. 2022 Sep 15;29(9):1409-1418.e6.

doi: 10.1016/j.chembiol.2022.06.004. Epub 2022 Jul 8.

Context-dependent regulation of ferroptosis sensitivity

Affiliations

¹ Department of Biology, Stanford University, Stanford, CA 94305, USA.
² Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA; UCLA Lipidomics Laboratory, University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ Department of Computer Science and Engineering, University of Minnesota-Twin Cities, 200 Union Street, Minneapolis, MN 55455, USA.
⁴ Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
⁵ Department of Computer Science and Engineering, University of Minnesota-Twin Cities, 200 Union Street, Minneapolis, MN 55455, USA; Program in Biomedical Informatics and Computational Biology, University of Minnesota-Twin Cities, 200 Union Street, Minneapolis, MN 55455, USA.
⁶ Departments of Molecular and Cell Biology and Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA 94720, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA.
⁷ Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA; UCLA Lipidomics Laboratory, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁸ Department of Biology, Stanford University, Stanford, CA 94305, USA. Electronic address: sjdixon@stanford.edu.

PMID: 35809566
PMCID: PMC9481678
DOI: 10.1016/j.chembiol.2022.06.004

Context-dependent regulation of ferroptosis sensitivity

Leslie Magtanong et al. Cell Chem Biol. 2022.

. 2022 Sep 15;29(9):1409-1418.e6.

doi: 10.1016/j.chembiol.2022.06.004. Epub 2022 Jul 8.

Authors

Affiliations

¹ Department of Biology, Stanford University, Stanford, CA 94305, USA.
² Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA; UCLA Lipidomics Laboratory, University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ Department of Computer Science and Engineering, University of Minnesota-Twin Cities, 200 Union Street, Minneapolis, MN 55455, USA.
⁴ Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
⁵ Department of Computer Science and Engineering, University of Minnesota-Twin Cities, 200 Union Street, Minneapolis, MN 55455, USA; Program in Biomedical Informatics and Computational Biology, University of Minnesota-Twin Cities, 200 Union Street, Minneapolis, MN 55455, USA.
⁶ Departments of Molecular and Cell Biology and Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA 94720, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA.
⁷ Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA; UCLA Lipidomics Laboratory, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁸ Department of Biology, Stanford University, Stanford, CA 94305, USA. Electronic address: sjdixon@stanford.edu.

PMID: 35809566
PMCID: PMC9481678
DOI: 10.1016/j.chembiol.2022.06.004

Erratum in

Context-dependent regulation of ferroptosis sensitivity.
Magtanong L, Mueller GD, Williams KJ, Billmann M, Chan K, Armenta DA, Pope LE, Moffat J, Boone C, Myers CL, Olzmann JA, Bensinger SJ, Dixon SJ. Magtanong L, et al. Cell Chem Biol. 2022 Oct 20;29(10):1568. doi: 10.1016/j.chembiol.2022.08.008. Cell Chem Biol. 2022. PMID: 36270234 No abstract available.

Abstract

Ferroptosis is an important mediator of pathophysiological cell death and an emerging target for cancer therapy. Whether ferroptosis sensitivity is governed by a single regulatory mechanism is unclear. Here, based on the integration of 24 published chemical genetic screens combined with targeted follow-up experimentation, we find that the genetic regulation of ferroptosis sensitivity is highly variable and context-dependent. For example, the lipid metabolic gene acyl-coenzyme A (CoA) synthetase long chain family member 4 (ACSL4) appears far more essential for ferroptosis triggered by direct inhibition of the lipid hydroperoxidase glutathione peroxidase 4 (GPX4) than by cystine deprivation. Despite this, distinct pro-ferroptotic stimuli converge upon a common lethal effector mechanism: accumulation of lipid peroxides at the plasma membrane. These results indicate that distinct genetic mechanisms regulate ferroptosis sensitivity, with implications for the initiation and analysis of this process in vivo.

Keywords: ACSL4; PUFA; ROS; cancer; ether lipid; ferroptosis; iron.

