Abstract
Ferroptosis is a form of regulated cell death that is caused by the iron-dependent peroxidation of lipids1,2. The glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols3,4. Ferroptosis has previously been implicated in the cell death that underlies several degenerative conditions2, and induction of ferroptosis by the inhibition of GPX4 has emerged as a therapeutic strategy to trigger cancer cell death5. However, sensitivity to GPX4 inhibitors varies greatly across cancer cell lines6, which suggests that additional factors govern resistance to ferroptosis. Here, using a synthetic lethal CRISPR–Cas9 screen, we identify ferroptosis suppressor protein 1 (FSP1) (previously known as apoptosis-inducing factor mitochondrial 2 (AIFM2)) as a potent ferroptosis-resistance factor. Our data indicate that myristoylation recruits FSP1 to the plasma membrane where it functions as an oxidoreductase that reduces coenzyme Q10 (CoQ) (also known as ubiquinone-10), which acts as a lipophilic radical-trapping antioxidant that halts the propagation of lipid peroxides. We further find that FSP1 expression positively correlates with ferroptosis resistance across hundreds of cancer cell lines, and that FSP1 mediates resistance to ferroptosis in lung cancer cells in culture and in mouse tumour xenografts. Thus, our data identify FSP1 as a key component of a non-mitochondrial CoQ antioxidant system that acts in parallel to the canonical glutathione-based GPX4 pathway. These findings define a ferroptosis suppression pathway and indicate that pharmacological inhibition of FSP1 may provide an effective strategy to sensitize cancer cells to ferroptosis-inducing chemotherapeutic agents.
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Data availability
All data that support the conclusions in this manuscript are available from the corresponding author upon reasonable request. Raw data for Fig. 1 are provided in Supplementary Table 1. Raw data for Fig. 3 are provided in Supplementary Table 3. Raw data for Fig. 4 are provided in Supplementary Table 4, and are publicly available from the Cancer Cell Line Encyclopedia (https://portals.broadinstitute.org/ccle) and CTRP databases.
Code availability
The casTLE statistical framework software for analysis of data from the CRISPR screen can be accessed at www.bitbucket.org/dmorgens/castle/. Bowtie software can be accessed at www.bowtie-bio.sourceforge.net/bowtie2/index.shtml. MATLAB image analysis software to analyse lipid droplet distributions can be obtained at www.dropletproteome.org.
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Acknowledgements
This research was supported by grants from the National Institutes of Health (R01GM112948 to J.A.O., 1R01GM122923 to S.J.D., P42 ES004705 to D.K.N. and 1DP2CA195761-01 to R.Z.). J.A.O. is a Chan Zuckerberg Biohub investigator. D.K.N. was supported by a Cancer Research ASPIRE award from the Mark Foundation. P.H.T. was supported by the Internal Research Fund of the Division of Pathology and Laboratory Medicine, Cincinnati Children’s Hospital Medical Center. We thank P.-J. Ko (Stanford) for assistance with confocal imaging, and D. Leto (Stanford) and R. Kopito (Stanford) for helpful discussions.
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Authors and Affiliations
Contributions
K.B. and J.A.O. conceived the project and designed the experiments. J.A.O. and K.B. wrote the manuscript. All authors read and edited the manuscript. K.B. performed the majority of the experiments. Z.L. and M.A.R. performed and analysed the CRISPR screen with guidance from M.C.B. K.B. prepared samples and R.Z. performed the TIRF microscopy, B.F. performed the lipidomics, and P.H.T. measured CoQ levels and redox state. J.H. performed the click chemistry myristoylation experiments. S.J.D. and L.M. performed the glutathione measurements and C11 experiments. J.M.H. generated the overexpression and knockout lung cancer lines and analysed ferroptosis in these lines. D.K.N., J.M.H., B.F., and M.A.R. performed the xenograft experiments. T.J.M. and B.T. synthesized IKE.
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S.J.D. is a member of the scientific advisory board for Ferro Therapeutics.
