriers to treatment of p­ancreatic ductal adenocarcinoma. Cancer Cell 21(3):418–429. https://doi.org/10.1016/j. ccr.2012.01.007 Rahib L, Smith BD, Aizenberg R, Rosenzweig AB, Fleshman JM, Matrisian LM (2014) Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States. Cancer Res 74(11):2913–2921. https://doi. org/10.1158/0008-­5472.CAN-­14-­0155 Rhim AD, Mirek ET, Aiello NM, Maitra A, Bailey JM, McAllister F et  al (2012) EMT and dissemination precede pancreatic tumor formation. Cell 148(1–2):349–361. https://doi.org/10.1016/j. cell.2011.11.025 Rhim AD, Oberstein PE, Thomas DH, Mirek ET, Palermo CF, Sastra SA et  al (2014) Stromal elements act to restrain, rather than support, pancreatic ductal adenocarcinoma. Cancer Cell 25(6):735–747. https://doi. org/10.1016/j.ccr.2014.04.021 Ricoult SJ, Manning BD (2013) The multifaceted role of mTORC1  in the control of lipid metabolism. EMBO Rep 14(3):242–251. https://doi.org/10.1038/ embor.2013.5 Robe PA, Martin D, Albert A, Deprez M, Chariot A, Bours V (2006) A phase 1-2, prospective, double blind, randomized study of the safety and efficacy of Sulfasalazine for the treatment of progressing malignant gliomas: study protocol of [ISRCTN45828668]. BMC Cancer 6:29. https://doi. org/10.1186/1471-­2407-­6-­29 Robe PA, Martin DH, Nguyen-Khac MT, Artesi M, Deprez M, Albert A et  al (2009) Early termination of ISRCTN45828668, a phase 1/2 prospective, randomized study of sulfasalazine for the treatment of progressing malignant gliomas in adults. BMC Cancer 9:372. https://doi. org/10.1186/1471-­2407-­9-­372 Sakorafas GH, Sarr MG (2003) Pancreatic cancer after surgery for chronic pancreatitis. Dig Liver Dis 35(7):482–485 Sasaki H, Sato H, Kuriyama-Matsumura K, Sato K, Maebara K, Wang H et al (2002) Electrophile response element-mediated induction of the cystine/glutamate exchange transporter gene expression. J Biol Chem 277(47):44765–44771. https://doi.org/10.1074/jbc. M208704200 Sato H, Shiiya A, Kimata M, Maebara K, Tamba M, Sakakura Y et al (2005) Redox imbalance in cystine/ glutamate transporter-deficient mice. J Biol Chem 280(45):37423–37429. https://doi.org/10.1074/jbc. M506439200

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Sato M, Kusumi R, Hamashima S, Kobayashi S, Sasaki S, Komiyama Y et al (2018) The ferroptosis inducer erastin irreversibly inhibits system xc- and synergizes with cisplatin to increase cisplatin’s cytotoxicity in cancer cells. Sci Rep 8(1):968. https://doi.org/10.1038/ s41598-­018-­19213-­4 Shibue T, Weinberg RA (2017) EMT, CSCs, and drug resistance: the mechanistic link and clinical implications. Nat Rev Clin Oncol 14(10):611–629. https://doi. org/10.1038/nrclinonc.2017.44 Sinha S, Leach SD (2016) New insights in the development of pancreatic cancer. Curr Opin Gastroenterol 32(5):394–400. https://doi.org/10.1097/ MOG.0000000000000295 Sperti C, Pasquali C, Piccoli A, Pedrazzoli S (1997) Recurrence after resection for ductal adenocarcinoma of the pancreas. World J Surg 21(2):195–200 Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF et al (2012) Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366(26):2443–2454. https://doi. org/10.1056/NEJMoa1200690 Tseng JF, Willett CG, Fernandez-del Castillo C, Ryan DP, Clark JW, Zhu AX et  al (2005) Patients undergoing treatment for pancreatic adenocarcinoma can mount an effective immune response to vaccinations. Pancreatology 5(1):67–74. https://doi. org/10.1159/000084492 Ursini F, Maiorino M, Valente M, Ferri L, Gregolin C (1982) Purification from pig liver of a protein which protects liposomes and biomembranes from peroxidative degradation and exhibits glutathione peroxidase activity on phosphatidylcholine hydroperoxides. Biochim Biophys Acta 710(2):197–211. https://doi. org/10.1016/0005-­2760(82)90150-­3 Viswanathan VS, Ryan MJ, Dhruv HD, Gill S, Eichhoff OM, Seashore-Ludlow B et al (2017) Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway. Nature 547(7664):453–457. https:// doi.org/10.1038/nature23007 Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr, Kinzler KW (2013) Cancer genome landscapes. Science 339(6127):1546–1558. https://doi. org/10.1126/science.1235122 Von Hoff DD, Ervin T, Arena FP, Chiorean EG, Infante J, Moore M et al (2013) Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine. N Engl J Med 369(18):1691–1703. https://doi.org/10.1056/ NEJMoa1304369 Vucetic M, Cormerais Y, Parks SK, Pouyssegur J (2017) The central role of amino acids in cancer redox homeostasis: vulnerability points of the cancer redox code. Front Oncol 7:319. https://doi.org/10.3389/ fonc.2017.00319 Wang W, Kryczek I, Dostal L, Lin H, Tan L, Zhao L et al (2016) Effector T cells abrogate stroma-mediated chemoresistance in ovarian cancer. Cell 165(5):1092– 1105. https://doi.org/10.1016/j.cell.2016.04.009 Wang W, Green M, Choi JE, Gijon M, Kennedy PD, Johnson JK et  al (2019) CD8(+) T cells regulate

24 tumour ferroptosis during cancer immunotherapy. Nature 569(7755):270–274. https://doi.org/10.1038/ s41586-­019-­1170-­y Wang-Gillam A, Li CP, Bodoky G, Dean A, Shan YS, Jameson G et  al (2016) Nanoliposomal irinotecan with fluorouracil and folinic acid in metastatic pancreatic cancer after previous gemcitabine-based therapy (NAPOLI-1): a global, randomised, open-label, phase 3 trial. Lancet 387(10018):545–557. https://doi. org/10.1016/S0140-­6736(15)00986-­1 Wei SC, Duffy CR, Allison JP (2018) Fundamental mechanisms of immune checkpoint blockade therapy. Cancer Discov 8(9):1069–1086. https://doi. org/10.1158/2159-­8290.CD-­18-­0367 Winograd R, Byrne KT, Evans RA, Odorizzi PM, Meyer AR, Bajor DL et al (2015) Induction of T-cell immunity overcomes complete resistance to PD-1 and CTLA-4 blockade and improves survival in pancreatic carcinoma. Cancer Immunol Res 3(4):399–411. https://doi.org/10.1158/2326-­6066.CIR-­14-­0215 Wu J, Minikes AM, Gao M, Bian H, Li Y, Stockwell BR et  al (2019) Intercellular interaction dictates cancer cell ferroptosis via NF2-YAP signalling. Nature 572(7769):402–406. https://doi.org/10.1038/ s41586-­019-­1426-­6 Yagoda N, von Rechenberg M, Zaganjor E, Bauer AJ, Yang WS, Fridman DJ et al (2007) RAS-RAF-MEK-­

M. Vucetic et al. dependent oxidative cell death involving voltage-­ dependent anion channels. Nature 447(7146):864–868. https://doi.org/10.1038/nature05859 Yamaguchi Y, Kasukabe T, Kumakura S (2018) Piperlongumine rapidly induces the death of human pancreatic cancer cells mainly through the induction of ferroptosis. Int J Oncol 52(3):1011–1022. https:// doi.org/10.3892/ijo.2018.4259 Yang WS, Stockwell BR (2008) Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem Biol 15(3):234–245. https://doi. org/10.1016/j.chembiol.2008.02.010 Yang WS, SriRamaratnam R, Welsch ME, Shimada K, Skouta R, Viswanathan VS et  al (2014) Regulation of ferroptotic cancer cell death by GPX4. Cell 156(1–2):317–331. https://doi.org/10.1016/j. cell.2013.12.010 Yauch RL, Gould SE, Scales SJ, Tang T, Tian H, Ahn CP et al (2008) A paracrine requirement for hedgehog signalling in cancer. Nature 455(7211):406–410. https:// doi.org/10.1038/nature07275 Zhang Y, Tan H, Daniels JD, Zandkarimi F, Liu H, Brown LM et  al (2019) Imidazole ketone erastin induces ferroptosis and slows tumor growth in a mouse lymphoma model. Cell Chem Biol 26(5):623–633. https:// doi.org/10.1016/j.chembiol.2019.01.008

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Iron Homeostasis and Metabolism: Two Sides of a Coin Vivek Venkataramani

Abbreviations ∙OH Hydroxyl Radical 4-HNE 4-hydroxynonenal 56 Ni Nickel-56 8-OHdG 8-hydroxy-2′-deoxyguanosine CO Carbon Monoxide DCYTB Duodenal Cytochrome B DMT1 Divalent Metal Transporter 1 Fe Ferrum Fe2+ Ferrous Iron Fe3+ Ferric Iron FeOH3 Ferric Hydroxides Fe-S Iron-Sulfur FNR Fumarate and Nitrate Reductase FPN1 Ferroportin FTH Ferritin Heavy Chain FTL Ferritin Light Chain GPX Glutathione Peroxidase GSH Glutathione HAMP Hepcidin HCP1 Heme Carrier Protein 1 HEPHL1 Hephaestin Like-1 HO, HMOX Heme Oxygenase HRG-1 Heme Responsive Gene-1

V. Venkataramani (*) Institute of Pathology, University Medical Center Göttingen (UMG), Göttingen, Germany e-mail: [email protected]

I ron-Refractory Iron Deficiency Anemia L∙ Lipidyl Radical LIP Labile Iron Pool LO∙ Alkoxyl Radical LOOH Lipid Hydroperoxides MDA Malondialdehyde Mk mice Microcytic Anemia Mice NTBI Non-Transferrin Bound Iron PUFA Polyunsaturated Fatty Acid ROS Reactive Oxygen Species sla Sex-Linked Anemia SoxR Redox-Sensitive Transcriptional Activator STEAP Six-Transmembrane Epithelial Antigen Of Prostate TFR1, CD71 Transferrin Receptor ZIP ZRT/IRT-like Protein IRIDA

3.1

Iron in Physiology and Disease

3.1.1 I ron: Origin, Chemical Properties and Evolution Iron is a chemical element with the symbol Fe (latin: ferrum) and atomic number 26. The first iron atoms in the universe popped into existence from dying stars that collapsed under their own gravity and became supernovae. In the course of

© Springer Nature Switzerland AG 2021 A. F. Florez, H. Alborzinia (eds.), Ferroptosis: Mechanism and Diseases, Advances in Experimental Medicine and Biology 1301, https://doi.org/10.1007/978-3-030-62026-4_3

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a supernova, carbon and oxygen were fused together generating huge amounts of radioactive Nickel-56 (56Ni), which subsequently decayed in an exothermic chain reaction finally to iron. This “iron core” of these collapsing stars is not the cause of the supernovae, but its presence marks the inevitable violent end of the star’s life cycle (Woosley and Heger 2015; Frey and Reed 2012). The released iron scattered into space to accumulate in rocky planets like Earth (Wallner et  al. 2016). Therefore it is not surprising that the transition metal iron is the fourth most abundant metal in Earth’s crust (∼5.6%) and oceans (Taylor 1964) and is fundamentally responsible for nearly all life on this planet that relies on oxygen-­ dependent energy production (Dixon and Stockwell 2014). Similar to other transition metals, iron exists in a wide range of oxidation states, from −II to +VII. However, chemically the most common and biologically, the most relevant oxidation states are +2 (Fe2+, ferrous iron) and +3 (Fe3+, ferric iron), although some iron-binding proteins generate high-valent Fe4+ and Fe5+ intermediates during their catalytic cycles (Ghosh 2006). All living beings -plants, bacteria, animals, humans- continuously rely on the interchangeability of the Fe2+/Fe3+ redox pair to facilitate many vital electron transfer and acid-­ base reactions necessary to propagate survival and growth. Both iron redox species readily form complexes with organic molecules, such as heme and iron-sulfur (Fe-S) clusters, with immense biological implications. For instance, heme (composed of protoporphyrin and Fe2+) is an essential co-factor involved in multiple biological processes, including oxygen carrying (hemoglobin, two-third of the human body’s iron) (Knutson and Wessling-­ Resnick 2003), oxygen storage (myoglobin), energy production (cytochrome c), detoxification (cytochrome P450, catalase) and host defense (nitric oxide synthase, NAPDH oxidase) (Muckenthaler et al. 2017). Iron-sulfur clusters are one of the ancient types of metalloprotein prosthetic groups. When early life forms began to evolve, about 3.5 billion years ago, our earth had a reductive (anaerobic) atmosphere, and iron was bioavailable to the first

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living organisms in its soluble reduced Fe2+ form (Wacey et al. 2011). It has been hypothesized that these favorable reducing conditions in combination with high hydrogen sulfide concentrations (e.g. near hydrothermal vents) resulted in the formation of iron-sulfur clusters of various stoichiometries, ranging from simple [2Fe–2S] diamonds and [4Fe–4S] distorted cubes to more complex structures, found in nitrogenase and molybdoenzymes. Accordingly, the “metabolism-­ first” theory of the origin of life posits that these spontaneously assembled iron-sulfur clusters may have acted as basic building blocks of life fueling the gradual conversion from abiotic mineral-­based catalysts to biotic protein-based biocatalysts (Rouault and Klausner 1997; Wächtershäuser 2008). This would explain why most components of the Fe-S cluster biogenesis machinery display versatile biological functions and are therefore essential for cell and organismal viability. These include crucial metabolic enzymes (aconitases), gene regulation (bacterial transcription factors FNR, SoxR), electron transfer reactions (ferredoxins, Rieske proteins), substrate activation (cyclooxygenase, lipoxygenase) and protein structure stabilization (bacterial endonuclease III) (Johnson 1998). As pointed out above, the unparalleled versatility of iron as a biological catalyst relies on the rich coordination chemistry of its two main ionic states, Fe2+ and Fe3+. However, exactly this versatility also poses two major challenges: The potential redox-activity of Fe2+ and the insolubility of Fe3+ necessitate iron to be constantly chaperoned in all higher organisms (Richardson et al. 2010).

3.1.2 I nsights into the Redox Chemistry of Iron Compared to other transition metal ions, such as zinc and manganese that have no or very little reducing potential, copper and iron are highly redox-active and hence become potentially toxic in excess (Crichton 2019). In 1894, H.J. Fenton first described that a mixture of acidified iron and H2O2 (Fenton’s reagent) generates a strong oxidant (Fenton 1894). However, it was 50  years

3  Iron Homeostasis and Metabolism: Two Sides of a Coin

later (1934) that Haber and Weiss proposed that highly reactive hydroxyl radicals (∙OH) are generated by the one-electron transfer from Fe2+ to H2O2, now known as the Haber-Weiss (Fenton) reaction (Fe2+ + H2O2→OH− + FeO2+ + H+→Fe3 +  + HO− + ∙OH) (Haber et al. 1934). This reaction does not occur with Fe3+, since this ionic form is not capable of participating in such a one-­electron process, highlighting the fact that free redox-­ cycling Fe2+ is predominantly responsible for iron-mediated cellular toxicity. Moreover, the oxidation of Fe2+ to Fe3+ is greatly pH-dependent (the reaction half-life of Fe2+ at pH  3.5 is 1000 days, while at pH 7.0 it is 8 min) (Koppenol and Liebman 1984). This has an important biological implication, since subcellular compartments, such as acidic lysosomes with a pH around 5.4, provide an optimal condition for maximal solubility and catalytic performance of iron-­ mediated free radical oxidation (Kurz et al. 2008; Huang 2003). Unrestricted Fe2+-catalyzed production of free hydroxyl radicals within a cell can cause irreversible damage leading to detrimental consequences for organisms (Hutchinson 1957). Hydroxyl radicals have a short half-life (10−9 s) and are the most potent oxidant formed from oxygen (with a high electrophilicity and thermokinetic reactivity) and can react with virtually all types of biomolecules, such as nucleic acids, lipids and amino acids, further underlining their extensive aggressiveness (Pryor 1988; Halliwell and Gutteridge 1995). Interestingly, of all the radicals and oxidants known under the collective term “reactive oxygen species” (ROS), only the hydroxyl radical readily attacks DNA.  Oxidative DNA damage includes covalent modification of bases, causing various DNA adducts that directly or indirectly (as a consequence of the DNA repair process) result in single- and double-strand breaks (Halliwell 1998; Breen and Murphy 1995; von Sonntag 1991). In particular, hydroxyl free radical-­ induced damages occur at the guanine base which is sensitive to oxidation, making the detection of 8-hydroxy-2′-deoxyguanosine (8-OHdG) a reasonable biomarker for oxidative DNA injury (Ames 1989; Shibutani et al. 1991). Experimental, epidemiological and clinical evidence highlight a close link between iron

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load and 8-OHdG (Tuomainen et  al. 2007). This suggests that free and harmful Fe2+ acts as a contributor to potentially mutagenic and carcinogenic, oxidatively modified DNA (Kato et al. 2001; Kuo et al. 2008; Nelson et al. 1994; Valk et al. 2000). Generally, highly reactive hydroxyl radicals attack the nearest stable molecule, gaining its electron and turning the “attacked” molecule into a radical itself. In biological systems, the most well-known example of such a radical chain reaction is the lipid peroxidation cascade. Lipid peroxidation occurs when oxidants, such as free radicals, attack lipids containing carbon-carbon double bonds, especially polyunsaturated fatty acids (PUFAs). The process of lipid oxidation includes three crucial steps with complex reactions and interactions between redox-cycling Fe2+ and its substrates, termed initiation, propagation and termination (Yin et  al. 2011). In the initiation step, pro-oxidants such as hydroxyl radicals subtract a hydrogen atom from a methylene carbon in the lipid substrate to generate a highly reactive lipidyl radical (L∙). In the propagation phase, this lipid radical reacts with oxygen in the presence of Fe2+ to form lipid peroxyl(LOO∙) and/or alkoxyl- (LO∙) radicals. This lipid peroxyl-radical can remove hydrogen from other lipid molecules to generate a new lipid radical and lipid hydroperoxides (LOOH), resulting in a fatal loop. In the presence of Fe2+ these preformed lipid hydroperoxides are unstable and participate in the formation of reactive lipid radicals, underlining that redox-active iron not only propagates but also amplifies the lipid peroxidation chain reaction. Compared to hydroxyl radicals that can only attack adjacent biomolecules, peroxidation-­ generated lipid radicals can easily diffuse across membranes far from their site of origin. The continued radical chain reaction alters membrane fluidity (promoting increased membrane rigidity due to loss of PUFAs) and thus inactivates membrane-­ bound proteins resulting in loss of membrane integrity and cell death (Halliwell and Gutteridge 1984; Negre-Salvayre et  al. 2008; Ayala et al. 2014; Wong-Ekkabut et al. 2007). Once the lipid peroxidation cascade is initiated and propagates autonomously, it can only be terminated by a reaction with other radicals or

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radical-trapping antioxidants (such as glutathione, vitamin E or coenzyme Q10). These donate a hydrogen atom to the LOO∙ and LO∙ radical species forming nonradical inactive products, terminating the lipid peroxidation cascade (Yin et al. 2011). Importantly, the kinetics of the initiation and propagation phase are pH-dependent, as an acidic pH increases the solubility and membrane permeability of iron (Schafer and Buettner 2000). Furthermore, a low environmental pH in combination with reductants (e.g. ascorbate) further enhances lipid peroxidation by releasing iron from safe storage proteins such as ferritin (Wills 1966; Schafer and Buettner 2000). Breakdown of lipids via lipid oxidation results in the formation of a wide array of primary (e.g. lipid hydroperoxides) and secondary oxidation products. Among these secondary products, malondialdehyde (MDA), propanal, hexanal, and 4-­hydroxynonenal (4-HNE) have been extensively studied in the context of Fe2+-induced lipid peroxidation (Esterbauer et al. 1982). In particular, 4-HNE and MDA have been linked to a wide array of diseases, including cancer, diabetes, neurodegenerative and cardiovascular diseases (reviewed in Ayala et al. 2014). It appears that the biological activities of MDA include cross-linking with DNA, highlighting its mutagenic potential and its high reactivity and toxicity (Esterbauer et  al. 1990). MDA has been widely used as convenient and reliable biomarker for lipid peroxidation (of omega-3 and -6-fatty acids) in pre-clinical and clinical settings, especially to determine the extent of oxidative stress elicited by iron exposure (Zager et al. 2004; Roob et al. 2000). Like MDA, 4-HNE has a high reactivity with multiple biomolecules, such as proteins and DNA that leads to the formation of function-altered adducts. High levels of 4-HNE can generate a variety of cytotoxic and genotoxic stress, inducing long-­ lasting biological consequences, including cell cycle arrest, senescence and programmed cell death (Ayala et  al. 2014). More recently, cell death mechanisms closely associated with lipid peroxidation have emerged (Conrad et al. 2016). Disrupted iron homeostasis that exacerbates the formation of redox-cycling Fe2+ and unchecked toxic lipid peroxidation are two key characteris-

V. Venkataramani

tics of ferroptosis (derived from Latin ferro, “ferrous iron” (Fe2+), and ptosis, from the Greek “to fall”), a term and concept first introduced 2012 by Dixon and Stockwell (Dixon et  al. 2012). Intriguingly, this mode of cellular sabotage is mechanistically, phenotypically and biochemically distinct from other cell death processes (Stockwell et al. 2017; Yang and Stockwell 2016; Yagoda et al. 2007). Since the formation of pro-oxidative species is an inevitable process in an oxygen-enriched environment, biological systems have evolved several regulatory strategies to prevent toxic ROS formation. One defense strategy is the reduction of peroxides or radical intermediates through multiple enzymes that metabolize the hydroxyl radical precursor H2O2, such as catalase and glutathione peroxidases (GPXs) (Day 2009). The two enzymes act in a complementary fashion that differ markedly in their ranges of efficacy and capacity to defend against toxic H2O2 buildup. Catalase is a ubiquitously expressed heme-­ containing enzyme that is mostly located in peroxisomes and mitochondria and is one of the most efficient antioxidative enzymes that can decompose millions of H2O2 to water and oxygen every second (~105  M−1  s−1) (Kirkman and Gaetani 2007). However, homozygous catalase knockout mice develop normally and show no gross abnormalities and even present a differential pattern of oxidant sensitivity in the types of tissue (Ho et  al. 2004). In line, acatalasia, an autosomal-recessive disorder with near-total deficiency of catalase activity, is considered a benign condition, implicating a compensatory increase in other ROS-detoxifying enzymes (Góth and Nagy 2013). GPX enzymes belong to the well-studied family of peroxidases and are widely distributed in mammalian tissues. They contain a redox-active selenocysteine in their active site where the sulfur is replaced by selenium, resulting in fast reaction kinetics (~108 M−1 s−1) (Flohe et al. 1972). These GPX enzymes reduce lipid peroxides to their corresponding non-reactive lipid alcohols and H2O2 to water, typically using glutathione (GSH) as a reductant (Day 2009). In mammals, there are eight different isoforms with specific

3  Iron Homeostasis and Metabolism: Two Sides of a Coin

subcellular localization in the cytosol, nuclei and mitochondria (Brigelius-Flohé and Maiorino 2013). In particular, GPX4 represents an essential gene, since mice genetically engineered to lack GPX4 present a lethal phenotype. Moreover, conditional GPX4 knockout in many tissues and cell types causes cell death in a pathological relevant form of ferroptosis (Seiler et al. 2008; Yoo et  al. 2012; Friedmann Angeli et  al. 2014; Linkermann et al. 2014). This critical importance of GPX4 may depend on its function as key GSH utilizing enzyme that reduces toxic lipid peroxidation and so preventing spontaneous ferroptosis execution (Friedmann Angeli et  al. 2014; Yang et al. 2014; Forcina and Dixon 2019). Besides the elimination of peroxides by antioxidant enzymes, the risk of toxic radical buildup is also mitigated by the sequestration of redox-­ active Fe2+. Organisms have evolved a variety of iron-binding proteins such as ferritin, transferrin, haptoglobin, hemopexin that maintain iron in a redox-inert state (Sullivan 1988). Ferritin proteins are conserved among most living organisms, from bacteria to mammals, and shield cells by catalytically oxidizing toxic Fe2+ and safely sequestering the resulting Fe3+ ions inside their ferritin mineral nanocages. The ferritin complex consists of a heavy chain (FTH) that retains Fe3+ via its enzymatic ferroxidase activity, and a light chain (FTL), which lacks this enzymatic activity and, participates in iron storage (Torti and Torti 2002). The iron molecules concentrated in ferritin (up to 4500) cannot participate in Fenton-mediated hydroxyl radical production, while iron remains bioavailable upon demand via lysosomal ferritin degradation, an autophagic process known as ferritinophagy. This process is responsible for delivering ferritin to the lysosome in iron-depleted conditions (see also Chap. 4: Ferritinophagy) (Eisenstein et  al. 1991; Mancias et al. 2014). In vertebrates, transferrin transports iron through the plasma without the risk of ROS formation (Enns et al. 1996). Each transferrin glycoprotein contains two tightly bound Fe3+ ions and transports them into cells for utilization (discussed later), mostly for heme and Fe-S biosynthesis, while excess intracellular iron is stored

29

within ferritin (Lambert et  al. 2005; Aisen and Listowsky 1980). Haptoglobin and Hemopexin act as soluble scavengers of free hemoproteins (hemoglobin, Hb; and myoglobin) and heme, respectively. Both plasma proteins represent a sequential protection system with Haptoglobin as the major and Hemopexin as backup defense attenuating Hb and heme-induced oxidative damage (Schaer et  al. 2014). Once released from ruptured red blood cells, unbound hemoproteins can cause severe lipid peroxidation as a consequence of the H2O2 mediated redox cycling between ferric [Hb (Fe3+)] and ferryl [Hb(Fe4+  =  O)] states. Lipid peroxidation drives this vicious ferric/ferryl redox cycle towards ferryl heme generation that further oxidizes residues within the Hb globin chains (protein radicals), ultimately resulting in Hb degradation and heme loss (Boutaud et  al. 2010). Haptoglobin binding does not alter the primary Hb reactivity with peroxides, but rather limits the secondary oxidative Hb reactions with globin amino acids and may also function as a free radical scavenger within the complex blocking heme release (Pimenova et al. 2010; Cooper et  al. 2013). Finally, the Haptoglobin-Hb complex is cleared by macrophages via CD163-­ mediated endocytosis followed by lysosomal proteolysis and heme breakdown to recycle iron (discussed later) (Kristiansen et al. 2001). In contrast to Haptoglobin, Hemopexin forms the backup line of defense during severe hemolysis. Hemopexin acts as an antioxidant by its high heme-binding affinity forming a redox-inert complex that is transported to the hepatocytes by receptor-mediated endocytosis for catabolism and excretion (Delanghe and Langlois 2001). The reason why there are multiple enzymes and strategies for regulating iron and redox homeostasis may lie in the evolving view that pro- and antioxidative activities are not strictly opposing processes. Both rather function as regulators to control biological and physiological processes as signaling molecules (“ROS signaling”); however, excessive accumulation of ROS can trigger programmed cell death, including apoptosis and ferroptosis (Mittler 2017; Schieber and Chandel 2014; Sies and Jones 2020).

