Abstract
Ferroptosis, a form of regulated cell death that is induced by excessive lipid peroxidation, is a key tumour suppression mechanism1,2,3,4. Glutathione peroxidase 4 (GPX4)5,6 and ferroptosis suppressor protein 1 (FSP1)7,8 constitute two major ferroptosis defence systems. Here we show that treatment of cancer cells with GPX4 inhibitors results in acute depletion of N-carbamoyl-l-aspartate, a pyrimidine biosynthesis intermediate, with concomitant accumulation of uridine. Supplementation with dihydroorotate or orotate—the substrate and product of dihydroorotate dehydrogenase (DHODH)—attenuates or potentiates ferroptosis induced by inhibition of GPX4, respectively, and these effects are particularly pronounced in cancer cells with low expression of GPX4 (GPX4low). Inactivation of DHODH induces extensive mitochondrial lipid peroxidation and ferroptosis in GPX4low cancer cells, and synergizes with ferroptosis inducers to induce these effects in GPX4high cancer cells. Mechanistically, DHODH operates in parallel to mitochondrial GPX4 (but independently of cytosolic GPX4 or FSP1) to inhibit ferroptosis in the mitochondrial inner membrane by reducing ubiquinone to ubiquinol (a radical-trapping antioxidant with anti-ferroptosis activity). The DHODH inhibitor brequinar selectively suppresses GPX4low tumour growth by inducing ferroptosis, whereas combined treatment with brequinar and sulfasalazine, an FDA-approved drug with ferroptosis-inducing activity, synergistically induces ferroptosis and suppresses GPX4high tumour growth. Our results identify a DHODH-mediated ferroptosis defence mechanism in mitochondria and suggest a therapeutic strategy of targeting ferroptosis in cancer treatment.
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Data availability
All data that support the conclusions in this manuscript are available from the corresponding author upon reasonable request. The source data of immunoblots are provided. The raw data used for generating Figs. 1–4 and Extended Data Figs. 1–9 are included in the Source Data. Source data are provided with this paper.
Change history
02 August 2021
A Correction to this paper has been published: https://doi.org/10.1038/s41586-021-03820-9
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Acknowledgements
We apologize to the colleagues whose relevant work cannot be cited here owing to space limitations. This research was supported by Institutional Research Fund from The University of Texas MD Anderson Cancer Center, and grants R01CA181196, R01CA190370, R01CA244144, R01CA247992 from the National Institutes of Health (to B.G.). PDX generation and annotation were supported by the University of Texas MD Anderson Cancer Center Moon Shots Program, Specialized Program of Research Excellence (SPORE) grant CA070907 and University of Texas PDX Development and Trial Center grant U54CA224065. This research was also supported by the National Institutes of Health Cancer Center Support Grant P30CA016672 to The University of Texas MD Anderson Cancer Center.
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Authors and Affiliations
Contributions
C.M. performed most of the experiments with assistance from X.L., Y.Z., G.L., Y.Y., H.L., P.K., S.W. and L.Z.; K.O. conducted all metabolomic analyses; B.F. provided PDXs used in this study; M.V.P. provided resources for the project; B.G., C.M., and K.O. designed the experiments; B.G. supervised the study, established collaborations, allocated funding for this study, and wrote most of the manuscript with assistance from K.O. and C.M.; and all authors commented on the manuscript.
Corresponding authors
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Competing interests
K.O. and M.V.P. are full-time employees of Kadmon Corporation, LLC. B.G., K.O., and M.C. have filed a patent application relating to the use of DHODH inhibitors to target ferroptosis in cancer therapy. Other authors declare no competing financial interests.
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Peer review information Nature thanks Kivanç Birsoy and Navdeep Chandel for their contribution to the peer review of this work. Peer reviewer reports are available.
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Extended data figures and tables
Extended Data Fig. 1 Pharmacological inhibition of GPX4 affects intermediate levels in the de novo pyrimidine biosynthesis pathway.
