Cytochrome P450 reductase (POR) as a ferroptosis fuel

Oxygen, iron, and polyunsaturated fatty acids (PUFAs; fatty acids containing more than one double bond) are all beneficial to our cellular lives. Incorporation of these components into cellular processes, however, comes at a cost: the bisallylic structure of PUFAs and the enrichment of cellular environments with iron and oxygen render PUFA-containing phospholipids (PUFA-PLs) particularly susceptible to peroxidation (Yang and Stockwell, 2016). Accumulation of lethal amounts of lipid peroxides in cell membranes leads to a form of cell death known as ferroptosis (Dixon et al., 2012; Stockwell et al., 2017; Stockwell and Jiang, 2020). Consequently, cells are equipped with strong antioxidant defense systems that constantly dissipate toxic lipid peroxides generated in cellular membranes, thereby maintaining cell viability and homeostasis (Zheng and Conrad, 2020). The most powerful anti-ferroptosis defense system is believed to be mediated by glutathione peroxidase 4 (GPX4), a glutathione peroxidase that uses glutathione as its cofactor to reduce lipid hydroperoxides to non-toxic lipid alcohols (Fig. 1) (Zheng and Conrad, 2020). A variety of ferroptosis inducers (FINs) act to inactivate GPX4 or deplete glutathione, causing an imbalance between the production and detoxification of lipid peroxides that subsequently induces ferroptotic cell death (Yang et al., 2014). Genetic ablation of GPX4 can have the same effect (Friedmann Angeli et al., 2014). Exactly how lipid peroxidation occurs in PUFA-PLs is less well understood, but lipid peroxidation is known to involve both non-enzymatic reactions (e.g., autoxidation, which requires iron and oxygen) and protein enzymes (Yin et al., 2011; Conrad and Pratt, 2019). Enzymatic lipid peroxidation was initially thought to be carried out mostly by lipoxygenases (ALOXs) (Yang et al., 2016; Wenzel et al., 2017). However, several lines of evidence from recent studies argue against ALOXs being essential drivers of lipid peroxidation. First, the ALOX inhibitors that were used to establish the role of ALOX in regulating ferroptosis turned out to also harbor a previously unrecognized radical-trapping antioxidant activity (Shah et al., 2018). Therefore, whether these compounds block ferroptosis by inhibiting ALOXs or by trapping lipid peroxyl radicals remains unclear. Further, ALOXs are expressed in extremely low levels in most cancer cell lines (Zou et al., 2020b), raising further questions about its role in regulating ferroptosis in broad contexts. Finally, depleting ALOXs in cancer cells that already have low ALOX expression did not affect the cells’ sensitivity to ferroptosis (Zou et al., 2020b). These findings suggest that one or more additional enzymes exist to mediate lipid peroxidation, the identity of which was revealed in two recent studies (Yan et al., 2020; Zou et al., 2020b). In both studies, CRISPR screening identified two genes, acyl-coenzyme A (CoA)-synthetase long-chain family member 4 (ACSL4) and cytochrome P450 reductase (POR), as top suppressor hits (whose inactivation suppressed FIN-induced ferroptosis, resulting in relative enrichment of their gRNAs in FIN-treated cells relative to that in control cells). ACSL4 is an acyl-CoA synthetase that converts free longchain fatty acids into fatty acyl-CoA esters, with a preference for PUFAs. ACSL4 deficiency is known to compromise PUFA-PL biosynthesis, leading to remarkable ferroptosis resistance in cells (Dixon et al., 2015; Doll et al., 2017; Kagan et al., 2017). Therefore, the identification of ACSL4 as a top suppressor hit validated these CRISPR screens. However, the potential role of POR in ferroptosis had been previously unknown. POR deletion was subsequently confirmed to promote strong resistance to ferroptosis induced by different FINs and in a wide range of cancer cell lines. POR deficiency did not affect levels of GPX4 or its cofactor glutathione, binding between GPX4 and its inhibitors, or phospholipid profiles, but POR deficiency significantly reduced the levels of peroxidized PL species resulting from treatment with FINs, indicating that POR promotes lipid peroxidation during ferroptotic stress (Yan et al., 2020; Zou et al., 2020b).