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3.1.3 Strategies for Iron Assimilation and Transport

V. Venkataramani

tured” Fe3+ by an iron-chelating molecule and the direct intracellular transport after reduction to Fe2+ by an enzyme capable of ferrireductase Earth’s atmosphere became oxidative about 2.5 activity. The proteins involved in iron transport billion years ago due to the continuous oxygen and regulation of iron metabolism are unique in emission by photosynthesis thus enabling the higher eukaryotes. In mammalian cells, dietary evolution of many higher organisms. The appear- iron comes in several forms such as heme, inorance of atmospheric oxygen coincided with Fe2+ ganic Fe3+ iron, ferritin and ferric-iron complexed oxidation to Fe3+ that precipitated as insoluble with other macromolecules. The primary sources ferric hydroxides (FeOH3) and so become inac- of heme-bound iron are hemoglobin and myoglocessible as a bioavailable source. Consequently, bin from meat consumption, whereas inorganic environments that are seemingly rich in iron non-heme iron is obtained from vegetables and actually contain concentrations of free Fe3+ ions fruits (Hurrell and Egli 2010). (10−9−10−18  M) which are too low to allow The inorganic Fe3+ form must first be reduced growth by aerobic organisms (Mittler 2017; to Fe2+ by a ferrireductase, such as the duodenal Chipperfield and Ratledge 2000). Responding to cytochrome b (DCYTB) that is present on the this challenge, organisms have evolved a variety apical membranes of intestinal gut epithelia. of chelation strategies to acquire and transport DCYTB is a di-heme transmembrane protein that iron in its ferric state. is normally expressed at low levels, but can be For instance, microorganisms (such as bacte- markedly induced under iron deficient or hypoxic ria and fungi) chelate Fe3+ from the extracellular stress conditions (McKie et al. 2001). However, environment by secreting small ferric iron-­ recent studies demonstrated that animals genetibinding molecules, known as siderophores (greek cally lacking DCYTB maintain iron stores and for “iron carrier”). These water-soluble Fe3+- hematologic parameters to a similar extent as siderophore complexes are internalized through wild-type mice, even under a long-term iron-­ siderophore-specific receptors (outer membrane deficient diet (Gunshin et  al. 2005). These data receptors). Bacterial siderophores are necessary argue against a major role for DCYTB in vivo, to hijack nutritional iron from the host, allowing raising the possibility that other ferrireductase pathogens to survive and thrive during infection. systems such as members of the STEAP (six-­ In a clinical context, some bacterial siderophores transmembrane epithelial antigen of prostate) are predictive of virulence and are involved in protein family, like STEAP3, provide the reducbiofilm formation that promotes antibiotic resis- ing power necessary for iron absorption (Ohgami tance (Harrison and Buckling 2009; Marti et al. et al. 2005). 2011). This highlights the therapeutic potential of After the obligatory redox conversion, free targeting the siderophore import system (via soluble Fe2+ can be imported into the cell by the gallium-­containing compounds, siderophore bio- divalent metal transporter 1 (DMT1). Besides its synthesis inhibitors or siderophore-antibiotic high plasma membrane expression in intestinal conjugates) as a novel and synergistic antimicro- cells, DMT1 is also abundant in erythrocyte prebial strategy (Wilson et al. 2016). In the intracel- cursors where it transports transferrin-bound iron lular environment, iron is predominantly present from endosomes to the cytoplasm (discussed in its reduced Fe2+ form and therefore, aerobic later). As implied by its name, DMT1 has no microbes use membrane-bound ferric reductases specificity for Fe2+ and also facilitates the import to directly reduce chelated Fe3+ to soluble Fe2+ for of other divalent metals. However, the identificatransport or intracellular incorporation into tion of spontaneous DMT1 missense mutations heme- and non-heme-containing proteins in mice (G185R, Mk mice) and rats (Belgrade rats), as well as human, highlight the in vivo rel(Schröder et al. 2003). Higher organisms use both iron assimilation evance of DMT1 in iron transport (Fleming et al. strategies: the receptor-mediated uptake of “cap- 1997, 1998; Mims et  al. 2005). These hypo-

3  Iron Homeostasis and Metabolism: Two Sides of a Coin

morphic DMT1 mutations result in severe microcytic anemia (deficient synthesis of hemoglobin) due to defective intestinal iron absorption and a decreased ability to utilize transferrin-bound iron. In humans, DMT1 mutations result also in hepatic iron overload, possibly due to paradoxical hyperabsorption of iron via residual DMT1 activity, compensatory induced alternative Fe2+ importer, such as ZRT/IRT-like proteins (ZIPs) or due to a greater proportion of heme iron import (Mims et al. 2005; Andrews and Schmidt 2007; Liuzzi et  al. 2006). High ZIP14 levels during early development have been proposed to contribute to the iron assimilation throughout the neonatal period. However, ZIP14 transports Fe2+ poorly in the acidic microenvironment of the duodenum, suggesting that DMT1 may play a more prominent role in Fe2+ transport during adulthood (Pinilla-Tenas et al. 2011; Wang et al. 2011). Like ingested inorganic Fe3+ iron, heme iron is largely derived from both meat (in the form of hemoglobin and myoglobin) and plants such as soybeans (in the form of leghemoglobin) (Theil 2004). Currently, there are two candidate transporters proposed to import heme into the cytoplasm. Heme carrier protein 1 (HCP1) was initially identified as an intestinal heme transporter (Shayeghi et  al. 2005). Although clearly capable of transporting heme, recent evidence suggests that HCP1 is not a physiologically relevant heme transporter, as it operates as a folate transporter that is mutated in patients with genetic folate deficiency (Qiu et  al. 2006). The other putative heme transporter is heme responsive gene-1 (HRG-1), a homolog to heme transporters identified in the natural heme auxotroph caenorhabditis elegans (Rajagopal et al. 2008). Since mammalian HRG-1 is localized in phagolysosomes of macrophages and is induced upon hemolysis (destruction of red blood cells), it is postulated that this putative heme importer is involved in heme recovery from senescent erythrocytes (Rajagopal et al. 2008; Zhang et al. 2018). The intracellular fate of heme is currently not fully elucidated. Under physiological conditions, it is inserted as a prosthetic group into the heme

31

pockets of hemoproteins (such as hemo-, myo-, neuroglobins and cytochromes) that tightly control the electron exchange rate between Fe2+heme and a variety of ligands (Dawson 1988). However, unbound free heme has the pro-­ oxidative chemical property of a Fenton reactor generating free radicals derived from H2O2 (Jeney et al. 2002). To avoid such a cytotoxic accumulation, imported free heme is catabolized by heme oxygenase (HO) enzymes, which consists of two forms, the inducible HO-1 and the constitutive isoform HO-2. Although these are two different gene products with limited sequence homology and different tissue distributions, both enzymes catalyze the first and rate-limiting step in heme degradation to generate equimolar amounts of redox-active Fe2+, billiverdin (which can be further converted to the antioxidant bilirubin) and the gasotransmitter carbon monoxide (CO). Both bilirubin and CO can protect cells from a multitude of stressors including iron. The presence of both HO-1- and HO-2-encoding genes (HMOX1 and HMOX2, respectively) in most living organisms implies that the need for heme metabolism occurred early during evolution (reviewed in Gozzelino et al. 2010). On the one hand, this conserved enzymatic strategy exerts cytoprotective effects by limiting the free pro-oxidant heme pool, while on the other it enables the extraction of redox-active Fe2+ so that it can enter the same storage, utilization and transport pathways taken by inorganic iron. Beside inorganic non-heme and heme-bound iron, other iron import mechanisms include ferritin (possibly imported via receptor-mediated endocytosis; (Han et al. 2011; San Martin et al. 2008)), lactoferrin (transferrin protein family member that is found in mucosal secretions, notably in human milk; reviewed in Kruzel et al. 2017) and the incorporation of siderophore-­ bound iron such as lipocalin 2 (secreted from neutrophil granulocytes preventing bacterial uptake of siderophore-bound iron; reviewed in Xiao et  al. 2017) that are less well studied but may become important in individuals with iron poor diets. Regardless the way how iron is extracted from the diet or transported across the enterocyte

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membrane, once iron is inside the enterocyte it is likely chelated by small-molecular-weight organic acids (e.g. citrate and GSH) and amino acids that is all part of the labile iron pool (LIP) (Hider and Kong 2011; Patel et  al. 2019). This transitory, catalytically active compartment of free and chelatable Fe2+ iron is thought to supply the metal for storage and for metabolic needs. Recently, iron chaperone poly(rC)-binding ­proteins (PCBP) could be identified as multifunctional adaptor protein that chaperons imported Fe2+ and delivers it to mitochondria (e.g. for heme, Fe-S cluster synthesis, cytochromes and mitochondrial ferritin), non-heme iron enzymes (such as lipoxygenases), iron-storage proteins or transfers it near the basolateral surface the enterocyte where the iron is released into circulation for systemic usage (Yanatori et  al. 2014). As mentioned earlier, excess iron is stored in its Fe3+ form inside ferritin nanocages. The iron retained in enterocytes stored as ferritin is rapidly lost since these cells turn over every two to 5 days in humans. The desquamation of epithelial surfaces (including shredded enterocytes, skin cells and menstrual loss) only results in a daily iron loss of 1–2  mg/day that is balanced by dietary iron absorption (Green et al. 1968). Notably, there is no active regulatory mechanism for excreting iron (probably due to its relatively low availability in nature), which is why iron balance is highly regulated by dietary iron absorption that involves transport across the apical membrane and through the basolateral membrane as discussed in the following section. Iron transported to the basolateral membrane exits the enterocyte via the transmembrane iron exporter ferroportin (FPN1, also known as SLC40A1). FPN1 appears to be the sole mammalian Fe2+ exporter and is vital for early embryonic development. Mice globally deficient in FPN1 are embryonically lethal and accumulate iron in cell types that essentially contribute to plasma iron levels. These include enterocytes (that traffic dietary iron into the bloodstream), hepatocytes (the major non-erythroid site of iron reservoir ranging from 300–1000 mg in humans and export iron as needed), and macrophages (phagocytic cells that recycle iron from old red

V. Venkataramani

blood cells). In line, intestine-specific FPN1 inactivation results in an anemic phenotype due to lack of intestinal iron absorption (Donovan et  al. 2005; Pietrangelo 2016). In mammals, FPN1 levels are controlled on the systemic level by hepcidin (HAMP), a circulating peptide hormone primarily synthesized in the liver. This bioactive 25-amino acid peptide essentially impacts dietary iron absorption and distribution by mediating the phosphorylation, internalizing and degrading of membrane-bound FPN1 resulting in intracellular iron retention. Transgenic hepcidin overexpression in mice results in iron deficiency anemia that is caused by inhibition of iron absorption and increased iron retention in storage sites (Nicolas et al. 2002). In a clinical context, inappropriate high hepcidin level contribute to various iron disorders, including inflammatory disorders, hepcidin-producing hepatic adenomas and hereditary iron-refractory iron deficiency anemia (IRIDA) (Nemeth and Ganz 2009; Ganz and Nemeth 2012). On the other side, hepcidin deficiency in mice causes hyperabsorption of iron resulting in parenchymal iron overload in liver, pancreas and heart. These are typical clinical manifestations in hereditary hemochromatosis (a disease caused by inactivating mutations in hepcidin regulatory genes or HAMP) and β-thalassemia (a disease associated with hepcidin suppression due to high erythropoietic activity) (Nemeth and Ganz 2009; Ganz and Nemeth 2012). After export via FPN1, Fe2+ is converted to 3+ Fe . This oxygen-dependent reaction is carried out by a family of membrane-bound and copper-­ containing ferroxidases. This includes the multicopper oxidase ceruloplasmin, the ceruloplasmin paralog hephaestin (human hephaestin is 50% identical and 68% similar to the amino acid sequence of human ceruloplasmin (Syed et  al. 2002)), as well as the newly discovered hephaestin like-1 (HEPHL1), also known as zyklopen (Cherukuri et al. 2005; Vulpe et al. 1999; Chen et  al. 2010). In enterocytes, membrane-bound hephaestin (named after Hephaestus, the Greek god of metal working) assists to export and oxidatively convert Fe2+ to Fe3+, thereby facilitating its uptake by apo-transferrin (highlighted later).

3  Iron Homeostasis and Metabolism: Two Sides of a Coin

This assures very low concentrations of Fe2+ at the cell surface creating a gradient that drives iron efflux. Genetic hephaestin ablation in mice (sex-linked anemia (sla) mice) results only in a mild systemic iron deficiency and anemia underlying that hephaestin is important for optimal iron absorption, but not essential (Bannerman and Cooper 1966; Fuqua et al. 2014). Other ferroxidases, such as ceruloplasmin may partially compensate the lack of hephaestin in the intestine. Ceruloplasmin (literally meaning “a blue substance from plasma”) is synthesized and highly expressed in the liver. Besides being the major copper-carrying protein secreted into plasma, it could be demonstrated that this ferroxidase can liberate iron from other tissues and can augment intestinal iron absorption after severe blood loss (Cherukuri et al. 2005). Genetic deficiency of ceruloplasmin in mice (Cp−/− mice) and patients (aceruloplasminemia) present signs of impaired iron mobilization causing a mild microcytic anemia in combination with massively iron depositions in liver (hepatocytes and liver resident macrophages, Kupffer cells), spleen (macrophages) and pancreas (Harris et al. 1999). Intriguingly, all known ferroxidases are also found in the brain, implicating their role in neuronal iron homeostasis and antioxidant defense. The importance of the neuroprotective role is supported by the fact that ceruloplasmin-deficient humans and mice result in the only known iron overload disorder in which brain and systemic iron overload are combined (Harris et  al. 1998; Patel et  al. 2002). Aceruloplasminemia patients frequently develop the classical triad of retinal degeneration, dementia and diabetes, while iron accumulation in the liver rarely leads to clinically overt manifestations such as cirrhosis and liver failure (reviewed in Marchi et al. 2019). In comparison, genetic deficiency of hephaestin has not yet been linked to a human disease. However, hephaestin-deficient sla mice also accumulate iron in distinct brain regions, but do not develop neurodegeneration (Jiang et al. 2015). Combined hephaestin-ceruloplasmin deficiency result in retinal defects and neurodegeneration in mice, suggesting that both ferroxidases are able to par-

33

tially compensate for each other’s brain iron transport functions (Zhao et al. 2015; Hahn et al. 2004; Chen et al. 2019). The recently identified zyklopen (named after Cyclops, the assistants of the smith-god Hephaestus) was demonstrated to be absent in intestinal and liver cells, but highly expressed in the placenta. However, the physiological relevance in placental iron efflux in vivo has not yet been reported (Chen et  al. 2010; Sangkhae et al. 2020). Once Fe3+ is successfully translocated from the enterocyte to the blood plasma, it is captured by apo-transferrin for delivery to all cells and tissues. Interestingly, only about 10–30% of transferrin is occupied by iron, providing a buffer for any further increases in plasma iron that could derive from high iron diet or increased turnover from iron recycling. Only when a transferrin saturation reaches 30–60% (due to e.g. chronic blood transfusions or genetic defects in iron metabolism genes), free iron in the circulatory system, also called Non-Transferrin Bound Iron (NTBI) becomes detectable in the plasma. NTBI includes also a fraction of redox-active toxic form potentially exerting cellular damage at the plasma membrane and towards intracellular organelles (Brissot et al. 2012). The importance of transferrin is strengthened by atransferrinemia/hypotransferrinemia, an extremely rare autosomal-recessive disorder caused by complete absence or insufficient expression of transferrin that is characterized by anemia in conjunction with high NTBI levels and iron overload in the heart and liver (Pantopoulos 2018). The high erythropoietic drive suppresses hepicidin levels that fuel increased iron absorption despite a systemic iron overload (Beutler et al. 2000; Athiyarath et al. 2013). Since 2/3 of body iron is present as hemoglobin, a high surface expression of transferrin receptor (TFR1, CD71) marks erythroid precursors as primary iron consumers (Marsee et al. 2010). The essential physiological function is highlighted by the early embryonic lethality resulting from loss of TFR1 in mice and the pathologies of several tissue-­ specific knockouts (Levy et  al. 1999; Barrientos et  al. 2015; Wang et  al. 2020). Iron-­

34

V. Venkataramani

loaded holo-transferrin binds with the corresponding TFR1 that is further internalized via receptor-mediated endocytosis. Once inside the cell, encapsulated Fe3+ is released from transferrin in the endosome due to the low pH.  It is thought to be immediately reduced to Fe2+ by the STEAP3 ferrireductase and then transported across the endosomal membrane via DMT1 and

so can contribute to the cytosolic LIP to meet the cell’s need or follow above described intracellular iron routes (Bali et  al. 1991; Ohgami et  al. 2005; Sendamarai et al. 2008). Finally, the emptied apo-transferrin is then exported for re-use into the bloodstream and TFR1 returns back to the plasma membrane via recycling endosomes. A summary of all these steps is depicted in Fig. 3.1.

Fig. 3.1  Cellular iron metabolism. Intracellular iron homeostasis is balanced by coordinated iron uptake, utilization, storage and export. (A) Absorbed inorganic ferric iron (Fe3+) must be first converted to ferrous iron (Fe2+) via ferrireductase STEAP3 (six-Transmembrane Epithelial Antigen of Prostate 3) and subsequently taken up by iron importer DMT1 (divalent metal transporter 1) and ZIP14 (ZRT/IRT-like protein 14), while (B) heme-bound iron can be transported by putative heme importers HCP1 (heme carrier protein 1) and/or HRG-1 (heme responsive gene-1). (C) Once inside the cell, heme is catabolized by HO-1 (heme oxygenase 1) to liberate Fe2+, CO (carbon monoxide) and biliverdin, which is subsequently converted to bilirubin. (D) The cumulative source of loosely bound Fe2+, termed LIP (labile iron pool), acts as crossroads in iron trafficking, providing iron for functional incorporation and directing excess towards storage or export. However, Fe2+ excess results in free radical production (ROS) that can cause lipid peroxidation as well as protein oxidation and/or oxidative DNA damage (not illustrated). Therefore, redox-active iron must be safely

transporter via chaperon PCBP (poly(rC)-binding protein) to (E) iron storage protein ferritin that converts Fe2+ back into redox-inert Fe3+ and safely stores them into “ferritin nanocages”. (F) Another route of bioavailable Fe2+ is to the mitochondria (mito) where it is used for various crucial biological processes, such as heme and Fe-S (iron-­ sulfur cluster) biosynthesis or stored as mitochondrial ferritin (FtMt). (G) At saturated intracellular Fe2+, excess iron must be exported via the sole mammalian Fe2+ exporter FPN1 (ferroportin) that is systematically regulated by FPN1-degrading hormone HAMP (hepcidin). (H) Once exported, Fe2+ must be oxidized to Fe3+ via multicopper oxidases Cp (ceruloplasmin), HEPH (hephaestin) and/or HEPHL1 (hephaestin like-1, zyklopen). (I) In the blood stream, converted Fe3+ is bound to transferrin (Holo-TF) and enters effector cells, such as erythroid precursors, via the TFR1 (transferrin receptor) endocytosis pathway. (J) Inside the endosomes, transported Fe3+ is reduced by STEAP3 and exported to the cytoplasm via DMT1 to begin the cycle again

3  Iron Homeostasis and Metabolism: Two Sides of a Coin

3.2

Concluding Remarks

35

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39 Stockwell BR, Friedmann Angeli JP, Bayir H, Bush AI, Conrad M, Dixon SJ et al (2017) Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease. Cell 171(2):273–285. https://doi. org/10.1016/j.cell.2017.09.021 Sullivan JL (1988) Iron, plasma antioxidants, and the ‘oxygen radical disease of prematurity’. Am J Dis Child 142(12):1341–1344. https://doi.org/10.1001/ archpedi.1988.02150120095048 Syed BA, Beaumont NJ, Patel A, Naylor CE, Bayele HK, Joannou CL et al (2002) Analysis of the human hephaestin gene and protein: comparative modelling of the N-terminus ecto-domain based upon ceruloplasmin. Protein Eng 15(3):205–214. https://doi.org/10.1093/ protein/15.3.205 Taylor SR (1964) Abundance of chemical elements in the continental crust: a new table. Geochim Cosmochim Acta 28(8):1273–1285. https://doi. org/10.1016/0016-­7037(64)90129-­2 Theil EC (2004) Iron, ferritin, and nutrition. Annu Rev Nutr 24(1):327–343. https://doi.org/10.1146/annurev. nutr.24.012003.132212 Torti FM, Torti SV (2002) Regulation of ferritin genes and protein. Blood 99(10):3505–3516. https://doi. org/10.1182/blood.v99.10.3505 Tuomainen T-P, Loft S, Nyyssönen K, Punnonen K, Salonen JT, Poulsen HE (2007) Body iron is a contributor to oxidative damage of DNA.  Free Radic Res 41(3):324–328. https://doi.org/ 10.1080/10715760601091642 Valk D, Addicks G, Lu H, Marx M (2000) Non-­ transferrin-­ bound iron is present in serum of hereditary haemochromatosis heterozygotes. Eur J Clin Investig 30(3):248–251. https://doi. org/10.1046/j.1365-­2362.2000.00628.x von Sonntag C (1991) The chemistry of free-­ radical-­ mediated DNA damage. Basic Life Sci 58:287–317; discussion 317-221. https://doi. org/10.1007/978-­1-­4684-­7627-­9_10 Vulpe CD, Kuo YM, Murphy TL, Cowley L, Askwith C, Libina N et  al (1999) Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse. Nat Genet 21(2):195–199. https://doi.org/10.1038/5979 Wacey D, Kilburn MR, Saunders M, Cliff J, Brasier MD (2011) Microfossils of sulphur-metabolizing cells in 3.4-billion-year-old rocks of Western Australia. Nat Geosci 4(10):698–702. https://doi.org/10.1038/ ngeo1238 Wallner A, Feige J, Kinoshita N, Paul M, Fifield LK, Golser R et  al (2016) Recent near-earth supernovae probed by global deposition of interstellar radioactive 60Fe. Nature 532(7597):69–72. https://doi. org/10.1038/nature17196 Wang B, He L, Dong H, Dalton TP, Nebert DW (2011) Generation of a Slc39a8 hypomorph mouse: markedly decreased ZIP8 Zn2+/(HCO3-)2 transporter expression. Biochem Biophys Res Commun 410(2):289–294. https://doi.org/10.1016/ j.bbrc.2011.05.134

40 Wang S, He X, Wu Q, Jiang L, Chen L, Yu Y et al (2020) Transferrin receptor 1-mediated iron uptake plays an essential role in hematopoiesis. Haematologica 105(8):2071–2082. https://doi.org/10.3324/ haematol.2019.224899 Wills ED (1966) Mechanisms of lipid peroxide formation in animal tissues. Biochem J 99(3):667–676. https:// doi.org/10.1042/bj0990667 Wilson BR, Bogdan AR, Miyazawa M, Hashimoto K, Tsuji Y (2016) Siderophores in iron metabolism: from mechanism to therapy potential. Trends Mol Med 22(12):1077–1090. https://doi.org/10.1016/j. molmed.2016.10.005 Wong-Ekkabut J, Xu Z, Triampo W, Tang IM, Tieleman DP, Monticelli L (2007) Effect of lipid peroxidation on the properties of lipid bilayers: a molecular dynamics study. Biophys J 93(12):4225–4236. https://doi. org/10.1529/biophysj.107.112565 Woosley SE, Heger A (2015) The remarkable deaths of 9–11 solar mass stars. Astrophys J 810(1):34. https:// doi.org/10.1088/0004-­637x/810/1/34 Xiao X, Yeoh BS, Vijay-Kumar M (2017) Lipocalin 2: an emerging player in iron homeostasis and inflammation. Annu Rev Nutr 37(1):103–130. https://doi. org/10.1146/annurev-­nutr-­071816-­064559 Yagoda N, von Rechenberg M, Zaganjor E, Bauer AJ, Yang WS, Fridman DJ et  al (2007) RAS–RAF–MEK-­ dependent oxidative cell death involving voltage-­ dependent anion channels. Nature 447(7146):865–869. https://doi.org/10.1038/ nature05859 Yanatori I, Yasui Y, Tabuchi M, Kishi F (2014) Chaperone protein involved in transmembrane transport of iron.

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4

The Role of NCOA4-Mediated Ferritinophagy in Ferroptosis Naiara Santana-Codina, Ajami Gikandi, and Joseph D. Mancias

Abbreviations 4-HNE 4-Hydroxynonenal AD Alzheimer’s Disease AP-MS Affinity-Purification Mass Spectrometry ATG8 Autophagy-Related Protein 8 BSO Buthionine Sulfoximine COPD  Chronic Obstructive Pulmonary Disease DFO Deferoxamine DMT1 Divalent Metal Transporter 1 DNA Deoxyribonucleic Acid ESCRT Endosomal Sorting Complex Required for Transport Fe Iron FPN Ferroportin FTH1 Ferritin Heavy Chain FTL Ferritin Light Chain GPX4 Glutathione Peroxidase 4 GSH Glutathione H2O2 Hydrogen Peroxide HD Huntington’s Disease

Authors Naiara Santana-Codina, Ajami Gikandi, and Joseph D.  Mancias have equally contributed to this chapter.

HERC2 HECT and RLD Domain Containing E3 Ubiquitin Protein Ligase 2 HSCs Hepatic Stellate Cells IREB2 Iron Response Element Binding Protein 2 LA α-Lipoic acid LIP Labile Iron Pool LOX Lipoxygenase MCAO Middle Cerebral Artery Occlusion MEFs Mouse Embryonic Fibroblasts NCOA4 Nuclear Receptor Co-Activator 4 NEURL4 Neuralized E3 Ubiquitin Protein Ligase 4 NF Neuroferritinopathy PD Parkinson’s Disease PUFA Polyunsaturated Fatty Acid ROS Reactive Oxygen Species RSL3 RAS-Selective Lethal 3 SILAC Stable Isotopic Labeling with Amino Acids in Cell Culture SLC7A11 Solute Carrier Family 7 Member 11 TF Transferrin TFRC Transferrin Receptor xCT Cystine/Glutamate Transporter

N. Santana-Codina · A. Gikandi · J. D. Mancias (*) Division of Radiation and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA, USA e-mail: [email protected] © Springer Nature Switzerland AG 2021 A. F. Florez, H. Alborzinia (eds.), Ferroptosis: Mechanism and Diseases, Advances in Experimental Medicine and Biology 1301, https://doi.org/10.1007/978-3-030-62026-4_4

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4.1

Introduction

Iron is an essential element required in biological processes such as oxygen and electron transport and DNA synthesis and repair (Pantopoulos et al. 2012; Bogdan et  al. 2016; Muckenthaler et  al. 2017). Accordingly, iron levels must be tightly regulated as iron deficiency can lead to several pathological conditions such as anemia (Camaschella 2015) while iron accumulation can lead to generation of potentially lethal reactive oxygen species (ROS) by the Fenton reaction (Fenton 1894). Iron-mediated ROS generation contributes to lipid peroxidation in a recently discovered form of cell death termed ‘ferroptosis’ (Dixon and Stockwell 2019). An increase or decrease in cellular free iron levels can therefore promote or inhibit ferroptosis, respectively (Dixon et  al. 2012; Friedmann Angeli et  al. 2014). Intracellular iron homeostasis is a highly regulated process (see Chap. 3) that depends on a number of proteins involved in iron transport (transferrin receptor (TFRC), transferrin (TF), ferroportin (FPN)) and storage (ferritin) (Pantopoulos et al. 2012). In particular, the ferritin complex is composed of 24 subunits of ferritin light and heavy chain subunits (FTL, FTH1) and is the main iron storage machinery of the cell with a capacity to sequester 4500 iron atoms per complex (Weis et al. 2017). By storing iron in a non-labile form, ferritin prevents excess free iron in the cell and also provides a store in times of decreased cellular iron availability. However, how iron is released from ferritin intracellularly to respond to changing iron availability in the cell was an unanswered question until recently. Our lab and others described a mechanism by which iron can be released from ferritin in a process known as “ferritinophagy” (Dowdle et al. 2014; Mancias et  al. 2014). This process requires NCOA4 (Nuclear Receptor Coactivator 4), a selective cargo receptor that binds to ferritin and mediates its delivery to the autophagosome and subsequently to the lysosome for ferritin degradation and concomitant iron release (Mancias et al. 2015). This process is tightly regulated by iron levels, which can modulate NCOA4 stability

and therefore iron release. When cellular iron levels are high, iron-bound NCOA4 interacts with the ubiquitin E3 ligase HERC2, targeting NCOA4 for proteasomal degradation thereby decreasing ferritinophagy. Conversely, when iron levels are low, the NCOA4 and HERC2 interaction is inhibited leading to stabilization of NCOA4, increased ferritinophagic flux and iron release in the lysosome (Mancias et  al. 2015). Given that NCOA4-mediated ferritinophagy can alter iron homeostasis, several studies have described a correlation between NCOA4 levels and sensitivity to ferroptosis inducing agents (Gao et al. 2016; Hou et al. 2016). Here, we will review the biochemical aspects of NCOA4 function and regulation as well as its role in iron homeostasis. Furthermore, we will highlight a role for NCOA4 in ferroptosis with a focus on cancer and neurodegeneration. Finally, we will discuss the therapeutic potential of modulating ferritinophagy in combination with ferroptotic agents.

4.2

NCOA4-Mediated Ferritinophagy

Autophagy is a conserved cellular process whereby misfolded proteins, damaged or superfluous organelles, intracellular pathogens, and other cytosolic constituents are delivered to the lysosome for degradation (Glick et al. 2010). The breakdown products from the degradation of certain substrates can be recycled to the cell and used as nutrients in energy production or macromolecule biosynthesis, thus autophagy is frequently described as a homeostatic and survival mechanism. In macroautophagy (hereafter referred to as autophagy), substrates are delivered to the lysosome via a double-membrane vesicle called the autophagosome (Fig.  4.1a). Autophagy can be a selective or non-selective process. In selective autophagy, selective autophagy receptors bind and target specific substrates (their cargo), such as mitochondria (mitophagy), pathogens (xenophagy), endoplasmic reticulum (ER-phagy), and protein aggregates (aggrephagy), for autophagic degradation (Mancias and

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Fig. 4.1  NCOA4-mediated ferritinophagy is required for ferroptosis induction. (a) Iron (Fe) is stored in ferritin complexes containing ferritin heavy (FTH1) and light (FTL) chains. NCOA4 binds to ferritin and mediates its delivery to the autophagosome. Fusion of autophagosomes with the lysosomes leads to degradation of ferritin and subsequent iron release. This process is known as “ferritinophagy.” (b) Exported iron can be used in physiological processes, or it can be used to generate reactive oxygen species via the Fenton Reaction which can lead to lipid peroxidation and susceptibility to ferroptosis. The major mechanisms modulating the labile iron iron pool (LIP) are iron import via transferrin receptor 1-mediated

(TFRC) endocytosis of holo-transferrin, ferritinophagic flux, and iron export through Ferroportin (FPN). DMT1 (in polarized cells) and ZIP14 (mainly in hepatocytes) are additional specialized non-transferrin iron transporters that can import iron and may therefore modulate ferroptosis sensitivity. SLC7A11 exports glutamate and imports cystine, a precursor for GSH synthesis. Glutathione peroxidase 4 (GPX4) mediates repair of lipid peroxidation and inhibits ferroptosis. Ferroptosis is a promising therapeutic target. Use of ferroptosis inducers (e.g. erastin, RSL3) can trigger tumour cell death, while use of ferroptosis inhibitors (e.g. liproxistatin-1, iron chelators) may protect against neurodegeneration

Kimmelman 2016). In non-selective autophagy, bulk cytosol is sequestered in autophagosomes, often in response to a cellular stressor like nutrient deprivation, and then delivered to the lysosome, resulting in the non-specific degradation of many substrates. Early evidence linking degradation of ferritin, the cellular iron-storage complex, to the lysosome and autophagy came from electron micrographs of ferritin particles in lysosomes and autophagic vesicles and in vitro assays demonstrating the ability of purified lysosomes to degrade endogenous ferritin (Trump et al. 1973; Radisky and Kaplan 1998). Corroborating evidence was provided by numerous studies showing the rescue of ferritin degradation by pharmacological inhibition of lysosome function and genetic ablation of autophagy-related genes (Kidane et  al. 2006; Zhang et  al. 2010; Asano et al. 2011). It is now well-established that ferritin lysosomal degradation can be induced by

various stressors, such as iron chelation (De Domenico et  al. 2006), amino acid starvation (Öllinger and Roberg 1997), doxorubicin treatment (Kwok and Richardson 2004), and Neisseria meningitidis infection (Larson et al. 2004). We and others identified nuclear receptor coactivator 4 (NCOA4) as the selective autophagy receptor for ferritin degradation (Dowdle et al. 2014; Mancias et al. 2014). We used mass spectrometry to identify enriched proteins in intact autophagosomes purified from cultured cells (Mancias et al. 2014). Consistently, NCOA4 was among the most highly enriched autophagosomal-­ associated proteins. NCOA4 association with autophagosomes was validated by its co-localization with autophagosomal markers (e.g. LC3B). Affinity-purification mass spectrometry (AP-MS) identified both components of the ferritin complex (FTH1 and FTL), which were also enriched in autophagosome datasets, as high-confidence interacting partners of NCOA4.

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Autophagosome and lysosome targeting of ferritin was abrogated by NCOA4 knockdown and rescued by ectopic expression of NCOA4, thereby implicating NCOA4 as the selective autophagy receptor for ferritinophagy (Fig. 4.1a). Lastly, NCOA4 depletion protected against H2O2-induced oxidative stress, presumably by decreasing the amount of lysosomal and cytosolic iron available for ROS-producing Fenton reactions, thus demonstrating a role for NCOA4-­ mediated ferritinophagy in both iron and redox homeostasis. Further biochemical investigations have identified molecular determinants of NCOA4 function and regulation. FTH1 binds the NCOA4α isoform at a conserved region of its C-terminus and that NCOA4 amino acid residues I489/W497 and the FTH1 surface residue R23 are essential for the NCOA4-FTH1 interaction (Mancias et al. 2015). A subsequent study identified that each ferritin complex can bind up to 24 NCOA4 molecules (Gryzik et al. 2017). Ferritinophagic flux is dependent on NCOA4 levels which are responsive to intracellular iron levels. Interaction proteomics (Mancias et  al. 2014) identified the HECT E3 ubiquitin ligase HERC2 as a high-­ confidence interacting partner of NCOA4. Under iron-replete conditions, NCOA4-HERC2 binding is stabilized. This promotes NCOA4 proteasomal turnover and lowers NCOA4 levels, thereby decreasing ferritinophagic flux and promoting ferritin accumulation and iron storage when iron is abundant (Mancias et al. 2015). Under conditions of iron-deficiency, NCOA4-HERC2 binding is weakened. Consequently, decreased proteasomal turnover of NCOA4 leads to increased NCOA4 levels that promote ferritinophagy and can increase free iron when cellular iron levels are low (Fig.  4.2) (Mancias et  al. 2015). We and others have shown that NCOA4-­ mediated ferritinophagy is important in physiological processes that require large amounts of iron, such as erythropoiesis (Bellelli et al. 2016; Ryu et  al. 2017; Santana-Codina et  al. 2019). While NCOA4 is important for systemic iron homeostasis, it was originally identified as a

nuclear coactivator of androgen receptors (Alen et  al. 1999) and was recently reported to be a negative regulator of DNA replication (Bellelli et al. 2014), both in a ferritinophagy-independent manner. Although recent work has provided important insight into the NCOA4-FTH1 interaction and its biochemical regulation, it has also raised a number of important questions that remain unanswered: (1) FTH1 and HERC2 bind to similar regions of NCOA4 (Mancias et  al. 2015). Is NCOA4 binding with FTH1 and HERC2 mutually exclusive? What spatial and temporal mechanisms govern these interactions? (2) Multiple selective autophagy receptors recognize more than one cargo (Mancias and Kimmelman 2016). Does NCOA4 similarly mediate the selective degradation of any other substrates? (3) NCOA4 co-purifies with iron atoms (Mancias et al. 2015). How does NCOA4 recognize iron? (4) What are the relative roles of classical and non-classical autophagy in NCOA4-dependent ferritinophagy (Goodwin et al. 2017; Mejlvang et al. 2018)? (5) How does the role of NCOA4 in ferritinophagy synchronize with NCOA4’s other reported roles in nuclear receptor coactivation and DNA replication (Alen et al. 1999; Bellelli et al. 2014; Gao et  al. 2017)? Answering these questions will allow us to better understand how NCOA4 modulates iron metabolism and contributes to the pathogenesis of diseases characterized by aberrations in iron homeostasis.