a–c, Volcano plots comparing metabolomic profiles from HT-1080 (a), A-498 (b) or RCC4 (c) cells treated with vehicle and the same cells treated with RSL3 (10 μM) or ML162 (10 μM) for 2 h. d, e, Fold change in C-Asp and uridine induced by RSL3 (10 μM) or ML162 (10 μM) treatment for 2 h compared with vehicle treatment in A-498 (d) or RCC4 (e) cells. f, Simplified schematic of de novo pyrimidine biosynthesis pathway. g, Fold change in intracellular DHO and OA levels upon treatment with vehicle, DHO (100 μM) or OA (100 μM), respectively, for 48 h in NCI-H226 cells. h, Fold change in intracellular C-Asp levels upon treatment with vehicle or C-Asp (100 μM) for 48 h in NCI-H226 cells. i, DHO activity in HT-1080 cells treated with RSL3 (10 μM) for 2 h, following pretreatment with vehicle, OA (100 μM) for 24 h, or Lip-1 (10 μM) for 48 h. j, GPX4 protein levels in different cell lines determined by western blotting. k, Cell viability in TK-10, UMRC2, A-498 and RCC4 cells treated with different doses of RSL3 for 4 h, following pretreatment with vehicle, C-Asp (100 μM), DHO (100 μM), OA (100 μM), or uridine (50 μM) for 48 h. l, Cell viability in SW620, U-87 MG, A549, NCI-H1437, MDA-MB-436 and MDA-MB-231 cells treated with different doses of RSL3 for 4 h, following pretreatment with vehicle, DHO (100 μM) or OA (100 μM) for 48 h. m, GPX4, DHODH, and FSP1 protein levels in different cancer cell lines determined by western blotting. n, Cell viability in GPX4high (HT-1080, A-498, RCC4, 786-O, and 769-P) and GPX4low (HCT-8, UMRC6, TK-10, UMRC2, and NCI-H226) cells treated with different doses of the DHODH inhibitors BQR, leflunomide (LFM), or teriflunomide (TF) for 4 h. Data are presented as mean ± s.d., n = 3 independent repeats; unpaired, two-tailed t-test. Western blots are representative of two biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant. Asp, aspartate; C-P, carbamoyl phosphate; P, phosphate; FMN, flavin mononucleotide; FMNH2, reduced flavin mononucleotide; PRPP, phosphoribosyl pyrophosphate; PPi, inorganic pyrophosphate; OMP, orotidine 5′-monophosphate; UMP, uridine 5′-monophosphate.
Extended Data Fig. 2 The effect of DHODH inhibitors on inducing ferroptosis in different cancer cells with differential expression of GPX4.
a, b, Cell survival fraction and PTGS2 mRNA levels in NCI-H226 (a) and HT-1080 (b) cells upon treatment with BQR (500 μM for NCI-H226 cells; 5 mM for HT-1080 cells), following pretreatment with vehicle, ZVF (10 μM), and/or Lip-1 (10 μM) for 24 h. c, Cell viability in HT-1080 cells treated with different doses of RSL3 and co-treated with LFM (100 μM) or TF (500 μM) for 4 h, following pretreatment with vehicle or Lip-1 (10 μM) for 24 h. d, Cell viability in HT-1080 cells treated with different doses of ML162 and co-treated with BQR (500 μM), LFM (100 μM), or TF (500 μM) for 4 h, following pretreatment with vehicle or Lip-1 (10 μM) for 24 h. e, Cell survival fraction and PTGS2 mRNA levels in HT-1080 cells upon treatment with RSL3 (1 μM) and/or BQR (500 μM) for 4 h, following pretreatment with vehicle or Lip-1 (10 μM) for 24 h. f, Cell viability in HT-1080 cells treated with different doses of sulfasalazine (SAS) and co-treated with BQR (500 μM), LFM (100 μM) or TF (500 μM) for 4 h, following pretreatment with vehicle or Lip-1 (10 μM) for 24 h. g, Cell viability in HT-1080 cells treated with different doses of erastin and co-treated with BQR (500 μM), LFM (100 μM) or TF (500 μM) for 4 h, following pretreatment with vehicle or Lip-1 (10 μM) for 24 h. h, mRNA levels of SLC7A11, GPX4, or ACSL4 (bar charts) and their protein expression (western blot), were measured in HT-1080 cells treated with BQR (500 μM), LFM (100 μM), or TF (500 μM) for 4 h. i, GSH level measurement in HT-1080 cells upon treatment with BQR (500 μM), LFM (100 μM), or TF (500 μM) for 2 h. Data are presented as mean ± s.d., n = 3 independent repeats; unpaired, two-tailed t-test. Western blot is representative of two biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.
