Metabolic regulation of ferroptosis in the tumor microenvironment

Ferroptosis is an iron-dependent, nonapoptotic form of regulated cell death triggered by impaired redox and antioxidant machinery and propagated by the accumulation of toxic lipid peroxides. A compendium of experimental studies suggests that ferroptosis is tumor-suppressive. Sensitivity or resistance to ferroptosis can be regulated by cell-autonomous and non-cell-autonomous metabolic mechanisms. This includes a role for ferroptosis that extends beyond the tumor cells themselves, mediated by components of the tumor microenvironment, including T cells and other immune cells. Herein, we review the intrinsic and extrinsic factors that promote the sensitivity of cancer cells to ferroptosis and conclude by describing approaches to harness the full utility of ferroptotic agents as therapeutic options for cancer therapy. Ferroptosis is an iron-dependent, nonapoptotic form of regulated cell death triggered by impaired redox and antioxidant machinery and propagated by the accumulation of toxic lipid peroxides. A compendium of experimental studies suggests that ferroptosis is tumor-suppressive. Sensitivity or resistance to ferroptosis can be regulated by cell-autonomous and non-cell-autonomous metabolic mechanisms. This includes a role for ferroptosis that extends beyond the tumor cells themselves, mediated by components of the tumor microenvironment, including T cells and other immune cells. Herein, we review the intrinsic and extrinsic factors that promote the sensitivity of cancer cells to ferroptosis and conclude by describing approaches to harness the full utility of ferroptotic agents as therapeutic options for cancer therapy. Cancer cells are subjected to and robustly adapt to more oxidative stress than nonmalignant cells (1Fiaschi T. Chiarugi P. Oxidative stress, tumor microenvironment, and metabolic reprogramming: A diabolic liaison.Int. J. Cell Biol. 2012; 2012: 762825Google Scholar). Higher oxidative stress in tumors is thought to be caused by altered mitochondrial function and increased activity of reactive oxygen species (ROS)-generating enzymes such as cyclooxygenases, lipoxygenases, and NADPH oxidases, most of which are modulated by tumor-intrinsic factors including increased growth factor and oncogenic signaling (e.g., Ras signaling) and loss of tumor suppressor function (e.g., p53). In addition, external factors such as chemotherapy and radiation also contribute to redox stress in cancer cells. However, if not carefully controlled, unabated ROS can damage macromolecular structures, including proteins and membrane lipids, which can result in cell death or senescence. Therefore, cancer cells rely on endogenous antioxidant networks to maintain redox homeostasis (2Trachootham D. Alexandre J. Huang P. 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Ferroptosis is a term that describes a nonapoptotic form of cell death associated with the perturbation of redox and antioxidant mechanisms, and propagation of lipid peroxidation reactions. This cell death requires labile active iron and is morphologically, phenotypically, and biochemically distinct from other cell death programs such as apoptosis, necroptosis, pyroptosis, and necrosis (6Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. Morrison B. Stockwell B.R. Ferroptosis: An iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Google Scholar, 7Cao J.Y. Dixon S.J. Mechanisms of ferroptosis.Cell. Mol. Life Sci. 2016; 73: 2195-2209Google Scholar, 8Conrad M. Kagan V.E. Bayir H. Pagnussat G.C. Head B. Traber M.G. Stockwell B.R. Regulation of lipid peroxidation and ferroptosis in diverse species.Genes Dev. 2018; 32: 602-619Google Scholar, 9Gaschler M.M. Andia A.A. Liu H. Csuka J.M. Hurlocker B. Vaiana C.A. Heindel D.W. Zuckerman D.S. Bos P.H. Reznik E. Ye L.F. Tyurina Y.Y. Lin A.J. Shchepinov M.S. Chan A.Y. et al.FINO2 initiates ferroptosis through GPX4 inactivation and iron oxidation.Nat. Chem. Biol. 2018; 14: 507-515Google Scholar). Dysregulated metabolism is at the core of ferroptosis, characterized by three hallmarks: (i) impaired antioxidant machinery, (ii) availability of redox-active iron, and (iii) the propagation of toxic lipid hydroperoxides (10Dixon S.J. Stockwell B.R. The hallmarks of ferroptosis.Annu. Rev. Cancer Biol. 2019; 3: 35-54Google Scholar). The importance of ferroptosis in cancer is indicated by its tumor-inhibitory capacity in both primary and drug-resistant cancer cells (11Hangauer M.J. Viswanathan V.S. Ryan M.J. Bole D. Eaton J.K. Matov A. Galeas J. Dhruv H.D. Berens M.E. Schreiber S.L. McCormick F. McManus M.T. Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition.Nature. 2017; 551: 247-250Google Scholar, 12Wu Y. Yu C. Luo M. Cen C. Qiu J. Zhang S. Hu K. Ferroptosis in cancer treatment: Another way to Rome.Front. Oncol. 2020; 10: 571127Google Scholar). Much of the now classic work on ferroptosis in cancer has focused on cell-autonomous effects in the tissue culture setting. These studies put forth the idea that ferroptosis may serve a tumor suppressor mechanism, akin to apoptosis or senescence. Contemporary studies have begun to test this idea in animal models, studying cancer ferroptosis and how this is impacted by the tumor microenvironment (TME). The TME contains a myriad of distinct cell types and factors including the tumor cells, stromal cells (e.g., cancer associated fibroblasts), immune cells, vasculature, extracellular matrix (e.g., collagen, fibronectin, laminin), and secreted molecules (e.g., metabolites, cytokines). This collective milieu influences all aspects of tumor biology. For example, oxidative stress is among the many factors influenced by the TME, and this cross talk promotes cancer cell proliferation and survival, cell migration and invasion, wound healing, tumor vascularization defects, and treatment resistance (4Reuter S. Gupta S.C. Chaturvedi M.M. Aggarwal B.B. Oxidative stress, inflammation, and cancer: How are they linked?.Free Radic. Biol. Med. 2010; 49: 1603-1616Google Scholar, 13Grivennikov S.I. Greten F.R. Karin M. Immunity, inflammation, and cancer.Cell. 2010; 140: 883-899Google Scholar). However, there is a delicate balance relating to the impact of ROS in the TME, wherein moderate levels promote tumor growth, while higher levels tilt the balance to increased oxidative damage to macromolecules and cell death (14Hayes J.D. Dinkova-Kostova A.T. Tew K.D. Oxidative stress in cancer.Cancer Cell. 2020; 38: 167-197Google Scholar). In line with this concept, the production of lipid ROS and, by extension the initiation of ferroptosis, could have multifaceted and complex impact on the TME, tumor growth, and therapeutic response. In this review, we start by setting the stage for ferroptosis as a metabolic form of cell death. We describe early studies that elucidated the mechanisms that form the basis of our understanding of cell-autonomous ferroptosis as tumor-suppressive. Further, we discuss recent studies describing the impact of ferroptosis on the TME. We round out the review by highlighting the therapeutic utility and current challenges mitigating the use of classic ferroptotic agents in preclinical and clinical setting and close by outlining perspectives for future investigation. Ferroptosis occurs when lipid ROS production exceeds the capacity of the lipid ROS detoxification machinery. Lipid ROS is generated by redox active labile iron via Fenton chemistry, and the availability of iron has been shown to be crucial to the initiation of ferroptosis (6Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. Morrison B. Stockwell B.R. Ferroptosis: An iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072Google Scholar) (Fig. 1). The lipid ROS are then spontaneously propagated across unsaturated lipids in the membrane. Specifically, peroxidation of membrane polyunsaturated fatty acid (PUFA) phospholipids (PUFA-PLs) drives the ferroptosis process (15Stockwell B.R. Friedmann Angeli J.P. Bayir H. Bush A.I. Conrad M. Dixon S.J. Fulda S. Gascón S. Hatzios S.K. Kagan V.E. Noel K. Jiang X. Linkermann A. Murphy M.E. Overholtzer M. et al.Ferroptosis: A regulated cell death nexus linking metabolism, redox biology, and disease.Cell. 2017; 171: 273-285Google Scholar). Studies have identified phosphatidylethanolamine (PE)-containing arachidonic acid (AA; C20:4) and adrenic acid (AdA; C22:4) phospholipids as key substrates of phospholipid peroxidation in ferroptosis (16Kagan V.E. Mao G. Qu F. Angeli J.P.F. Doll S. Croix C.S. Dar H.H. Liu B. Tyurin V.A. Ritov V.B. Kapralov A.A. Amoscato A.A. Jiang J. Anthonymuthu T. Mohammadyani D. et al.Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis.Nat. Chem. Biol. 2017; 13: 81-90Google Scholar). The presence of highly oxidizable methylene double bonds makes them major substrates for phospholipid peroxidation (17Yin H. Xu L. Porter N.A. Free radical lipid peroxidation: Mechanisms and analysis.Chem. Rev. 2011; 111: 5944-5972Google Scholar). The direct connection between accumulation of lipid peroxides and cell death manifests in the fact that ferroptosis can be effectively inhibited by lipophilic antioxidants such as alpha-tocopherol (vitamin E) and its analogs, ferrostatin-1, and liproxstatin-1, all of which serve as radical trapping antioxidants that neutralize lipid ROS and therefore prevent cell death (18Gaschler M.M. Hu F. Feng H. Linkermann A. Min W. Stockwell B.R. Determination of the subcellular localization and mechanism of action of ferrostatins in suppressing ferroptosis.ACS Chem. Biol. 2018; 13: 1013-1020Google Scholar, 19Skouta R. Dixon S.J. Wang J. Dunn D.E. Orman M. Shimada K. Rosenberg P.A. Lo D.C. Weinberg J.M. Linkermann A. Stockwell B.R. Ferrostatins inhibit oxidative lipid damage and cell death in diverse disease models.J. Am. Chem. Soc. 2014; 136: 4551-4556Google Scholar). Detoxification of cellular lipid ROS, which consequently inhibits ferroptosis, can occur through multiple pathways. In fact, many of these pathways have been illuminated by observing that their inhibition promotes ferroptotic cell death. For example, a small-molecule screen for compounds selectively lethal in oncogenic Ras-expressing cells identified what is now considered to be a classic inducer of ferroptosis: i.e., Erastin, a system xC− inhibitor (20Yang W.S. Stockwell B.R. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells.Chem. Biol. 2008; 15: 234-245Google Scholar). System xC− is a heterodimeric cystine-glutamate antiporter encoded by SLC3A2 (CD98 subunit) and the transporter-specific gene SLC7A11 (xCT subunit) (21Bassi M.T. Gasol E. Manzoni M. Pineda M. Riboni M. Martín R. Zorzano A. Borsani G. Palacín M. Identification and characterisation of human xCT that co-expresses, with 4F2 heavy chain, the amino acid transport activity system xc-.Pflugers Arch. 2001; 442: 286-296Google Scholar, 22Bannai S. Kitamura E. Transport interaction of L-cystine and L-glutamate in human diploid fibroblasts in culture.J. Biol. Chem. 1980; 255: 2372-2376Google Scholar). Cystine is the dimeric and oxidized form of cysteine, a nonessential amino acid and the rate-limiting component of glutathione (GSH). GSH is an antioxidant tripeptide and a primary mediator of cellular redox balance. Extracellular cysteine in serum (and tissue culture media) is predominantly present as cystine, and cancer cells employ a variety of mechanisms to increase levels of xCT to obtain cysteine (23Koppula P. Zhang Y. Zhuang L. Gan B. Amino acid transporter SLC7A11/xCT at the crossroads of regulating redox homeostasis and nutrient dependency of cancer.Cancer Commun. 2018; 38: 12Google Scholar). This provides the rationale as to why cancer cells are more vulnerable to ferroptosis initiated by system xC− inhibition. Shortly after the description of system xC− inhibitors (24Dolma S. Lessnick S.L. Hahn W.C. Stockwell B.R. Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells.Cancer Cell. 2003; 3: 285-296Google Scholar), a second class of ferroptosis-inducing compounds were described that inhibit glutathione peroxidase 4 (GPX4) (20Yang W.S. Stockwell B.R. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells.Chem. Biol. 2008; 15: 234-245Google Scholar). GPX4 is a GSH-dependent and selenocysteine-containing lipid peroxidase, in which direct or indirect inhibition, e.g., GSH depletion, leads to ferroptosis (20Yang W.S. Stockwell B.R. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells.Chem. Biol. 2008; 15: 234-245Google Scholar, 25Yang W.S. SriRamaratnam R. Welsch M.E. Shimada K. Skouta R. Viswanathan V.S. Cheah J.H. Clemons P.A. Shamji A.F. Clish C.B. Brown L.M. Girotti A.W. Cornish V.W. Schreiber S.L. Stockwell B.R. Regulation of ferroptotic cancer cell death by GPX4.Cell. 2014; 156: 317-331Google Scholar). A notable inhibitor of GPX4 is the small-molecule compound, (1S,3R)-Ras Selective Lethal 3, abbreviated as RSL3. RSL3 acts by binding covalently to GPX4 and inducing the latter’s inactivation and consequent accumulation of lipid ROS in the cell (25Yang W.S. SriRamaratnam R. Welsch M.E. Shimada K. Skouta R. Viswanathan V.S. Cheah J.H. Clemons P.A. Shamji A.F. Clish C.B. Brown L.M. Girotti A.W. Cornish V.W. Schreiber S.L. Stockwell B.R. Regulation of ferroptotic cancer cell death by GPX4.Cell. 2014; 156: 317-331Google Scholar). Thus, these two nodes (system xC− and GPX4) converge to promote ferroptosis by way of cysteine depletion and GPX4 inhibition. More recently, other mechanisms have been uncovered that arise from genetic, transcriptional, or metabolic dysregulation that predict sensitivity or resistance to ferroptosis. Some of these directly act on the cysteine-GSH-GPX4 axis, and others work entirely in parallel. In addition, there is now a growing appreciation that the mechanisms regulating ferroptosis in isolated cancer cells in culture are not readily recapitulated in vivo. Put another way, the in vivo tumor microenvironment can protect cancer cells from ferroptosis through non-cell-autonomous functions and activities. In the following sections, we will discuss these metabolic factors and pathways from a cell-autonomous perspective followed by a description of the ways these pathways are circumvented by factors in the TME. The importance and therapeutic potential of ferroptosis in cancer and other diseases have recently received considerable attention. In line with this, so has the list of cell autonomous mechanisms reported to govern ferroptotic sensitivity and resistance (Fig. 2). The section that follows provides a succinct overview of the cell autonomous mechanisms that regulate ferroptosis, as more detailed descriptions have been provided in several excellent reviews (10Dixon S.J. Stockwell B.R. The hallmarks of ferroptosis.Annu. Rev. Cancer Biol. 2019; 3: 35-54Google Scholar, 15Stockwell B.R. Friedmann Angeli J.P. Bayir H. Bush A.I. Conrad M. Dixon S.J. Fulda S. Gascón S. Hatzios S.K. Kagan V.E. Noel K. Jiang X. Linkermann A. Murphy M.E. Overholtzer M. et al.Ferroptosis: A regulated cell death nexus linking metabolism, redox biology, and disease.Cell. 2017; 171: 273-285Google Scholar, 26Jiang X. Stockwell B.R. Conrad M. Ferroptosis: Mechanisms, biology and role in disease.Nat. Rev. Mol. Cell Biol. 2021; 22: 266-282Google Scholar, 27Kim M.J. Yun G.J. Kim S.E. Metabolic regulation of ferroptosis in cancer.Biology (Basel). 2021; 10: 83Google Scholar). Notably, we highlight the literature examples that form the basis of the well-appreciated mechanisms regulating ferroptosis in order to prepare the reader for our more detailed discussion of the non-cell-autonomous mechanisms that follow. Two of the earliest characterized genes in the regulation of GPX4-inhibition mediated ferroptosis are the lipid metabolic genes: acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3) (16Kagan V.E. Mao G. Qu F. Angeli J.P.F. Doll S. Croix C.S. Dar H.H. Liu B. Tyurin V.A. Ritov V.B. Kapralov A.A. Amoscato A.A. Jiang J. Anthonymuthu T. Mohammadyani D. et al.Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis.Nat. Chem. Biol. 2017; 13: 81-90Google Scholar, 28Doll S. Proneth B. Tyurina Y.Y. Panzilius E. Kobayashi S. Ingold I. Irmler M. Beckers J. Aichler M. Walch A. Prokisch H. Trümbach D. Mao G. Qu F. Bayir H. et al.ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition.Nat. Chem. Biol. 2017; 13: 91-98Google Scholar, 29Yuan H. Li X. Zhang X. Kang R. Tang D. Identification of ACSL4 as a biomarker and contributor of ferroptosis.Biochem. Biophys. Res. Commun. 2016; 478: 1338-1343Google Scholar, 30Dixon S.J. Winter G.E. Musavi L.S. Lee E.D. Snijder B. Rebsamen M. Superti-Furga G. Stockwell B.R. Human haploid cell genetics reveals roles for lipid metabolism genes in nonapoptotic cell death.ACS Chem. Biol. 2015; 10: 1604-1609Google Scholar). ACSL4 and LPCAT3 were found to be critical proferroptotic genes, which encode proteins that remodel the cellular membrane lipid architecture to execute ferroptosis. The protein encoded by ACSL4 functions in the acylation of long-chain fatty acids, a step that is crucial for the biosynthesis of long-chain PUFA-CoA, including arachidonic acid-CoA (AA-CoA) and adrenic acid-CoA (AdA-CoA). While the protein encoded by LPCAT3, on the other hand, mediates the incorporation of these and other acylated AA into membrane phospholipids (16Kagan V.E. Mao G. Qu F. Angeli J.P.F. Doll S. Croix C.S. Dar H.H. Liu B. Tyurin V.A. Ritov V.B. Kapralov A.A. Amoscato A.A. Jiang J. Anthonymuthu T. Mohammadyani D. et al.Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis.Nat. Chem. Biol. 2017; 13: 81-90Google Scholar, 28Doll S. Proneth B. Tyurina Y.Y. Panzilius E. Kobayashi S. Ingold I. Irmler M. Beckers J. Aichler M. Walch A. Prokisch H. Trümbach D. Mao G. Qu F. Bayir H. et al.ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition.Nat. Chem. Biol. 2017; 13: 91-98Google Scholar, 29Yuan H. Li X. Zhang X. Kang R. Tang D. Identification of ACSL4 as a biomarker and contributor of ferroptosis.Biochem. Biophys. Res. Commun. 2016; 478: 1338-1343Google Scholar, 30Dixon S.J. Winter G.E. Musavi L.S. Lee E.D. Snijder B. Rebsamen M. Superti-Furga G. Stockwell B.R. Human haploid cell genetics reveals roles for lipid metabolism genes in nonapoptotic cell death.ACS Chem. Biol. 2015; 10: 1604-1609Google Scholar). ACSL4 expression directly correlates with sensitivity of cancer cells to ferroptosis; deletion of ACSL4 suppresses ferroptosis sensitivity, while overexpression sensitizes cells to ferroptosis. Accordingly, ACSL4 transcript levels were found to be downregulated in ferroptosis-resistant cancer cell lines (29Yuan H. Li X. Zhang X. Kang R. Tang D. Identification of ACSL4 as a biomarker and contributor of ferroptosis.Biochem. Biophys. Res. Commun. 2016; 478: 1338-1343Google Scholar). Deletion of ACSL4 and LPCAT3 prevented the integration of PUFA into the membrane bilayer, thereby depleting the substrate for oxidative lipid damage (30Dixon S.J. Winter G.E. Musavi L.S. Lee E.D. Snijder B. Rebsamen M. Superti-Furga G. Stockwell B.R. Human haploid cell genetics reveals roles for lipid metabolism genes in nonapoptotic cell death.ACS Chem. Biol. 2015; 10: 1604-1609Google Scholar). Remarkably cells with ACSL4 and GPX4 codeleted maintained their viability (29Yuan H. Li X. Zhang X. Kang R. Tang D. Identification of ACSL4 as a biomarker and contributor of ferroptosis.Biochem. Biophys. Res. Commun. 2016; 478: 1338-1343Google Scholar), indicating the interconnection between the two enzymes. Hence, the membrane remodeling enzymes are key regulatory nodes of ferroptosis susceptibility. PUFAs are the site of oxidative lipid damage and are required for the execution of ferroptosis (31Yang W.S. Kim K.J. Gaschler M.M. Patel M. Shchepinov M.S. Stockwell B.R. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E4966-E4975Google Scholar). The abundance of PUFAs determines the extent of available lipid peroxidation sites and thus ferroptosis susceptibility. Moreover, high concentration of PUFAs on lipid membranes has been shown to be positively correlated with increased dependency on GPX4 and enhanced sensitivity to iron-dependent oxidative lipid damage (32Tousignant K.D. Rockstroh A. Poad B.L.J. Talebi A. Young R.S.E. Taherian Fard A. Gupta R. Zang T. Wang C. Lehman M.L. Swinnen J.V. Blanksby S.J. Nelson C.C. Sadowski M.C. Therapy-induced lipid uptake and remodeling underpin ferroptosis hypersensitivity in prostate cancer.Cancer Metab. 