4.3

NCOA4-Mediated Ferritinophagy and Ferroptosis

Ferroptosis is an iron-dependent non-apoptotic type of cell death triggered by peroxidation of polyunsaturated fatty acids (PUFAs) (Dixon et al. 2012; Dixon and Stockwell 2019). This type of cell death is different from apoptosis and necrosis at the biochemical (lack of bioenergetic failure) and morphological level (absence of plasma membrane rupture, blebbing or chromatin condensation along with presence of small mito-

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Fig. 4.2  HERC2-mediated NCOA4 degradation is iron-dependent. The E3 ubiquitin ligase HERC2 interacts with NCOA4 via a CUL7 domain in an iron-­ dependent manner. Under iron-replete or acute iron overload conditions, NCOA4-HERC2 binding is stabilized. HERC2-mediated ubiquitylation facilitates NCOA4 turnover by targeting it to the proteasome for degradation.

When cellular iron levels are low, NCOA4-HERC2 binding is decreased. NCOA4 liberated from HERC2 participates in the trafficking of iron-laden ferritin to the lysosome for degradation (ferritinophagy). The subsequent release of lysosomal iron into the cellular labile iron pool helps restore cellular iron homeostasis

chondria with reduced cristae) (Dixon et  al. 2012). The canonical pathway of ferroptosis induction requires three elements considered the hallmarks of ferroptosis (Dixon and Stockwell 2019): oxidation of PUFAs, presence of redox-­ active iron and loss of lipid peroxide repair by glutathione peroxidase 4 (GPX4) inactivation (Brigelius-Flohé and Maiorino 2013; Yang et al. 2016) (Fig. 4.1b). GPX4 can be inhibited directly with compounds such as RAS-selective lethal (RSL3) or indirectly by targeting SLC7A11 (erastin) to inhibit cystine metabolism and glutathione synthesis, a necessary cofactor required for GPX4 function (Yang et al. 2014; Xie et al. 2016; Yang and Stockwell 2016; Stockwell et al. 2017; Hassannia et al. 2019) (Fig. 4.1b). Recently, two studies described a new mechanism by which ferroptosis can be regulated independently of

GPX4 and glutathione. In this complementary model, FSP1 (Ferroptosis Suppressor protein 1) mediates the reduction of CoQ10 (ubiquinone) to ubiquinol, trapping lipid peroxyl radicals and inhibiting lipid peroxidation and ferroptosis (Bersuker et al. 2019; Doll et al. 2019). Iron accumulation is a key regulator of ferroptosis leading to free radical formation and lipid peroxidation. Therefore, limiting intracellular iron availability with iron chelators like deferoxamine (DFO) can inhibit ferroptosis (Friedmann Angeli et  al. 2014; Yang et  al. 2014) while increasing iron levels via exogenous iron or a high-iron diet can induce ferroptosis in vitro and in vivo (Dixon et  al. 2012; Wang et  al. 2017). Likewise, ferroptosis sensitivity can also be regulated by enzymes in the iron regulatory network with the ability to modulate the labile iron pool

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(LIP), such as IREB2 and FBXL5, and other proteins involved in iron import and storage, such as TFRC, FTH1 and FTL (Yang and Stockwell 2008; Sun et  al. 2015). For instance, increased expression of TFRC (Gao et al. 2016) or HMOX1 (Chang et al. 2018) have been shown to enhance ferroptosis by increasing the LIP.  Conversely, increased expression of FPN, the primary cellular iron export channel, reduces ferroptosis sensitivity by decreasing free iron levels (Ma et al. 2016). As cellular iron importers, DMT1 and ZIP14 may also modulate ferroptosis sensitivity by altering the LIP (Liuzzi et  al. 2006; Kajarabille and Latunde-Dada 2019). Regulation of cellular ferritin levels can also modulate sensitivity to ferroptosis. Indeed, ferroptosis induction stimulates exocytosis of iron-laden ferritin via prominin-2, which acts as a suppressor of ferroptosis by limiting availability of cellular iron derived from ferritin (Brown et al. 2019). Given the role of NCOA4-mediated ferritinophagy in modulating iron homeostasis, several studies have identified NCOA4 as a key regulator of ferroptosis (Gao et al. 2016; Hou et al. 2016). NCOA4 depletion decreases intracellular free iron and oxidative stress (Mancias et  al. 2014) while increasing glutathione levels (Hou et  al. 2016). These changes result in reduced sensitivity to ferroptosis-inducing compounds in cell culture systems (Gao et al. 2016; Hou et al. 2016). Recently, Yoshida et al. described a functional in vivo role of NCOA4-mediated ferritinophagy in ferroptotic lung epithelial cell death in response to cigarette smoke (Yoshida et al. 2019). In this study, NCOA4 ablation in in vitro and in vivo models of chronic obstructive pulmonary disease (COPD) attenuated ferroptosis induced by GPX4 knockdown (Yoshida et  al. 2019). Similarly, impaired ferritinophagy as a consequence of lysosomal dysfunction promoted resistance to ferroptosis in senescent fibroblasts (Masaldan et al. 2018). In this complex model, iron-trapping in ferritin led to a perceived iron deficiency which was compensated by activation of IRP2 and iron import through TFRC (Masaldan et  al. 2018). Conversely, increasing ferritinophagic flux and iron release from ferritin degradation via exogenous NCOA4 over-expression leads to increased

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sensitivity to ferroptosis (Hou et  al. 2016). Whether there are specific cellular, tissue, physiologic, or pathophysiologic contexts in which modulation of ferritinophagy has no effect on ferroptosis is unclear and is an ongoing area of research. NCOA4 levels vary in response to ferroptosis-­ inducing agents, with reports of increased expression after sorafenib treatment in hepatic stellate cells (HSCs) (Zhang et al. 2018b), no differential expression after erastin treatment in pancreatic cancer cells (Hou et al. 2016), and even decreased levels after erastin treatment in MEFs or artesunate in HSCs, consistent with NCOA4 autophagic degradation (Gao et al. 2016; Kong et al. 2019). These data suggest a complex temporal, cell-specific and compound-specific regulation of NCOA4 levels and ferritinophagic flux as a preceding event or as a result of ferroptosis induction. Despite the biochemical connection between NCOA4-mediated ferritinophagy and ferroptosis, the link between ferroptosis and autophagy in general is controversial, with results showing a positive correlation (Gao et al. 2016; Hou et al. 2016; Torii et al. 2016) and others suggesting that ferroptosis is an autophagy-independent process (Dixon et  al. 2012). Nevertheless, in the cases where genetic autophagy suppression reduces ferroptosis, cells are still able to partially induce ferroptosis (Gao et  al. 2016; Hou et  al. 2016; Dixon and Stockwell 2019). Gao et al. show that effects of autophagy inhibition on ferroptosis are more obvious at early timepoints, therefore suggesting autophagy is required for initiation of the ferroptotic cascade (Gao et al. 2016). It is likely that autophagy contributes to ferroptosis by promoting ferritinophagy and iron release but further work is required to assess the temporal requirement of NCOA4 in ferroptosis initiation and/or propagation of down-stream effects. Finally, despite some studies suggesting that NCOA4 modulation in vivo can alter ferroptosis and expression of enzymes required to cope with oxidative stress (Bellelli et  al. 2016; Yoshida et al. 2019), additional in vivo studies will clarify the mechanism(s) of ferroptosis regulation by NCOA4 as well as any therapeutic potential in disease.

4  The Role of NCOA4-Mediated Ferritinophagy in Ferroptosis

4.4

Ferritinophagy and Ferroptosis in Cancer

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induced ferroptosis in pancreatic cancer cells (Hou et al. 2016). Therefore, increasing ferritinophagic flux and iron levels could be an efficient Inducing ferroptosis in cancer has recently gained way of sensitizing cells to ferroptosis-inducing interest as an anti-cancer therapeutic strategy. agents as a possible anti-tumoural strategy. Triggering other types of cell death could be a Increased ferritinophagic flux usually correway of overcoming resistance to apoptosis lates with activation of autophagy, a common induced by conventional therapies (Mohammad process in a variety of cancers (Yang et al. 2011; et al. 2015), a strategy that has proven successful Lazova et  al. 2012; Huo et  al. 2013; Xie et  al. in a variety of cancer models such as in therapy-­ 2015; Santanam et  al. 2016). Despite the comresistant neuroblastoma (Hassannia et  al. 2018) plex role of autophagy as a tumour promoter or or cisplatin-resistant osteosarcoma (Liu and tumour suppressor (Santana-Codina et al. 2017), Wang 2019). Interestingly, resistance to therapy activation of autophagy generally supports correlates with presence of mesenchymal mark- tumour growth by promoting protein and iron ers and increased susceptibility to GPX4 inhibi- turnover as well as recycling of impaired mitotion (Hangauer et  al. 2017; Viswanathan et  al. chondria to maintain redox homeostasis. 2017) mediated by NF2-YAP signaling (Wu et al. Interestingly, activation of oncogenes like KRAS 2019a), suggesting this subset of persister cells drive autophagy (Wu et al. 1999; Kakhlon et al. are ideal candidates for ferroptotic agents. Further 2002; Guo et al. 2011; Lock et al. 2011) as well rationale for targeting ferroptosis in cancer is as other features required for ferroptotic cell provided by long-standing observations that can- death such as activation of iron import by cer cells prime an aberrant iron metabolism con- increased TFRC expression (Ryschich et  al. sisting of increased iron import and decreased 2004; Yang and Stockwell 2008). Other oncoexport (Pinnix et  al. 2010; Basuli et  al. 2017), genes and tumour suppressors have been shown which leads to an increase in the pool of bioavail- to modulate ferroptosis sensitivity like NRF2/ able redox-active iron that may promote ferropto- KEAP1, p53 and BAP1. For in-depth reviews on sis when GSH is limiting (Daher et  al. 2019; the subject, readers are referred to recent reviews Dixon and Stockwell 2019; Badgley et al. 2020). (Dixon and Stockwell 2019; Hassannia et  al. The role of NCOA4-mediated ferritinophagy 2019). in tumour progression is largely unknown. These studies, together with the fact that some Several studies have identified an increase in anti-cancer therapies can sensitize tumours to a NCOA4 mRNA and NCOA4α protein levels after ferroptotic cell death (Dixon and Stockwell, transformation induced by overexpression of 2019), suggest targeting ferroptosis in combinaoncogenes (MYC, H-Ras, p53 inactivation) in tion with anti-cancer therapies might be a useful normal endometriotic cells (Shaw et  al. 2001; therapeutic approach (See Chap. 2). In fact, ferRockfield et al. 2018). These studies suggest that roptosis has been reported as a more immunoNCOA4 isoforms might have opposing roles in genic type of cell death than apoptosis due to survival, with NCOA4α acting as a tumour pro- delivery of chemoattractant signals and activamoter and NCOA4β a tumour suppressor in ovar- tion of immune cells (Mou et al. 2019) suggestian cancer, while these roles would be reversed in ing a potential for useful combinations with prostate and breast cancer (Peng et al. 2008; Wu immunotherapy. A recent study unexpectedly et al. 2011). Despite a limited understanding of identified ATM (Ataxia-telangiectasia mutated) the role of NCOA4 in tumourigenesis or in other as a critical genetic determinant of ferroptosis particular cancer contexts, pancreatic cancer cells through regulation of iron metabolism. ATM present increased NCOA4 expression and ferri- inhibition reduced labile iron pools by modulatinophagic flux (Mancias et  al. 2014) which tion of MTF1 activity, which increased mRNA would likely make them susceptible to ferropto- expression of FTH1, FTL and FPN1 as well as sis. In fact, NCOA4 depletion reduced erastin-­ protein levels of FTH1, FTL, GPX4 and

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SLC7A11 (also known as xCT) leading to reduced sensitivity to ferroptosis. These data suggest ionizing radiation, which activates ATM, could be combined with ferroptosis inducers as a new therapeutic strategy (Chen et al. 2019b). In fact, radiotherapy-activated tumoural ATM and immunotherapy-activated CD8+ T cells can both trigger ferroptosis by inhibition of SLC7A11, a unit of the system xc- (Wang et  al. 2019; Lang et  al. 2019). Given the variety of recent studies that describe a positive correlation between modulation of redox/iron metabolism and ferroptotic agents in cancer (Alvarez et  al. 2017; Bersuker et al. 2019; Chen et al. 2019a; Daher et al. 2019; Doll et al. 2019; Zou et al. 2019), it is tempting to speculate that modulation of ferritinophagy might synergize with ferroptosis inducers in a therapeutic context. The development of new, more potent ferroptotic agents that target the tumour with high specificity (Zhang et al. 2019) and could ultimately work in patients is an exciting first step towards developing combinatorial strategies to target ferroptosis in cancer.

4.5

Ferritinophagy and Ferroptosis in Neurodegeneration

The identification of ferroptosis as a mediator of glutamate neurotoxicity by Dixon et al. raised the exciting possibility of using ferroptosis inhibition to prevent cell death in other neuropathologies (Dixon et  al. 2012). Indeed, a growing body of evidence now links ferroptosis to the pathophysiology of a number of neurological disorders. Targeting the ferroptosis pathway may be a viable therapeutic approach to delay neurodegeneration and mitigate other neurological insults. Given that ferroptosis is an autophagic cell death process and depends on ferritinophagic flux (Gao et al. 2016; Hou et al. 2016), it is therefore likely that NCOA4-mediated ferritinophagy also modulates ferroptosis sensitivity in certain neurological contexts.

4.5.1 Neuroferritinopathy The most direct evidence of a relationship between ferritinophagy, ferroptosis, and neurodegeneration was recently shown in the setting of neuroferritinopathy (NF), a genetic disorder characterized by dominant negative mutations in the FTL gene that prevent the ferritin complex (FTH1/FTL) from assembling and storing iron properly, thus leading to an increase in cytosolic iron (Cozzi et al. 2010). Cozzi et al. showed that primary neurons derived from patients with NF have elevated levels of cytosolic iron and ferritin aggregation compared to control neurons derived from patients without NF (Cozzi et al. 2019). In addition, NF-derived neurons demonstrated increased levels of lipid ROS and decreased levels of GSH (Cozzi et  al. 2019). Together, these observations suggested a role for ferroptosis in NF pathology, a hypothesis which was supported by their observation that ferrostatin-1 increased cell viability in NF-derived neurons, but not isogenic controls that were generated by CRISPR-­ Cas9-­ mediated knock-in of the wildtype FTL gene (Cozzi et al. 2019). Interestingly, they also observed decreased levels of NCOA4 in NF-derived neurons. They concluded that decreased NCOA4 leads to decreased ferritinophagy, which results in an accumulation of defective ferritin that can’t store iron properly. The resultant increase in the cytosolic iron pool favors Fenton chemistry and lipoxygenase (LOX) activity that could lead to lipid peroxidation and eventually ferroptosis, a hypothesis that supports their prior work which showed cell death in NF to be dependent on dysregulation of iron homeostasis and oxidative stress (Cozzi et al. 2010). Given recent studies that implicate NCOA4-­ mediated ferritinophagy in ferroptosis (Gao et al. 2016; Yoshida et  al. 2019), and the presence of brain iron accumulation and autophagy dysfunction in a number of neurological diseases, such as static encephalopathy of childhood with neurodegeneration in adulthood caused by pathogenic mutations in the autophagy-related gene WDR45 (Saitsu et  al. 2013), it is likely that NCOA4-­

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mediated ferritinophagy also contributes to ferroptosis demonstrated in additional neurological pathologies (Santana-Codina and Mancias 2018). As no studies have directly examined this hypothesis, further research is necessary to fully understand the role of NCOA4-mediated ferritinophagy in ferroptosis-mediated neuropathologies. Below, we review the evidence to date for ferroptosis and general iron homeostasis dysregulation in frequently diagnosed neurodegenerative conditions. Notably, as these diseases are highly complex and our review focuses solely on the potential role of ferroptosis in their pathology, we refer the reader to excellent comprehensive reviews on the overall disease pathogenesis process (Querfurth and LaFerla 2010; Bates et  al. 2015; Kalia and Lang 2015; Scheltens et  al. 2016; Dugger and Dickson 2017)

4.5.2 Alzheimer’s Disease Alzheimer’s disease (AD) is the most common form of neurodegeneration (ND) and dementia and is characterized by the presence of insoluble amyloid plaques and neurofibrillary tangles formed from an accumulation of amyloid-beta peptide (Aβ) and tau protein aggregate (Bloom 2014; Atri 2019). The accumulated evidence supports iron and ferroptosis playing an important role in AD pathogenesis. For example, the Alzheimer’s risk gene amyloid precursor protein (APP) plays an important role in iron homeostasis by stabilizing the iron export protein FPN1 (Lumsden et al. 2018), brain iron has been shown to co-localize with the AD pathological marker amyloid-beta (Aβ) (van Bergen et al. 2016), and elevated levels of lipid peroxidation markers have been measured in postmortem brains of AD patients (Montine et  al. 2002). Moreover, baseline levels of ferritin in cerebrospinal fluid are negatively correlated with cognitive functional decline in AD patients (Ayton et  al. 2015) and clinical trials using the iron chelator PBT2 have yielded promising results (Lannfelt et al. 2008). Recent studies have further strengthened the link between ferroptosis and AD.

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Early degeneration of neurons in the basal forebrain is a hallmark of AD.  Recently, Hambright et  al. generated a forebrain neuron specific inducible Gpx4 KO mouse (Gpx4BIKO) model to observe whether forebrain neurons were susceptible to ferroptosis (Hambright et al. 2017). Induced Gpx4 ablation led to hippocampal neurodegeneration and decreased cognitive function. Moreover, upon Gpx4 ablation, there was absence of the apoptosis marker cleaved caspase-3, elevated levels of the lipid peroxidation marker 4-hydroxynonenal (4-HNE), exacerbation of neurodegeneration with a diet deficient in the antioxidant vitamin E, and attenuation of neurodegeneration with liproxstatin-1, all supporting ferroptosis being the predominant cell death mechanism contributing to forebrain neurodegeneration in AD pathogenesis (Hambright et al. 2017). High demands on mitochondrial metabolism for energy production, use of nitric oxide in signal transduction, and high abundance of PUFAs in neuronal membranes make the brain susceptible to oxidative stress and lipid ROS formation (Cardoso et al. 2017). Thus, it is not surprising that loss of GPX4 leads to ferroptosis, as loss of lipid peroxide repair is a hallmark of ferroptosis (Dixon and Stockwell 2019), and GPX4 is one of the main enzymes required for removing dangerous lipid ROS. In fact, low levels of GPX4 and other selenoproteins have been observed in postmortem brain tissues obtained from patient’s with neurodegenerative diseases (Gao et  al. 2007; Bellinger et  al. 2011). Interestingly, although the total volume fraction of GPX4 was lower in postmortem brain tissues obtained from patients with neurodegeneration compared to controls, surviving neurons in these affected brain regions showed a higher volume density of GPX4 compared to these same brain regions in control samples, suggesting that GPX4 upregulation is a protective mechanism against ferroptosis, or that neurons with high basal levels of GPX4 are less sensitive to ferroptosis (Bellinger et al. 2011). In the APP/PS1 transgenic mouse model of AD, a long-term diet high in iron leads to elevated levels of hyperphosphorylated tau (p-Tau),

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a well-established biomarker of AD pathology (Guo et al. 2013). Importantly, treatment with the iron chelator DFO suppresses this tau hyperphosphorylation, suggesting that iron-dependent processes contribute to AD pathology (Guo et  al. 2013). Dietary α-Lipoic acid (LA) works by chelating intralysosomal iron, therefore LA supplementation could protect against ferroptosis-mediated neuronal death by preventing iron from being utilized by LOXs and Fenton chemistry in the formation of lipid ROS (Persson et  al. 2001). Indeed, dietary supplementation with LA was shown to decrease iron overload, ROS, lipid peroxidation, and levels of the pathological AD marker hyperphosphorylated Tau in the Alzheimer’s-like tauopathy P301S mouse model (Zhang et  al. 2018a). Additionally, LA supplementation led to downregulation of TFRC, and upregulation of FPN1, SLC7A11, and GPX4 (Zhang et  al. 2018a). Finally, LA treatment has been shown to stabilize the cognitive functions of patients with AD and to the delay progression of the disease (Hager et  al. 2007). Together, these results suggest that Tau-induced neurodegeneration is iron and lipid ROS dependent, and therefore at least partially mediated by ferroptosis. Thus, these studies support the continued investigation of iron chelation, antioxidant supplementation, and future anti-ferroptotic therapies in the treatment of tauopathies like AD.

4.5.3 Parkinson’s Disease Similar to AD, evidence also supports a role for ferroptosis in Parkinson’s disease (PD) pathogenesis, which is characterized by the loss of substantia nigra dopaminergic neurons (Kalia and Lang 2015). Postmortem substantia nigra tissue from patients with PD shows elevated lipid peroxidation (Dexter et al. 1994), depleted GSH (Sian et  al. 1994), and high iron content (Dexter et al. 1989), while clinical trials with the iron chelator deferiprone and the antioxidant NAC have shown promise, with patients receiving these drugs demonstrating decreased brain iron levels, increased dopamine transporter bind-

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ing, and higher scores on motor function and quality of life assessments (Monti et  al. 2016; Martin-­Bastida et al. 2017). In the MPTP mouse, the most commonly used mouse model of PD, induction of dopamine neuron degeneration with the chemical neurotoxin MPTP leads to upregulation of DMT1, which can facilitate neuronal iron import, and elevated iron content in brain regions experiencing dopaminergic cell loss (Salazar et  al. 2008). Importantly, this dopaminergic cell loss was partially rescued by inactivating mutations in DMT1, suggesting that an iron-­dependent form of regulated cell death is responsible for the neuronal cell loss seen in PD pathology (Salazar et al. 2008). Recently, ferrostatin-1 treatment was shown to reduce dopaminergic neuronal cell death in the MPTP mouse (Do Van et  al. 2016). In another recent study using this mouse model, dietary supplementation with the iron chelator lactoferrin was also shown to alleviate MPTP-­induced neurodegeneration (Xu et al. 2019). Interestingly, lactoferrin treatment was shown to protect dopaminergic neurons by suppressing the elevated levels of iron, TFRC, DMT1, ROS, and inflammation that are typically seen after MPTP induction (Xu et al. 2019). In addition, elevated levels of GPX4 and FTH1 are associated with reduced neuronal cell death in the substantia nigra of rats with PD (Lu et al. 2019). Together, these results support there being a ferroptotic contribution to the neurodegeneration seen in PD.

4.5.4 Huntington’s Disease Emerging evidence also links ferroptosis to the pathogenesis of Huntington’s disease (HD). In HD, mutations in the multifunctional Huntingtin protein make it susceptible to fragmentation (Bates et al. 2015). These fragments can lead to nerve damage and death when they misfold and aggregate (Bates et  al. 2015). Samples from patients with HD show elevated levels of brain iron and low GSH levels (Klepac et  al. 2007; Muller and Leavitt 2014), while elevated levels of the lipid peroxidation marker 4-HNE and

4  The Role of NCOA4-Mediated Ferritinophagy in Ferroptosis

abnormal mitochondrial morphology that are rescued by treatment with the lipid peroxidation inhibitor NGDA are observed in mouse models of HD (Lee et  al. 2011). In the R6/2 mouse model of HD, an accumulation of redoxactive iron is observed in striatal neurons (Chen et  al. 2013). This mouse model also exhibits compensatory changes in iron homeostasis that favor iron export, such as increased FPN expression, and decreased IRP-1 and TFRC expression (Chen et al. 2013). One hypothesis is that these changes are occurring to protect against ferroptosis, as decreasing the labile iron pool mitigates formation of lipid ROS via Fenton chemistry and LOX activity. Furthermore, in accordance with an iron-­ dependent mechanism of cell death, intraventricular delivery of the iron chelator DFO attenuated cognitive decline in the R6/2 mouse model (Chen et al. 2013). Further evidence for a role of ferroptosis in HD came from in vitro experiments where ferrostatin-1 treatment was shown to decrease death in rat corticostriatal brain slices transfected with the pathogenic huntingtin gene (htt) exon 1 variant (Skouta et  al. 2014). The master antioxidant regulator NRF2 may also mediate ferroptosis. NRF2 regulates the expression of many genes involved in ferroptosis, such as SLC7A11, GPX4, TFRC and FTH1 (Abdalkader et  al. 2018), and elevated NRF2 signaling has recently been shown to protect against ferroptosis in several contexts (Hou et  al. 2016; Sun et  al. 2016; Roh et  al. 2017; Fan et al. 2017). This raises the possibility that the elevated NRF2 signaling seen in HD brains is a compensatory mechanism protecting against ferroptosis-­associated oxidative stress (Browne et  al. 1999). Therefore, an impaired ability to upregulate NRF2 activity could lead to increased ferroptosis susceptibility and neuronal cell death, a hypothesis that is supported by there being suppressed NRF2 activation in neural stem cells obtained from patient’s with HD compared to nondiseased controls (Quinti et  al. 2017). Thus, current research suggests that ferroptosis inhibition might be effective in treating Huntington’s disease.

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4.5.5 Amyotrophic Lateral Sclerosis Mitochondrial dysfunction is one of the first pathophysiological signs of amyotrophic lateral sclerosis (ALS) (Smith et al. 2017), while motor neuron tissue from patients with ALS shows elevated levels of lipid peroxidation markers (Pedersen et al. 1998). Furthermore, ALS patients who received the iron chelator deferiprone in an early phase clinical trial showed decreased brain iron levels, reduced oxidative stress, and increased functionality compared to patients who did not receive the therapy (Moreau et al. 2018). Together, these data suggest that iron and oxidative stress play an important role in ALS pathogenesis. In the Gpx4 neuronal inducible knockout (Gpx4NIKO) mouse, induced Gpx4 ablation resulted in spinal motor neuron degeneration and paralysis (Chen et  al. 2015). The presence of inflammation, lipid peroxidation, and mitochondrial dysfunction that was rescued by treatment with the ferroptosis inhibitor vitamin E, and the absence of apoptosis markers like caspase-3 and TUNEL, suggests that this motor neuron degeneration is caused by ferroptosis (Chen et  al. 2015). Interestingly, induced Gpx4 ablation in other neurons (e.g. cerebral cortex, hippocampus) did not produce the same phenotype, which suggests that certain neuronal populations are more susceptible to ferroptosis (Chen et al. 2015). Recent evidence suggests that ferroptosis also contributes to the neurodegeneration seen in other movement disorders, such as Friedrich’s ataxia and neuroferritinopathy (Cozzi et al. 2019; Grazia Cotticelli et al. 2019).

4.5.6 Brain Injury Accumulating evidence also links ferroptosis to acute brain injury. In a mouse model of ischemic stroke, middle cerebral artery occlusion (MCAO) induced an ischemia-reperfusion injury that resulted in Tau-suppression and led to an accumulation of brain iron (Tuo et al. 2017). Parenteral delivery of the ferroptosis inhibitors liproxstatin­1 and ferrostatin-1, as well as the lipoxygenase

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inhibitor M1351, reduced infarct size, neuronal loss, cognitive impairments, and functional decline that resulted from reperfusion injury, suggesting that ferroptosis mediates the neurodegeneration seen in ischemic stroke injury (Tuo et al. 2017). In accordance, ferrostatin-1 treatment reduced neuronal cell death, decreased brain injury volume, reduced neurological deficit, and decreased lipid ROS levels in a mouse model of collagenase-induced intracerebral hemorrhage (Li et al. 2017). Importantly, both studies showed that ferroptosis inhibition can be protective against brain injury caused by hemorrhage even when therapy is delayed. These results could be particularly important for treating patients whose brain injury presents late, such as those with delayed traumatic intracerebral hemorrhage. Ferroptosis is also implicated in other acute CNS insults, such as peripartum asphyxia and traumatic spinal cord injury (Wu et  al. 2019b; Yao et al. 2019). It is clear that ferroptosis makes different contributions to different neurological disorders. Continued research will provide further insight into the relationship between ferroptosis and ­neurodegeneration and allow us to better understand the therapeutic contexts in which ferroptosis inhibition might be most beneficial. Likewise, the role of NCOA4 in physiologic neuronal and pathophysiologic neurodegenerative conditions is an unexplored area of research. The model systems and tools for studying NCOA4 in these contexts are now becoming available and should provide insight into NCOA4 function in the brain.

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regulate interaction with FTH1 or HERC2 remain unknown. Second, the NCOA4 residues that mediate iron binding need to be defined. Third, the determinants of NCOA4 localization (nucleus, cytoplasm, autophagosomes) are unclear. Further studies are also required to understand the importance of NCOA4 in physiological and pathological conditions with a focus on its contribution to ferroptosis. Similar to how Gpx4 transgenic mouse models have provided important insights into the mechanisms driving ferroptosis (Chen et al. 2015; Ingold et al. 2018), acute and long-term Ncoa4 KO mouse models might provide further insight into how NCOA4-­ mediated ferritinophagy contributes to ferroptosis in neurodegeneration and other pathological contexts (Bellelli et  al. 2016; Santana-Codina et al. 2019). NCOA4 depletion in cell culture systems leads to decreased ferritinophagy, reduced free iron and thereby ROS production all leading to a protective role against ferroptosis inducers. Paradoxically, mice with long-term Ncoa4 KO demonstrate increased tissue iron and an iron overload phenotype (Bellelli et al. 2016; Santana-­ Codina et  al. 2019). Interestingly, when compared to wild-type mice, long-term Ncoa4 KO mice have decreased survival when fed a high iron diet, likely because Ncoa4 KO leads to saturation of ferritin, increased LIP, and subsequent ROS production via Fenton reactions (Bellelli et  al. 2016). These results suggest that while acute loss of NCOA4 in cellular models and in vivo might be protective against ferroptosis by limiting iron availability (Gao et  al. 2016; Hou et al. 2016; Yoshida et al. 2019), long-term loss of NCOA4 might increase ferroptosis susceptibility 4.6 Conclusions and Future by saturating ferritin deposits leading to increased intracellular free iron pools. Importantly, differDirections ent tissues demonstrate differing levels of ferriThe role and regulation of NCOA4-mediated fer- tinophagic flux (Santana-Codina et  al. 2019) ritinophagy in intracellular and systemic iron although the relative levels are still unknown; homeostasis has been established in the last sev- therefore, any change in ferroptosis susceptibility eral years. However, many molecular aspects are after NCOA4 loss is likely to be tissue-specific still unclear. First, how NCOA4 is regulated post-­ and this is subject of ongoing research in translationally and how these modifications may our laboratory. Furthermore, given that the

4  The Role of NCOA4-Mediated Ferritinophagy in Ferroptosis

contribution of autophagy to ferroptosis seems to be more obvious at different time points (Gao et  al. 2016), further studies will be required to assess the temporal and tissue-specific role of NCOA4 in modulation of ferroptosis and if this might be exploited for therapeutic purposes. The use of ferroptosis-inducing agents in cancer therapy or ferroptosis inhibitors in neurodegeneration is an exciting therapeutic opportunity. However, the translation to in vivo systems and ultimately to patients still presents some difficulties such as reaching therapeutic concentrations in tumour tissues with minimal toxicity. For instance, targeting GPX4, which is required for development and tissue homeostasis (Brigelius-­ Flohé and Maiorino 2013), might lead to off-­ target effects. The identification of new targets in the ferroptosis pathway, like FSP1 (Bersuker et al. 2019; Doll et al. 2019), opens a promising direction for development of novel ferroptosis inducers in order to reach an adequate therapeutic index in tumours compared to normal tissues. On the other hand, identifying novel biomarkers of ferroptosis to predict and assess drug efficacy will be key to define the potential use of ferroptosis-­targeted therapies in cancer patients. It remains to be seen whether NCOA4 levels could be used as a biomarker of ferroptosis sensitivity. The discovery of NCOA4-mediated ferritinophagy in 2014 has opened up multiple exciting new areas of iron biology. The next era of studies will identify the role of NCOA4 in the context of multiple disease processes such as cancer and neurodegeneration as well as its role in regulating sensitivity to ferroptosis. Acknowledgements This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01DK124384, a Burroughs Wellcome Fund Career Award for Medical Scientists, and a Brigham and Women’s Hospital MFCD Award to J.D.M.  Portions of the illustration were generated using Biorender. Conflicts of Interest  J.D.M. is an inventor on a patent pertaining to the autophagic control of iron metabolism.