Extended Data Fig. 3 DHODH deletion sensitizes GPX4high cancer cells to ferroptosis or induces ferroptosis in GPX4low cancer cells.
a, DHODH protein levels in Cas9 control and DHODH KO GPX4high cancer cell lines. b, DHO activity in Cas9 control and DHODH KO HT-1080 cells. c, Cell survival fraction in Cas9 control and DHODH KO HT-1080 cells upon treatment with vehicle or uridine (50 μM). d, PTGS2 mRNA levels in Cas9 control and DHODH KO HT-1080 cells. e, Lipid peroxidation in Cas9 control and DHODH KO GPX4high cell lines as indicated. f, Cell viability in Cas9 control and DHODH KO HT-1080 cells treated with different doses of ML162 for 4 h. g, Cell survival fraction and PTGS2 mRNA levels in Cas9 control and DHODH KO HT-1080 cells upon treatment with RSL3 (1 μM) for 4 h. h, Western blot analysis of DHODH and ACSL4 protein levels in HT-1080 cells with indicated genotypes. i, Cell viability measurement in HT-1080 cells with indicated genotypes treated with different doses of RSL3 for 4 h. j, Measurement of SLC7A11, GPX4, and ACSL4 mRNA (bar charts) and protein levels (western blot) in Cas9 control and DHODH KO HT-1080 cells. k, GSH levels in Cas9 control and DHODH KO HT-1080 cells. l, DHODH protein levels in Cas9 control and DHODH KO GPX4low cell lines. m, DHO activity in Cas9 control and DHODH KO NCI-H226 cells. n, Cell proliferation of Cas9 control and DHODH KO NCI-H226 cells. o, PTGS2 mRNA levels in Cas9 control and DHODH KO NCI-H226 cells. p, Lipid peroxidation in Cas9 control and DHODH KO GPX4low cells. Cells were grown in medium supplemented with Lip-1 (10 μM) (l, m) and/or uridine (50 μM) (a, b, d–p). Data are presented as mean ± s.d., n = 3 independent repeats; unpaired, two-tailed t-test. Western blots are representative of two biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.
Extended Data Fig. 4 Analyses of genetic interactions between DHODH and GPX4 (or FSP1).
a, Western blotting analysis of GPX4 and DHODH protein levels in shControl and shGPX4 HT-1080 cells. b, Cell proliferation of shControl and shGPX4 HT-1080 cells. c, Cell viability of shControl and shGPX4 HT-1080 cells treated with different doses of LFM or TF for 4 h. d, Cell survival fraction and PTGS2 mRNA levels in shControl and shGPX4 HT-1080 cells upon treatment with BQR (500 μM) for 4 h. e, Western blot analysis of GPX4 and DHODH protein levels in HT-1080 cells with indicated genotypes. f, PTGS2 mRNA levels in HT-1080 cells with indicated genotypes. g, Cell proliferation of HT-1080 cells with DHODH KO and shControl or shGPX4. h, Western blot analysis of DHODH and FSP1 protein levels in HT-1080 cells with indicated genotypes. i, Cell viability in Cas9 control or DHODH KO HT-1080 cells with indicated genotypes treated with different doses of RSL3 for 4 h, following pretreatment with vehicle or Lip-1 (10 μM) for 24 h. j, Western blot analysis of DHODH and FSP1 protein levels in HT-1080 cells with indicated genotypes. k, Cell viability in Cas9 control or DHODH KO HT-1080 cells with indicated genotypes treated with different doses of RSL3 for 4 h, following pretreatment with vehicle or Lip-1 (10 μM) for 24 h. l, Cell viability in Cas9 control or FSP1 KO HT-1080 cells treated with vehicle or BQR (500 μM), and different doses of RSL3 for 4 h. m, Simplified schematic of DHODH protein and its mutants. n, Western blotting showing DHODH protein levels in cytosolic and mitochondrial fractions from DHODH KO HT-1080 cells that express the indicated DHODH constructs. o, DHO activity in DHODH KO HT-1080 cells that express the indicated DHODH constructs. p, Cell viability in DHODH KO HT-1080 cells that express the indicated DHODH constructs treated with different doses of ML162 for 4 h. q, Cell survival fraction, lipid peroxidation and PTGS2 mRNA levels in DHODH KO HT-1080 cells that express the indicated DHODH constructs upon treatment with RSL3 (1 μM). Cells were grown in medium supplemented with uridine (50 μM) (e–l, n–q). Data are presented as mean ± s.d., n = 3 independent repeats (b–d, f, g, i, k, l, o–q); unpaired, two-tailed t-test. Western blots are representative of two biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant. MTS, mitochondrial targeting sequence; DHOD domain, dihydroorotate dehydrogenase domain.