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Peer review information Nature thanks Kivanc Birsoy, Navdeep S. Chandel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Fig. 1 Synthetic lethal screen coverage and validation.
a, Distribution of counts across all sgRNA elements from the CRISPR–Cas9 screen. b, Western blot of control and FSP1KO cells. c, d, Western blot analysis (c) and dose response of RSL3-induced death (d) of FSP1KO cells that express doxycycline-inducible, untagged FSP1. e, Time-lapse analysis of cell death of FSP1KO cells that express inducible, untagged FSP1. f, Flow cytometric analysis of caspase 3/7 activity in FSP1KO cells that express inducible, untagged FSP1, treated with doxycycline for 48 h. As a positive control, non-induced cells were treated with 50 μM etoposide for 24 h before analysis. g, Western blot analysis of lysates from control cells treated with 10 μM nutlin-3 for 48 h. h, Dose response of ML162 and erastin2-induced cell death. i, j, Dose response of rotenone-induced death of control (i) and FSP1KO (j) cells. k, l, Dose response of hydrogen-peroxide-induced death of control (k) and FSP1KO (l) cells. m, Dose response of RSL3-induced cell death in the presence of inhibitors of apoptosis (ZVAD(OMe)-FMK, 10 μM) and necroptosis (necrostatin-1, 1 μM). n, Western blot analysis of lysates from ACSL4KO and FSP1KO ACSL4KO cells. o, Schematic of domains present in AIF and FSP1. In d, i–m, shading indicates 95% confidence intervals for the fitted curves and each data point is the average of three technical replicates. Panels are representative of two biological replicates, except panels c–e and k, l, which show single experiments.
Extended Data Fig. 2 Subcellular distribution of FSP1.
a, Inducible FSP1–GFP cells were transiently transfected with LYN11–mCherry–FRB for 24 h, induced with doxycycline for 48 h and fixed before imaging. b, FSP1–GFP cells were treated with 200 μM oleate for 24 h to induce lipid droplets and treated with 100 μM AutoDOT to label lipid droplets before imaging. c, Line intensity plots showing colocalization between FSP1–HaloTag and organelle markers. d–f, Confocal and TIRF microscopy of FSP1–HaloTag (d), and inducible FSP1(WT)–GFP (e) and FSP1(G2A)–GFP (f) cells. g, FSP1–HaloTag cells were transiently transfected with BFP–Sec61 for 48 h before imaging to label the endoplasmic reticulum. h, FSP1–HaloTag cells were incubated with 100 nM MitoTracker Green FM to label mitochondria. i, Plasma-membrane subdomains from control cells were enriched by OptiPrep gradient centrifugation. Endo., endogenous FSP1. Western blot is representative of two biological replicates. Images are representative of at least n = 10 imaged cells. Scale bars, 10 μm.
Extended Data Fig. 3 Myristoylation and lipid droplet localization of FSP1.
a, Schematic showing the procedure for metabolic labelling of cells with the myristate-alkyne YnMyr and conjugation of YnMyr-labelled proteins with TAMRA-azide-PEG-biotin using click chemistry. b, Analysis of FSP1 myristoylation in buoyant fractions enriched in lipid droplets, by streptavidin enrichment of YnMyr-labelled proteins, click chemistry and SDS–PAGE. Cells were treated with 200 μM oleate to induce lipid droplets and with 100 μM YnMyr or 100 μM myristate for 24 h. c, FSP1–GFP was induced with doxycycline for 24 h and cells were incubated with 100 μM YnMyr for an additional 24 h to label proteins in the presence or absence of 75 μM emetine. YnMyr-labelled proteins were affinity-purified and analysed by click chemistry and SDS–PAGE. d, Buoyant fractions enriched in lipid droplets, from cells expressing inducible FSP1–GFP, were isolated by sucrose gradient fractionation and analysed by western blot. Endo., endogenous FSP1. e, Inducible FSP1–GFP cells were treated with 200 μM oleate in the presence or absence of 10 μM NMT inhibitor, fixed and stained with anti-PLIN2 antibody before imaging. Images are representative of at least n = 10 imaged cells. Scale bar, 10 μm. f, Western blot analysis of FSP1KO cells induced for 48 h with doxycycline to express the indicated proteins. All panels are representative of two biological replicates.