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Emerging Role for Ferroptosis in Infectious Diseases Eduardo Pinheiro Amaral and Sivaranjani Namasivayam

Abbreviations •OH 12-LOX 5-LO AA-PE ASC ATP cGAMP

cGAS COVID-19 COX-2 DAMPs ER Fe2+ Fe3+ Fer-1 GPX4 GSDMD GSDME GSH

Hydroxyl Radicals 12-Lipoxygenase 5-Lipoxygenase A rachidoyl-Phosphatidylethanoamine Apoptosis-Associated Speck Like Protein Adenosine Triphosphate Cyclic Guanosine Monophosphate-Adenosine Monophosphate Cyclic GMP-AMP Synthase Coronavirus Disease 2019 Cyclooxygenase-2 Damage-Associated Molecular Patterns Endoplasmic Reticulum Ferrous Iron Ferric Iron Ferrostatin-1 Glutathione Peroxidase 4 Gasdermin D Gasdermin E Glutathione

E. P. Amaral (*) · S. Namasivayam Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA e-mail: [email protected]

H2O2 HMGB1 HO-1 HSV-1 HXA3 IFN IL IV LCMV L-O− L-OOH LTB4 LXA4 MLKL Mtb NAC NADPH NCOA4 NF-kB NO NOS2 O2 O•2- OONO− PAMPs PGE2 PLC

Hydrogen Peroxide High Mobility Group Box-1 Heme Oxygenase-1 Herpes Simplex Virus-1 Hepoxilin A3 Interferon Interleukin Intravenous Lymphocytic Choriomeningitis Virus Alkoxyl Radicals Lipid Hydroperoxides Leukotriene B4 Lipoxin A4 Mixed Lineage Kinase Domain-Like Protein Mycobacterium tuberculosis N-Acetylcysteine Nicotinamide Adenine Dinucleotide Phosphate Nuclear Receptor Coativator 4 Nuclear Factor Kappa-LightChain-Enhancer of Activated B Nitric Oxide Nitric Oxide Synthase 2 Oxygen Superoxide Peroxynitrite P a t h o g e n - A s s o c i a t e d Molecular Patterns Prostaglandin E2 Phospholipase C

© Springer Nature Switzerland AG 2021 A. F. Florez, H. Alborzinia (eds.), Ferroptosis: Mechanism and Diseases, Advances in Experimental Medicine and Biology 1301, https://doi.org/10.1007/978-3-030-62026-4_5

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E. P. Amaral and S. Namasivayam

60

pLoxA PUFA RIPK

Pseudomonas Lipoxygenase A Polyunsaturated Fatty Acids Receptor-Interacting Serine/ Threonine-Protein Kinase Reactive Nitrogen Species Reactive Oxygen Species Serum Amyloid A Severe Acute Respiratory Syndrome Coronavirus 2 Solute Carrier Family 7 Member 11 Superoxide Dismutase Signal Transducer and Activator of Transcription 3 Stimulator of Interferon Genes Tuberculosis Toll Like Receptor Tumour Necrosis Factor Receptor 1 Tumour Necrosis Factor Alpha Tryparedoxin Peroxidases Viral Accessory Protein Xanthine Oxidase

synthase 2 (NOS2), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase (XO), lipoxygenase and cyclooxygenase (Betteridge 2000; Iovine et al. 2008). Peroxynitrite RNS (OONO−), an example of RNS, can modify ROS molecular structures of DNA and proteins by SAA reacting with important catalytic or binding sites, SARS-Cov-2 which in turn may affect function and/or biological properties of these molecules (Pacher et  al. SLC7A11 2007). For instance, peroxynitrite-derived radicals are highly reactive with proteins containing SOD transition metal center, including iron and this STAT3 reaction can in turn result in intracellular iron accumulation (Bartesaghi and Radi 2018; STING Ischiropoulos and al-Mehdi 1995; Shu et  al. TB 2015; Slovakova and Mraz 1991). Elevated intraTLR cellular iron level has been shown to be detrimenTNFR1 tal for the host in several diseases (Dev and Babitt 2017). In the case of infectious diseases, accumuTNF-α lation of free iron favors pathogen growth and Tpx can also trigger an iron-dependent form of Vpr necrotic cell death named ferroptosis (Amaral XO et al. 2019; Costa et al. 2016). Physiologically optimal concentrations of ROS are critical for normal cell function and survival. However, excessive and uncontrolled pro5.1 Introduction duction and accumulation of ROS due to insufficient cellular antioxidant capacity can be Cellular stress is the first effector mechanism in detrimental to host cells. Important ROS molethe host response to non-self-antigens, self-­ cules include superoxide, peroxides and hydroxyl danger signals (also known as damage-associated radicals. Superoxide anion ( O•2- ) is a free oxygen molecular pattern, DAMPs) and pathogens. radical resulting from the addition of an electron Reactive nitrogen species (RNS) and reactive to oxygen (Miller et al. 1990). The generation of oxygen species (ROS) are a family of antimicro- this free radical mostly occurs in the mitochonbial molecules generated by intracellular chemi- dria as a result of spontaneous mitochondrial cal reactions and/or by enzymatic activity. electron transport chain, but can also be derived Although well-known for their importance in as a byproduct of other enzymatic reactions, such controlling infection, these compounds also have as lipoxygenases (McIntyre et  al. 1999). a role in regulating several physiological and cel- Superoxide cannot penetrate into biological lular functions including vascular smooth muscle membranes and is rapidly converted into hydrotone, blood pressure, platelet activation, vascular gen peroxide (H2O2) and oxygen (O2) by supercell signaling, modulation of immune response oxide dismutase (SOD) or XO (Loschen et  al. and cell death in homeostatic conditions (Guzik 1974). H2O2 can easily pass through biological et  al. 2002; Roy et  al. 2017; Sena and Chandel lipid membranes and interact with DNA-bound 2012; Vara and Pula 2014). transition metal ions or with ferric iron in the RNS and ROS are molecules derived from cytosol, culminating in the generation of highly nitric oxide (NO) or oxygen generated through toxic hydroxyl free radicals via Fenton reaction the enzymatic activity of inducible nitric oxide (Blakely et  al. 1990; Halliwell et  al. 2000;

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Phaniendra et  al. 2015). The generation of hydroxyl radicals is detrimental to host cells causing damage to proteins and DNA. It also initiates lipid peroxidation, which in turn promotes inflammation, fibrosis and ferroptosis, a form of regulated cell death (Dias et  al. 2020; Albano et al. 2005; Yin et al. 2015; Dixon et al. 2012). All of the above-mentioned reactive species are highly induced following pathogen infection, leading to ROS-dependent host cell death (Ivanov et al. 2016; Lee et al. 2011; Maurice et al. 2019; Miotto et al. 2019; Paiva and Bozza 2014; Shin et  al. 2010). We will discuss how these signals can modulate cell fate and pathogenesis in detail in this chapter. Regulation of both cell death and inflammation are critical during host immune response against invading pathogens. Apoptosis and necrosis are two major forms of cell death that display completely different outcomes. Apoptosis is a caspase-dependent and non-inflammatory form of programmed cell death. This form of programmed death has been suggested to facilitate the control of most intracellular pathogen infections via an active process called efferocytosis (Briken 2012; Martin et al. 2012). Efferocytosis is an innate mechanism of engulfment of apop-

totic bodies as a result of programmed tissue repair. Thus, clearance of apoptotic bodies containing infective organisms facilitates pathogen killing without causing pathogen dissemination or triggering strong inflammatory response (Martin et al. 2012). On the other hand, necrosis is a lytic form of cell death known to be highly inflammatory as all intracellular contents including enzymes, DNA as well as the pathogen are released to the extracellular milieu. Loss of plasma membrane integrity is the most defining feature of necrosis, a process that is not observed in apoptosis. The release of large amounts of danger signals triggers a massive inflammatory response leading to extensive immune cell migration into the affected tissue, which results in tissue damage and in some cases favors the spread of infection. The major features that distinguish apoptosis and necrosis are listed in the Table 5.1. In this chapter, we review the mechanisms of necrotic cell death and highlight how imbalance between oxidative stress and cellular antioxidants may limit or favor pathogen dissemination and host cell death. Specifically, we discuss the role of ferroptosis, its induction, regulation and molecular components involved in the context of pathogenesis of infectious diseases.

Table 5.1  Differences in features of apoptosis and necrosis Feature Cause Trigger Pathogenesis Cell size Energy balance Annexin V/PI staining assay Plasma membrane

Morphology Organelles

Nuclei Release of cytoplasm content Inflammatory response

Apoptosis Programmed Intrinsic and extrinsic Physiological and pathologic Reduced Retained ATP production Annexin Vpositive/PInegative

Necrosis Damage/injury Extrinsic Pathologic Increased ATP depletion Annexin Vpositive/PIpositive

Intact displaying changes in lipid orientation; extensive membrane blebbing; retraction of membrane projections Spherical shape of cells Intact

Disrupted

Condensation and DNA fragmentation in intranucleosomal fragments No Non-inflammatory

Increased translucent cytoplasm Swelling of ET and loss of ribosomes; swollen mitochondria with altered density; lysosomal rupture Random DNA fragmentation Yes Acute inflammation

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5.2

Necrosis in Infectious Diseases

release of DAMP signals from dying cells that promotes extensive cellular migration into the tissue as well as sensitizing neighboring cells to Necrosis is an irreversible form of cell death die. In general, DAMPs are endogenous host resulting from physical/mechanical and/or chem- molecules that alert the immune response to cell ical stimuli. Such stimuli include oxygen depri- death, microbial infection or cellular stress, vation or hypoxia, extreme environmental including high mobility group box-1 (HMGB1), conditions such as temperature variation, radia- S100A8 (also known as MRP8 or calgranulin A), tion, toxins, and infectious agents such as bacte- S100A9 (also known as MRP9 or calgranulin B), ria, viruses, fungi and parasites (Belavgeni et al. serum amyloid A (SAA) and ATP (Gong et  al. 2020; Sarhan et al. 2018b; Tang et al. 2019). The 2020; Matzinger 2002). In infectious diseases, major feature of necrotic cells is the loss of the recognition of extracellular ATP (eATP) plasma membrane integrity leading to release of released from necrotic cells by a purinergic all intracellular products as well as intracellular receptor named P2x7 has been described to trigpathogens to the extracellular space. Along with ger extensive inflammation in several models of plasma membrane disruption, typical features of infection (Amaral et  al. 2014; Bomfim et  al. necrotic cell death include oxidative burst, mito- 2017; Chaves et  al. 2019; Santana et  al. 2015). chondrial membrane hyperpolarization, lyso- Depending on the concentration of eATP, P2x7 somal membrane permeabilization and receptor can play a dual role in infectious generalized swelling of membranous organelles disease. In mycobacterial infections involving such as mitochondria (Table 5.1). hypervirulent strains, P2x7 receptor facilitates For a long time, necrosis was considered to be development of necroinflammation as a consean uncontrolled passive form of cell death. quence of large amount of eATP released from However, a number of recent studies have dem- dead cells (Amaral et  al. 2014; Bomfim et  al. onstrated that this process is carried out by dis- 2017). On the other hand, less virulent mycobactinct and complex molecular mechanisms. terial infections result in low concentration of Necrotic cell death can be divided in two groups: eATP which in turn can trigger apoptosis, favoraccidental cell death and regulated necrotic cell ing bacterial control and promote tissue repair death. Accidental cell death is a form of lytic (Hill et al. 2010). In most bacterial infections, the death usually triggered by extreme physical and/ establishment of a necroinflammatory response or chemical stimuli leading to uncontrolled death facilitates bacterial dissemination to other host (Tang et al. 2019). On the other hand, regulated cells and organs (Amaral et al. 2015). In this secnecrotic cell death, which includes pyroptosis, tion, we present a brief description of these cell necroptosis (See Chap. 7) and ferroptosis, are death pathways and introduce the key enzymes controlled by intracellular components that and main regulatory mechanisms involved in induce plasma membrane rupture through pore-­ these processes. formation or via oxidation and/or degradation of lipids on plasma membrane (Conrad et al. 2016). The molecular mechanism of these distinct forms 5.2.1 Pyroptosis and Necroptosis in Infectious Diseases of regulated necrotic cell death will be discussed in detail in this book. Necroinflammatory response is a term used to Pyroptosis and necroptosis, both forms of regudescribe an amplification loop of cell death lated necrosis, depend on the activation of a numdriven by necrosis (accidental necrosis and/or ber of enzymes and oligomerization of regulated necrotic cell death) promoting a wide- pore-forming proteins on the plasma cell memspread inflammatory response, which in most brane. Pyroptosis is a caspase-dependent form of cases lead to organ failure (Fig. 5.1). This gener- necrosis and requires the activation of effector alized inflammatory response is associated with caspases such as caspase-1 and caspase-11 asso-

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Fig. 5.1  Necroinflammation as a result of a widespread cellular necrosis promoting tissue damage. Necrotic cell death is marked by loss of plasma membrane integrity. In contrast to apoptosis, necrosis is a proinflammatory form of cell death. As a result of necrosis of plasma membrane rupture, proinflammatory mediators including cytokines, ATP, DNA, oxidized lipids and others are released into extracellular milieu. All forms of regulated necrotic cell

death including pyroptosis, necroptosis and ferroptosis have a potent capacity to trigger proinflammatory response, culminating in an extensive cellular infiltration into the affected tissue. Since these forms of necrotic death release DAMP signals, they may sensitize neighboring cells to undergo necrosis, promoting a widespread cellular necrosis and tissue damage

ciated with the inflammasome complex and result in production of the highly pro-inflammatory cytokines, IL-1β and IL-18. Inflammasomes are a macromolecular complex composed by a sensor protein, NLRP3, an adaptor protein named apoptosis-­ associated speck like protein (ASC) that contains a caspase recruitment domain and caspase-1/11. Different stimuli may trigger the activation of inflammasomes such as pathogen-­ associated molecular patterns (PAMPs), DAMPs, Ca2+ influx and K+ efflux (Lamkanfi and Dixit 2014). Once activated, caspase-1/11 cleave pro-­ IL-­1β and pro-IL-18 generating mature forms of IL-1β and IL-18 (Dinarello 2009). In addition, active caspase-1/11 induce the cleavage of gasdermin D (GSDMD) which oligomerizes, forming a pore structure on plasma membrane (Ding et  al. 2016; Liu et  al. 2016). In certain cases,

caspase-­ 3 activated during apoptosis cleaves gasdermin E resulting in a form of secondary pyroptotic cell death due to gasdermin E (GSDME)-dependent pore-formation on the plasma membrane (Mai et  al. 2019; Rathkey et al. 2018). As pore formation triggered by gasdermins is an important feature of pyroptosis, this pathway is also defined as gasdermin-­ dependent form of cell death (Fig. 5.2). Pyroptosis has been reported to occur in the context of several bacterial and viral infections, such as those due to Salmonella sp. (Miao et al. 2010), Shigella sp. (Kobayashi et  al. 2013), Listeria monocytogenes (Sauer et al. 2010), Pseudomonas aeruginosa (Balakrishnan et al. 2018), Yersinia (Sarhan et  al. 2018a), influenza virus (Kuriakose et  al. 2016), as well as during infection by the parasite Toxoplasma gondii (Fisch et al. 2019).

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Fig. 5.2  Regulated necrotic cell death can be classified by its requirement or not of caspase activity. Pyroptosis is a caspase-dependent form of necrosis characterized by IL-1β and IL-18 generation as well as gasdermin-­ dependent pore formation on plasma membrane. Necroptosis and ferroptosis are both forms of regulated necrotic cell death which do not require activation of caspases. Necroptosis is regulated by the phosphorylation of RIPK1, RIPK3 and MLKL, which in turn oligomerizes and forms a pore on plasma membrane. Ferroptosis is triggered by intracellular iron accumulation occurs when host antioxidant response is found to be  insufficient, marked by low levels of glutathione as well as reduced glutathione

peroxidase 4 expression and activity. In ferroptosis, plasma membrane rupture is induced by the accumulation of toxic lipid peroxides. Accidental cellular necrotic death is a typical unregulated form of necrosis triggered by mechanical or chemical stimuli. IL interleukin, IFN interferon, DAI DNA-dependent activator of IFN-regulatory factors, PKR protein kinase R, pMLKL phosphorylated mixed lineage kinase domain-like protein, RIPK receptor-­ interacting serine/threonine-protein kinase, STAT3 signal transducer and activator of transcription 3, TLR Toll like receptor, GSH glutathione, GPX4 glutathione peroxidase 4

The second form of regulated necrotic cell death necroptosis is caspase-independent. This form of regulated necrosis shares morphological similarities with pyroptosis, such as plasma membrane rupture induced by pore-formation. However, the molecular pathway leading to the pore formation in necroptosis is different from that observed in pyroptosis and occurs when capase-8 is found to be inhibited or non-­ functional and is triggered by type 1 interferons or binding of the cytokine tumour necrosis factor (TNF) to its receptor TNFR1 (Robinson et  al.

2012). Necroptosis is regulated by the activation of enzymes called RIP kinases, such as RIPK1 and RIPK3 (Degterev et al. 2008; He et al. 2009; Lin et al. 2004). Deubiquitylated RIPK1 interacts with RIPK3 promoting autophosphorylation of RIPK3 and subsequently phosphorylation of mixed lineage kinase domain-like protein (MLKL). Phosphorylated MLKL oligomerizes and binds to the plasma membrane resulting in pore formation and membrane permeabilization leading to lytic cell death (Fig.  5.2). Similar to pyroptosis, this form of cell death has also been

5  Emerging Role for Ferroptosis in Infectious Diseases

implicated in several models of infection including Salmonella Typhimurium (Robinson et  al. 2012), E. coli (Sridharan and Upton 2014), Staphylococcus aureus (Kitur et  al. 2015), Mycobacterium tuberculosis (Roca and Ramakrishnan 2013), Yersinia pestis (Weng et al. 2014) and influenza (Kuriakose et al. 2016).

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death (Fig. 5.3) (Miotto et al. 2019; Soustre and Rakotonirina 1981). In addition, lipid peroxidation can also be induced by enzymes called lipoxygenases via degradation of PUFA. As discussed previously, the lytic cell process seen in pyroptosis and necroptosis is driven by a protein pore formation mediated by gasdermins or MLKL oligomerization, respectively. On the other hand, the nature of lysis in ferroptosis is different when 5.3 Ferroptosis in Infectious compared to other forms of regulated necrotic cell death. In ferroptosis, it is hypothesized that Diseases the accessibility of oxidants to PUFA-enriched Ferroptosis is a regulated form of lytic cell death membranes increases cellular membrane curvacharacterized by accumulation of lipid peroxides ture allowing more oxidation of those lipids. The on biological membranes generally in an iron-­ failure of GPX4 activity in removing lipid hydrodependent manner (Dixon et al. 2012; Stockwell peroxides from biological membranes lead to et  al. 2020). This form of necrotic cell death is pore generation and micellization, culminating in finely regulated by glutathione peroxidase 4 irreversible damage to membrane integrity (GPX4) enzymatic activity (Ingold et al. 2018). (Agmon et  al. 2018). Nevertheless, ferroptosis Lipid peroxides are commonly induced in steady has also been shown to play an important role in state. However, GPX4 utilizes glutathione (GSH) diseases caused by M. tuberculosis (Amaral et al. to reduce oxidized lipids on membranes made up 2019), P. aeruginosa (Dar et al. 2018), Leishmania of polyunsaturated fatty acids (PUFA), prevent- major (Matsushita et  al. 2015), Plasmodium sp ing plasma membrane destabilization. (Kain et  al. 2019) and polymicrobial-induced Dysregulation in cellular labile iron levels can be sepsis (Kang et al. 2018). Additionally, modulatriggered by several factors such as via increased tion of ferroptotic components has been described heme oxygenase 1 (HO-1) (Kwon et  al. 2015), in Salmonella Typhimurium (Agbor et al. 2014) transferrin uptake (Gao et  al. 2015), reduced and HIV (Morris et al. 2013) infections. expression of ferroportin (Geng et al. 2018) and depletion of ferritin via ferritinophagy (Hou et al. 2016; Mancias et al. 2014; Yoshida et al. 2019). 5.3.1 Pathogen-Induced Ferroptotic Cell Death The mechanism of intracellular labile iron accumulation by ferritinophagyhas been discussed in depth in Chap. 4 of this book. Further, mitochon- Infectious agents usually trigger an oxidative drial superoxide has been shown to play an stress response in host cells, which in some cases important role in removing ferric iron (Fe3+) may favor the pathogen and in other models facilbound to ferritin resulting in the release of fer- itate the killing of these invasive agents by modurous iron (Fe2+) into the cytosol (Biemond et al. lating host cell death response. In this section we 1988). Fe2+ feeds the Fenton reaction and gener- will discuss the role of ferroptosis in some infecates hydroxyl radicals as a bioproduct. Hydroxyl tious diseases as well as examine the regulation radicals are potent reactive molecules and react of molecular components involved in the ferropwith PUFA and promote oxidation of these lipids totic pathway. (Lloyd et al. 1997). Fe2+/Fe3+ disbalance associated with deregula- 5.3.1.1 Bacterial Infections tion of cellular antioxidative capacity allow the Bacterial pathogenesis is not strictly dependent generation of toxic lipid peroxides called alkoxyl on host cell viability, since a number of bacteria radicals. This lipid peroxidation eventually desta- are able to replicate in either or both intracellular bilizes membrane integrity resulting in lytic cell and extracellular environments. However, there

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Fig. 5.3  Ferroptosis is an iron-mediated form of regulated necrotic cell death characterized by accumulation of lipid peroxides on biological membranes. Labile iron reacts with H2O2 generating hydroxyl radicals, which in turn triggers the oxidation of polyunsaturated lipids leading to the formation of toxic lipid peroxides (alkoxyl radicals). In steady state, GPX4 oxidizes glutathione to reduce lipid peroxides, avoiding plasma membrane rupture. However, under stress conditions, cells fail in detoxifying

are some species that survive and proliferate better intracellularly evading host cellular defense. In this case, host cell integrity plays a dual role. One, preventing bacterial spread and necroinflammatory response and two, facilitating migration of effector cells to the site of infection that in turn results in bacterial killing.

E. P. Amaral and S. Namasivayam

lipid peroxides on biological membranes due to loss of GPX4 activity as a result of glutathione depletion. The mechanism by which GSH levels and GPX4 expression/ activity are found downregulated in ferroptosis is unclear. Recently, GPX4-independent mechanisms of ferroptosis regulation have been described, which are associated with the engagement of FSP1 and GCH1. •OH hydroxyl radicals, L-OOH lipid hydroperoxides, L-O− alkoxyl radicals, GSH glutathione, GPX4 glutathione peroxidase 4

for ferroptosis in this infectious disease. Interestingly, neutrophils migration was significantly increased when GPX4 mRNA was knocked down in intestinal epithelial cells whereas GPX4 overexpression in these epithelial cells completely abrogated the migration of polymorphonuclear cells. Moreover, S. Typhimurium infection triggered high levels of ROS which was Salmonella Typhimurium Infection associated with reduced expression and activity Several hallmarks of ferroptosis are observed of GPX4. Decreased GPX4 activity favored augduring Salmonella infection in an animal model. mented 12-LOX expression leading to increased In this model of bacterial infection, migration of arachidonic acid metabolism, HXA3 synthesis neutrophils across mucosal surfaces has been and neutrophil migration across the epithelial implicated in the dysfunction of epithelial barrier barrier. Interestingly, it seems that bacterial facproperties (Lee et al. 2000). It is known that an tor SipA appears to be involved in the downreguendogenous product of 12-lipoxygenase (12-­ lation of GPX4 expression and activity. This was LOX), hepoxilin A3 (HXA3), is secreted from the the first study demonstrating that bacteria can intestinal epithelium eliciting the migration of subvert the antioxidant system by diminishing neutrophils across the epithelial surface (Agbor both expression and activity of GPX4. More et  al. 2014). Also, a significant reduction of recently, administration of a dichloromethane GPX4 expression at both mRNA and protein lev- fraction of Dichrocephala integrifolia to animals els was observed during S.  Typhimurium infec- infected with S.  Typhimurium was found to tion by an unclear mechanism, suggesting a role increase the levels of glutathione, superoxide

5  Emerging Role for Ferroptosis in Infectious Diseases

d­ ismutase and catalase along with a reduction in lipid peroxide accumulation and tissue damage. This treatment significantly enhanced mouse resistance against S.  Typhimurium infection by improving antioxidant status and reducing lipid peroxidation, unquestionably indicating the importance of ferroptosis in this disease model and warranting further investigation (Fankem et al. 2019). Mycobacterium tuberculosis Infection M. tuberculosis is the etiological agent of tuberculosis (TB). A prolonged struggle between the host and pathogen emerges when the infection is established in the lung. Macrophages are the first cells that encounter the pathogen and are required for bacterial control. Nevertheless, M. tuberculosis modulates macrophage defense as well as cell fate. Exacerbated host response against M. tuberculosis may contribute to mycobacterial sterilization but may also be detrimental for the host by promoting a necroinflammatory response resulting in uncontrolled immunopathology. The balance between apoptosis and necrosis in tuberculosis determines the disease outcome, since apoptosis has been associated with bacterial control whereas necrosis is reported to facilitate mycobacterial dissemination to other cells as well as to other organs (Amaral et  al. 2016b). These distinct cell death modalities promote a wide spectrum of immunopathology, ranging from asymptomatic infection to disseminated disease and ultimately patient death. Bacterial virulence is thought to play a role in the modulation of macrophage death. Avirulent mycobacteria have been shown to modulate macrophage death towards apoptosis whereas virulent strains are prone to trigger necrotic death (Divangahi et al. 2009). Two groups of enzymes, cyclooxygenases (COX) and lipoxygenases, have been shown to play an important role in modulating host defense and cell death following M. tuberculosis infection. Both enzymes compete for arachidonic acid and cyclooxygenases induce the synthesis of prostaglandins (PGE2), while lipoxygenases cleave polyunsaturated fatty acid generating

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lipoxins ad leukotrienes (Divangahi et al. 2009). Interestingly, lipoxygenases have been reported to be involved in ferroptosis by inducing peroxidation of PUFA whereas prostaglandins are shown to play an important role in promoting cell viability by inducing GPX4 activity (Karuppagounder et al. 2018; Yang et al. 2014). High levels of lipoxin A4 (LXA4) have been reported in patients with acute TB disease as well as in mice infected with virulent strains (Mayer-­ Barber et al. 2014). Elevated LXA4 has deleterious effect on host cell response against M. tuberculosis by promoting macrophage necrosis. In contrast, PGE2 has been proposed to protect macrophages from necrosis thus favoring host resistance to M. tuberculosis. Indeed, 5-­ lipoxygenase (5-LO) deficient mice display increased resistance to M. tuberculosis infection, whereas mice deficient in PGE2 exhibit high susceptibility to infection (Bafica et al. 2005; Behar et  al. 2010; Chen et  al. 2008). Moreover, IL-1 signaling has been implicated to play a role in the regulation of PGE2, since mice deficient in IL-1β exhibit profound reduction of PGE2 levels along with accumulation of LXA4 and LTB4, the latter two being products of 5-LO activity (Mayer-­ Barber et al. 2014). Furthermore, GPX4 has been shown to inhibit lipoxygenase activity and increase the expression of COX-2  in different models of pathologic diseases (Barriere et  al. 2004; Imai et al. 2017). These findings argue in favor of a role of ferroptosis in M. tuberculosis infection. Indeed, recently, ferroptosis has been shown to be involved in M. tuberculosis-induced necrotic cell death. Increased M. tuberculosis replication in macrophages is associated with elevated levels of intracellular labile iron, increased mitochondrial superoxide, accumulation of lipid peroxides and cellular necrosis. Furthermore, highly infected macrophages display reduced intracellular GSH levels and GPX4 expression which were both correlated with cells undergoing necrosis. All of these parameters are together important hallmarks of ferroptosis. Interestingly, following treatment with either ferrostatin-­1 (Fer-1), a potent inhibitor of lipid

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E. P. Amaral and S. Namasivayam

peroxidation, or an iron chelator, M. tuberculosis-­ the activity of lipoxygenases which in turn poteninfected macrophages displayed resistance to tiates the deleterious effect of lipid peroxidation necrotic cell death in vitro, suggesting ferroptosis (Dufrusine et al. 2019). as a major form of regulated necrotic cell death in Finally, several reports have associated vitaM. tuberculosis infection. Notably, Fer-1 treat- min E (an antioxidant that dampens lipid ROS) ment resulted in remarkable reduction of lung tis- deficiency with host susceptibility and disease sue necrosis as well as lowered bacterial burden severity, supporting the hypothesis that loss of in lungs and spleen (Amaral et  al. 2019). antioxidant response and excessive ROS generaHowever, the exact mechanism by which M. tion are both detrimental to the host (Aibana et al. tuberculosis triggers ferroptosis is still unclear. 2018; Dalvi et  al. 2013; Lamsal et  al. 2007; There is some evidence to suggest accumulation Madebo et al. 2003; Vijayamalini and Manoharan of intracellular iron via overexpression of HO-1 2004). A number of clinical studies that have and ferritinophagy following infection as a pos- shown that patients given vitamin E (Dixon et al. sible mechanism. HO-1 cleaves intracellular 2012), selenium (important for function of some heme generating three byproducts, carbon mon- antioxidant enzymes such as GPX4) (Ingold oxide, biliverdin and labile iron (Chung et  al. et al. 2018) and/or N-acetylcysteine (NAC; a pre2009). Elevated plasma levels of HO-1, increased cursor of GSH, an important antioxidant) expression of HO-1 and iron overload have been (Amaral et al. 2016a) as adjunct to M. tuberculoreported in the lungs of active TB patients as well sis antibiotic therapy  displayed improved host as in a murine model of pulmonary M. tuberculo- response to treatment when compared to those sis infection (Andrade et  al. 2013, 2015; Costa receiving placebo (Campa et al. 2017; Cao et al. et  al. 2016; Rockwood et  al. 2017). Thus, aug- 2018; Hernandez et al. 2008; Kranzer et al. 2015; mented HO-1 activity may favor the accumula- Mahakalkar et  al. 2017; Seyedrezazadeh et  al. tion of labile iron, an important fuel for Fenton 2008). reaction and ferroptosis. Another possible mechanism for intracellular iron overload following Pseudomonas aeruginosa Infection M. tuberculosis infection is via the induction of Ferroptosis has also been recently implicated in ferritin degradation, the cellular cytosolic iron Pseudomonas aeruginosa infection. This opporstorage complex, via autophagy. Ferritinophagy tunistic bacterium secretes lipoxygenase is mediated by the activation of nuclear receptor (pLoxA) and oxidizes host arachidoyl-­ coativator 4 (NCOA4). This molecule induces phosphatidylethanoamine (AA-PE), a process selective autophagic degradation of ferritin required for the transition of P. aeruginosa thereby affecting the regulation of intracellular growth. Strikingly, epithelial cells in the presence and systemic iron homeostasis. NCOA4-­ of secreted pLoxA undergo necrotic cell death. mediated ferritinophagy have been shown to Increased levels of lipid peroxidation and reduced increase susceptibility to ferroptosis in several intracellular GSH levels were associated with models (Gao et  al. 2016; Hou et  al. 2016). epithelial cell death. Interestingly, this necrotic Interestingly, mice deficient in ferritin are cell death was inhibited by treating cells with extremely susceptible to M. tuberculosis infec- Fer-1 but not following treatment with drugs that tion, displaying increased bacterial loads in lungs prevent apoptosis or pyroptosis (z-VAD-fmk, pan and spleens and massive cellular infiltration and caspase inhibitor), necroptosis (necrostatin-1) or tissue damage. Also, iron-associated proteins autophagic death (bafilomycin-A1). Moreover, P. including HO-1 and ferroportin are upregulated aeruginosa-induced ferroptosis was also associin the lungs of mice lacking ferritin upon M. ated with reduced protein expression of GPX4. tuberculosis infection, which in turn favor the Further, redox phospholipidomics revealed eleaccumulation of labile iron in system (Reddy vated levels of oxidized AA-PE in airway tissues et al. 2018). Intriguingly, labile iron also increases from patients with cystic fibrosis, which was not

5  Emerging Role for Ferroptosis in Infectious Diseases

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found in samples from patients with emphysema or cystic fibrosis negative for P. aeruginosa (Dar et al. 2018). Thus, inhibiting pLoxA-driven ferroptotic epithelial cell death has been suggested as a potential target for therapeutic intervention against P. aeruginosa-associated diseases.

glutathione levels and reduced availability of free iron (Dal-Pizzol et  al. 2010; Moch et  al. 1995; Ritter et al. 2004; Vlahakos et al. 2012). Although the activation of components of pyroptotic pathway is thought to play a role in inducing septic lethality in mice, these findings suggest that the induction of ferroptotic death pathway by failure of GPX4 activity is an important trigger of caspase-­11/GSDMD-mediated pyroptosis.

