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

Selenoproteins are rare proteins among all kingdoms of life containing the 21 amino acid, selenocysteine. Selenocysteine resembles cysteine, differing only by the substitution of selenium for sulfur. Yet the actual advantage of selenolate- versus thiolate-based catalysis has remained enigmatic, as most of the known selenoproteins also exist as cysteine-containing homologs. Here, we demonstrate that selenolate-based catalysis of the essential mammalian selenoprotein GPX4 is unexpectedly dispensable for normal embryogenesis. Yet the survival of a specific type of interneurons emerges to exclusively depend on selenocysteine-containing GPX4, thereby preventing fatal epileptic seizures. Mechanistically, selenocysteine utilization by GPX4 confers exquisite resistance to irreversible overoxidation as cells expressing a cysteine variant are highly sensitive toward peroxide-induced ferroptosis. Remarkably, concomitant deletion of all selenoproteins in Gpx4 cells revealed that selenoproteins are dispensable for cell viability provided partial GPX4 activity is retained. Conclusively, 200 years after its discovery, a specific and indispensable role for selenium is provided.

High-risk neuroblastoma is a devastating malignancy with very limited therapeutic options. Here, we identify withaferin A (WA) as a natural ferroptosis-inducing agent in neuroblastoma, which acts through a novel double-edged mechanism. WA dose-dependently either activates the nuclear factor-like 2 pathway through targeting of Kelch-like ECH-associated protein 1 (noncanonical ferroptosis induction) or inactivates glutathione peroxidase 4 (canonical ferroptosis induction). Noncanonical ferroptosis induction is characterized by an increase in intracellular labile Fe(II) upon excessive activation of heme oxygenase-1, which is sufficient to induce ferroptosis. This double-edged mechanism might explain the superior efficacy of WA as compared with etoposide or cisplatin in killing a heterogeneous panel of high-risk neuroblastoma cells, and in suppressing the growth and relapse rate of neuroblastoma xenografts. Nano-targeting of WA allows systemic application and suppressed tumor growth due to an enhanced accumulation at the tumor site. Collectively, our data propose a novel therapeutic strategy to efficiently kill cancer cells by ferroptosis.

The design of cancer-targeting particles with precisely-tuned physiocochemical properties may enhance delivery of therapeutics and access to pharmacological targets. However, molecular level understanding of the interactions driving the fate of nanomedicine in biological systems remains elusive. Here, we show that ultrasmall (< 10 nm in diameter) poly(ethylene glycol) (PEG)-coated silica nanoparticles, functionalized with melanoma-targeting peptides, can induce a form of programmed cell death known as ferroptosis in starved cancer cells and cancer-bearing mice. Tumor xenografts in mice intravenously injected with nanoparticles using a high-dose multiple injection scheme exhibit reduced growth or regression, in a manner that is reversed by the pharmacological inhibitor of ferroptosis, liproxstatin-1. These data demonstrate that ferroptosis can be targeted by ultrasmall silica nanoparticles and may have therapeutic potential.