Extended Data Fig. 4 Targeting of FSP1 to subcellular compartments.
a, Western blot analysis of FSP1KO cells induced for 48 h with doxycycline to express the indicated proteins. b, Live-cell microscopy of cells that express the indicated FSP1(G2A)–GFP constructs, incubated with 100 nM Mitotracker Orange to label mitochondria, 1 μM BODIPY 558/568 C12 to label lipid droplets or 5 μg ml-1 Cell Mask to label the plasma membrane. To label the endoplasmic reticulum, cells were transiently transfected with BFP–Sec61 48 h before imaging. Images are representative of at least n = 10 imaged cells. Line intensity plots show colocalization between FSP1 and organelle markers. Scale bar, 10 μm. c, Plasma-membrane subdomains from FSP1KO cells that express inducible LYN11–FSP1(G2A)–GFP were enriched by OptiPrep gradient centrifugation. The densitometry plot indicates the distribution of overexpressed and endogenous proteins. Panels are representative of two biological replicates except for c, which shows a single experiment.
Extended Data Fig. 5 Lipid droplets are not required for inhibition of ferroptosis by FSP1.
a, Control cells were treated with inhibitors of DGAT1 (20 μM T863) and DGAT2 (10 μM PF-06424439) for 48 h, stained with 1 μM BODIPY 493/503 and imaged by fluorescence microscopy. The image is representative of n = 50 imaged fields. Scale bar, 10 μm. b, The size and number of lipid droplets were quantified from cells (n > 5,000) in a. c, Dose response of RSL3-induced cell death of control cells pretreated for 48 h with 20 μM T863 and 10 μM PF-06424439 before addition of RSL3. Each data point is the average of three technical replicates. All panels are representative of two biological replicates.
Extended Data Fig. 6 Analysis of lipid peroxidation, glutathione and lipid levels in FSP1KO cells.
a, b, Ratio of oxidized-to-total BODIPY 581/591 C11 from images in Fig. 3a, at the plasma membrane (a) or at internal membranes (b). Each data point represents an individual cell quantified in one of two biological replicates. For a, Cas9ctl DMSO, n = 34; Cas9ctl RSL3, n = 45; FSP1KO DMSO, n = 30; FSP1KO RSL3, n = 33; ***P < 0.001 by one-way ANOVA. For b, Cas9ctl DMSO, n = 33; Cas9ctl RSL3, n = 45; FSP1KO DMSO, n = 30; FSP1KO RSL3, n = 33; ***P < 0.001 by one-way ANOVA. Error bars show mean ± s.d. c, Total intracellular glutathione (GSH + GSSG) levels in control and FSP1KO were determined following treatment with 250 nM RSL3 or 1 μM erastin2. The graph shows mean ± s.d. of three biological replicates. n.s., FSP1KO DMSO versus RSL3, P = 0.7278; n.s., FSP1KO RSL3 versus Cas9ctl RSL3, P = 0.1522, **P = 0.0072 by one-way ANOVA. d, e, GSH and GSSG levels in control and FSP1KO cells were measured. Where indicated, cells were treated with 1 μM erastin2. The graph shows mean ± s.d. of three biological replicates. n.s., GSH P = 0.6269; n.s., GSSG P = 0.8284 by two-tailed t-test. f, The plot shows the average of the fold change in lipids measured in two FSP1KO cell lines generated using FSP1 sgRNA no. 1 and FSP1 sgRNA no. 2 (labelled KO1 and KO2, respectively), relative to control cells. Cas9ctl, n = 5; KO1, n = 4; KO2, n = 5 biological replicates (Supplementary Table 3). g, Levels of select lipid species in biological replicates of control and FSP1KO cells measured in f. The average values are indicated. 16:0 20:4 PE, **P = 0.0017; 18:0 20:4 PE, **P = 0.0011; 18:0 LPE, KO2 **P = 0.0036, KO1 **P = 0.0019; 16:0 LPE, KO2 *P = 0.0133 and KO1 *P = 0.0335 by two-tailed t-test.