Polymicrobial Sepsis Sepsis is a common lethal complication of bacterial infections. This process occurs due to excessive activation of innate immune cells leading to tissue damage and multiple organ failure, accounting for 250,000 deaths per year only in USA (Singer et  al. 2016). It has been reported that pyroptotic cell death is a crucial event in the lethal sepsis cascade, triggered by bacterial infection as a result of GSDMD cleavage by caspase-1 and caspase-11 (Kayagaki et al. 2015; Shi et al. 2015). Kayagaki and colleagues showed that animals deficient in GSDMD are more resistant to lethal sepsis induced by high concentration of LPS compared to WT mice. More recently, a crosstalk between two necrotic death pathways was elucidated in a sepsis model. Kang and colleagues showed that GPX4 negatively modulates macrophage pyroptosis and prevents septic lethality in mice (Kang et  al. 2018). GPX4 depletion in myeloid cells increases lipid peroxidation triggering excessive caspase-11 activation and ultimately enhanced GSDMD cleavage, suggesting that GPX4 activity by inhibiting lipid peroxidation prevents activation of caspase-11/GSDMD-dependent pyroptosis. Moreover, vitamin E, a ferroptosis inhibitor, when given to mice protected the animals from lethal sepsis. In addition, increased activation of phospholipase C (PLC) was observed in macrophages deficient in GPX4 and the same was significantly inhibited in mice receiving vitamin E. Importantly, drug inhibition or genetic ablation of PLC prevented GSDMD-­ mediated pyroptosis. Interestingly, animals receiving NAC (an glutathione precursor) and deferoxamine (an iron chelator), both compounds described to prevent ferroptosis, also displayed increased resistance against lethal sepsis and this effect was found to be mediated through improved

5.3.1.2 Viral Infection The role of ferroptosis in viral infections is only beginning to be documented. Some hallmarks of ferroptosis are induced during HIV infection, which may increase host susceptibility to secondary infections by opportunistic pathogens. Infection of macrophages by HIV was first described in the 1980s and a recent study revealed that tissue macrophages are found infected at all stages of disease (Cory et al. 2013). It has been shown that monocytes/macrophages and T cells isolated from HIV+ individuals display lowered levels of reduced GSH favoring M. tuberculosis co-infection (de Quay et al. 1992; Eck et al. 1989; Guerra et  al. 2011; Morris et  al. 2013). Spontaneous H2O2 production, an important ROS component of the Fenton reaction, in monocytes from HIV-infected individuals is associated with high viral load (Elbim et al. 1999). HIV envelope glycoprotein gp41 and viral accessory protein (Vpr) can also increase ROS levels in monocyte-­ derived macrophages causing cell death (Garg and Blumenthal 2006; Hoshino et  al. 2010). Further, high levels of lipid peroxidation has also been found in HIV+ patients (Anderson et  al. 2018; Favier et  al. 1994; Lopez et  al. 1996; Mebrat et  al. 2017; Sonnerborg et  al. 1988). Interestingly, vitamin E, selenium and glutathione peroxidase levels are all reduced in plasma of HIV+ patients when compared with healthy individuals (Favier et  al. 1994; Malvy et  al. 1994). Supplementation of vitamin E in the diet of HIV+ patients reduced plasma lipid peroxides and was associated with lowered viral load (Allard et al. 1998). In addition, GSH supplementation has also been reported to improve macrophage and T cell function in HIV+ patients. Furthermore, this

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supplementation decreased the intracellular pathogen growth in macrophages obtained from these HIV+ patients (Guerra et  al. 2011; Morris et al. 2013). A similar profile of host protective response was observed in patients receiving selenium supplementation. Se-treated patients displayed significantly lower risk of developing incident TB disease compared with placebo (Campa et al. 2017). Effect of selenium was also examined in animal models of AIDS.  Monkeys infected with SIV displayed lowered levels of blood selenium and the lowest levels correlated with the highest peak of viral. Significant reduction in GPX1 and GPX4 expression was also observed following SIV infection, suggesting that low levels of both selenium and glutathione peroxidase are associated with progression of AIDS (Xu et al. 2002). A murine model of AIDS using LP-BM5 murine leukemia virus (MuLV) was also employed to investigate the effect of selenium on host resistance. Infected animals displayed reduced levels of selenium, glutathione and glutathione peroxidase along with increased levels of lipid peroxidation. Interestingly, selenium treatment reversed this phenotype and improved the antioxidant status in these animals (Chen et al. 1998). Similarly, a study utilizing the acute lymphocytic choriomeningitis virus (LCMV) model of infection has revealed a role for GPX4 in T cell immunity and protection against LCMV infection. While expression of GPX4 in T cells was not required for thymic development and maturation, it was found to be essential for maintenance of CD8 T cells in the periphery and expansion of both CD4 and CD8 T cells following LCMV infection. The absence of GPX4 results in an increased accumulation of lipid peroxides in T cells and ferroptotic cell death. In addition, the viability and expansion of GPX4-deficient T cells was restored via dietary vitamin E supplementation (Matsushita et al. 2015). The importance of redox homeostasis maintained by GPX4 in modulating the host immune response against herpes simplex virus-1 (HSV-1) was recently demonstrated (Jia et al. 2020). It is known that production of type I IFNs by innate

E. P. Amaral and S. Namasivayam

immune cells via activation of cGAS-STING pathway is crucial for the protective host response against HSV-1 (Stempel et al. 2019). This signaling pathway is initiated by sensing of cytosolic pathogen-derived DNA or self-DNA from genomic DNA damage mediated by cGAS, generating cyclic guanosine monophosphate-­ adenosine monophosphate (cGAMP). Binding of cGAMP triggers STING translocation from endoplasmic reticulum (ER) to Golgi apparatus, which is required for optimal activation of STING.  By infecting GPX4-deficient macrophage in vitro or LysMcre+GPX4fl/fl mice with HSV-1, Jia and colleagues showed that in both experimental settings the host immune response against this pathogen was compromised by the lack of GPX4. The enhanced host susceptibility to HSV-1 infection seen in the absence of GPX4 was marked by reduced type I IFN production, inactivation of STING, increased levels of lipid peroxidation, elevated pathogen burden, and high animal lethality when compared with HSV-1-­ infected WT mice. Mechanistically, the accumulation of lipid peroxides found in absence or inactivation of GPX4 led to STING carbonylation at C88, and thus prevented STING trafficking from ER to Golgi as well as STING activation. Importantly, it was shown that STING inactivation mediated by lipid peroxides was not associated with cell death at earlier time-points (Jia et al. 2020). However, it is still unclear whether HSV-1 infected cells undergo death through ferroptosis at later time-points in vitro and in vivo as a consequence of lack of GPX4 activation. A new strain of coronavirus named “severe acute respiratory syndrome coronavirus 2” (SARS-Cov-2) has been responsible for the most recent pandemic due to its high infectivity, which reflects in elevated numbers of deaths. The major feature of COVID-19, the disease caused by SARS-Cov-2, is an exacerbated and uncontrolled inflammatory cytokine production (also known as “cytokine storm”) associated with increased vascular permeability and oxidative stress, promoting acute lung injury or acute respiratory distress syndrome (Guan and Zhong 2020; Moore and June 2020). In this regard, oxidative stress in

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the lung may form a positive feedback loop to exacerbate pro-inflammatory cytokine production, leading to deterioration of some patients. Several reports have pointed out oxidative stress as a key player in severe COVID-19 ­(Delgado-­Roche and Mesta 2020; Ntyonga-Pono 2020), suggesting a possible role for ferroptosis in this disease. It is not clear whether SARS-Cov2-­infected cells undergo regulated necrosis such as pyroptosis or ferroptosis. However, histological analysis showed that COVID-19 patients display injury to the alveolar epithelial cells, lung consolidation accompanied with abundant intra-­ alveolar neutrophilic infiltration, and fibrinoid vascular necrosis of the small vessels (Tian et al. 2020). As mentioned previously GPX4, a selenoenzyme that utilizes selenium for its activity, is an important player in regulating ferroptotic cell death (Ingold et  al. 2018). Strikingly, a recent report revealed a strong association between selenium status and severe outcome of COVID-19 in patients in China. The authors showed that COVID-19 patients displaying low levels of selenium (potent antioxidant micronutrients) are more susceptible to severe forms of the disease. On the other hand, COVID-19 patients receiving high levels of selenium in their diet were more likely to recover from SARS-Cov-2 infection (Zhang et  al. 2020). Furthermore, endogenous deficiency of glutathione has been suggested as the most plausible explanation for severe manifestation and death in COVID-19 patients. Briefly, patients with moderate/severe disease displayed lowered levels of GSH and higher levels of ROS when compared with patients with mild disease (Polonikov 2020). Interestingly, a case report published recently showed that two COVID-19 patients receiving oral or intravenous (IV) glutathione or glutathione precursors (N-acetyl-cysteine, NAC) exhibited immediate improvement in their symptoms (Horowitz et al. 2020). Despite the small sample size, the authors suggested that NAC/GSH may represent a novel therapeutic approach for blocking exacerbated NF-kB activation and cytokine storm syndrome in patients with COVID-19 pneumonia. In addition, another study showed that IV administration

of NAC blocked hemolysis and elevated inflammatory immune response in patients with severe forms of COVID-19, suggesting NAC as a potential candidate for therapeutic interventions in COVID-19 (Ibrahim et  al. 2020). Moreover, Ebselen, a synthetic organoselenium drug that acts as a mimic of glutathione peroxidase in inhibiting ferroptosis (Dixon et  al. 2012), has been shown to exhibit inhibitory activity against COVID-19 in a cell-based setting, indicating potential beneficial effects of this drug in COVID-­19 therapy (Sies and Parnham 2020; Jin et al. 2020). Despite evidence supporting a possible role for redox homeostasis and ferroptosis in COVID-19, more studies investigating the role of oxidative stress as well as ferroptotic cell death in severe forms of COVID-19 are needed. Together, these studies provide evidence for the importance of ferroptosis in host resistance against viral infections and warrants further investigation.

5.3.1.3 Parasitic Infection Eukaryotic parasitic infections, unlike that due bacterial and viral pathogens, present a unique scenario in the study of cell death. Specifically, with regards to ferroptosis, the lipids involved in lipid peroxidization and ferroptotic cell death could arise either from the parasite or host cell. Indeed, recent work in Trypanosoma brucei, a parasite that causes African trypanosomiasis or sleeping sickness, identified the presence of GPX4 homologs, tryparedoxin peroxidases (Tpx) in these pathogens (Bogacz and Krauth-Siegel 2018). The insect stage of this parasite requires oxidative phosphorylation by the mitochondrion for energy production. Tpx deficiency is detrimental to the parasite in the insect stage and this lethal phenotype can be rescued via treatment with ferroptosis inhibitors such as Fer-1 or iron chelators. However, it should be noted that T. brucei display a decrease in ATP and loss of mitochondrial membrane potential that precede any plasma membrane leakage, two phenomena that are not essential in the ferroptosis mechanism observed in mammalian cells. Nevertheless, this evidence suggests that lipid peroxides gener-

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ated by the parasites need to be considered when studying ferroptosis in host cells infected with eukaryotic pathogens. In this regard, a study investigating the importance of ferroptosis in malaria identified that increased lipid peroxides in hepatocytes is, interestingly, beneficial to the host in the liver stage of the malaria parasite Plasmodium yoelii (Kain et  al. 2019). In an in vitro infection model, a decreased parasite load was observed in hepatocytes when GPX4 or the upstream gene in the pathway, a solute carrier family 7 member 11 (SLC7A11, also known as xCT), were knocked down or inhibited, whereas treatment with Fer-1 resulted in decreased parasite-localized lipid peroxides. This finding was corroborated in in vivo studies, where mice treated with a SLC7A11 inhibitor Erastin prior to P. yoelii challenge displayed up to a 2-day delay in the onset of blood stage infection. One could hypothesize that death of the hepatocytes is a mechanism to curtail parasite expansion in the liver stage which is essential for blood stage infection and thus is beneficial to the host. On the contrary, another study reported that GPX4 is essential in the resistance to Leishmania major infection via its important role in T cell function (Matsushita et al. 2015). T cell specific knockout of GPX4 caused a defect in the expansion of both antigen-specific CD4 and CD8 T cells following L. major infection resulting in

persistent parasite loads. Although in this scenario the importance of the ferroptosis relevant gene GPX4 is via ensuring optimal expansion of T cells during infection rather than a direct role in regulating death of an infected cell, these studies, nevertheless, highlight the need to further investigate lipid peroxidation and ferroptosis as a mechanism of cell death in parasitic infections.

5.4

Concluding Remarks

Ferroptosis is a newly described form of necrotic cell death and its role has been reported in several pathological diseases. A major role for ferroptosis was clearly described in M. tuberculosis infection and may provide a new perspective for host-directed therapy in TB. Ferroptosis has also been associated with the progression of disease in Leishmania and Pseudomonas murine infection models. In addition, lipid peroxidation-­ induced ferroptosis has also been implicated in sepsis, which represent an opportunity for developing novel strategies to prevent lethal sepsis (Fig. 5.4). Despite multiple lines of evidence suggesting that ferroptosis may play an important role in oxidative-induced cell death in many infectious diseases, the involvement of ferroptotic cell death in pathogen infection needs more formal and thorough investigation.

Fig. 5.4  Summary of potential effects of ferroptosis in promoting or controlling pathogen infection

5  Emerging Role for Ferroptosis in Infectious Diseases Acknowledgments This work was supported by the Intramural Research Program of the NIAID, NIH.

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NF-kappaB pathway in rheumatoid arthritis synovial cells. Mediat Inflamm 2015:460310. https://doi. org/10.1155/2015/460310 Yoshida M, Minagawa S, Araya J, Sakamoto T, Hara H, Tsubouchi K et  al (2019) Involvement of cigarette smoke-induced epithelial cell ferroptosis in COPD pathogenesis. Nat Commun 10(1):3145. https://doi. org/10.1038/s41467-­019-­10991-­7 Zhang J, Taylor EW, Bennett K, Saad R, Rayman MP (2020) Association between regional selenium status and reported outcome of COVID-19 cases in China. Am J Clin Nutr 111(6):1297–1299. https://doi. org/10.1093/ajcn/nqaa095

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Small Molecule Regulators of Ferroptosis Sylvain Debieu, Stéphanie Solier, Ludovic Colombeau, Antoine Versini, Fabien Sindikubwabo, Alison Forrester, Sebastian Müller, Tatiana Cañeque, and Raphaël Rodriguez

Abbreviations 12-HPETE 15-HPETE 15-LO 5-HPETE 5-LO ABCB10 ABCB7/8 ACACA ACAT ACSL4 AIFM2 ALOX15 AOA

12-­hydroperoxyeicosatetraenoic acid 15-­hydroperoxyeicosatetraenoic acid Arachidonate 15-­Lipoxygenase 5(S)-­H ydroperoxy-­6 -­t rans-­­ 8,11,14-­cis-­eicosatetraenoic acid Arachidonate 5-­Lipoxygenase ATP Binding Cassette Subfamily B Member 10 ATP Binding Cassette Subfamily B Member 7/Menber 8 Acetyl-­CoA Carboxylase Alpha Acetyl-­CoA Acetyltransferase Acyl-­CoA Synthetase Long Chain Family Member 4 Apoptosis-­Inducing Factor Mitochondria-­Associated 2 Arachidonate 15-­Lipoxygenase Aminooxyacetic Acid

S. Debieu · S. Solier · L. Colombeau · A. Versini F. Sindikubwabo · A. Forrester · S. Müller T. Cañeque · R. Rodriguez (*) Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France PSL Université Paris, Paris, France Chemical Biology of Cancer Laboratory, CNRS UMR 3666, INSERM U1143, Paris, France e-mail: [email protected]

ARE ATG5/7 ATP BAP1

Antioxidant Response Element Autophagy-­Related Gene 5 / 7 Adenosine Triphosphate Breast Cancer Susceptibility Gene 1-­Associated Protein 1 BECNP Phosphorylated Beclin 1 c-LIP cytosolic Labile Iron Pool c-Myb MYB Proto-­Oncogene, Transcription Factor c-Myc MYC Proto-­ Oncogene, BHLH Transcription Factor CARS Cysteinyl-­TRNA Synthetase CBS Cystathionine-­Beta-­Synthase CD44 Cluster of Differentiation 44 protein CDC Cinnamyl-­3 ,4-­d ihydroxy-­α cyanocinnamate CDO1 Cysteine Dioxygenase Type 1 CGL Cystathionine Gamma-­Lyase CISD1 CDGSH Iron Sulfur Domain 1 CMA Chaperone-­Mediated Autophagy CoA Coenzyme A CoQ10 Coenzyme Q 10 CP Ceruloplasmin DFO Deferoxamine D-PUFA Deuterated Polyunsaturated Fatty Acids DMT1 Divalent Metal Transporter 1 DPP4 Dipeptidyl Peptidase 4 EGCG Epigallocatechin Gallate EGLN1 Egl-­9 Family Hypoxia Inducible Factor 1

© Springer Nature Switzerland AG 2021 A. F. Florez, H. Alborzinia (eds.), Ferroptosis: Mechanism and Diseases, Advances in Experimental Medicine and Biology 1301, https://doi.org/10.1007/978-3-030-62026-4_6

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ER ERK

Endoplasmic Reticulum Extracellular Signal-­Regulated Kinase ETosis Extracellular Traps formation FAC Ferrous Ammonium Citrate FADS2 Fatty Acid Desaturase 2 FDA Food and Drug Administration FDPS Farnesyl Diphosphate Synthase FECH Ferrochelatase FLAP 5 -­L i p o x y g e n a s e -­A c t iva t i n g Protein FRDA Friedreich Ataxia FSP1 Ferroptosis Suppressor Protein 1 FTH1 Ferritin Heavy Chain 1 FtMt Mitochondrial Ferritin FVLCR1b Feline Leukemia Virus, subgroup C and receptor 1 isoform b FXN Frataxin G6PD Glucose-­6-­Phosphate Dehydroge­ nase GCL Glutamate-­Cysteine Ligase GLS2 Glutaminase 2 GLUT1 Glucose Transporter 1 GOT1 Glutamic-­Oxaloacetic Transami­ nase 1 GPX4 Glutathione Peroxidase 4 GPXs Glutathione Peroxidases GSH Glutathione GSR Glutathione-­Disulfide Reductase GSS Glutathione Synthetase GSTP1 Glutathione-­S-­transferase Pi 1 HETE Hydroxyeicosatetraenoic Acid HMG-CoA  3-­H ydroxy-­3 -­M ethylglutaryl Coenzyme A HMGC 3-­Hydroxy-­3-­Methylglutaryl-CoA Synthase HMGCR 3-­Hydroxy-­3-­Methylglutaryl-CoA Reductase HO-1 Heme Oxygenase 1 HSP90 Heat Shock Protein 90 P HSPB1 Phosphorylated Heat Shock Protein Family B (Small) Member 1 HTS High-­throughput screening IGF-1 Insulin-­Like Growth Factor 1 IKE Imidazole Ketone Erastin IPP Isopentenyl pyrophosphate

IRP1/2

Iron Regulatory Protein 1 (Aconitase 1 with Fe4S4 cluster)/ Iron Regulatory Protein 2 ISC Iron-­Sulfur Cluster ISCU Iron-­Sulfur Cluster Assembly Enzyme ISD11 LYR Motif Containing 4 JNK Jun N-­Terminal Kinases KEAP1 Kelch-­like ECH-­Associated Protein 1 l-LIP lysosomal Labile Iron Pool LC3 Microtubule-­Associated Protein 1 Light Chain 3 LOX Lipoxygenase LPCAT3 Ly s o p h o s p h a t i d y l - c h o l i n e Acyltransferase 3 LSH Helicase, Lymphoid Specific m-LIP mitochondrial Labile Iron Pool MAP2K1/2 Mitogen-­Activated Protein Kinase Kinase 1/2 MAPK Mitogen-­Activated Protein Kinase MCAT Malonyl-­CoA-­Acyl Carrier Protein Transacylase MCU Mitochondrial Calcium Uniporter ME1 Malic Enzyme 1 MEF Mouse Embryonic Fibroblasts MoA Mechanism of Action MUC1-C Mucin 1 C-­Terminal Subunit NAC N-­Acetylcysteine NADP+ Reduce Nicotinamide Adenine Dinucleotide Phosphate NADPH Oxidized Nicotinamide Adenine Dinucleotide Phosphate NAPQI N-­acetyl-­p-­benzoquinone imine NCOA4 Nuclear Receptor Coactivator 4 NDGA Nordihydroguaiaretic Acid Nf-kB Nuclear Factor  Kappa-­Light-­­ Chain-­ Enhancer of Activated B Cells NFS1 NFS1 Cysteine Desulfurase NLRP3 NACHT, LRR and pyrin domain-­­containing protein 3 NOX NADPH Oxidase NOX1 NADPH Oxidase 1 NOX2 NADPH Oxidase 2

6  Small Molecule Regulators of Ferroptosis

NQO1

NAD(P)H Quinone Dehydroge­ nase 1 NrF2 Nuclear Factor Erythroid-­2-­­ Related Factor 2 P53 Tumour Protein p53 PAOX Polyamine Oxidase PCBP1/2 Poly(RC) Binding Protein 1 / 2 PE-PUFAs Phosphatidylethanolamine-­­ Polyunsaturated Fatty Acids PDK1 3-­P hosphoinositide-­D ependent Protein Kinase-­1 PGD Phosphogluconate Dehydroge­ nase PHKG2 Phosphorylase Kinase Catalytic Subunit Gamma 2 PKC Protein Kinase C PPARγ Peroxisome Proliferator-­­ Activated Receptor -­γ PPIX Protoporphyrin IX PRNP Prion Protein RAF Rapidly Accelerated Fibrosar­ coma ROS Reactive Oxygen Species SAT1 Spermidine/Spermine N1-­ Acetyltransferase 1 SCD1 Stearoyl-­CoA Desaturase 1 SLC1A5 Solute Carrier Family 1 Member 5 SLC25A28 Mitoferrin-­2 SLC25A37 Mitoferrin SLC3A2 Solute Carrier Family 3 Member 2 SLC7A11 Solute Carrier Family 7 Member 11 SQS Squalene Synthase STEAP3/4 Six-­Transmembrane Epithelial Antigen of Prostate 3/4 Metalloreductase TF Transferrin TFR1 Transferrin Receptor 1 THN Tetrahydro-­1,8-­naphthyridinol TrxR1 Thioredoxin Reductase 1 VDAC2/3 Voltage Dependent Anion Channel 2/3 ZIP8/14 Solute Carrier Family 39 Member 8 / Member 14 σ1R Sigma-­1 Receptor σ2R Sigma-­2 Receptor

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6.1

Introduction

Ferroptosis is currently described as an iron-­­ dependent form of regulated cell death initiated by oxidative perturbations of the intracellular microenvironment that is under constitutive control by the enzyme glutathione peroxidase 4 (GPX4) (Galluzzi et al. 2018). GPX4 is a phospholipid hydroperoxidase that protects cells against peroxidation of lipids caused by reactive oxygen species (ROS), and uses Glutathione (GSH) as a substrate. Initial studies aimed at understanding ferroptosis and exploitation of underlying pathways thus focused on reducing levels of GPX4 or on inhibition of its activity. Based on this research, it became evident that both lipid metabolism and mechanisms involving reactive ROS were central to the mechanisms revealed by GPX4 depletion. Indeed, it was found that specialized cellular ROS detoxification systems can protect cells from ferroptosis, adding another layer of complexity. The name ferroptosis is derived from its relation to iron. This metal can be a major contributor of ROS formation in cells via Fenton chemistry, putting iron homeostasis and labile iron(II) pools as central players in the mechanisms underlying ferroptosis. Each of these players can potentially be targeted by small molecules. Taken together, ferroptosis is revealed as a dedicated cell death pathway that is modulated by positive and negative biological regulators, whose regulatory molecular machinery is a potential target for the development of drugs (Lei et al. 2019; Conrad and Pratt 2019). In this chapter, we describe the recent developments of small-­molecule-­regulators of ferroptosis and what is known about their mechanisms of action (MoA). These molecules will be classified according to their biological target or MoA and depicted in known metabolic pathways related to ferroptotic cell death. Scheme 6.1 describes the pathways related to GSH and GPX4, while Scheme 6.2 describes cellular iron homeostasis and its implication in ferroptosis. This chapter raises questions and opportunities for further research around ferroptosis, which was coined a cell death pathway in 2012. However, prior metabolic studies had already reported some features

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Scheme 6.1  Schematic illustration of the GPX4-­related pathways and their contributions to Ferroptosis. When activated, GPX4 translocates to membranes; ACACA: Acetyl-­CoA Carboxylase Alpha; ACAT: Acetyl-­CoA Acetyltransferase; ACSL4: Acyl-­CoA Synthetase Long Chain Family Member 4; BAP1: Breast Cancer susceptibility gene 1-­Associated Protein 1; BECNP: Phosphorylated Beclin 1; CARS: Cysteinyl-­TRNA Synthetase; CBS: Cystathionine-­Beta-­Synthase; CD44: CD44 Molecule (Indian Blood Group); CDO1: Cysteine Dioxygenase Type 1; CGL: Cystathionine Gamma-­Lyase; CMA: Chaperone-­mediated autophagy; CoA: Coenzyme A; CoQ10: Coenzyme Q10 (Red.: reduced form, Ox.: oxidized form); c-­Myb: MYB Proto-­Oncogene, Transcription Factor; DPP4: Dipeptidyl Peptidase 4; FDPS: Farnesyl Diphosphate Synthase; FSP1: ferroptosis suppressor protein 1; G6PD: Glucose-­6-­­ Phosphate Dehydrogenase; GCL: Glutamate-­Cysteine Ligase catalytic subunit; GLS2: Glutaminase 2; GOT1: Glutamic-­Oxaloacetic Transaminase 1; GPX4: Glutathione Peroxidase 4; GSR: Glutathione-­Disulfide Reductase; GSS: Glutathione Synthetase;

HMGC: 3-­ Hydroxy-­3-­Methylglutaryl-­CoA Synthase; HMGCR: 3-­Hydroxy-­3-­­Methylglutaryl-­CoA Reductase; KEAP1: Kelch-­like ECH-­Associated Protein 1; LOXs: Lipoxygenases (mainly Arachidonate 15-­ Lipoxygenase and Arachidonate 5-­Lipoxygenase); LPCAT3: Lysophosphatidylcholine Acyltransferase 3; MCAT: Malonyl-­CoA-­Acyl Carrier Protein Transacylase; ME1: Malic Enzyme 1; MUC1-­C: Mucin 1 C-­Terminal Subunit; NADP+: oxidized Nicotinamide Adenine Dinucleotide Phosphate; NADPH: Reduced Nicotinamide Adenine Dinucleotide Phosphate; NOX1: NADPH Oxidase 1; NOXs: NADPH Oxidases; NRF2: Nuclear Factor Erythroid-­2-­­Related Factor 2; P53: Tumour Protein p53; PE-­PUFAs: phosphatidylethanolamine-­Polyunsaturated Fatty Acids; PGD: Phosphogluconate Dehydrogenase; ROS: Reactive Oxygen Species; SAT1: Spermidine/Spermine N1-­ Acetyltransferase 1; SLC1A5: Solute Carrier Family 1 Member 5; SLC3A2: Solute Carrier Family 3 Member 2; SLC7A11: Solute Carrier Family 7 Member 11; SQS: Squalene Synthase; VDAC2/3: Voltage Dependent Anion Channel 2/3

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Scheme 6.2  Schematic illustration of the iron homeostasis and its implication in Ferroptosis. ABCB7/8: ATP Binding Cassette Subfamily B Member 7 / Member 8; ABCB10: ATP Binding Cassette Subfamily B Member 10; ATG5/7: Autophagy-­ Related Gene 5 / 7; CISD1: CDGSH Iron Sulfur Domain 1; CoQ10: Coenzyme Q10; c-­Myc: MYC Proto-­ Oncogene, BHLH Transcription Factor; CP: Ceruloplasmin; DMT1: Solute Carrier Family 11 Member 2; EGLN1: Egl-­9 Family Hypoxia Inducible Factor 1; FADS2: Fatty Acid Desaturase 2; FECH: Ferrochelatase; FTH1: Ferritin Heavy Chain 1; FtMt: Mitochondrial Ferritin; FVLCR1b: Feline Leukemia Virus, subgroup C and receptor 1 isoform b; FXN: Frataxin; GLUT1: Solute Carrier Family 2 Member 1; GSH: Glutathione; HA: Hyaluronic acid; HO-­1: Heme Oxygenase 1; HSPB1P: Phosphorylated Heat Shock Protein Family B (Small) Member 1; IRP1/2: Iron Regulatory Protein 1 (Aconitase 1 with Fe4S4 cluster)/ Iron Regulatory Protein 2; ISC: Iron-­Sulfur Cluster; ISCU: Iron-­Sulfur Cluster Assembly Enzyme; ISD11: LYR

Motif Containing 4; LC3: Microtubule-­Associated Protein 1 Light Chain 3; c-­LIP: cytosolic Labile Iron Pool; l-­LIP: lysosomal Labile Iron Pool; m-­LIP: mitochondrial Labile Iron Pool; LSH: Helicase, Lymphoid Specific; MCU: Mitochondrial Calcium Uniporter; NCOA4: Nuclear Receptor Coactivator 4; NFS1: NFS1 Cysteine Desulfurase; NQO1: NAD(P)H Quinone Dehydrogenase 1; NRF2: Nuclear Factor Erythroid-­2-­Related Factor 2; PCBP1/2: Poly(RC) Binding Protein 1 / 2; PHKG2: Phosphorylase Kinase Catalytic Subunit Gamma 2; PKC: Protein Kinase C; PPIX: Protoporphyrin IX; PRNP: Prion Protein; ROS: Reactive Oxygen Species; SCD1: Stearoyl-­ CoA Desaturase 1; SLC25A28: Mitoferrin-­2; SLC25A37: Mitoferrin; STEAP3/4: Six-­ Transmembrane Epithelial Antigen Of Prostate 3 / STEAP4 Metalloreductase; TF: Transferrin; TFR1: Transferrin Receptor; ZIP8/14: Solute Carrier Family 39 Member 8 / Member 14

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of ferroptosis without pairing them under a collective umbrella. Notably, cystine deprivation leading to unique morphological features of cell death compared to deprivation of other amino acids was shown back in the early 1950s. For further historical consideration about research on ferroptosis, we direct the reader to some available reviews (Doll and Conrad 2017; Hirschhorn and Stockwell 2019).