2020; 8: 11Google Scholar). Exogenous supplementation with long chain PUFAs enhances ferroptosis sensitivity, even under conditions of ACSL4 deletion (28Doll S. Proneth B. Tyurina Y.Y. Panzilius E. Kobayashi S. Ingold I. Irmler M. Beckers J. Aichler M. Walch A. Prokisch H. Trümbach D. Mao G. Qu F. Bayir H. et al.ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition.Nat. Chem. Biol. 2017; 13: 91-98Google Scholar). Additionally, exogenous PUFA in the form of DGLA (dihomo-γ-linolenic acid) was recently described as a metabolic inducer of ferroptosis in human cancer cells, and this could be blocked by ferrostatin-1 or endogenous ether lipids (33Perez M.A. Magtanong L. Dixon S.J. Watts J.L. Dietary lipids induce ferroptosis in caenorhabditiselegans and human cancer cells.Dev. Cell. 2020; 54: 447-454.e4Google Scholar). The proferroptotic activity of DGLA appeared to be unique to this class of PUFA, as a similar phenotype was not shared by other PUFAs such as AA, eicosapentaenoic acid (EPA). and docosahexaenoic acid (DHA) (33Perez M.A. Magtanong L. Dixon S.J. Watts J.L. Dietary lipids induce ferroptosis in caenorhabditiselegans and human cancer cells.Dev. Cell. 2020; 54: 447-454.e4Google Scholar). Exogenous monounsaturated fatty acids (MUFAs), such as oleic acid (OA, C18:1), display the opposite effect(s) of PUFAs by potently suppressing ferroptosis. Mechanistically, this involves the competitive decrease of PUFA incorporation into membranes, which blocks the propagation of lipid ROS at the plasma membrane (31Yang W.S. Kim K.J. Gaschler M.M. Patel M. Shchepinov M.S. Stockwell B.R. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E4966-E4975Google Scholar, 34Magtanong L. Ko P.-J. To M. Cao J.Y. Forcina G.C. Tarangelo A. Ward C.C. Cho K. Patti G.J. Nomura D.K. Olzmann J.A. Dixon S.J. Exogenous monounsaturated fatty acids promote a ferroptosis-resistant cell state.Cell Chem. Biol. 2019; 26: 420-432.e9Google Scholar). This lipid peroxidation-suppressing and ferroptosis-inhibitory role of MUFA is dependent on the activity of the protein encoded by the acyl-coenzyme A synthetase long-chain family member 3 (ACSL3) gene, which allows for the selective incorporation of MUFAs into membrane lipids. Treating cells with exogenous MUFAs decreased cellular PUFA-PLs levels, ultimately leading to ferroptosis resistance. Accordingly, low expression of ACSL3 was correlated with increased ferroptosis sensitivity (34Magtanong L. Ko P.-J. To M. Cao J.Y. Forcina G.C. Tarangelo A. Ward C.C. Cho K. Patti G.J. Nomura D.K. Olzmann J.A. Dixon S.J. Exogenous monounsaturated fatty acids promote a ferroptosis-resistant cell state.Cell Chem. Biol. 2019; 26: 420-432.e9Google Scholar). Therefore, the differential incorporation of PUFAs or MUFAs into membranes increases or decreases sensitivity to ferroptosis, respectively. The ferroptosis suppressor protein 1 (FSP1) was recently characterized as a novel lipid peroxidation neutralizing pathway distinct and parallel to the GPX4 pathway. FSP1 was identified in genetic screens from two independent research groups that sought to determine pathways that govern resistance to GPX4 inhibition-induced ferroptosis in cancer lines. It was discovered that FSP1 provided resistance to ferroptosis under conditions of GPX4 deletion (35Bersuker K. Hendricks J.M. Li Z. Magtanong L. Ford B. Tang P.H. Roberts M.A. Tong B. Maimone T.J. Zoncu R. Bassik M.C. Nomura D.K. Dixon S.J. Olzmann J.A. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis.Nature. 2019; 575: 688-692Google Scholar, 36Doll S. Freitas F.P. Shah R. Aldrovandi M. da Silva M.C. Ingold I. Goya Grocin A. Xavier da Silva T.N. Panzilius E. Scheel C.H. Mourão A. Buday K. Sato M. Wanninger J. Vignane T. et al.FSP1 is a glutathione-independent ferroptosis suppressor.Nature. 