Extended Data Fig. 7 Analysis of the FSP1 oxidoreductase mutant.
a, FSP1KO cells were treated with 250 nM RSL3 and 10 μM idebenone or 50 μM DFO for 75 min, labelled with BODIPY 581/591 C11 and fixed before imaging. Images are representative of at least n = 10 cells imaged for each treatment condition. Scale bar, 20 μm. b, Sequence alignment showing residues conserved between AIF and FSP1. The arrow points to E313 in AIF (aligns to E156 in FSP1) that functions in binding to flavin adenine dinucleotide. c, Structural alignment between the crystal structure of mouse AIF (RCSB Protein Data Bank code (PDB) 1GV4) and the Phyre2-generated model of FSP1. d, Live-cell microscopy of FSP1KO cells expressing inducible FSP1(E156A)–GFP labelled with 5 μg ml-1 Cell Mask. The image is representative of at least n = 10 imaged cells. Scale bar, 10 μm. e, Plasma-membrane subdomains from FSP1KO cells that express FSP1(E156A)–GFP were enriched by OptiPrep gradient centrifugation. f, SDS–PAGE and Coomassie brilliant blue stain of recombinant His–FSP1(WT) and His–FSP1(E156A) purified with Ni-NTA agarose beads. g, Reduction of resazurin by recombinant FSP1 in the presence of NADH. h, Oxidation of NADH by recombinant FSP1 in the presence of coenzyme Q1. Panels g and h are representative of two biological replicates, and e shows a single experiment.
Extended Data Fig. 8 Lipid peroxidation in CoQ-depleted cells.
a, Total CoQ levels in control and FSP1KO cells treated for 48 h with 3 mM 4-CBA. The graph shows mean ± s.d. of three biological replicates. ***P = 0.0007, *P = 0.0132 by two-tailed t-test. b, Dose response of RSL3-induced death of inducible FSP1–GFP cells pretreated for 48 h with 3 mM 4-CBA and doxycycline before addition of RSL3. Shading indicates 95% confidence intervals for the fitted curves and each data point is the average of three technical replicates. The panel is representative of two biological replicates. c, Genomic sequencing of the COQ2 gene in COQ2KO and FSP1KO COQ2KO cells. The ATG start codon is boxed in the COQ2 consensus sequence. d, Control and COQ2KO cells treated with 250 nM RSL3 for 3 h were labelled with BODIPY 581/591 C11 and fixed before imaging. e, COQ2KO cells were treated with 250 nM RSL3 and 10 μM idebenone or 50 μM DFO for 3 h, labelled with BODIPY 581/591 C11 and fixed before imaging. In panels d, e, images are representative of at least n = 10 cells imaged for each treatment condition. Scale bars, 20 μm.
Extended Data Fig. 9 Role of NQO1 in ferroptosis resistance.
a, Western blot analysis of lysates from NQO1KO and NQO1KO FSP1KO cells. b, Dose response of RSL3-induced death of control and NQO1KO cells. c, Dose response of RSL3-induced death of FSP1KO and NQO1KO FSP1KO cells. Cells in b and c were generated using NQO1 sgRNA 1. d, Western blot analysis of lysates of FSP1KO cells that express doxycycline-inducible NQO1–GFP. e, Live-cell microscopy of inducible NQO1–GFP cells labelled with 5 μg ml-1 Cell Mask. f, Plasma-membrane subdomains from FSP1KO cells that express NQO1–GFP were enriched by OptiPrep gradient centrifugation. g, Dose response of RSL3-induced death of FSP1KO cells expressing the indicated inducible constructs. h, Live-cell microscopy of FSP1KO cells that express inducible LYN11–NQO1–GFP labelled with 5 μg ml-1 Cell Mask. i, Dose response of RSL3-induced death of FSP1KO cells that express the indicated inducible constructs. For panels b, c, g, i, shading indicates 95% confidence intervals for the fitted curves and each data point is the average of three technical replicates. Panels are representative of two biological replicates except for f and i, which show the results of single experiments. In e and h, the images are representative of at least n = 10 imaged cells. Scale bars, 10 μm.