6.2

Activators of Ferroptosis

Many studies have pointed out the vulnerability of cancer cells to ferroptosis compared to normal cells. Among them, persister cancer cells, drug-­­ tolerant cancer cells or tumour-­ initiating cells have been shown to be vulnerable to this form of cell death (Hangauer et al. 2017; Liu et al. 2018b; Mai et  al. 2017). In particular, the iron-­­ dependency of these cells (Basuli et  al. 2017; Torti et al. 2018) could hint towards the development of a strategy that employs ferroptosis inducers as novel therapeutic indications. Indeed, ferroptosis research and the development of ferroptosis activators could have a profound impact (Shen et al. 2018; Friedmann Angeli et al. 2019), both on our understanding of the disease and for the development of potential drug candidates.

6.2.1 T  argeting GPX4 Activity and Lipid Production A central cellular protector against oxidative stress is GPX4, which uses GSH to reduce hydrogen peroxide, lipid peroxide and other oxidized cysteine-­bearing proteins. In mammals, GPX4 is an essential selenoprotein for embryo development and cell homeostasis. In cells, GPX4 is the central enzyme preventing accumulation of peroxidized lipids, key regulators of ferroptosis. Thus, inactivation of GPX4 is an effective method to induce accumulation of lipid peroxides and thus ferroptosis.

6.1.1.1 GPX4 inhibitors (Scheme 6.1; Fig. 6.1) The small molecule RSL3 has been identified as an inducer of ferroptosis and its MoA, that involves binding and inhibition of GPX4, was recently reported (Yang et  al. 2014) as determined by affinity-­based proteomics. GPX4 could also be inhibited upstream as a result of the inhibition of selenoprotein (SELT), for instance by directly chelating selenium, which is now the focus of current active research. As RSL3 drives ferroptosis through a mechanism that is distinct from another ferroptosis inducer Erastin (described later, see Sect. 6.2.2.1), this opens the possibility to investigate different parts of mechanisms leading to or involving ferroptosis. Thus, this newly recognized class of GPX4 inhibitors has become an area of interest. A small molecule ML-­ 162 with a similar structure to RSL3, was identified as a ferroptosis inducer by high-­throughput screening (HTS) followed by a structure-­ activity relationship study (Weïwer et  al. 2012). This molecule potentially acts through the same MoA as RSL3 itself. Altretamine, a small molecule previously used as an antineoplastic agent, inhibits GPX4 activity (Woo et  al. 2015; Yang and Stockwell 2008) although its precise MoA remains elusive. Isoflurane, a Food and Drug Administration (FDA) approved general anesthetic, was associated with ferroptosis through impairment of GPX4 activity in the context of neurotoxicity (Xia et  al. 2019). Its MoA may be complex though as it has been reported to affect gamma-­­ aminobutyric acid, glutamate, glycine receptors, sodium and potassium channels, although how these players connect in a unifying mechanism is unclear.

6.2.1.1 Deficiency of GPX4 Production (Scheme 6.1; Fig. 6.2) GPX4 activity relies on the incorporation of a selenocysteine into its active site, which depends on isopentenyl pyrophosphate (IPP) and Sec-­­ tRNA[Ser]Sec. The selenium atom allows enzymatic break of the peroxyl bond by undergoing oxida-

6  Small Molecule Regulators of Ferroptosis Fig. 6.1  Inhibitors of GPX4 activity

89 (1S,3R)-RSL3

ML-162

O

Cl

H N

O

O

NH

N N

O O O Isoflurane F F F O F F Cl

tion, resulting in an inactive hydroxyselenocysteine and a harmless hydroxylipid. The regeneration of the active site is done by GSH in a two-­step sequence: the first step involves the loss of a hydroxyl group releasing water, and the second step releases the active GPX4 enzyme and glutathione disulphide (GSSG). IPP itself is generated by the mevalonate pathway from acetyl-­­ CoA.  Statin drugs such as Cerivastatin and Simvastatin have been described to inhibit β-­Hydroxy β-­methylglutaryl Coenzyme A (HMG-­CoA) reductase, which participates in the mevalonate pathway and sensitizes cells to ferroptosis, by altering the production of Coenzyme Q 10 (CoQ10) and squalene, which both play key

O

S

Cl

N N N

O

Cl

Altretamine N N

N

roles in antioxidant cell defense, but also potentially by inhibiting the biosynthesis of GPX4 (Fradejas et  al. 2013; Viswanathan et  al. 2017; Shimada et al. 2016).

6.2.1.2 Boost of Lipid Production (Scheme 6.1; Fig. 6.3) While reduction of lipid production negatively influences ferroptosis, increased production of fatty acids or fatty acid supplementation can also trigger cell death. Eleostearic acid is a conjugated fatty acid and is one of the isomers of octadecatrienoic acid. Eleostearic acid was identified as a suppressor of tumour growth via lipid peroxidation (Tsuzuki et al. 2004). Importantly, oxidation-­­

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90 F

Cerivastatin

Simvastatin HO

O

O

O

OH OH O OH

O

O

H

N

Fig. 6.2  Inhibitors of GPX4 biosynthesis by targeting the mevalonate pathway

dependent mechanisms of eleostearic acid-­induced cell death were also described in breast cancer (Grossmann et  al. 2009; Beatty et al. 2019) and its induction of the peroxisome proliferator-­activated receptor γ (PPARγ) in bladder cancer(Sun et al. 2012). However, cell death induced by eleostearic acid can be blocked by α-­Tocopherol (a component of Vitamin E, see Sect. 6.3.1.3), which inhibits the kinase responsible for the phosphorylation and subsequent activation of dual specificity mitogen-­activated protein kinase kinase 1 (MAP 2K1) (Kondo et al. 2010). Reported caspase activation (Tsuzuki et al. 2004) indicates that the cell death triggered by eleostearic acid could be apoptosis. However, taken together it appears that eleostearic acid can activate different cell death pathways in different cell types, but this warrants more thorough investigations. This also highlights that the distinction between ferroptosis and other cell death pathways is challenging and requires further work

and clarification (Conrad and Pratt 2019). On another note, the small molecule CIL56 and its derivative FIN56 were reported to act on the enzymatic activity of acetyl-­ CoA carboxylase (ACACA), which positively contributes to ferroptosis by altering cellular lipid composition (Feng and Stockwell 2018; Dixon et al. 2015). A second pathway, related to ferroptosis, also seems to be affected by this class of molecules, which involves squalene synthase and subsequently leads to depletion of CoQ10 (Tsuzuki et al. 2004).

6.2.2 G  SH Depletion and Nuclear Factor (Erythroid-Derived 2)-like 2 (NrF2) Inhibition Since inhibition of GPX4 represents an efficient mechanism of ferroptosis induction, methods to deprive cells of GSH represent a useful alternative approach.

6  Small Molecule Regulators of Ferroptosis

91

FIN56 HO O HN S O

N

CIL56 HO O N S O

HN S O O

N

O S N O

Eleostearic acid (example of conjugated linolenic fatty acid)

O HO

Fig. 6.3  Inducers of lipid production and their identified targets

6.2.2.1 System Xc- Inhibition (Scheme 6.1; Fig. 6.4) Historically, ferroptosis was closely associated with the system Xc− and its inhibition. The system Xc− is composed of the following subunits: a) solute carrier family 7 member 11 (SLC7A11), a cystine/glutamate transporter and b) solute carrier family 3 member 2 (SLC3A2), which plays a role in the uptake of cysteine (as an oxidized dimer named cystine) through exchange with glutamate. This protein complex also regulates cellular GSH levels and subsequently antioxidant defense. Erastin was identified early on by the Stockwell group through a HTS to identify small molecule inducers of death in cancer cells (Dolma

et al. 2003). Induction of non-­apoptotic cell death by Erastin was examined and initially attributed to its interactions with voltage dependent anion channels 2 and 3 (VDAC2/3) (Yagoda et  al. 2007). This was shown in oncogenic Ras-­­ expressing cancer cells, which exhibit high levels of transferrin receptor 1, low levels of ferritin and an increased pool of labile iron (Xia et al. 2019). Another study showed that the inhibitory effects of Erastin on system Xc− were shown to be concomitant with endoplasmic reticulum (ER) stress (Lange and Proft 1970), but further investigation is required to elucidate this mechanistic detail. Erastin has been identified as a potential therapeutic candidate in a drug discovery study, therefore numerous analogs have been designed to

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92 N O

Erastin N N N O

O

O Cl

N

N O

O

O HN S O

N N

Sulfasalazine N

Imidazole Ketone Erastin (IKE) O

N

OH HO

O

OH

O

Sorafenib N

H N

NH2

O

OH

O

HO

O

O

Cl

O

O

O

Glutamic acid /Glutamate

N N

O

O

N

O

Pseudolaric Acid B

O O

H N

H N O

Cl F

F

F

Fig. 6.4  Inhibitors of system Xc− function and identified targets of Erastin

exhibit improved pharmacological properties. Two molecules in particular can be highlighted: (a) a piperazine-­ containing analog of Erastin, which was developed to increase water-­solubility and metabolic stability, operating via the same MoA (Yang et al. 2014) and (b) Imidazole Ketone Erastin (IKE), an Erastin derivative bearing an imidazole to improve water solubility (Zhang et al. 2019). This imidazole was added at the para position of the phenol, which limits its degradation by oxidative mechanisms, increasing its inhibition of system Xc−. A supplementary methyl group on the alpha position of the ethoxy group also increases metabolic stability. After the discovery of the mechanisms underlying Erastin-­­

induced cell death, the Stockwell group described a number of molecules that also acted on the same metabolic pathway as Erastin, namely DPI2 or DPI10 (Stockwell et al. 2014). Another molecule, Sorafenib, was developed as an inhibitor of rapidly accelerated fibrosarcoma (RAF) kinase, from Onyx Pharmaceuticals (Lyons et al. 2001) and has since been used to treat hepatocellular carcinoma. Its impact on ferroptosis was highlighted in two cases of melanoma (Yagoda et al. 2007) and one case of hepatocellular carcinoma (Louandre et al. 2013). Its activity as an inhibitor of system Xc− was reported (Lange and Proft 1970) and Sorafenib has since been used to study the system Xc− (Chen et  al. 2017; Dahlmanns

6  Small Molecule Regulators of Ferroptosis Acetaminophen

Cisplatin

Buthionine sulfoximine (BSO)

Cl Cl Pt NH2 H2 N

OH

O

93

N H

O S NH

NH2

OH

O

BAY 87-2243 N

O N

N

N

N

N

F3CO

N

Fig. 6.5  Inhibitors of GSH metabolism and their identified targets

et al. 2017; Song et al. 2018). Sulfasalazine has been described as a potential treatment for ulcerative colitis (Svartz 1948), and its specific inhibitory effect on system Xc− has been reported more recently (Gout et  al. 2001). Furthermore, Pseudolaric acid B was found to trigger ferroptosis in glioma cells and remarkably, cell death could be prevented by using the ferroptosis inhibitor Ferrostatin-­1 or GSH in these cells (Wang et  al. 2018). The MoA involves activation of NADPH oxidase 4 and inhibition of system Xc−. Finally, Glutamate has been identified as an inducer of cell death in the central nervous system through iron-­dependent oxidation (Tan et al. 2001; Dixon and Stockwell 2014). Interestingly, glutamate toxicity can occur when calcium influx is caused by glutamate receptor activation (Murphy et al. 1989) or when glutamate accumu-

lates inside the cell during system Xc− dysfunction, which interferes with cellular cysteine supply leading to GSH deficiency (Bannai and Kitamura 1980).

6.2.2.2 Alteration of GSH Metabolism (Scheme 6.1; Fig. 6.5) Downstream of system Xc−, cysteine is converted to γ-­glutamyl cysteine, then GSH. Altering this pathway by enzymatic inhibition or decreasing GSH availability can lead to GSH depletion and subsequent cell death. Buthionine sulfoximine was identified (Griffith and Meister 1979) as an inhibitor of GSH synthesis through irreversible inhibition of glutamate cysteine ligase (GCL). Thus, following reduced GSH levels GCL inhibition increases ROS levels in cells and ultimately results in ferroptosis. How exactly and where in

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94

the cells ROS are produced needs to be investigated to understand this mechanism better. Interestingly, analogs of cisplatin widely used in cancer therapy (Rancoule et  al. 2017) are approved by the FDA, regardless of their pleiotropic effects, including causing DNA-­damage (Müller 2017). However, their efficacy can be decreased by high expression of GSH in their target cells, which protects the cells from oxidation-­­ induced death. It will be interesting to study the direct effects of cisplatin and its derivatives on GSH levels. In this situation, depletion of GSH and subsequent inactivation of Glutathione peroxidases (GPXs) could induce ferroptosis, re-­­ sensitizing the cells to therapy (Guo et al. 2018). The molecule BAY 87-­2243 has been developed by Bayer as part of a screening program designed to identify molecules targeting the hypoxia-­­ inducible factor (HIF) pathway (Ellinghaus et al. 2013). Induction of ferroptosis by BAY 87-­ 2243  in melanoma cells promotes inhibition of mitochondrial complex I, leading to depolarization of the mitochondrial membrane potential, which in turn increases cellular ROS levels, lipid peroxidation and decreases GSH levels (Schöckel et al. 2015; Basit et al. 2017). A common moleArtesunate H O

O

O O H

H

O

H O

N O

O

O

O H

O O

O

Brusatol

OH

OH O HO

H

H HO

H

O O

O

O H

OH

Fig. 6.6  Inhibitors of NrF2 activity

6.2.2.3 NrF2 Inhibition (Scheme 6.1; Fig. 6.6) NrF2 is a transcription factor regulating the expression of antioxidant proteins that protect cells against oxidative damage triggered by injury and inflammation. In the context of ferroptosis, a key interest in NrF2 inhibitors is linked to the capacity of these molecules to block antioxidant defense when ROS levels in cells are high enough to provoke cell death. To this end, artesunate is an antimalarial drug for which the pro-

Withaferin A

Trigonelline OH

cule of interest here is acetaminophen, also known as paracetamol, which in overdose provokes hepatotoxicity subsequent to oxidative stress and lipid peroxidation (Jaeschke and Ramachandran 2018). In 2015, a study showed that a metabolite of paracetamol called N-­acetyl-­­ p-­benzoquinone imine (NAPQI) causes ferroptosis via GSH deprivation (Lőrincz et al. 2015). In this context, the ferroptosis activator Piperlongumine (Chatterjee and Dutta 1967) can be metabolized and then interact with GSH (Prejanò et al. 2018). However, its MoA remains elusive and will be further discussed in detail in paragraph Sect. 6.2.3.2.

H

H O

O

O

6  Small Molecule Regulators of Ferroptosis

posed MoA involves the production of ROS (Cui and Su 2009; Chen et al. 2019). Artesunate has been shown to interact with membrane bound glutathione S-­transferase, leading to the reduction of the amount of GSH in Plasmodium falciparum (Lisewski et  al. 2014). This mechanism appears to activate ferroptosis in pancreatic ductal adenocarcinoma cell lines (Eling et al. 2015) and in head & neck cancer (Roh et al. 2017). This effect seems to be modulated by Nrf2-­antioxidant response element (ARE) pathway activation. The same study also describes the use of Trigonelline, which has been reported to inhibit Nrf2 activation (Boettler et al. 2011; Arlt et al. 2012), exacerbating Artesunate induced-­ferroptosis. Another compound named Brusatol has been shown to act as an inhibitor of Nrf2. This molecule was identified as an antitumour agent (Hall et al. 1979) and has been used to antagonize the protective effect of Nrf2  in intestinal ischemia/reperfusion-­­ induced injury (Olayanju et al. 2015) by enhancing Nrf2 ubiquitination and degradation. Withaferin A is a natural steroidal lactone isolated from Indian winter cherry. Numerous pharmacological effects of withaferin A have been reported (Lee and Choi 2016; Chirumamilla et al. 2017) and it has been shown to cause ferroptosis in neuroblastoma (Dhami et al. 2017; Hassannia et al. 2018) by acting on the NrF2 pathway and by inhibiting GPX4.

6.2.3 Iron and ROS Production As lipid peroxidation occurs after GPX4 depletion, another strategy to promote ferroptotic cell death involves accelerated lipid peroxidation that outruns GPX4 activity. This can occur through direct iron-­catalyzed lipid oxidation. Overloading ROS and/or labile iron(II) pools in a cellular environment is a characteristic of numerous diseases and can potentially be exploited to selectively kill distinct populations of cells.

6.2.3.1 Labile iron Pool Enrichment (Scheme 6.2; Fig. 6.7) Promoting the accumulation of labile cellular iron can lead to an increased rate of Fenton chem-

95

istry, subsequent production of ROS and lipid peroxidation. Iron-­related Fenton chemistry naturally occurs in cells and is required to facilitate iron storage by ferritin under its oxidized form iron(III). The rate of the Fenton reaction is dependent on labile iron concentrations. It forms free radicals, leading to ROS in cells. The mechanisms surrounding the Fenton reaction in cells are complex, and a general reaction scheme can be found in Fig. 6.8. Fenton chemistry can occur after supplementing cells with ferric ammonium citrate (FAC), or physiologically through abnormal ferritinophagy or heme degradation. FAC has been used to increase cellular iron load, which can promote ferroptosis in numerous biological systems. This led primarily to the identification of new inhibitors of ferroptosis (Guerrero-­Hue et  al. 2019; Kose et  al. 2019; Fang et  al. 2018; Mattera et al. 2001; Camiolo et al. 2019; Cotticelli et  al. 2019). Furthermore, these studies also helped establish the kinetics of ferroptotic cell death (Adedoyin et  al. 2018). FINO2 (1,2-­­dioxolane derivative) is an organic peroxide that was found in a high-­ throughput screen designed to identify inducers of ferroptosis (Abrams et  al. 2016). The MoA of FINO2 was later described as acting both through inhibition of GPX4 function and direct oxidation of iron, which leads to uncontrolled lipid peroxidation (Gaschler et al. 2018a). Interestingly, monocyclic peroxide has previously been linked to arachidonic acid-­initiated aggregation of human platelets (Menzel et al. 1976). Importantly, the natural product Salinomycin, isolated from Streptomyces albus, has been shown to specifically kill cancer stem cells (Gupta et  al. 2009). The Rodriguez group found that this molecule and its synthetic derivative Ironomycin cause specific sequestration of iron in lysosomes (Mai et al. 2017). The subsequent iron depletion in other cell compartments induced lysosomal ferritin degradation, leading to further iron loading in lysosomes. Altogether, this leads to lysosomal ROS production, lysosomal and mitochondrial lipid membrane permeabilization and cell death with features reminiscent of ferroptosis. In addition, Magnesium isoglycyrrhizinate is a terpene saponin, which has been shown to exhibit

S. Debieu et al.

96 Salinomycin OH O

Ironomycin OH O

O

O

O O

OH

O

O

O O

O

O

HO

OH

O HO

Magnesium isoglycyrrhizinate (example of triterpene saponin)

O OH

O xFe3+

O

OH O

yNH3

O H

H

H

O

O H O

OH

O

O

Fig. 6.7  Small molecules capable of interfering with the labile iron pool

O

OH OH OH Mg2+ 4H2O OH

H

HO

Fig. 6.8 Fenton reaction and its plausible mechanism(s) (Salgado et al. 2013)

OH

O

OH

Ferric ammonium citrate (FAC)

FINO2

O NH

O

OH

tBu

O

OH

O

6  Small Molecule Regulators of Ferroptosis Ferroptocide

Piperlongumine O

O

O

O

O

N

O

O

F3 C

O Cadmium

O

Lanperisone

O N

N N N

Cl

97

Paraquat

Cd

HO

N+

OH O

6-Hydroxydopamine

2Cl-

NH2

HO HO

OH

N+

Fig. 6.9  Inducers of ROS production and their identified targets

­anti-­­inflammatory and antioxidant properties (Xie et al. 2015; Xu et al. 2016; Zhao et al. 2017). However, in the context of liver fibrosis, Magnesium isoglycyrrhizinate acts as a ferroptosis inducer though ER stress (Bian et  al. 2017) and heme oxygenase-­1 (HO-­1) activation, which in turn leads to heme degradation, labile iron accumulation and subsequent Fenton chemistry in cells (Sui et al. 2018).

6.2.3.2 Inducing ROS (Scheme 6.2; Fig. 6.9) Several small molecules have been described to promote the production of ROS at lethal levels. Lanperisone was first developed as a muscle relaxant (Sakitama et  al. 1997) via a combined action on voltage-­ gated sodium and calcium

channels (Kocsis et al. 2005). It was later identified to induce cell death, by synthetic lethal chemical screening, in mouse embryonic fibroblasts (MEFs) expressing oncogenic Ras, which have an elevated vulnerability to oxidative stress. The MoA of Lanperisone has been described to be iron-­and Ras/MAPK-­dependent, even if the mechanism of increased ROS production has not yet been fully elucidated (Shaw et  al. 2011). Interestingly, Piperlongumine has drawn considerable attention among the scientific community. It was isolated from Piper Longum Linn (Chatterjee and Dutta 1967) and has been shown to induce ferroptosis by elevating ROS levels in a number of cancer cell types. Glutathione-­ S-­­ transferase Pi 1 (GSTP1) and thioredoxin reductase 1 (TrxR1) have been reported as possible

98

targets of Piperlongumine in the context of ferroptosis. Although the actual MoA remains ­elusive, one hypothesis involves the formation of a conjugate between GSH and the product of hydrolysis of Piperlongumine, which helps explain GSTP1 inhibition and the decrease of GSH upon Piperlongumine treatment (Karki et  al. 2017; Yamaguchi et  al. 2018; Kumar and Agnihotri 2019; Prejanò et al. 2018; Wang et al. 2019). A study reported that Piperlongumine binds to a cysteine residue of the active site of TrxR1 (Zou et al. 2016). Furthermore, ferroptocide is a small molecule obtained by means of complexity-­to-­diversity optimization of the natural product of diterpene, Pleuromutilin, which was first identified in a screen for cytotoxic compounds and was reported to be an inhibitor of thioredoxin. Thioredoxin is a redox protein that reduces proteins via thiol-­disulfide exchange on cysteine residues. It normally regulates ROS homeostasis in cells and thus, its inhibition can lead to ROS accumulation and eventually ferroptosis (Llabani et  al. 2019). Other work showed that the small molecule 6-­Hydroxydopamine is toxic to sympathetic nerve cells (Angeletti and Levi-­Montalcini 1970) and adrenergic nerve endings (Malmfors and Sachs 1968), and it has been used as a model to mimic Parkinson’s disease in rats (Cashin and Sutton 1973; Li et al. 2019b). In this context, several studies have described the effects of 6-­ Hydroxydopamine on biological mechanisms associated with ferroptosis, such as cysteine uptake by system Xc− and the generation of ROS reliant on free iron (Do Van et  al. 2016; Youdim et  al. 2004; Massie et  al. 2011; Guiney et al. 2017). The compound Paraquat is an herbicide infamously toxic to humans, with a MoA well studied since the 1960s (Clark et  al. 1966). Some studies linked Paraquat to ferroptosis in neurodegeneration through elevation of NADPH-­ oxidase (NOX) levels, and NACHT, LRR and pyrin domain-­ containing protein 3 (NLRP3) inflammasome activation by Paraquat induced-­mitochondrial ROS (Chen et  al. 2015). Finally, cadmium-­ induced neurotoxicity has been reported to operate through oxidative cell death in microglia (Yang et al. 2007). Cadmium-­­ induced cell death has been reported in renal

S. Debieu et al.

cells, and it will be interesting to see if there is a ferroptotic component to this mechanism, or if another cell death pathway is involved. Since the pan-­caspase inhibitor Z-­VAD-­FMK markedly suppressed cadmium chloride-­induced cell death, apoptosis could be the presumed mechanism (Lee et al. 2017; Fujiki et al. 2019). This opens up exciting new avenues of future research.

6.2.3.3 A Small molecule Inducer of Membrane Leakage (Scheme 6.2; Fig. 6.10) One of the hallmarks of ferroptosis is lipid peroxidation which disrupts lipid membranes, and in the cellular environment can cause organelle damage, such as lysosomal and mitochondrial membrane permeabilization. Thus, lysosomotropic or mitotropic detergents can potentially activate ferroptosis. Siramesine (also called Lu 28-­179) was reported to exhibit high affinity for the sigma-­1 receptor (σ1R) and sigma-­2 receptor (σ2R) (Perregaard et al. 1995). σ2R is involved in cholesterol homeostasis, calcium signaling and lipid regulation and can be found in the ER (Ahmed et  al. 2012). Siramesine penetrates the blood-­brain barrier and is a selective and potent σ2R agonist. This small molecule causes lysosomal leakage, which leads to cell death (Ostenfeld et  al. 2005; Jensen et  al. 2017; Das et al. 2018) and this effect can be counteracted by α-­tocopherol. The exact MoA between its localization to the ER and resulting lysosomal leakage is not known and requires further investigation. In addition, mitochondrial destabilization has been reported to trigger mechanisms of Siramesine-­ induced cell death (Česen et  al. 2013). Interestingly, in some models Siramesine does not induce membrane leakage effectively enough to cause ferroptosis. In one report it was shown that co-­ treatment with tyrosine kinase inhibitor Lapatinib exacerbates induction of ferroptosis. This work suggested that the co-­­ treatment using these two small molecules had an impact on iron endocytosis, which could explain the observed synergistic effect. However, the MoA still requires additional investigation (Ma et al. 2016).

6  Small Molecule Regulators of Ferroptosis

99

Siramesine

O

N

N

F

Fig. 6.10  Structure and MoA of Siramesine

6.3

Inhibitors of Ferroptosis

The proposed involvement of ferroptosis in neuronal cell death, Alzheimer’s disease, Parkinson’s disease and tissue injuries has generated an increasing interest in the development of inhibitors of this cell death pathway for clinical purposes in this context (Angeli et al. 2017; Wu et al. 2018). Numerous inhibitors of ferroptosis have been described and can be ranked according to their respective targets and MoA.

6.3.1 L  ipoxygenases (LOXs), Lipid peroxidation and Lipid Production A suitable strategy to inhibit ferroptosis is to prevent the accumulation of ROS and lipid peroxidation by inhibiting the lipoxygenases that initiate peroxidation. In the context of ferroptosis, arachidonate 15-­lipoxygenase (15-­LO) and arachidonate 5-­lipoxygenase (5-­LO) appear to be the major drivers of lipid peroxidation after lipid autoxidation, which is the reaction between oxygen and unsaturated lipids to form lipid hydro-­­ peroxides (Shah et al. 2018). In addition to 5-­LO

and 15-­LO, the LOX family encompasses other important enzymes such as arachidonate 12-­lipoxygenase, arachidonate 15-­lipoxygenase type B and arachidonate lipoxygenase 3. Arachidonic acid metabolism produces eicosanoids such as prostaglandins, thromboxanes, leukotrienes, lipoxins, resolvins, eoxins and hydroxyeicosatetraenoic acids (HETE). These molecules participate in paracrine and autocrine signaling and induce changes in cells by modulating pro-­inflammatory pathways. The equilibrium between eicosanoids can be important for both cell survival and death. The following inhibitors have been shown to block cell death through their interactions with LOXs or by preventing the accumulation of lipid hydroperoxides.

6.3.1.1 Arachidonate 5-Lipoxygenase Inhibitors (Scheme 6.1; Fig. 6.11) 5-­LO is a non-­heme iron-­containing dioxygenase, which catalyzes the conversion of arachidonic acid to 5(S)-­hydroperoxy-­6-­trans-­8, 11,14-­cis-­­eicosatetraenoic acid (5-­HPETE). This protein needs arachidonate 5-­ lipoxygenase-­ activating protein (FLAP) for its function.

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100 Zileuton S HO

N

AA-861 O

O

BW A4C

HO

O N OH

NH2

O O

HO

Cinnamyl-3,4-dihydroxy-alphacyanocinnamate (CDC) O

Nordihydroguaiaretic acid (NDGA)

O

HO

CN

HO

OH

OH

OH

Fig. 6.11  Inhibitors of arachidonate 5-­lipoxygenase activity

Interestingly, a study on polymorphism of the genes encoding these two proteins suggested that they contribute to Alzheimer’s disease and could be important factors in vascular pathology (Manev and Manev 2006). An active inhibitor of 5-­LO, Zileuton, has been introduced by Abbott Laboratories as an inhibitor of leukotriene formation. It has a well-­­established role in the treatment of asthma. However, another possible use as a neuroprotective agent against ferroptosis has recently been put forward. Its action against glutamate oxidative damage in the HT22 neuronal cell line is caspase-­ independent and acts through the same signaling cascade as the ferroptosis inhibitor Ferrostatin-­1 (Liu et al. 2015). Other studies have also reported the ferroptosis inhibitory effects of Zileuton (Yang et al. 2016; Li et  al. 2017a). Cinnamyl-­3,4-­dihydroxy-­α-­ cyanocinnamate (CDC) and Nordihydroguai­

aretic acid (NDGA) are known lipoxygenase inhibitors capable of protecting against ferroptosis. NDGA has already been described as a protector of neurons against free radical and calcium accumulation (Goodman et al. 1994) and can also act on the Insulin-­like growth factor 1 (IGF-­1) receptor (Blecha et al. 2007; Youngren et  al. 2005). Studying the MoA of CDC has helped decipher the role of lipoxygenase in ferroptosis (Shah et al. 2018; Yang et al. 2016) and it was shown that both have potent inhibitory effects on 5-­LO (Sud'ina et al. 1993; West et al. 2004; Pergola et al. 2011). The small molecules termed AA-­ 861 (Yoshimoto et  al. 1982) and BW A4C (Higgs et  al. 1988) have also been identified as 5-­LO inhibitors capable of inhibiting ferroptosis (Li et al. 2017b; Karuppagounder et al. 2018).

