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. 2020 Mar;16(3):302-309.
doi: 10.1038/s41589-020-0472-6. Epub 2020 Feb 17.

Cytochrome P450 oxidoreductase contributes to phospholipid peroxidation in ferroptosis

Yilong Zou #  1   2 , Haoxin Li #  3   4 , Emily T Graham  3 , Amy A Deik  5 , John K Eaton  3 , Wenyu Wang  3 , Gerardo Sandoval-Gomez  3 , Clary B Clish  5 , John G Doench  6 , Stuart L Schreiber  7   8
Affiliations

Cytochrome P450 oxidoreductase contributes to phospholipid peroxidation in ferroptosis

Yilong Zou et al. Nat Chem Biol. 2020 Mar.

Erratum in

Abstract

Ferroptosis is widely involved in degenerative diseases in various tissues including kidney, liver and brain, and is a targetable vulnerability in multiple primary and therapy-resistant cancers. Accumulation of phospholipid hydroperoxides in cellular membranes is the hallmark and rate-limiting step of ferroptosis; however, the enzymes contributing to lipid peroxidation remain poorly characterized. Using genome-wide, CRISPR-Cas9-mediated suppressor screens, we identify cytochrome P450 oxidoreductase (POR) as necessary for ferroptotic cell death in cancer cells exhibiting inherent and induced susceptibility to ferroptosis. By genetic depletion of POR in cancer cells, we reveal that POR contributes to ferroptosis across a wide range of lineages and cell states, and in response to distinct mechanisms of ferroptosis induction. Using systematic lipidomic profiling, we further map POR's activity to the lipid peroxidation step in ferroptosis. Hence, our work suggests that POR is a key mediator of ferroptosis and potential druggable target for developing antiferroptosis therapeutics.