Extended Data Fig. 5 DHODH cooperates with mitochondrial GPX4 to suppress ferroptosis.
a, Western blotting analysis of GPX4 levels in cytosolic and mitochondrial fractions in a panel of cancer cell lines. b, Simplified schematic of cytosolic and mitochondrial GPX4 protein constructs. c, Western blotting showing GPX4 protein levels in cytosolic and mitochondrial fractions from shGPX4 HT-1080 cells that express the indicated GPX4 constructs. d, Cell viability in shGPX4 HT-1080 cells that express the indicated GPX4 constructs treated with different doses of LFM or TF for 4 h. e, Cell survival fraction, lipid peroxidation and PTGS2 mRNA levels in shGPX4 HT-1080 cells that express the indicated GPX4 constructs upon treatment with BQR (500 μM). f, Western blotting showing GPX4 protein levels in shGPX4 cells that express the indicated GPX4 constructs in a variety of cell lines. g, Cell viability measurement in various shGPX4 cells that express the indicated GPX4 constructs treated with different doses of BQR for 4 h. Data are presented as mean ± s.d., n = 3 independent repeats (d, e, g); unpaired, two-tailed t-test. Western blots are representative of two biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.
Extended Data Fig. 6 Inactivation of DHODH and GPX4 induces mitochondrial lipid peroxidation.
a, Western blot showing GPX4 protein levels in cytosolic and mitochondrial fractions from NCI-H226 cells that express the indicated GPX4 constructs. b, Cell proliferation of NCI-H226 cells that express the indicated GPX4 constructs. c, Cell viability in NCI-H226 cells that express the indicated GPX4 constructs treated with different doses of BQR, LFM or TF for 4 h. d, Cell survival fraction, lipid peroxidation and PTGS2 mRNA levels in NCI-H226 cells that express the indicated GPX4 constructs upon treatment with BQR (500 μM). e, Cell viability in Cas9 control and DHODH KO HT-1080 cells treated with different doses of ML162 for 4 h, following pretreatment with vehicle, TEMPO (10 μM), MitoTEMPO (10 μM), or Lip-1 (10 μM) for 24 h. f, Cas9 control and DHODH KO HT-1080 cells were treated with RSL3 (1 μM) for 2 h, then stained with mito-BODIPY. Oxidized mito-BODIPY (green) indicates mitochondrial lipid peroxidation (scale bar, 5 μM). g, Mitochondrial lipid peroxidation in Cas9 control and DHODH KO HT-1080 cells upon treatment with RSL3 (1 μM) for 2 h. h, Mitochondrial lipid peroxidation in shControl and shGPX4 HT-1080 cells upon treatment with BQR (500 μM) for 2 h. i, Mitochondrial lipid peroxidation in HT-1080 cells upon treatment with RSL3 (1 μM) and/or BQR (500 μM), LFM (100 μM), or TF (500 μM) for 2 h, following pretreatment with vehicle or Lip-1 (10 μM) for 24 h. j, Mitochondrial lipid peroxidation in HT-1080 cells upon treatment with ML162 (1 μM) and/or BQR (500 μM), LFM (100 μM), or TF (500 μM) for 2 h, following pretreatment with vehicle or Lip-1 (10 μM) for 24 h. k, Mitochondrial lipid peroxidation in DHODH KO HT-1080 cells that express the indicated DHODH constructs upon treatment with RSL3 (1 μM) for 2 h. l, m, Mitochondrial lipid peroxidation in Cas9 control and DHODH KO HT-1080 cells with indicated genotypes upon treatment with RSL3 (1 μM) for 2 h, following pretreatment with vehicle or Lip-1 (10 μM) for 24 h. n, Mitochondrial lipid peroxidation in Cas9 control and FSP1 KO HT-1080 cells upon treatment with RSL3 (1 μM) and/or BQR (500 μM) for 2 h. o, Western blot analysis of DHODH and FSP1 protein levels in cytosolic and mitochondrial fractions of HT-1080 cells with indicated genotypes. p, Cell viability in Cas9 control and DHODH KO HT-1080 cells with indicated genotypes treated with different doses of RSL3 for 4 h, following pretreatment with vehicle or Lip-1 (10 μM) for 24 h. q, Mitochondrial lipid peroxidation in Cas9 control and DHODH KO HT-1080 cells with indicated genotypes upon treatment with RSL3 (1 μM) for 2 h, following pretreatment with vehicle or Lip-1 (10 μM) for 24 h. r, Mitochondrial lipid peroxidation in shGPX4 HT-1080 cells that express the indicated GPX4 constructs upon treatment with BQR (500 μM) for 2 h. s, Mitochondrial lipid peroxidation in NCI-H226 cells that express the indicated GPX4 constructs upon treatment with BQR (500 μM) for 2 h. Cells were grown in medium supplemented with uridine (50 μM) (e–g, k–q). Data are presented as mean ± s.d., n = 3 independent repeats (b–e, g–n, p–s); unpaired, two-tailed t-test. Western blots are representative of two biological replicates. Images are representative of at least n = 5 imaged cells (f). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns. Not significant. Mito-C11, fluorescent mitochondria-targeted lipid peroxidation probe.