2019; 575: 693-698Google Scholar). Specifically, FSP1 is recruited to the plasma membrane where it uses NAD(P)H to reduce coenzyme Q10 (CoQ10) to its radical trapping antioxidant form, ubiquinol (CoQ10-H2). This then blocks oxidative phospholipid damage and directly mitigates lipid peroxidation. Deletion of FSP1 in cancer cells results in increased lipid ROS even in the presence of functional GPX4, and overexpression of FSP1 blocks propagation of lipid ROS in RSL3-treated cells. These collective results illustrate that FSP1 serves as a complementary pathway to GPX4 to block lipid peroxidation (Fig. 2). A recent metabolism-centered genetic screen identified a novel role of the cofactor tetrahydrobiopterin (BH4) and the enzyme dihydrofolate reductase (DHFR) in the protection against ferroptosis (37Soula M. Weber R.A. Zilka O. Alwaseem H. La K. Yen F. Molina H. Garcia-Bermudez J. Pratt D.A. Birsoy K. Metabolic determinants of cancer cell sensitivity to canonical ferroptosis inducers.Nat. Chem. Biol. 2020; 16: 1351-1360Google Scholar). BH4 was found to be a potent endogenous radical-trapping antioxidant that regulates sensitivity to ferroptosis induced by GPX4-inhibition, but not cysteine depletion. The radical-trapping antioxidant role of BH4 was distinct from its function as a cofactor to enzymes involved in hydroxylation of aromatic amino acids. The biosynthesis of BH4 from GTP involves several steps catalyzed by the rate-limiting enzyme, GTP cyclohydrolase 1 (GCH1). Genetic deletion of GCH1 reduced intracellular levels of BH4 and decreased the antioxidant capacity of the cells. These BH4-deficient cells showed enhanced sensitization to GPX4 inhibition-induced ferroptosis. Consistently, treating BH4-deficient cells with exogenous BH4 in the form of dihydrobiopterin (BH2), the dehydrogenated product of BH4, protected against and rescued RLS3 and ML162-induced ferroptosis, but not Erastin-induced ferroptosis. In this setting, DHFR was involved in the restoration of BH4 from BH2 (37Soula M. Weber R.A. Zilka O. Alwaseem H. La K. Yen F. Molina H. Garcia-Bermudez J. Pratt D.A. Birsoy K. Metabolic determinants of cancer cell sensitivity to canonical ferroptosis inducers.Nat. Chem. Biol. 2020; 16: 1351-1360Google Scholar) (Fig. 2). Contrasting results have been presented on the role of PUFA-ether phospholipids (PUFA-ePLs) in ferroptosis regulation, thus suggesting the involvement of various cellular and genetic contexts in ferroptosis. Perez et al. (33Perez M.A. Magtanong L. Dixon S.J. Watts J.L. Dietary lipids induce ferroptosis in caenorhabditiselegans and human cancer cells.Dev. Cell. 2020; 54: 447-454.e4Google Scholar) showed that ether lipids resist the induction of lipid peroxidation and ferroptosis induced by exogenous dihomo-γ-linolenic acid (DGLA) in Caenorhabditis elegans and human cancer cells (33Perez M.A. Magtanong L. Dixon S.J. Watts J.L. Dietary lipids induce ferroptosis in caenorhabditiselegans and human cancer cells.Dev. Cell. 2020; 54: 447-454.e4Google Scholar). This observation was consistent with the role of endogenous ether lipids as antioxidants that protect PUFAs from lipid peroxidation (38Shi X. Tarazona P. Brock T.J. Browse J. Feussner I. Watts J.L. A Caenorhabditis elegans model for ether lipid biosynthesis and function.J. Lipid Res. 2016; 57: 265-275Google Scholar, 39Engelmann B. Plasmalogens: Targets for oxidants and major lipophilic antioxidants.Biochem. Soc. Trans. 2004; 32: 147-150Google Scholar). In contrast, Zou et al. (40Zou Y. Henry W.S. Ricq E.L. Graham E.T. Phadnis V.V. Maretich P. Paradkar S. Boehnke N. Deik A.A. Reinhardt F. Eaton J.K. Ferguson B. Wang W. Fairman J. Keys H.R. et al.Plasticity of ether lipids promotes ferroptosis susceptibility and evasion.Nature. 2020; 585: 603-608Google Scholar) found that PUFA-ePLs, synthesized by peroxisomes, have a proferroptotic function. In this study (40Zou Y. Henry W.S. Ricq E.L. Graham E.T. Phadnis V.V. Maretich P. Paradkar S. Boehnke N. Deik A.A. Reinhardt F. Eaton J.K. Ferguson B. Wang W. Fairman J. Keys H.R. et al.Plasticity of ether lipids promotes ferroptosis susceptibil