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Figures

Figure 1.
Figure 1.. Genome-wide CRISPR screens identify POR as a mediator of ferroptosis.
a. Viability curves for UACC-257-Cas9 melanoma cells pre-treated with bovine serum albumin (BSA)-containing vehicle or indicated fatty acids for 3 days, then treated with indicated concentrations of GPX4 inhibitors ML210/RSL3. Abbreviations: OA, oleic acid (C18:1); LA, linoleic acid (C18:2); ALA, ɑ-linolenic acid (C18:3); AA, arachidonic acid (C20:4); EPA, eicosapentaenoic acid (C20:5); DPA, docosapentaenoic acid (C22:5); DHA, docosahexaenoic acid (C22:6). n=4, error bars: mean±s.d. Representative plot of experiments performed in triplicate. b. Partial volcano plots showing the top hits in AA, DPA or DHA treated screening conditions in UACC-257-Cas9 cells, highlighting POR and ACSL4. Target genes of sgRNAs enriched in the ML210-treated condition are presented. c. Partial volcano plots showing the top hits in the 786-O-Cas9 screen, highlighting POR and ACSL4. The top left corner indicates how many days of ML210 treatment was used in each condition. Target genes of sgRNAs enriched in the ML210-treated condition are presented. d. Brief scheme summarizing the structure and functions of the cytochrome P450 enzyme (POR / CYP) system. NADPH, nicotinamide adenine dinucleotide phosphate; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide.
Figure 2.
Figure 2.. POR contributes to ferroptotic cell death in multiple cancer lineages.
a. Viability curves for sgNC or sgPOR-expressing UACC-257-Cas9 melanoma cells pre-treated with vehicle (veh) or arachidonic acid (AA, C20:4) for 3 days, then treated with indicated concentrations of ML210. b. Viability curves of 786-O-Cas9 cells expressing sgNC or POR-targeting sgRNAs treated with indicated concentrations of ML210. c. Immunoblot analysis of POR protein levels in wildtype 786-O-Cas9 cells, and POR−/− single cell progenies (SCP) 1-4. β-Actin was used as a loading control. Representative plot of experiment performed twice. See Supplementary Figure 10 for the original blots. d. Viability curves of 786-O-Cas9 cells expressing control sgRNA (WT), or POR−/− SCP1 and SCP4 treated with indicated concentrations of ML210. e. Viability curves of POR+/+ and POR−/− 786-O cells expressing GFP or POR-GFP cDNAs treated with indicated concentrations of ML210. f. Viability curves of ES-2-Cas9 ovarian carcinoma cells expressing sgNC or POR-targeting sgRNA treated with indicated concentrations of ML210. g. Viability curves of HuH-7 hepatocellular carcinoma cells expressing shNC or POR-targeting shRNA treated with indicated concentrations of ML210. h. Viability curves of SW-1463-Cas9 colorectal adenocarcinoma cells expressing sgNC or POR-targeting sgRNA treated with indicated concentrations of ML210. Viability experiments were independently performed three times and representative plots are presented. In each plot, n=4 for each data point, data and error bars indicate mean±s.d.
Figure 3.
Figure 3.. POR is not required for GPX4-ML210/RSL3 binding.
a.Chemical structure of RSL3/ML210 alkyne analogs and ML210-yne (compound 3) and RSL3-yne (compound 4). b. Viability curves of POR+/+ and POR−/− 786-O cells expressing GFP or POR-GFP cDNAs treated with indicated concentrations of ML210-yne or RSL3-yne. n=4, error bar: mean±s.d. c. Immunoblot analysis showing the interaction between GPX4 and ML210-yne/RSL3-yne in 786-O-Cas9 or 769-P-Cas9 cells expressing sgNC (WT) or POR-sg2. Representative plot of experiment performed twice. d. Immunoblot analysis of POR protein levels in 786-O-Cas9 cells that are GPX4+/+, GPX4−/−-sgNC or GPX4−/−-POR-sg2. Representative plot of experiment performed three times. e. Viability time course of 786-O-Cas9 cells that are GPX4+/+ (wildtype, WT), GPX4−/−-sgNC (single knockout, sKO) or GPX4−/−-POR-sg2 (double knockout, dKO) after withdrawing ferrostatin-1 (Fer-1). n=4, error bar: mean±s.d. β-Actin was used as a loading control in immunoblotting analyses. See Supplementary Figure 10 for the original blots.
Figure 4.
Figure 4.. POR contributes to ferroptosis induced by distinct mechanisms.
a. Viability curves of 786-O-Cas9 cells expressing control sgRNA (WT) or POR-sg2, and POR−/− single cell clones SCP1 and SCP4 cells treated with the indicated concentrations of ferroptosis inducers including erastin, FIN56, FINO2 or BSO. b. Viability curves of 769-P-Cas9 cells expressing control sgRNA (WT, POR+/+) or POR-sg2 treated with the indicated concentrations of ferroptosis inducers. Viability experiments were independently performed three times and representative plots are presented. In each plot, n=4 for each data point, data and error bars indicate mean±s.d.
Figure 5.
Figure 5.. POR mediates ferroptosis by facilitating lipid peroxidation.
a. Volcano plot showing the lipidomics analysis of 786-O-Cas9 cells expressing control sgRNA (sgNC) or POR-sg2. n=3, two-tailed T-test, p values were adjusted for multi-testing using the Benjamini-Hochberg method. b. Flow cytometry analyses of lipid peroxidation levels and cytosolic reactive oxygen species (ROS) levels reported respectively by BODIPY-C11 and H2-DCFDA in 786-O-Cas9 cells expressing sgNC or POR-sg2 and treated with ML210. Representative plots of experiments repeated three times. c. Fluorescent microscope images of BODIPY-C11 (oxidized: green, reduced, orange) in control or POR-depleted 786-O-Cas9 cells. Scale bar, 25μm. Representative images of experiments repeated three times. d. Bar plot showing the malondialdehyde (MDA) levels in control or POR-depleted 786-O-Cas9 cells treated with DMSO, ML210 or ML210+Lip-1. n=3, error bar: mean±s.d. Two-tailed T-tests. Representative plot of experiments repeated twice. e. Bar plots showing the hydroperoxyl (−OOH) or hydroxyl (−OH) derivatives of indicated polyunsaturated phosphatidylethanolamine (PUFA-PE) species in WT (sgNC, POR+/+) or POR-depleted 786-O-Cas9 and 769-P-Cas9 cells treated with DMSO, ML210 or ML210+Lip-1 and analyzed by redox-lipidomics. n=3, error bar: mean±s.d. Two-tailed T-test; ns, not significant. f. Schematic diagram summarizing the role of POR in lipid peroxidation and ferroptosis. Abbreviations: L-H, symbol for polyunsaturated- (PUFA-) phospholipid; CYP, cytochrome P450 proteins; POR, cytochrome P450 oxidoreductase; LOOH, lipid hydroperoxides; GPX4, glutathione peroxidase 4; Vit. E, vitamin E; Lip-1, liproxstatin-1; Fer-1, ferrostatin-1.
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References

    1. Viswanathan VS et al. Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway. Nature 547, 453–457 (2017). - PMC - PubMed
    1. Wenzel SE et al. PEBP1 Wardens Ferroptosis by Enabling Lipoxygenase Generation of Lipid Death Signals. Cell 171, 628–641.e26 (2017). - PMC - PubMed
    1. Hangauer MJ et al. Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition. Nature 551, 247–250 (2017). - PMC - PubMed
    1. Alim I et al. Selenium Drives a Transcriptional Adaptive Program to Block Ferroptosis and Treat Stroke. Cell 177, 1262–1279.e25 (2019). - PubMed
    1. Li Q et al. Inhibition of neuronal ferroptosis protects hemorrhagic brain. JCI Insight 2, e90777 (2017). - PMC - PubMed

Methods-only References

    1. Hannah VC, Ou J, Luong A, Goldstein JL & Brown MS Unsaturated fatty acids down-regulate srebp isoforms 1a and 1c by two mechanisms in HEK-293 cells. J. Biol. Chem 276, 4365–4372 (2001). - PubMed
    1. Doench JG et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol 34, 184–191 (2016). - PMC - PubMed
    1. Hegde M, Strand C, Hanna RE & Doench JG Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens. PLoS One 13, e0197547 (2018). - PMC - PubMed
    1. Najm FJ et al. Orthologous CRISPR–Cas9 enzymes for combinatorial genetic screens. Nat. Biotechnol 36, 179–189 (2018). - PMC - PubMed
    1. Sanson KR et al. Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat. Commun 9, 5416 (2018). - PMC - PubMed

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