Extended Data Fig. 7 DHODH regulation of ferroptosis relates to its function to reduce CoQ to CoQH2 in mitochondria.
a, Cell viability in HT-1080 cells treated with different doses of FIN56 and co-treated with BQR (500 μM), LFM (100 μM) or TF (500 μM) for 4 h, following pretreatment with vehicle or Lip-1 (10 μM) for 24 h. b, Cell survival fraction, mitochondrial lipid peroxidation and PTGS2 mRNA levels in HT-1080 cells upon treatment with vehicle, FIN56 (50 μM) and/or BQR (500 μM), following pretreatment with vehicle or Lip-1 (10 μM) for 24 h. c, Western blot analysis of COQ2 and DHODH protein levels in HT-1080 cells with indicated genotypes. d, Total CoQ in Cas9 control and COQ2 KO HT-1080 cells. e, Total CoQ in HT-1080 cells that were treated with vehicle or 4-CBA (5 mM) for 24 h. f, Cell viability measurement in Cas9 control and DHODH KO HT-1080 cells with indicated genotypes treated with different doses of RSL3 for 4 h, following pretreatment with vehicle or Lip-1 (10 μM) for 24 h. g, Cell viability in Cas9 control and DHODH KO HT-1080 cells with indicated genotypes treated with different doses of ML162 for 4 h, following pretreatment with vehicle or Lip-1 (10 μM) for 24 h. h, Cell viability in Cas9 control and DHODH KO HT-1080 cells treated with different doses of RSL3 for 4 h, following pretreatment with vehicle, 4-CBA (5 mM), or 4-CBA (5 mM) + Lip-1 (10 μM) for 24 h. i, Cell viability in Cas9 control and DHODH KO HT-1080 cells treated with different doses of ML162 for 4 h, following pretreatment with vehicle, 4-CBA (5 mM) or Lip-1 (10 μM) for 24 h. j, Mitochondrial lipid peroxidation in Cas9 control and DHODH KO HT-1080 cells upon treatment with RSL3 (1 μM), following pretreatment with vehicle, 4-CBA (5 mM), or 4-CBA (5 mM) + Lip-1 (10 μM) for 24 h. k, Simplified schematic showing how DHODH couples the oxidation of DHO to OA to the reduction of CoQ to CoQH2 in the mitochondrial inner membrane. l, CoQ/CoQH2 ratio in NCI-H226 cells that were treated with BQR (1 mM) for 2 h. m, Cell viability in Cas9 control and DHODH KO HT-1080 cells treated with different doses of ML162 for 4 h, following pretreatment with vehicle, MitoQ (10 μM), MitoQH2 (10 μM), or Lip-1 (10 μM) for 24 h. n, Mitochondrial lipid peroxidation in Cas9 control and DHODH KO HT-1080 cells upon treatment with RSL3 (1 μM) for 2 h, following pretreatment with vehicle, MitoQ (10 μM), MitoQH2 (10 μM), or Lip-1 (10 μM) for 24 h. o, Lipid peroxidation in Cas9 control and DHODH KO HT-1080 cells upon treatment with RSL3 (1 μM) for 2 h, following pretreatment with vehicle, MitoQ (10 μM), MitoQH2 (10 μM), or Lip-1 (10 μM) for 24 h. Cells were grown in medium supplemented with uridine (50 μM) (c, d, f–j, m–o). Data are presented as mean ± s.d., n = 3 independent repeats (a, b, d–j, l–o); unpaired, two-tailed t-test. Western blot is representative of two biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant. OCR, oxygen consumption rate; MitoQ, [10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)decyl] triphenyl-phosphonium, monomethanesulfonate; MitoQH2, [10-(2,5-dihydroxy-3,4-dimethoxy-6-methylphenyl)decyl] triphenyl-phosphonium, monomethanesulfonate.