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Conflict of interest statement

Declaration of interests J.A.O. is a member of the scientific advisory board of Vicinitas Therapeutics. S.J.D. is a co-founder of Prothegen and a member of the scientific advisory boards for Ferro Therapeutics and Hillstream BioPharma. J.A.O. and S.J.D. hold patents related to ferroptosis.

Figures

**Figure 1.. A literature-curated regulatory genetic network of ferroptosis suppressors.**
(A) A consensus ferroptosis regulatory network integrating results from 24 loss of function genetic suppressor screens. Each genetic screen is indicated by a large, uniquely colored circular node that corresponds to the cell line and ferroptosis inducing condition employed. Each gene is represented by a small black node. Edges connect individual screens to specific genes. (**B-D**) Network gene characterization using the database for annotation, visualization and integrated discovery (DAVID) bioinformatic resource. **(E)** Aggregate reported gene ranks for genes identified in ≤2 or ≥3 conditions in the consensus network in A. Values closer to zero indicate the gene was a stronger hit. **(F)** Number of times a given gene was identified in the network in A, overlayed with number of abstracts reported in PubMed that mention the gene name and ferroptosis together. See also Figure S1 and Tables S1,2.

**Figure 2.. ACSL4 is required for ferroptosis in response to GPX4 inhibitors.**
**(A)** Results of a HAP1 CRISPR screen. Individual genes (n = 17,800) are plotted as circles. Select sensitizer genes are indicated. qGI: quantitative genetic interaction. Horizontal dotted line: FDR q < 0.05. **(B)** Select gene qGI scores from A. **(C)** HT-1080^N cells imaged after 48 h treatment. Scale bar = 50 μm. Ct: Control. Representative of three experiments. (**D,E**) Lethal fraction dose response curves over time for (D) RSL3- or (E) erastin2-treated cells. For lethal fraction, 0 = all cells alive, 1 = all cells dead. (F,G) Lethal fraction of cells pre-treated for 24 h ± rosiglitazone (ROSI) prior to lethal compound treatment. Data in (D-F) are mean ± SD of three independent experiments. Each data point in (G) is from one independent experiment. See also Figures S2,3 and Table S3.

**Figure 3.. Ether lipids are dispensable for ferroptosis induction.**
**(A)** Protein expression in Control (Ct) and gene-disrupted (KO) cell lines. **(B)** Relative lipid abundance in HT-1080^N Control versus *AGPS*^KO1/2 cells for significantly altered lipids (P < 0.05, Students t-test). PE: phosphatidylethanolamine, O: ether-linked, P: vinyl ether-linked. (C). Lethal fraction dose response curves over time. FIN: ferroptosis inducer. (D) Lethal fraction dose response curves over time of cells pre-treated for 24 h ± rosiglitazone (ROSI, 25 μM) prior to lethal compound treatment. Data in (B) are mean of four independent experiments. Data in (C,D) are mean ± SD from three independent experiments. See also Figure S4.

**Figure 4.. Context-dependent role for ACSL4 in lipid peroxidation spreading during ferroptosis.**
(A) HT-1080 Control and *ACSL4*^KO1 cells ± erastin2 (1 μM) for 10 h (Control) or 13 h (*ACSL4*^KO1). C11: C11 BODIPY 581/591, Non-ox: non-oxidized, Ox: oxidized. Scale bar = 20 μm. (B-D) HT-1080 Control and *ACSL4*^KO1 cells ± RSL3 (0.5 μM) for 2 h (B) or the indicated time points (C,D) prior to labeling with C11 and Hoechst. For (A-D), two or more independent experiments were performed, and representative images from one experiment are shown. See also Figure S4.

See this image and copyright information in PMC

Comment in

Ferroptosis execution: Is it all about ACSL4?
Lee H, Gan B. Lee H, et al. Cell Chem Biol. 2022 Sep 15;29(9):1363-1365. doi: 10.1016/j.chembiol.2022.08.002. Cell Chem Biol. 2022. PMID: 36113403

References

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Context-dependent regulation of ferroptosis sensitivity

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Context-dependent regulation of ferroptosis sensitivity

Authors

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Erratum in

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Conflict of interest statement

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