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Cytochrome P450 oxidoreductase contributes to phospholipid peroxidation in ferroptosis

Nature Chemical Biology volume 16, pages 302–309 (2020)Cite this article

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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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Fig. 1: Genome-wide CRISPR screens identify POR as a mediator of ferroptosis.
Fig. 2: POR contributes to ferroptotic cell death in multiple cancer lineages.
Fig. 3: POR is not required for GPX4-ML210/RSL3 binding.
Fig. 4: POR contributes to ferroptosis induced by distinct mechanisms.
Fig. 5: POR mediates ferroptosis by facilitating lipid peroxidation.

Data availability

Raw sequencing data for CRISPR–Cas9 screens in UACC-257 melanoma cells are deposited at the Gene Expression Omnibus (accession number GSE130982). Processed CRISPR screening data are supplied as Supplementary Dataset 1. The global lipidomics analysis results are presented in Supplementary Dataset 2, and the redox-lipidomics analysis results are presented in Supplementary Dataset 3. All original data that support the findings of this study are available upon request.

Code availability

All computational code that support the findings of this study are available upon request.

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Acknowledgements

We thank R. Aoyagi, M. Arita, P. Kennedy, M. Gijón and T. Zhao for assisting with the redox-lipidomics analyses. This work was supported in part by the NCI’s Cancer Target Discovery and Development (CTD2) Network (grant No. U01CA217848, awarded to S.L.S.), and in part by the National Institute of General Medical Sciences (grant No. R35GM127045, awarded to S.L.S.). G.S.-G. is supported by a fellowship from the Howard Hughes Medical Institute Exceptional Research Opportunities Program.

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Author notes

  1. These authors contributed equally: Yilong Zou, Haoxin Li.

Authors and Affiliations

  1. Chemical Biology and Therapeutics Science Program, Broad Institute, Cambridge, MA, USA

    Yilong Zou, Haoxin Li, Emily T. Graham, John K. Eaton, Wenyu Wang, Gerardo Sandoval-Gomez & Stuart L. Schreiber

  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Yilong Zou, Haoxin Li & Stuart L. Schreiber

  3. Metabolomics Platform, Broad Institute, Cambridge, MA, USA

    Amy A. Deik & Clary B. Clish

  4. Genetic Perturbation Platform, Broad Institute, Cambridge, MA, USA

    John G. Doench

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Contributions

Y.Z. and S.L.S. conceived the project and wrote the manuscript. Y.Z. and H.L. designed and performed the experiments and data analyses. H.L. and J.G.D. performed the CRISPR screens in UACC-257 cells. A.A.D. and C.B.C. performed the lipidomics analysis. J.K.E. performed chemical synthesis, as well as the GPX4 and ML210/RSL3 interaction analysis. W.W. assisted the chemical treatment experiments and data interpretations. E.T.G. and G.S-G. assisted the cellular experiments. All authors interpreted data, discussed results and contributed to writing the manuscript.

Corresponding authors

Correspondence to Yilong Zou or Stuart L. Schreiber.

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Competing interests

S.L.S. serves on the Board of Directors of the Genomics Institute of the Novartis Research Foundation (GNF); is a shareholder and serves on the Board of Directors of Jnana Therapeutics; is a shareholder of Forma Therapeutics; is a shareholder and advises Decibel Therapeutics and Eikonizo Therapeutics; serves on the Scientific Advisory Boards of Eisai Co., Ltd., Ono Pharma Foundation, Exo Therapeutics, and F-Prime Capital Partners; and is a Novartis Faculty Scholar. The remaining authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–10.

Reporting Summary

Supplementary Dataset 1

Top hits from genome-wide CRISPR screening in UACC-257-Cas9 cells treated with ML210 and linoleic acid (LA, C18:2), α-linolenic acid (ALA, C18:3), arachidonic acid (AA, C20:4), eicosapentaenoic acid (EPA, C20:5), docosapentaenoic acid (DPA, C22:5n-3) or docosahexaenoic acid (DHA, C22:6).

Supplementary Dataset 2

Global lipidomics analysis for WT and POR-depleted 786-O-Cas9 and 769-P-Cas9 cells.

Supplementary Dataset 3

Redox-lipidomics analysis for WT and POR-depleted 786-O-Cas9 and 769-P-Cas9 cells.

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Zou, Y., Li, H., Graham, E.T. et al. Cytochrome P450 oxidoreductase contributes to phospholipid peroxidation in ferroptosis. Nat Chem Biol 16, 302–309 (2020). https://doi.org/10.1038/s41589-020-0472-6

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