Summary Ferroptosis is a form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides to lethal levels. Emerging evidence suggests that ferroptosis represents an ancient vulnerability caused by the incorporation of polyunsaturated fatty acids into cellular membranes, and that cells have developed complex systems that exploit and defend against this vulnerability in different contexts. The sensitivity to ferroptosis is tightly linked to numerous biological processes, including amino acid, iron and polyunsaturated fatty acid metabolism, and the biosynthesis of glutathione, phospholipids, NADPH and coenzyme Q10. Ferroptosis has been implicated in the pathological cell death associated with degenerative diseases (i.e., Alzheimer's, Huntington's, and Parkinson's diseases), carcinogenesis, stroke, intracerebral hemorrhage, traumatic brain injury, ischemia-reperfusion injury, and kidney degeneration in mammals and is also implicated in heat stress in plants. Ferroptosis may also have a tumor suppressor function that could be harnessed for cancer therapy. This Primer reviews the mechanisms underlying ferroptosis, highlights connections to other areas of biology and medicine, and recommends tools and guidelines for studying this emerging form of regulated cell death.

Ferroptosis is a form of regulated necrotic cell death controlled by glutathione peroxidase 4 (GPX4). At present, mechanisms that could predict sensitivity and/or resistance and that may be exploited to modulate this form of cell death are needed. We applied two independent approaches, a genome-wide CRISPR-based genetic screen and microarray analysis of ferroptosis-resistant cell lines to uncover acyl-CoA synthetase long-chain family member 4 (Acsl4) as an essential component for ferroptosis execution. Specifically, Gpx4/Acsl4 double knockout cells presented an unprecedented resistance to ferroptosis. Mechanistically, Acsl4 enriches cellular membranes with long polyunsaturated ω6 fatty acids. Moreover, Acsl4 is preferentially expressed in a panel of basal-like breast cancer cell lines and predicts their sensitivity to ferroptosis. We further demonstrate that pharmacological targeting of Acsl4 with the antidiabetic compound class, thiazolidinediones, ameliorates tissue demise in a murine model of ferroptosis, suggesting that Acsl4 inhibition is a viable therapeutic approach to prevent ferroptosis-related diseases.

Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids 1,2. To date, ferroptosis has been believed to be restrained only by the phospholipid hydroperoxide (PLOOH)-reducing enzyme glutathione peroxidase 4 (GPX4) 3,4 and radicaltrapping antioxidants (RTAs) 5,6. The factors which underlie a given cell type's sensitivity to ferroptosis 7 is, however, critical to understand the pathophysiological role of ferroptosis and how it may be exploited for cancer treatment. Although metabolic constraints 8 and phospholipid composition 9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been yet been identified that account for ferroptosis resistance. We undertook an expression cloning approach to identify genes able to complement GPX4 loss. These efforts uncovered the flavoprotein "apoptosis inducing factor mitochondria-associated 2 (AIFM2)" as a previously unrecognized anti-ferroptotic gene. AIFM2, hereafter renamed "ferroptosis-suppressor-protein 1" (FSP1), initially described as a pro-apoptotic gene 11 , confers an unprecedented protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that ferroptosis suppression by FSP1 is mediated via ubiquinone (CoQ10): its reduced form ubiquinol traps lipid peroxyl radicals that mediate lipid peroxidation, while FSP1 catalyses its regeneration by using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. Conclusively, FSP1/CoQ10/NAD(P)H exists as a standalone parallel system, which cooperates with GPX4 and glutathione (GSH) to suppress phospholipid peroxidation (pLPO) and ferroptosis. program NEUROPROTEKT (03VP04260), as well as the m4 Award provided by the Bavarian Ministry of Economic Affairs, Regional Development and Energy (StMWi) to M.C., the Cancer Research UK

Ferroptosis is a non-apoptotic form of cell death induced by small molecules in specific tumour types, and in engineered cells overexpressing oncogenic RAS. Yet, its relevance in non-transformed cells and tissues is unexplored and remains enigmatic. Here, we provide direct genetic evidence that the knockout of glutathione peroxidase 4 (Gpx4) causes cell death in a pathologically relevant form of ferroptosis. Using inducible Gpx4−/− mice, we elucidate an essential role for the glutathione/Gpx4 axis in preventing lipid-oxidation-induced acute renal failure and associated death. We furthermore systematically evaluated a library of small molecules for possible ferroptosis inhibitors, leading to the discovery of a potent spiroquinoxalinamine derivative called Liproxstatin-1, which is able to suppress ferroptosis in cells, in Gpx4−/− mice, and in a pre-clinical model of ischaemia/reperfusion-induced hepatic damage. In sum, we demonstrate that ferroptosis is a pervasive and dynamic form of cell death, which, when impeded, promises substantial cytoprotection.

Enigmatic lipid peroxidation products have been claimed as the proximate executioners of ferroptosis - a specialized death program triggered by insufficiency of glutathione peroxidase 4 (GPX4). Here, by using quantitative redox lipidomics, reverse genetics, bioinformatics and systems biology we discovered that execution of ferroptosis involves a highly organized oxygenation center, whereby only one class of phospholipids, phosphatidylethanolamines (PE), undergoes oxidation in the ER-associated compartments with the specificity towards two fatty acyls – arachidonoyl (AA) and adrenoyl (AdA). Suppression of AA or AdA esterification into PE by genetic or pharmacological inhibition of acyl-CoA synthase 4 acts as a specific anti-ferroptotic rescue pathway. Lipoxygenases (LOX) generate doubly- and triply-oxygenated (15-hydroperoxy)-di-acylated PE species which act as death signals while tocopherols and tocotrienols suppress LOX and protect against ferroptosis suggesting an unforeseen homeostatic physiological role of vitamin E. This oxidative PE death pathway may also represent a target for drug discovery.