6  Small Molecule Regulators of Ferroptosis Fig. 6.12  Inhibitor of arachidonate 15-­lipoxygenase activity

101

PD-146176 NH S

6.3.1.2 Arachidonate 15-Lipoxygenase 6.3.1.3 Unspecified Lipoxygenase Inhibitor (Scheme 6.1; Fig. 6.12) Inhibitors (Scheme 6.1; Fig. 6.13) Interestingly, 15-­LO, which is encoded by the ALOX15 gene, is a non-­heme iron-­containing Trolox is a water-­soluble derivative of Vitamin E, dioxygenase that acts on various polyunsaturated which can limit oxidative stress damage. This substrates. Its described activities include conver- lipophilic antioxidant was used to prevent cell sion of arachidonic acid into death induced by Erastin or RSL3 in the fibrosar12-­­hydroperoxyeicosatetraenoic acid (12-­­ coma HT1080 and pancreatic cancer PANC1 cell HPETE) and 15-­ hydroperoxyeicosatetraenoic lines (Shimada et al. 2016). Vitamin E, which is a acid (15-­HPETE), and the conversion of linoleic mixture of four Tocopherols and four Tocotrienols, acid into 13-­ hydroperoxyoctadecadienoic acid. also shows regulating properties of ferroptosis by The small molecule PD-­146176, developed by interfering with iron loading in the active site of Pfizer, was identified by in vitro chemical library 15-­ LO (Hinman et  al. 2018). Metabolites of screens aimed to identify small molecules capa- Vitamin E have also shown the potential to limit ble of inhibiting the activity of 15-­LO in rabbit inflammation by targeting 5-­ LO (Pein et  al. reticulocytes (Sendobry et al. 1997). In addition, 2018). Importantly, α-­ Tocopherol is the most PD-­146176 prevents Erastin-­and RSL3-­induced abundant tocopherol in Vitamin E and has been ferroptosis in HT1080 fibrosarcoma cells used as an inhibitor of ferroptosis that is induced (Shintoku et  al. 2017) and inhibits ferroptosis by lipid peroxidation in T-­cells (Matsushita et al. induced by Golgi disrupters Brefeldin A and 2015). It was identified by the Stockwell group in Golgicide A, which cause Golgi stress and redox a HTS to identify molecules that induced cell imbalance (Alborzinia et  al. 2018). PD-­146176 death in Ras-­expressing tumour cells (Yagoda can also counteract the cell death triggered by et  al. 2007). Tetrahydro-­1,8-­naphthyridinol p53-­ induced spermidine/spermine N1-­ (THN) derivatives were designed as α-­Tocopherol acetyltransferase 1 (SAT1) activation (acetyla- analogs and share the same MoA, inhibiting fertion of polyamine with H2O2 production by the roptosis due to their antioxidative properties polyamine oxidase (PAOX) enzyme), supporting (Nam et al. 2007). Baicalein has been identified a correlation between ALOX15 and SAT1 expres- as a ferroptosis inhibitor by the Tang group, in a screen for natural products that protect against sion (Ou et al. 2016). Erastin-­induced cell death in PANC1 cells (Xie

S. Debieu et al.

102 alpha-tocopherol (Vitamin E)

Tocotrienol

HO R2

HO

R1

O O

XJB-5-131

R3 Trolox HO

Baicalein O

O

HO OH

O

O

NH

O O

HO OH

1,2,3,4-tetrahydro-1,8-naphthyridin-2-ol (THN exemple)

N

O

N H

OH

O O N

H N O

N

NH HN

O

H N

O

O

Fig. 6.13  Inhibitors of lipoxygenase activity

et  al. 2016). The efficacy of baicalein to rescue cell death was proposed to be due to its activity as an inhibitor of lipoxygenases, although it is documented to have a number of other activities in the cell; inhibition of α-­synuclein fibrillation (Zhu et  al. 2004), inhibition of platelet lipoxygenase (Sekiya and Okuda 1982) and activation of NrF2/ HO-­1 signaling (Yeh et al. 2015; Li et al. 2019a; Jeong et al. 2019). XJB-­5-­131 is a mitochondrial antioxidant derived from the stable nitroxide radical called TEMPO and was first used against cardiolipin oxidation (Ji et al. 2012), then against

motor decline in a mouse model of Huntington’s disease (Xun et al. 2012) and finally against ionizing radiation injury (Greenberger et al. 2015). The Stockwell group deciphered its MoA using synthetic analogs and described it as an inhibitor of ferroptosis. They reported that this class of molecules essentially preserves lipids of the mitochondrial membrane (Krainz et  al. 2016), suggesting that mitochondria may have a contributing role to ferroptotic cell death, supporting the discussions in the earlier section (Sect. 6.2.3.3).

6  Small Molecule Regulators of Ferroptosis Fig. 6.14 Lipid peroxidation inhibitors act via GPX4 activity

103

Selenium

Ebselen

Se

O N Se

CDDO HN

O

H2 N Cl

6.3.1.4 Lipid peroxidation Blockers – Selenium-Based Compounds Mimic GPX4 Activity and Stabilize GPX4 (Scheme 6.1; Fig. 6.14) Another elegant method to avoid the accumulation of lipid peroxidation and thus prevent ferroptosis involves modulation of the expression or activity of phospholipid hydroperoxidase GPX4, that protects cells against ferroptosis by removing peroxylated lipids. To this end, Ebselen is a seleno-­ organic compound with an antioxidant capacity. It has been thoroughly described and its glutathione peroxidase-­ like activity has been deciphered (Müller et  al. 1984; Wendel et  al. 1984; Parnham and Kindt 1984). Since then, Ebselen has been reported to also impact the thioredoxin system, lipid peroxidation and ROS formation, among other processes (Azad and Tomar 2014). For instance, Ebselen exhibits a neuroprotective effect associated with reversion of the suppression of Bcl-­2 and inhibition of Bax over-

expression induced by glutamate toxicity (Xu et  al. 2006), and rescues T-­cells from GPX4-­­ deficiency-­­ induced death (Matsushita et  al. 2015). Recently, an elegant link has been made between inhibition of heat shock protein 90 (HSP90), a mediator of chaperone-­ mediated autophagy (CMA) and ferroptosis. Through inhibition of HSP90, CDDO downregulates CMA, decreasing degradation of GPX4, thus increasing ferroptosis (Wu et al. 2019). As discussed previously (see Sects. 6.2.1.1 and 6.2.1.2), selenium plays a crucial role in the antioxidant system of cells, and GPX4 is a selenoprotein playing a major role in blocking ferroptosis by reducing peroxyl groups on lipids into alcohols (Ingold et al. 2018). Consistently, selenium deficiency is associated with oxidative stress, muscular dystrophy, lipid peroxidation and imbalance of calcium homeostasis (Tapiero et al. 2003; Xu et al. 2013; Conrad et al. 2018). It has also been reported that selenium can block ferroptosis through transcriptional regulation (Alim et al. 2019).

S. Debieu et al.

104 Ferrostatin-1 NH

UAMC-2418

NH2

O NH S O

NH O

UAMC-3203

O

NH

NH

Butylated Butylated hydroxytoluene hydroxyanisole OH OH

11,11-d2-Linoleic acid (example of D-PUFA) O OH D

N

NH2

S

S NH2

N

NH2

N N

Cl

NH

R=

H

R O

Squalene

CoQ10

10

O O

O U0126

O

O

NH

NH N

N NH N

Liproxstatin-1

Beta-Carotene

D

S

N

O NH S O

NH

NH

DT-PTZ-C

OH 5 5

Idebenone

+

PPh3 Mitoquinone

NH2

Fig. 6.15  Inhibitors of lipid peroxidation through the trapping of free radicals

6.3.1.5 Lipid peroxidation Blockers – Inhibition of Propagation of Free Radicals (Scheme 6.2; Fig. 6.15) Another strategy to suppress ferroptosis linked to lipid peroxide accumulation involves the use of molecules able to trap radical oxidants. In this context, Ferrostatin-­1 and Liproxstatin-­1 operate as potent inhibitors of ferroptosis in numerous disease models (Linkermann et al. 2014; Do Van et  al. 2016; Gascón et  al. 2016; Guiney et  al.

2017; Hambright et al. 2017; Zille et al. 2017) by preventing oxidative lipid damage (Skouta et al. 2014). Ferrostatin-­1 is a well-­established inhibitor of Erastin-­induced ferroptosis. The Stockwell group (Gaschler et al. 2018b) set out to determine the localization of this inhibitor and found it to accumulate in mitochondria, lysosomes and in the ER.  Surprisingly, they postulated that the contribution of the mitochondria and lysosomes to ferroptosis was very little. This conclusion requires further investigation, as the data did not

6  Small Molecule Regulators of Ferroptosis

fully support this hypothesis. However, the involvement of the ER, as a source of lipids as well as an oxidative environment, provides a base on which to form exciting hypotheses; more work is required to define to what extend the ER and other organelles could be involved in ferroptosis. New generations of Ferrostatin-­1 derivatives (UAMC-­2418 and UAMC-­3203) have been reported to exhibit a protective activity in vivo against tissue injury (Hofmans et  al. 2016; Devisscher et al. 2018). Liproxstatin-­1 has been described as a radical trapping antioxidant (Zilka et al. 2017; Sheng et al. 2017), acting by preventing non-­enzymatic lipid peroxidation, which can drive ferroptosis. The same study pointed out the important role of phosphoethanolamine-­­esterified polyunsaturated fatty acids (PE-­PUFAs) in ferroptosis. Cell death in GPX4−/− cells is prevented by Liproxstatin-­1, which counteracts ROS accumulation. It is noteworthy that this molecule also counteracts the effects of some ferroptosis-­­ inducing agents, including RSL3, and prevents tissue injury (Friedmann Angeli et al. 2014; Tuo et  al. 2017). Moreover, deuterated polyunsaturated fatty acids (D-­PUFA) have shown anti-­­ ferroptotic properties due to the deuterium atoms substituting the proton in the bis-­allylic position, which is the preferential site of peroxidation. This, in turn, decreases the rate of radical generation. In kidney rhabdoid tumour G-­401 cells, preincubation with d-­Linoleate has been shown to prevent Erastin-­and RSL3-­induced ferroptosis (Yang et  al. 2016). D-­ Linoleate also rescued fibroblasts from increased cell death associated with Friedreich ataxia (FRDA) (Cotticelli et  al. 2013; Zesiewicz et al. 2018), blocking ferroptosis by inhibiting lipoxygenases (Shah et al. 2018). Moreover, butylated Hydroxytoluene and butylated Hydroxyanisole exhibit antioxidant properties and prevented Erastin-­ induced ferroptosis acting as radical trapping oxidants (Yagoda et al. 2007; Kain et  al. 2018). β-­Carotene has been identified in a screen as an anti-­ferroptotic molecule (Yagoda et al. 2007) and has been shown by the Stockwell group to counteract lipid peroxidation induced by the molecule methyl mercuric chloride (Andersen and Andersen 1993). Squalene, which has a skeleton structure similar

105

to β-­Carotene, is a product of the mevalonate pathway and acts as a natural antioxidant preventing oxidative cell death (Garcia-­Bermudez et  al. 2019). Importantly, CoQ10 is a natural metabolite acting as an endogenous lipid-­soluble antioxidant, which prevents initiation and propagation of lipid peroxidation. Reducing CoQ10 levels can enhance ferroptosis (Shimada et al. 2016) as demonstrated by the use of the small molecule FIN56. In case of GPX4 deficiency, another protein named ferroptosis suppressor protein 1 (FSP1), also known as apoptosis-­inducing factor mitochondria-­associated 2 (AIFM2) can protect cells from death by regeneration of CoQ10 using NAD(P)H (Doll et al. 2019; Bersuker et al. 2019). Idebenone is a structurally related antioxidant to CoQ10, whose difference in tail structure causes significant changes in terms of its pharmacokinetics and bioactivation (Gueven et  al. 2015). Mitoquinone is also based on the same chemical structure containing a positive charge at the end of its aliphatic chain and has been shown to antagonize RSL3-­ induced cell death (Jelinek et  al. 2018) by trapping ROS in mitochondria. Furthermore, U0126 is a selective inhibitor of both MAP 2 K1 and MAP 2 K2, which inhibits extracellular signal-­regulated kinase (ERK) signaling. U0126 inhibits severe cold-­induced ferroptosis (Hattori et  al. 2017), Erastin-­induced cell death (Yagoda et al. 2007; Dixon et al. 2012) and neuronal cell death following injury (Magtanong and Dixon 2019) linked to antioxidant activity through suppressing ROS. Trolox and derivatives of Vitamin E (also see Sect. 6.3.1.3) also operate via a similar MoA due to their antioxidant properties. Lastly, DT-­PTZ-­C is a recently developed molecule, which has shown promising results in the protection of brain tissue against oxidative stress. DT-­PTZ-­C belongs to the N10-­carbonyl-­substituted phenothiazine family, scavenging hydroxyl radicals and blocking lipid peroxidation by formation of a stable phenothiazine radical cation (Keynes et al. 2019).

6.3.1.6 Lipid Production Blockers (Scheme 6.1; Fig. 6.16) Inhibitors of lipid biosynthesis have shown interesting anti-­ ferroptotic properties, mainly by

S. Debieu et al.

106 Aminooxyacetic acid (AOA)

Zaragozic acid O O COONa O OH COONa O COONa OH

O H 2N

O

OH

YM-53601 F

O

N

H

O

O

S

O N

O

N

Pioglitazone

Troglitazone O

H N

O

O

HCl

HO

Rosiglitazone

O S

NH

O

N O

O

H N

O

S

Fig. 6.16  Inhibitors of lipid biosynthesis and their identified targets

inhibiting Acyl-­ CoA Synthetase Long Chain Family Member 4 (ACSL4) (Yuan et al. 2016b). Aminooxyacetic acid (AOA) is a transaminase inhibitor (Wise et  al. 2008), which prevents the formation of α-­ketoglutarate from glutamine by inhibiting glutamic-­ oxaloacetic transaminase 1 (GOT1). Evidence of its inhibitory effects on Erastin-­induced cell death has previously been reported (Dixon et al. 2012). Furthermore, YM-­

53601 and Zaragozic acid A (Squalestatin 1) are squalene synthase (SQS) inhibitors. YM-­53601 was reported to reduce levels of cholesterol and triglyceride in vivo (Ugawa et al. 2000) and also to act as a suppressor of lipogenic biosynthesis (Ugawa et al. 2003). Zaragozic Acid A was discovered in a screen to identify ­inhibitors of SQS (Dawson et  al. 1992) and its binding mode to SQS (Liu et  al. 2012) has now been reported.

6  Small Molecule Regulators of Ferroptosis

These two molecules have been shown to protect cells from the ferroptosis inducer FIN56 (Shimada et  al. 2016). The thiazolidinedione class of small molecules called Glitazones were first used as antidiabetic drugs. These molecules canonically act as PPARγ agonists, activated by free fatty acids and eicosanoids, to allow its transactivation leading to an increase in fatty acid storage in adipocytes, causing an increased reliance on glycolysis in these cells. When activated, PPARγ causes a decrease of transcription of various pro-­inflammatory genes such as those encoding for nuclear factor kappa-­light-­chain-­­enhancer of activated B cells (Nf-­kB) (Yki-­­Järvinen 2004). Three Glitazones, namely Pioglitazone, Troglitazone and Rosiglitazone, were used to study and dissect the mechanisms of ferroptosis. By inhibiting ACSL4, these molecules prevented the accumulation of lipid peroxidation products (Dixon et  al. 2015; Doll et  al. 2017; Li et  al. 2019c). This inhibition was reported to be independent of the PPARγ pathway (Askari et  al. 2007). Pioglitazone also inhibits mitochondrial iron uptake and therefore limits ferroptosis by stabilizing the iron-­sulfur cluster of CDGSH iron sulfur domain 1 protein (CISD1) (Yuan et  al. 2016a).

6.3.2 Iron and ROS Production Cells produce basal levels of endogenous ROS. NOXs, which in normal circumstances produce ROS to fend off bacterial infections, are implicated in ROS production. These enzymes can be dysregulated in various diseases, such as Parkinson, Alzheimer’s and alcohol-­induced liver injury, leading to increased ROS and cell death. Thus, inhibiting NOXs represents another potential strategy to inhibit ferroptosis. Elevated free iron levels can also lead to increased ROS production, and thus decreasing the labile iron pool can prevent ferroptotic cell death.

6.3.2.1 Decreasing the Labile iron Pool (Scheme 6.2; Fig. 6.17) Labile iron can be a catalyst of free radical production by reacting with H2O2. These free radi-

107

cals can react with proteins, lipids or DNA and cause lethal cellular injuries. Importantly, iron(II) and H2O2 can also act directly on lipids to provoke peroxidation. Iron chelators can be used to decrease the pool of free iron. Alternatively, this pool can be modulated by interfering with the functions of proteins involved in uptake, export or management of iron (Bogdan et  al. 2016; Anderson and Frazer 2017; Müller et al. 2020). 2,2′-­ bipyridyl (2,2-­ BP) is a liposoluble iron bidentate chelator that is efficient in reducing the effect of ferroptosis following hemorrhagic brain injury (Wu et al. 2012; Imai et al. 2019). Chelating iron can interfere with Fenton chemistry and subsequent lipid peroxidation and in some circumstances can rescue cell viability, depending on the strength of chelation and in which organelle it occurs. Other iron chelators have also played a pivotal role in dissecting and controlling the cellular iron pool in numerous studies. Deferiprone was the first drug to be approved that decreases iron overload in thalassemia patients. Deferoxamine (DFO) is a bacterial siderophore that was approved to treat iron poisoning and iron overload related diseases. It has been shown to have a positive effect on recovery after traumatic spinal cord injury (Yao et al. 2019). Importantly, Deferiprone and DFO have both been shown to chelate iron(III) and thus prevent ROS accumulation (Do Van et al. 2016; Friedmann Angeli et al. 2014; Dixon et  al. 2012; Yang et  al. 2014). In addition, other molecules that control the cellular iron pool via direct chelation or an alternative mechanism are Ciclopirox, Puerarin, Curcumin and Epigallocatechin gallate (EGCG), which is the most abundant catechin in tea. Ciclopirox olamine was first developed as an antifungal agent, which exerts its activity by chelating iron. It was identified as an inhibitor of Erastin-­induced ferroptosis (Dixon et  al. 2012) and acts against lysosomal rupture (Skouta et  al. 2014). Furthermore, Puerarin is an isoflavone exhibiting several biological effects (Zhou et al. 2014); the most relevant here is its inhibition of ferroptosis in the context of heart failure (Liu et al. 2018a), where Puerarin administration blocks iron overload and lipid peroxidation in vivo. Curcumin has also been described as an inhibitor of fibrosis

S. Debieu et al.

108 2,2'-bipyridine (2,2-BP) Deferiprone O

H2N

HO

N N

Deferoxamine (DFO) OH O N O

OH N

N O

Epigallocatechin gallate (EGCG) OH OH HO

O

HO

Puerarin

HO

OH

O O

O

OH

OH

HO OH

OH

OH

O

GSK2334470

Dopamine

NH2

N N

NH2

N OH

O

OH

NH

O

O Curcumin

OH

O

OH HO N O

O

OH

O

N H

O

O OH

H N

Ciclopirox olamine

N

N H

HO

N

NH2

HO

HN

Fig. 6.17  Small molecules interfering with the labile iron pool

(Zhang et al. 2013; Kheradpezhouh et al. 2016) and more recently as an inhibitor of Erastin and iron accumulation-­ induced ferroptosis (Guerrero-­­Hue et  al. 2019; Kose et  al. 2019). EGCG is a polyphenol showing promising results in cancer chemoprevention (Du et al. 2012) and

treatment of various diseases (Chu et  al. 2017), and moreover its anti-­ferroptotic properties have been reported (Kose et al. 2019). Dopamine is a neurotransmitter produced by dopamine-­ secreting neurons in the substantia nigra, and its decreased production is linked to

6  Small Molecule Regulators of Ferroptosis

109

Diphenyleneiodonium

GKT137831 O Cl

I

N HN

N

O N

N

N N

O

N

N

N

HO

NH2

N

CN N

N O

Linagliptin

Alogliptin

O Vildagliptin O H N

N

O

CN N

NH2

Fig. 6.18  Small molecule inhibitors of NOXs activity

Parkinson’s disease. Anti-­ferroptotic activity of dopamine, due to its stabilization of GPX4 and the prevention of iron(II) accumulation, was highlighted by the Tang group (Wang et al. 2016) in multiple cell lines, such as PANC1, HEY, MEF and HEK293. Furthermore, GSK2334470 is a recently characterized specific inhibitor of 3-­Phosphoinositide-­dependent protein kinase-­1 (PDK1) (Najafov et  al. 2011). High levels of PDK1 are associated with vulnerability to ferroptosis inducers (Viswanathan 2015) and PDK1 also plays a role in iron-­induced toxicity by the sphingolipid/PDK1/MEF2 signaling cascade described in the neurodegenerative disease Friedreich’s ataxia (Rockfield et  al. 2018). The inhibitory effect of GSK2334470 was demonstrated in a study using a model of iron overload leading to ferroptosis induced by FAC (Fang et al. 2018).

6.3.2.2 NOXs-Related Inhibitors (Scheme 6.1; Fig. 6.18) Many processes in cells can lead to the production of ROS. Interestingly, there is a group of NADPH-­ oxidases termed NOXs, employed in respiratory bursts to release ROS as a defense against bacterial or fungal invasions. NOXs are sparingly used by human cells under normal conditions, but NOX is deregulated in ischemia and some neurodegenerative diseases (Rastogi et  al. 2017), forming interesting links between these enzymes and ferroptosis. Diphenyleneiodonium (DPI) is a NOX inhibitor, which also inhibits mitochondrial ROS production (Li and Trush 1998; Massart et  al. 2014). Furthermore, it is a nitric oxide synthetase inhibitor. It was shown to exhibit an inhibitory effect against Erastin-­­ induced cell death (Dixon et al. 2012) and other studies have used this molecule to decipher the mechanism of hypersensitive cell death response to fungal invasion in rice leaf sheath tissues

S. Debieu et al.

110

(Dangol et al. 2019), which is reminiscent of ferroptosis in plants. Other studies have shown that this molecule can counteract the effect of pesticides in a model of neurodegeneration (Andersen and Andersen 1993). Interestingly, another NOX inhibitor called GKT137831 has been described to decrease Erastin-­ induced ferroptosis. This molecule was developed by Genkyotex for use in fibrosis treatment. NOX1 itself can be activated by dipeptidyl peptidase-­4 (DPP4). Interestingly, the binding of DPP1 to NOX1 can be blocked by the protein TP53, which then leads to suppression of ferroptosis in colorectal cancer cells (Xie et al. 2017). In addition, Gliptin is a class of antidiabetic drug which acts by inhibiting DPP4, and additionally three molecules of this class named Vildagliptin, Alogliptin, and Linagliptin were shown to suppress DDP4-­mediated lipid peroxidation (Stockwell et al. 2017).

6.3.3 Increasing GSH and Modulating MAPK Expression Erastin acts on system Xc− and thus induces GSH depletion, while GSH depletion induces MAPK activation. Therefore, inhibiting MAPK activation can antagonize ferroptosis.

6.3.3.1 By-Pass of System Xc− Inhibition (Scheme 6.1; Fig. 6.19) System Xc− regulates cellular GSH levels and subsequent antioxidant defense. In vivo, cysteine is internalized by cells through multiple processes, including system Xc−, introducing complexity into the system. Despite this, studying inhibition of system Xc− in cellulo improves understanding of the link between GSH and ferroptosis. For example, β-­Mercaptoethanol can

Beta-mercaptoethanol

6-Aminonicotinamide N-acetylcysteine SH O (6-AN) O H HS O H2 N N H OH N NH 2 Glutathione SH O H HOOC COOH N N H NH2 O OH

Fig. 6.19  Small molecules counteracting the inhibition of system Xc− and their MoA

6  Small Molecule Regulators of Ferroptosis

111

act as a biological antioxidant. However, the main interest in this molecule in the context of ferroptosis, is linked to its ability to bypass system Xc− and instead to transport cysteines into the cell by forming mixed disulfides which favor the use of a distinct transporter (Ishii et al. 1981). This property counteracts both Sulfasalazine and Erastin-­induced ferroptosis (Dixon et  al. 2014; Dixon et  al. 2012). However, the effects of this pathway are cell specific; 6-­Aminonicotinamide (6-­AN) prevents Erastin-­induced ferroptosis in Calu-­1 and BJeLR cells, but only to a limited extent in HT-­ 1080 cells (Dixon et  al. 2012). N-­Acetylcysteine (NAC) and GSH are both scavengers of ROS. Interestingly, NAC is a metabolic precursor of GSH and they have both been shown to inhibit Erastin-­ induced ferroptosis. Importantly, GSH is required for the reduction of peroxylated lipids (Codenotti et  al. 2018; Yang et al. 2014). A global role of GSH is to maintain redox homeostasis in cells and its depletion is associated with elevated iron(II) levels and cellular aging (Jenkins et  al. 2019). NAC also targets toxic lipids following hemorrhagic stroke and improves functional recovery after intracerebral hemorrhage in mice (Karuppagounder et al. 2018).

6.3.3.2 Inhibitors of MAPK-Related Pathway Activation (Fig. 6.20) MAPK activation is often linked to antioxidant activity, but in the context of Erastin-­mediated cell death, MAPK activation is required for cell death. SU6656 is a selective Src family kinase

H N

SU6656

SB202190

O

N

S

6.3.4 Miscellaneous (Fig. 6.21) Cycloheximide is a natural bacterial fungicide that has been widely used in cell biology to block protein synthesis. It was identified as a potent inhibitor of ferroptosis occurring as a result of system Xc− inhibition (Ratan et  al. 1994), and has since been used in numerous studies in this context (Shimada et  al. 2016; Dixon et  al. 2012). However, Cycloheximide does not block RSL3-­­induced ferroptosis (Yang and Stockwell 2008).

OH

Anthrapyrazolone (SP600125) N

NH O

inhibitor (Blake et al. 2000), which blocks RSL5 and RSL3-­ induced cell death (Yang and Stockwell 2008) presumably acting through attenuation of Ras signaling, leading to reduced activation of the MAPK pathway. SP600125 is an anthrapyrazolone inhibitor that prevents activation of inflammatory genes via inhibition of Jun N-­terminal kinases (JNK), which are encoded by the MAPK8, MAPK9 and MAPK10 genes (Bennett et al. 2001). Importantly, this molecule may be used for the treatment of ischemic stroke. In addition, antagonizing effects of Erastin-­­ induced ferroptosis through inhibition of JNK phosphorylation have been observed with SP600125 (Yu et  al. 2015). Finally, SB202190 binds within the Adenosine triphosphate (ATP) pocket of active MAPK and selectively inhibits the p38α and β isoforms. This molecule has been reported to decrease Erastin-­induced cytotoxicity (Yu et al. 2015).

HN N

F

O

O N

Fig. 6.20  Inhibitors of MAPK activation

NH

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Cycloheximide O

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7

Necroptosis, the Other Main Caspase-Independent Cell Death Larissa C. Zanetti and Ricardo Weinlich

Abbreviations 4HB AML ATP BSO CDC37 cFLIP

N-terminal four-helix-bundle Acute myeloid leukemia Adenosine triphosphate buthionine sulfoximine Cell Division Cycle 37 CASP8 and FADD-like Apoptosis Regulator cIAPs Cellular Inhibitor of Apoptosis CLL Chronic lymphocytic leukemia CrmA Cytokine Response Modifier A CYLD Lysine 63 Deubiquitinase DAI DNA-dependent activator of IFN regulatory factors DAMPs Damage-Associated Molecular Patterns DR Death Receptor ESCRT-III Endosomal Sorting Complexes Required for Transport III FADD Fas-Associated Protein with Death Domain FasL Fas ligand FIN56 Ferroptosis inducer 56 FINO2 Ferroptosis inducer endoperoxide GPX4 Glutathione peroxidase 4 GSH Glutathione

L. C. Zanetti (*) · R. Weinlich Hospital Israelita Albert Einstein. Av. Albert Einstein, São Paulo, SP, Brazil e-mail: [email protected]

HGMB-1 High-Mobility Group Box 1 protein HSP90 Heat-Shock Protein 90 HSV Herpes Simplex virus IAV Influenza A virus ICP-6/10 Infected cell protein 6/10 IE1 Immediate-Early gene product 1 LUBAC Linear Ubiquitin Chain Assembly Complex M45/vIRA Viral inhibitor of RIP activation MAPK Mitogen-activated protein kinases MCMV murine cytomegalovirus MDA-5 Melanoma differentiation-­ associated protein 5 MLKL Mixed Lineage Kinase Domain Like Pseudokinase NADPH Nicotinamide adenine dinucleotide phosphate Nec-1 Necrostatin-1 NF-κB Nuclear factor kappa-light-chain-­ enhancer of activated B cells NS1 Non-structural protein 1 PI3K Phosphoinositide 3-kinases Ppm1b Phosphatase 1b PRR Pattern Recognition Receptors PTGS2 Prostaglandin-endoperoxide synthase 2 RHIM RIP Homology Motifs RIPK1 Receptor-Interacting Serine/ Threonine Protein Kinase 1 RIPK3 Receptor-Interacting Serine/ Threonine Protein Kinase 3

© Springer Nature Switzerland AG 2021 A. F. Florez, H. Alborzinia (eds.), Ferroptosis: Mechanism and Diseases, Advances in Experimental Medicine and Biology 1301, https://doi.org/10.1007/978-3-030-62026-4_7

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RSL3 TLR3/4 TNF TNFR1

RAS-selective lethal Toll-like receptor-3/4 Tumour Necrosis Factor Tumour Necrosis Factor Receptor 1 TNFRSF1A-associated via death domain TNF receptor associated factors 2/5 TNF-related apoptosis-inducing ligand Transient receptor potential melastatin Vaccinia virus

(Vercammen et  al. 1998a; Vercammen et  al. 1998b; Kawahara et al. 1998). The Receptor-Interacting Serine/Threonine Protein Kinase 1 (RIPK1), a molecule known to be involved in cell survival, was identified as the TRADD first member of this pathway (Holler et al. 2000; Degterev et al. 2005; Degterev et al. 2008). Later, TRAF2/5 its kinase activity was shown to be indispensable for DR-induced necroptosis, as necrostatin-1 TRAIL (Nec-1), a small molecule that targets RIPK1 kinase activity was able to suppress cell death TRPM induced by caspase inhibition (Degterev et  al. 2008; Degterev et  al. 2012). RIPK3, another VV member of the RIP kinase family, had been previously shown to interact with RIPK1 through their respective RIP Homology Motifs (RHIM) (Sun et al. 1999), so it did not take long to demonstrate 7.1 A Brief History that RIPK3 was a downstream mediator of this cell death mode (Cho et al. 2009; He et al. 2009; of Necroptosis Zhang et al. 2009). The exciting quest to deterNecroptosis was first reported in 1988 when it mine which was the effector molecule that was was shown that Tumor Necrosis Factor (TNF) activated by RIPK3 to execute the necroptotic could induce both apoptosis and necrosis, phenotype ended in 2012, when the Mixed although at that time it was identified as “pro- Lineage Kinase Domain Like Pseudokinase grammed cell death” (Laster et  al. 1988). Ten (MLKL), was found to exert this role (Sun et al. years later, the same outcome was observed for 2012; Choi et al. 2018; Murphy et al. 2013; Wu CD95 ligand, another classical apoptosis inducer et al. 2013). The effector mechanisms by which (Vercammen et  al. 1998a). Interestingly, both MLKL kills a cell are still elusive, and they are pathways could trigger cell death in a caspase-­ discussed later in this chapter. independent manner, with morphological feaGenetic studies using deficient animals were tures similar to necrosis: cell swelling, organelle unambiguous to demonstrate that Caspase-8 and dysfunction and rupture of the plasma membrane FADD keep the necroptotic pathway in check (Vercammen et al. 1998a; Laster et al. 1988). both during development and in adult tissues. The initial insights into its mechanism came Both Caspase-8- and FADD-knockout animals with the observation that cowpox virus infection die at embryonic day 10.5 with an excess of induced a necrosis-like cell death only upon cas- necrotic cells in the yolk sac (Varfolomeev et al. pase inhibition by the viral caspase inhibitor 1998; Yeh et al. 1998) a phenotype that is fully Cytokine Response Modifier A (CrmA) or pan-­ rescued with the concomitant ablation of RIPK3 caspases inhibitors, such as z-VAD-fmk (Ray and or MLKL (Oberst et al. 2011; Kaiser et al. 2011; Pickup 1996; Vercammen et  al. 1998b). Alvarez-Diaz et al. 2016). Inducible deficiency of Subsequent studies showed that Fas-Associated Caspase-8 in adult tissues also causes increased Protein with Death Domain (FADD) deficient cell death, which is rescued by concurrent deficells as well as caspase-8-deficient were sensitive ciency of either RIPK3 or MLKL (Ch’en et  al. to TNF- or CD95L-induced necrosis, indicating 2011; Weinlich et al. 2013). The details on how that an alternative cell death pathway could be Caspase-8 controls necroptosis are still not fully activated when the proximal events of the Death understood, although it is quite clear that it Receptor (DR) signaling were defective depends on the presence of CASP8 and FADD-­ like Apoptosis Regulator (cFLIP) (Weinlich et al.