Extended Data Fig. 10 The role of FSP1 in cancer.
a, b, A high level of expression of FSP1 is correlated with resistance to the GPX4 inhibitors ML210 (a) and ML162 (b) in non-haematopoietic cancer cells. Plotted data were mined from the CTRP database that contains correlation coefficients between gene expression and drug sensitivity for 907 cancer cell lines treated with 545 compounds. Plotted values are z-scored Pearson’s correlation coefficients. c, Western blot of FSP1 expression in a panel of lung cancer lines. d, Western blot of lysates from control and FSP1KO H460 cells. e, EC50 RSL3 dose for the indicated H460 cell lines was calculated from the results in Fig. 1d. Bars indicate 95% confidence intervals. f, Western blot of lysates from control and H1703 cells. g, EC50 RSL3 dose for the indicated H1703 cell lines was calculated from the results in Fig. 1e. Bars indicate 95% confidence intervals. h, Western blot analysis of H446 cells that express doxycycline-inducible FSP1–GFP. i, Dose response of RSL3-induced death of control and FSP1–GFP H446 cells. j, Western blot analysis of GPX4KO and GPX4KO FSP1KO H460 cells. k, GPX4KO H460 tumour xenograft cells were initiated in immune-deficient SCID mice (n = 16). Following 5 days of daily Fer1 injections (2 mg kg−1 body weight) to allow lines to develop tumours, 1 set of mice (n = 8) continued to receive daily Fer1 injections and a second set (n = 8) received vehicle injections for the remaining 17 days. The distribution of fold changes in sizes of individual tumours during the treatment is shown. GPX4KO (−) Fer1, n = 7; GPX4KO (+) Fer1, n = 7. l, Dose response of IKE-induced death of control and FSP1KO U-2 OS cells. m, Dose response of IKE-induced death of control and FSP1KO H460 cells. n, o, Control (n) and FSP1KO (o) H460 tumour xenografts were initiated in immune-deficient SCID mice (n = 16). After 10 days, each group of mice (n = 8) was injected daily with IKE or vehicle (40 mg kg−1 body weight). The distribution of fold changes in sizes of individual tumours during the treatment is shown. Cas9ctl (−) IKE, n = 4; Cas9ctl (+) IKE, n = 4; FSP1KO (−) IKE, n = 7; FSP1KO (+) IKE, n = 4. In k, n, o, box plots show median, 25th and 75th percentiles, minima and maxima of the distributions. Panels are representative of two biological replicates except l, m, which show the results of single experiments. In i, l, m, shading indicates 95% confidence intervals for the fitted curves and each data point is the average of three technical replicates.
Supplementary information
Supplementary Figure
This file contains the uncropped blots for the main figures and extended data figures
Supplementary Table 1 | CRISPR synthetic lethal screen data
Full casTLE results for the synthetic lethal CRISPR screen employing the “Apoptosis and Cancer” sublibrary of sgRNAs
Supplementary Table 2 | EC50 values for all cell lines and experiments
Table listing the EC50 values determined from the dose response analyses
Supplementary Table 3 | Lipidomic analysis of Cas9ctl and FSP1KO cells
Lipid levels in Cas9ctl and two-independent FSP1KO cell lines measured by mass spectrometry. Tables show fold change in lipid levels in FSP1KO lines relative to Cas9ctl, and the integrated peak intensity values corresponding to the lipid species in each sample. Cas9ctl, n = 5; FSP1KO #1, n = 4; FSP1KO #2, n = 5 biological replicates. P values were determined by two-tailed t-test
Supplementary Table 4 | Cell line specific FSP1 expression and sensitivity to GPX4 inhibitors
FSP1 mRNA expression values and AUC values for cell lines treated with RSL3, ML210, or ML162. FSP1 mRNA data were from the CCLE database (portals.broadinstitute.org). AUC data were mined from the CTRP database (portals.broadinstitute.org). AUC, area under the dose response curve
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Bersuker, K., Hendricks, J.M., Li, Z. et al. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature 575, 688–692 (2019). https://doi.org/10.1038/s41586-019-1705-2
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DOI: https://doi.org/10.1038/s41586-019-1705-2
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