Extended Data Fig. 8 The effects of mitoQ and mitoQH2 on RSL3- and BQR-induced ferroptosis in a variety of cell lines.
a, GPX4, DHODH and FSP1 protein levels in indicated cell lines determined by western blotting. b–j, Cell viability in 293T (b), Hela (c), Jurkat (d), SW620 (e), U-87 MG (f), A549 (g), NCI-H1437 (h), MDA-MB-436 (i), and MDA-MB-231 (j) cells treated with different doses of RSL3 with vehicle or BQR (500 μM) for 4 h, following pretreatment with vehicle, MitoQ (10 μM), MitoQH2 (10 μM), or Lip-1 (10 μM) for 24 h. k, CoQ/CoQH2 ratio in HT-1080 cells that were treated with myxothiazol (10 μM) for 2 h. l, Cell viability in Cas9 control and DHODH KO HT-1080 cells treated with different doses of RSL3 for 4 h, following pretreatment with vehicle or myxothiazol (1 μM) for 24 h. m, CoQ/CoQH2 ratio in A549 cells that were treated with myxothiazol (10 μM) for 2 h. n, Cell viability in A549 cells treated with different doses of RSL3 with or without BQR (500 μM) for 4 h, following pretreatment with vehicle or myxothiazol (1 μM) for 24 h. o, Western blot analysis of DHODH and CiAOX protein levels in HT-1080 cells with indicated genotypes. p, Mitochondrial lipid peroxidation in HT-1080 cells with indicated genotypes upon treatment with RSL3 (1 μM) for 2 h. Cells were grown in medium supplemented with uridine (50 μM) (l, o, p). Data are presented as mean ± s.d., n = 3 independent repeats (b–n, p); unpaired, two-tailed t-test. Western blots are representative of two biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.
Extended Data Fig. 9 DHODH inhibitor selectively suppresses GPX4low tumour growth.
a, Weights of shControl and shGPX4 HT-1080 xenograft tumours with the indicated treatments. b–d, Representative immunochemical images from shControl and shGPX4 HT-1080 xenograft tumours with the indicated treatments (b; scale bars, 20 μM), and staining scores of cleaved-caspase 3 (c) and ki67 (d). e, Weight measurements of NCI-H226 xenograft tumours with the indicated treatments. f, Weight measurements of TC632, TC629, or TC494 PDX tumours with the indicated treatments. g, Volumes of Cas9 control and DHODH KO NCI-H226 xenograft tumours with the indicated treatments at different time points (days). h, Weights of Cas9 control and DHODH KO NCI-H226 xenograft tumours with the indicated treatments. i, Weight measurements of HT-1080 xenograft tumours with the indicated treatments. j–l, Representative immunochemistry images of HT-1080 xenograft tumours with the indicated treatments (j; scale bars, 20 μM) and staining scores of cleaved-caspase 3 (k) and ki67 (l). m, Volumes of TC629 PDX tumours with the indicated treatments at different time points (days). n, Weights of TC632 and TC629 PDX tumours with the indicated treatments. o, Weights of mice for all cell line xenografts or PDXs with different treatments at different time points (days). Box plots indicate median, minima and maxima of the distributions, and with whiskers from minimum to maximum. Data are presented as mean ± s.d., n = 8 (a, e, g–i), n = 5 (c, d, k, l) or n = 6 independent tumours (f, m, n). n = 4 for nude mouse weights and n = 8 for NSG mouse weights (o). Unpaired, two-tailed t-test. Images are representative of n = 5 images. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant. 4-HNE, 4-hydroxynonenal.
Extended Data Fig. 10 Working model depicting how GPX4, FSP1, and DHODH suppress ferroptosis in different subcellular compartments.
See main text for a detailed description. PLOOH, phospholipid hydroperoxide; PLOO·, phospholipid hydroperoxyl radical; GSSH, oxidized glutathione; NAD(P)H, reduced nicotinamide adenine dinucleotide (phosphate); NAD(P)+, oxidized nicotinamide adenine dinucleotide (phosphate).
Supplementary information
Supplementary Figure
This file contains a figure exemplifying the gating strategy.
Supplementary Table 1
A list of sequences and primers used in the study.
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Mao, C., Liu, X., Zhang, Y. et al. DHODH-mediated ferroptosis defence is a targetable vulnerability in cancer. Nature 593, 586–590 (2021). https://doi.org/10.1038/s41586-021-03539-7
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DOI: https://doi.org/10.1038/s41586-021-03539-7
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