7  Necroptosis, the Other Main Caspase-Independent Cell Death

2013; Oberst et  al. 2011; Matsuda et  al. 2014). Most studies suggests that it is the proteolytic activity of the Caspase-8:cFLIP heterodimer that hinders the formation of the necroptotic signaling complex, probably due to the inactivating cleavage of RIPK1 and RIPK3 (Lin et al. 1999; Feng et al. 2007; Weinlich et al. 2013). Alternatively, other reports suggest that Caspase-8:cFLIP can cleave Lysine 63 Deubiquitinase CYLD, limiting the transition from Complex I to Complex II (see below) (O’Donnell et al. 2011).

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prompts RIPK3 recruitment via RHIM homotypic interactions (Sun et  al. 2012; Wu et  al. 2014a). Upon recruitment to this complex, termed necrosome, RIPK3 molecules auto-­ phosphorylate and oligomerize while also phosphorylating RIPK1, leading to the formation of a heterodimeric amyloid structure (Li et al. 2012) that recruits and phosphorylates MLKL (Morris et  al. 2012). RIPK3-mediated phosphorylation results in a conformational change that exposes MLKL N-terminal four-helix-bundle (4HB) domain, allowing its oligomerization and translocation to the plasma membrane (Quarato et  al. 7.2 Molecular Mechanisms 2016; Petrie et al. 2018). MLKL binds to phosphatidylinositol phosphates in the plasma memof Necroptosis brane, which induces further conformational Necroptosis can be initiated by a variety of sig- changes, stabilizing it (Dondelinger et al. 2014; nals. The classical necroptosis activators are the Hildebrand et  al. 2014; Dovey et  al. 2018) members of the Death Receptor family, being the (Fig.  7.1). Distinct mechanisms were suggested TNFR1 the most studied so far. Upon its ligation for the MLKL-mediated plasma membrane disby TNF, TNFR1 typically assembles a multimo- ruption, which includes direct pore or cation lecular complex composed by TNFRSF1A-­ channel formation, or indirectly, by activation of associated via death domain (TRADD), RIPK1, TRPM or other ion channels (Cai et al. 2014; Xia Cellular Inhibitor of Apoptosis (cIAPs) and et al. 2016; Huang et al. 2017; Liu et al. 2017). Linear Ubiquitin Chain Assembly Complex Nonetheless, regardless of the mechanism itself, (LUBAC) (Kelliher et  al. 1998; Vandenabeele MLKL-induced plasma membrane disruption et al. 2010) (Fig. 7.1). This complex is stabilized leads to full extravasation of intracellular compoby LUBAC and cIAPs-mediated linear and Lys-­ nents, including but not limited to lysosomal 63 ubiquitination, respectively, which licenses enzymes, ATP, mitochondrial DNA, Reactive the docking of TRAF2 and TRAF5, initiating Oxygen Species (ROS), HGMB-1, IL-1α and NF-κB and MAP kinase pathways and priming IL-33, all of them capable of eliciting a strong the cell to survival and proliferation. However, if inflammatory response (Amarante-Mendes et al. the TNF signal is strong or long enough, it 2018). induces CYLD-mediated deubiquitination, Apart from death receptors, necroptosis can allowing the formation of a second complex in also be triggered by many other stimuli. Pattern the cytosol, composed by TRADD, RIPK1 and Recognition Receptors (PRR) such as Toll-like FADD (Complex IIa) (Fig. 7.1). FADD recruits receptor-3 (TLR3) and TLR4 (He et  al. 2011; Caspase-8, which induces apoptosis either by Kaiser et al. 2013; Amarante-Mendes et al. 2018), cleavage of downstream effector caspases, such DAI (Upton et  al. 2012), MDA-5 (Loo et  al. as Caspase-7 and Caspase-3 or by cleaving Bid, 2008; Ding et al. 2015) and Interferon type I and initiating the mitochondrial-mediated apoptotic II (Downey et  al. 2017; Li et  al. 2018; Stetson pathway (Kelliher et  al. 1998; O’Donnell et  al. et al. 2018; Knuth et al. 2019) can directly acti2007; Mahoney et al. 2008; Vandenabeele et al. vate RIPK3, dispensing RIPK1 activity. 2010). Interestingly, in some of these instances, RIPK1 In the absence of FADD or functional can even exert an opposite role, i.e., it decreases Caspase-8, deubiquitinated RIPK1 auto-­or halts RIPK3-mediated cell death. In the phosphorylate its serine residue 161 (Degterev absence of RIPK1, RIPK3 recruits MLKL, rapet  al. 2008; Morris, et  al. 2012) an event that idly inducing necroptosis. When RIPK1 is pres-

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TNF-R1/CD95

TRADD RIPK1

(a) Complex I

TRADD

(b) Complex II

cIAP1/2 TRAF2/5

FADD

RIPK1

CYLD

FADD

(c) Complex IIb

Pro Caspase 8

Nec-1 FADD

Z-vad -fmk

TLR IF TR I DA

RIPK1 RIPK1 RIPK1 RIPK1 RIPK3 RIPK3 RIPK3 RIPK3 MLKL

MLKL

?

IFNAR

MLKL MLKL MLKL

MLKL

MLKL

PI

DAM

ines ytok C d Ps an

Fig. 7.1  Necroptosis pathway. (a) Upon Death Receptor stimulation, a complex containing TRADD, FADD, cIAPs, TRAF and RIPK1 is formed (Complex I) and signals to proliferative and survival responses. (b) Following Complex I deubiquitination, TRADD/FADD and RIPK1 forms the complex II at the cytosol, which can activate Caspase-8, thus inducing apoptosis. (c) Upon Caspase-8 inhibition, RIPK1 recruits RIPK3 that, in turn, recruits

and activates MLKL. Phosphorylated MLKL oligomerizes and migrates to the plasma membrane where it binds to phosphatidyl inositols. The accumulation of MLKL in the plasma membrane destabilizes it, killing the cells. During this process, intracellular contents are released, inducing an inflammatory response. TLR3 and TLR4 (via TRIF), DAI, and IFN can directly activate RIPK3

ent, it is also recruited by RIPK3 to the necrosome via its RHIM domain, bringing together FADD and Caspase-8:cFLIP heterodimers, known to act as negative regulators of necroptosis (Dillon et al. 2014). DNA damage, UV irradiation, oxidative stress, hypoxia, ischemia/reperfusion injuries and chemotherapeutic drugs can also trigger necroptosis, albeit it is yet to be fully determined whether RIPK1 is fundamentally required (Xu et al. 2010; Fulda 2014; Moriwaki et al. 2015). Additional molecules were associated with necrosome regulation. Heat-Shock Protein 90 (HSP90) and CDC37 interact with RIPK3 and MLKL, facilitating their oligomerization and activation (Li et al. 2015; Bigenzahn et al. 2016; Jacobsen et al. 2016). Ubiquitin-editing enzyme

A20, replaces Lys63-linked with Lys48-linked ubiquitin chains in Complex I, sending it to proteasomal degradation, thus avoiding its progression to Complex II (Jacobson et  al. 2009). A20 also restricts RIPK3 ubiquitination and its stable interaction with RIPK1 (Onizawa et  al. 2015). Additionally, the protein Phosphatase 1b (Ppm1b) can dephosphorylate RIPK3, reducing the levels of active RIPK3 molecules (Chen et  al. 2015). Finally, recent reports have suggested uncovered mechanisms that reduce the levels of active MLKL in the plasma membrane. Through a flotillin-­ 1/2-mediated endocytosis, phosphorylated MLKL is targeted for lysosomal degradation (Fan et  al. 2019). Also, cells can shed out plasma membrane areas that are rich in phospho-­

7  Necroptosis, the Other Main Caspase-Independent Cell Death

MLKL through the ESCRT-III pathway (Gong et al. 2017; Yoon et al. 2017; Fan et al. 2019).

7.3

Differences and Similarities Between Necroptosis and Ferroptosis

As pointed out in the other chapters, ferroptotic death is considered a biochemically controlled cell death with necrotic and pro-inflammatory features, like necroptosis and pyroptosis. Nonetheless, different from what was observed with necroptosis and pyroptosis, which present biochemical crosstalks (Frank and Vince 2019), and with necroptosis and apoptosis, which can be induced by common stimuli (Vanlangenakker et al. 2012), necroptosis and ferroptosis are quite distinct both in their triggers and their signaling pathways (Table 7.1 and Fig. 7.2). As both necroptosis and ferroptosis are necrotic-like cell death modes, they share a few

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phenotypic characteristics, such as plasma membrane damage and the release of Damage-­ Associated Molecular Patterns (DAMPs), which are intracellular components that once in the extracellular space can be sensed by surrounding cells and induce inflammatory responses. However, in a closer look, their morphological hallmarks are quite different. For instance, while in ferroptosis the mitochondria shrink and lose their cristae, during necroptosis this organelle swells and ruptures (Laster et  al. 1988; Vercammen et  al. 1998a; Dixon et  al. 2012). Initially, mitochondria were considered to be crucial for necroptosis execution, as recombinant MLKL could target mitochondria by binding to mitochondria phospholipids and also permeabilize cardiolipin-containing liposomes (Petrie et  al. 2017). However, mitochondria-depleted cells showed the same kinetics of necroptosis, demonstrating that this organelle is dispensable for this process (Moquin and Chan 2010; Marshall and Baines 2014). Still, in some cell

Table 7.1  Ferroptosis and Necroptosis similarities and differences regarding their inducers, features and involvement in diseases Inducers

Morphological hallmarks

Biochemical features

DAMPs released Related diseases

Ferroptosis Erastin, BSO, SRS, RSL3, FIN56, sorafenib, artemisinin, glutamate, FINO2 (Cao and Dixon 2016; Stockwell et al. 2017; Lei et al. 2019) Small mitochondria, high mitochondria membrane density, reduction or vanishing of mitochondrial cristae, rupture of the mitochondrial outer membrane (Dixon et al. 2012; Dixon et al. 2014; Cao and Dixon 2016) Iron-dependent accumulation of lipid peroxide, depletion of intracellular ATP, reduced GSH, inhibition of GPX4 of PTGS2, decrease of nicotinamide adenine dinucleotide phosphate (NADPH) (Cao and Dixon 2016; Brent R. Stockwell 2017; Sato et al. 2018) HMGB1 (Wen et al. 2019) Cancer, neurological disorders, cardiovascular diseases, kidney disease, bacterial infection, hepatic inflammation (Müller et al. 2017; Sato et al. 2018; Mou et al. 2019; Weiland et al. 2019)

Necroptosis TNF, FasL, TRAIL, IFNs, DAI, MDA-5, TLR3, TLR4, UV radiation, chemotherapy drugs (Kelliher et al. 1998; Zhang et al. 2009; Vandenabeele et al. 2010; Thapa et al. 2013; Moriwaki et al. 2015; Stetson et al. 2018) Cell and organelle swelling, loss of plasma membrane integrity (Laster et al. 1988; Vercammen et al. 1998a; Vercammen et al. 1998b)

Depletion of intracellular ATP, ROS generation, disturbed calcium homeostasis, loss of mitochondrial permeability (Kelliher et al. 1998; Garg et al. 2012; Zhang et al. 2017)

HMGB1, Heat shock proteins, mtDNA, IL-33 (Amarante-­Mendes et al. 2018) Viral and bacterial infection, neurological disorders, cancer, cardiovascular diseases, hepatocellular diseases, kidney disease, sepsis (Dara et al. 2016; Daniels et al. 2017; Stoll et al. 2017; Shan et al. 2018; Gong et al. 2019; Nailwal and Chan 2019)

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Death Receptor

FERROPTOSIS Erastin Sorafenib

Glutamate System Xc-

Complex I

NECROPTOSIS

RIPK1

Cystine Complex II

GSH GPX4

Ferrostatin Vit. E CoQ10

RIPK1

RSL3

Lipid ROS

Fe2+,O2

Complex IIb RIPK1 RIPK3 MLKL

Lipids Fig. 7.2  Necroptosis and ferroptosis have distinct signaling pathways. Necroptosis is dependent on RIPK3 activity. RIPK3 is activated by auto-phosphorylation when recruited to necroptosis-inducing platforms and then recruits and phosphorylates MLKL. Active MLKL migrates to and induces the rupture of the plasma membrane, releasing intracellular contents and inducing an inflammatory reaction. Ferroptosis, on the other hand, is

initiated by a failure or the pharmacological inhibition of the glutathione pathway that causes ROS accumulation. In the presence of free iron, ROS induces lipid peroxidation, thus initiating the ferroptotic cell death. Erastin and sorafenib inhibit cystine uptake by the cystine/glutamate antiporter (system x-), dramatically reducing antioxidant mechanisms and ultimately leading to the iron-dependent, oxidative cell death

types, mitochondrial ROS that is generated during necroptotic signaling can function as a feed forward mechanism by promoting autophosphorylation of RIPK1 (Zhang et al. 2017; Yang et al. 2018). Similarly, the importance of mitochondria in ferroptosis is still under debate, with evidence suggesting that they are not required at all and evidence that suggest that they can, in some cases, enhance ferroptotic signaling (Gao et  al. 2019). High-Mobility Group Box 1 protein (HMGB1) was characterized as the most relevant DAMP released following plasma membrane damage in ferroptosis (Wen et  al. 2019) while necroptosis presents a broader described group of DAMPs that include HMGB1 itself as well as IL-6, IL-1α, IL-33, ATP, histones and mtDNA, among others (Amarante-Mendes et al. 2018). HMGB1 seems

to play a central role in inflammation generated by both cell death types as it binds to specific receptors, mainly on endothelial and macrophages, inducing the production and release of pro-inflammatory cytokines (Yamada and Maruyama 2007; Ranzato et al. 2015). ROS production is a common biochemical feature for ferroptosis and necroptosis. In ferroptosis, defective glutathione pathway leads to the accumulation of ROS that, in the presence of free iron, causes lipid peroxidation, a key event to the execution of this cell death program (Cao and Dixon 2016). On the other hand, although ROS production is often observed during necroptosis, its role is considered to be only accessory. As mentioned earlier, in certain cell types, ROS promote RIPK1 autophosphorylation via oxidation of specific cysteines present in RIPK1, thus pro-

7  Necroptosis, the Other Main Caspase-Independent Cell Death

moting its interaction with RIPK3 (Zhang et al. 2017). Importantly, ROS production during necroptosis can be further increased by RIPK3-­ mediated activation of metabolic enzymes related with aerobic respiration (Qiu et  al. 2018). Moreover, in neutrophils, RIPK3-MLKL signaling increases ROS production through the activation of p38, MAPK and PI3K pathways, further suggesting an intricate relationship among these molecules (Wang et al. 2016). As necroptosis and ferroptosis are necrotic-­ like cell death programs, their involvement in diseases in which inflammation plays a big role should not come as a surprise. Indeed, both cell deaths were shown to contribute to the onset and/ or progression of several inflammation-related diseases, such as infection, cancer, cardiovascular diseases and kidney injury (Shan et al. 2018; Li et  al. 2020). Interestingly, although both necroptosis and ferroptosis can simultaneously occur in some cases, such as hemorrhagic stroke and doxorubicin-induced cell death (Fang et  al. 2019; Zille et al. 2017) it seems that in other diseases, they behave as alternative forms of cell death, one being induced when the other is inhibited (Müller et al. 2017).

7.4

Role of necroptosis in Human Diseases

One of the main hallmarks in cancer is the resistance to apoptosis. Likewise, many virus and bacteria had evolved to express apoptotic inhibitors or to present mechanisms to avoid apoptosis triggering (Orzalli et  al. 2018). As most of the initial necroptotic stimuli were common to apoptosis induction, and necroptosis was only observed when Caspase-8 or FADD were inhibited or absent, this cell death pathway was originally considered to be a backup mechanism in cases of failed apoptosis. However, it is now clear that necroptosis plays major roles in many pathophysiological conditions, mostly due to its ability to induce potent inflammatory and immune responses. Below, we illustrate these roles using three different groups of diseases, showing how necroptosis can be detrimental, promoting overt

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inflammatory responses, but also beneficial, by killing infected or transformed cells and also eliciting protective immune responses.

7.4.1 Viral Infection Viruses are known to harbor mechanisms that inhibit apoptosis, maintaining the replicative niche for prolonged periods of time, thus facilitating the propagation of the infection and increasing the risk to the host (Orzalli et  al. 2018). Keeping with that notion, the impairment of necroptosis signaling can be also detrimental to the virus-infected host. Indeed, RIPK3 deficient mice and RIPK1 kinase-dead mice fail to control Vaccinia virus replication in vivo (Cho et  al. 2009). Similarly, mice lacking RIPK3 are more susceptible to Influenza A virus (IAV) infection (Nogusa et al. 2016). Interestingly, the MLKL/FADD-double deficient mice recapitulate the RIPK3-deficient phenotype, suggesting that both apoptosis and necroptosis are important for restricting IAV infection and that RIPK3 can initiate, in some cases, the apoptotic signaling (Nogusa et  al. 2016). Additionally, pandemic IAVs, such the 1918 Spanish Flu and the 2009 Swine Flu, but not seasonal IAVs, cannot induce RIPK3-dependent immunogenic cell death, indicating that necroptosis has indeed an important role to limit IAV infection (Hartmann et al. 2017). Recently, it was shown that IAV encodes the viral protein NS1, which interacts with MLKL and is responsible for increasing its oligomerization and membrane translocation, promoting necroptosis in both macrophages and epithelial cells (Gaba and Fang 2019). The murine cytomegalovirus (MCMV) can also subvert the necroptotic pathway. It encodes the M45/vIRA gene that promotes viral replication and cell survival upon infection (Upton et al. 2008). M45/vIRA contains a N-terminal RHIM domain that allosterically interferes with RIPK3 recruitment to RIPK1 and DAI, thus inhibiting necroptosis (Upton et  al. 2008). Moreover, MCMV expressing RHIM-mutant M45 fails to replicate in cells due to RIPK3 activation (Pham et al. 2019). A similar mechanism that suppresses

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necroptosis in human cells was found in Herpes Simplex virus 1 (HSV-1) and HSV-2, which encode RHIM-containing proteins ICP-6 and ICP-10, respectively (Gu et  al. 2015). Also, an Immediate-Early gene product 1 (IE1) expressed by HCMV can block the recruitment of MLKL by RIPK3 in a RHIM-independent fashion (Omoto et  al. 2015). Vaccinia virus (VV) expresses E3L, a protein that inhibits DNA Activator of Interferon (DAI) ability to recruit RIPK3 (Koehler et  al. 2017). VV harboring a defective E3L presents a milder infection in animal models, which is restored in animals lacking RIPK3 (Koehler et al. 2017). Finally, viral MLKL homologs were recently found in poxviruses and they subvert necroptosis by RIPK3 sequestration (Petrie et al. 2019). However, in specific situations, virus-induced necroptosis can be detrimental to the host, especially when it kills cells that are central for the immune response against that pathogen. The best example is the HIV-induced necroptosis of HIV-­ specific CD8+ T cells, a population that is vital to the control of this infection (Gaiha et al. 2015). HIV-infected CD4+ T cells, but not bystander T cells, also die by necroptosis, contributing to the cytopathic effects observed in this disease (Pan et al. 2014).

7.4.2 Cancer As already mentioned, one of the cancer hallmarks is the resistance to apoptosis. One way of achieving it is via downmodulation of molecules that are important for apoptosis triggering and/or execution (Wong 2011). Unsurprisingly, the same mechanism was also found for necroptosis. RIPK3 is reduced or absent in many cancer types, including breast cancer (Koo et  al. 2015; Stoll et al. 2017), colorectal cancer (Feng et al. 2015; Koo et al. 2015), acute myeloid leukemia (AML) (Nugues et  al. 2014) and melanoma (Jönsson et al. 2010; Geserick et al. 2015). Reduced levels of MLKL were observed in gastric cancer (Sun et al. 2019), ovarian carcinoma (He et al. 2013), cervical squamous cell carcinoma (Ruan et  al. 2015) and colon cancer (Koo et al. 2015; Li et al.

L. C. Zanetti and R. Weinlich

2017). Also, a cohort of chronic lymphocytic leukemia (CLL) patients presented diminished CYLD expression (Liu et al. 2012). Importantly, lower expression of necroptotic molecules is usually associated with worse prognosis. Patients bearing breast cancer tumors with below-median RIPK3 expression have reduced disease-free survival (Koo et  al. 2015) and decreased RIPK3 expression in colorectal cancer negatively impacts both overall and disease-free survival (Feng et  al. 2015) . Likewise, cervical squamous cell, ovarian or gastric cancer patients presented worse overall survival risks when MLKL expression in the tumor was decreased (He et  al. 2013; Ruan et  al. 2015; Ertao et  al. 2016). Finally, low expression of CYLD is also associated with a group of CLL patients with poorer overall survival (Wu et al. 2014b). Of note, reduced expression of necroptotic molecules not only prolong the lifespan of the transformed cells but can also avoid the proper assembly of an immune response. In support of this idea, injection of necroptotic cells in mice generates higher levels of CD8+ T cell cross-­ priming and increased tumor immunity when compared with apoptotic cells injection (Aaes et  al. 2016; Yatim et  al. 2015). PolyI:C-induced necroptosis in cervical cancer cells triggered the production of IL-1α, an important activator of dendritic cells and IL-12 release (Schmidt et al. 2015). Also, RIPK3 or MLKL-deficient breast cancer cell lines render reduced tumorigenicity. RIPK3 may also have even unforeseen mechanisms to counter cancer, such as its role in restricting leukemogenesis in mice bearing AML-inducing mutations (Hockendorf et al. 2016). Nonetheless, there is growing evidence that necroptosis can assume the opposite function, i.e. to foster tumorigenesis. (Wang et  al. 2017; Lee et  al. 2018). Due to its pro-inflammatory ­characteristics, necroptosis can promote neoangiogenesis, induce tumor proliferation, facilitate metastasis and create an immunosuppressed environment (Garg and Agostinis 2017; Wang et  al. 2017; Ando et  al. 2020). Moreover, increased production of ROS triggered by necroptotic stimuli may exacerbate genomic instability in tumor cells, increasing the odds for

7  Necroptosis, the Other Main Caspase-Independent Cell Death

mutations that favor cancer progression, metastasis and severity (Dayal et al. 2008). Consequently, as necroptosis may be a double-edged sword in cancer, deep understanding of its roles in the specific tumor type is paramount to define whether and how this pathway should be used as a therapeutic target.

7.4.3 Inflammatory Diseases As one of the main outcomes of necroptosis is the generation of a strong inflammatory response, it should not come as a surprise that in several inflammatory diseases RIPK3 and MLKL are expressed in aberrant levels and increased necroptotic rates are observed. In fact, increased expression of RIPK3 and MLKL were found in biopsy samples from children with Inflammatory Bowel Disease and Crohn’s Disease (Pierdomenico et  al. 2014). A subsequent study also reported increased RIPK3 levels in Paneth cells in the terminal ileum of patients with Crohn’s disease, accompanied by increased RIPK3 in the mucosa and TNF in the serum (Garcia-Carbonell et al. 2019). RIPK3 deficient mice are more resistant to the development of atherosclerosis, with fewer necrotic areas and lower pro-inflammatory cytokine profile (Meng et al. 2015). Loss of MLKL decreases inflammation and the size of the necrotic core in atherosclerosis plates despite the puzzling increase of lipid accumulation in macrophages (Rasheed et al. 2019). MLKL, RIPK1 and RIPK3 expression levels were shown to be increased in the joints of mice with collagen-­ induced arthritis and ex vivo stimulation of cartilage explants with necroptotic triggers induced release of DAMPs and the increase of pro-­ inflammatory markers (Riegger and Brenner 2019). Furthermore, RIPK3-mediated necroptosis is associated with poorer prognosis in patients with alcoholic cirrhosis, and MLKL mediates RIPK3-independent hepatocellular necroptosis during hepatitis (Günther et  al. 2016). Interestingly, murine acute liver injury induced by acetaminophen does not seem to be mediated by necroptosis but concurs with a substantial

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decrease in glutathione levels, suggesting that ferroptosis may participate in this disease (Davis et  al. 1974; Schneider et  al. 2016; Dara et  al. 2016). Necroptosis also plays a role in inflammation-­ based neurological disorders, mediating TNF-­ induced oligodendrocyte degeneration, promoting cell death and inflammation in both in vitro and in vivo models of multiple sclerosis (Ofengeim et al. 2016), amyotrophic lateral sclerosis (Morrice et al. 2017), Parkinson’s disease (Yuan et al. 2019; Oñate et al. 2019) and Alzheimer’s disease (Shan et al. 2018; Choi et al. 2019).

7.5

Concluding Remarks

As shown above, necroptosis can markedly influence both the susceptibility to and the progression of viral infections, cancer and inflammatory diseases, sometimes being protective and in some instances being detrimental to the host. The same relationship is also observed for other diseases, including bacterial infection, ischemia-­ reperfusion injuries and cardiovascular disorders. (Zheng et al. 2011; Shan et al. 2018; Choi et al. 2019). In many of these cases, we have not yet fully uncovered all of the implications, mechanisms and consequences that are involved in the activation of RIPK1, RIPK3 and MLKL. Clearly, necroptotic cell death mediated by these molecules has a central role, not only by killing the affected cells but also modulating inflammation and/or immune response. Nonetheless, mounting evidence has demonstrated that RIPK1, RIPK3 and MLKL can crosstalk with many other biochemical pathways, such as pyroptosis, NF-κB, MAPK signaling, mitochondrial metabolism and apoptosis (Davidovich et al. 2014; Marshall and Baines 2014; Kondylis et al. 2017; Orzalli et al. 2018; Frank and Vince 2019). Strikingly, there is no evidence so far that necroptosis and ferroptosis signaling pathways interfere with each other. However, as the regulated necrotic-like cell death field is still fairly young and very fast-paced, we most definitely will learn a lot in the coming years and likely witness several revolutions in our concepts regarding these pathways.

132 Acknowledgements  This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), Brazil, through the grants #2016/17628-0 and #2017/25009-1. Conflict of Interest  The authors declare no conflict of interest.

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Index

A Apoptosis, v, 1, 29, 44, 47, 49, 51, 61–63, 67, 68, 90, 98, 124–127, 129–131 Autophagy, 1, 2, 42–44, 46–48, 53, 68, 85, 103

H Hallmarks of ferroptosis, 3, 4, 45, 66, 67, 69, 98 Heme, 11, 26, 29–32, 34, 65, 68, 87, 95, 97 Host defense, 26, 67

C Cancers, v, ix, 1, 3, 8–19, 28, 35, 42, 46–48, 53, 85, 88, 90, 91, 94, 95, 97, 101, 108, 110, 112, 127, 129–131 Cell death, v, vi, ix, 1–3, 9, 11, 13, 15, 16, 19, 27–29, 35, 42–44, 46–52, 60–72, 83, 88–95, 97–99, 101, 102, 105–107, 109, 111, 112, 123–131 Coronavirus disease 2019 (COVID-19), 70, 71 Cystine/Glutamate Transporter (xCT), 91 Cysteines, 3, 4, 9–12, 16–19, 85, 87, 91, 93, 98, 110, 111, 128

I Infectious diseases, v, 59–73 Inflammation, 50, 51, 61, 62, 94, 101, 127–129, 131 Iron, v, 2, 3, 10, 11, 17, 25–35, 42–53, 60, 64–69, 71, 83, 87, 91, 95–99, 101, 107–111, 128 Iron assimilation, 30–35 Iron storage, v, 11, 29, 32, 34, 42–44, 68, 95 Iron-sulfur-cluster, 34, 87, 107 Iron transport, 30, 33, 42

E Epithelial-to-mesenchymal transition (EMT), 14, 35 Erastin, 2, 3, 9, 11, 13–15, 43, 45, 46, 72, 88, 91, 92, 101, 108, 110, 127, 128 F Fenton reactions, 2, 10, 42–44, 52, 60, 65, 68, 69, 95, 96 Ferric (Fe³+), 26, 29, 30, 34, 60, 65, 95 Ferritin, 2, 3, 11, 28–32, 34, 35, 42–46, 48, 49, 52, 65, 68, 87, 91, 95 Ferritinophagy, 2, 3, 11, 29, 35, 41–53, 65, 68, 95 Ferroptosis, v, vi, ix, 1–3, 9–19, 28, 29, 35, 41–53, 59–73, 81–112, 127–129, 131 Ferrous (Fe²+), 26, 28, 34, 65 G Glutamate/Cystine antiporter, 128 Glutathione (GSH), 2–4, 9–12, 17, 18, 28, 29, 32, 43, 45–48, 50, 64–71, 83, 85, 87–95, 98, 103, 110–112, 127, 128, 131 Glutathione Peroxidase 4 (GPx4), 9–12, 14, 19, 29, 43, 45–47, 49–53, 64–72, 83, 85, 88–90, 95, 103–105, 109, 127

L Lipid peroxidation, 2, 3, 13, 27–29, 34, 42, 43, 45, 48–51, 61, 65, 67–70, 72, 89, 94, 95, 98–107, 110, 128 M Mixed Lineage Kinase Domain-Like Protein (MLKL), 64, 65, 124–131 N Necroptosis, v, vi, 1, 2, 62–65, 68, 123–131 Neurodegeneration, 2, 33, 35, 42, 48–53, 98, 110 Nuclear Receptor Coativator 4 (NCOA4), 2, 42–48, 52, 53, 68, 87 O Oxidative stresses, 28, 44, 46, 48, 49, 51, 61, 65, 70, 71, 88, 94, 97, 101, 103, 105, 126 P Pancreatic ductal adenocarcinoma (PDAC), 7–19, 95 Pyroptosis, v, vi, 2, 62–65, 68, 69, 71, 127, 131

© Springer Nature Switzerland AG 2021 A. F. Florez, H. Alborzinia (eds.), Ferroptosis: Mechanism and Diseases, Advances in Experimental Medicine and Biology 1301, https://doi.org/10.1007/978-3-030-62026-4

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Index

140 R Reactive oxygen species (ROS), 2, 3, 16, 27–29, 34, 42, 43, 48–52, 60, 66, 68, 69, 71, 83, 85, 87, 93–99, 103, 105, 107–111, 125, 127–130 Receptor Interacting Protein Kinase 1 (RIPK1), 64, 124–126, 128, 129, 131 Receptor Interacting Protein Kinase 3 (RIPK3), 64, 124–126, 128–131

S Small molecules, vi, 2, 9, 81–112, 124 T Transferrin Receptor Protein 1 (TFR1), 4, 10, 11, 33–35, 87 Transsulfuration, 3 Tuberculosis, 65, 67–69, 72 Tumour necrosis factor (TNF), 64, 124, 125, 127, 131