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Data availability
All data are available within the article and the Supplementary Information. Gel source images are shown in Supplementary Fig. 1. Source data are provided with this paper.
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Acknowledgements
The authors would like to thank A. Wahida for critical reading of the manuscript. This work was supported by funding from the Deutsche Forschungsgemeinschaft (DFG) (CO 291/7-1) and the DFG Priority Program SPP 2306 (CO 291/9-1, 461385412 and CO 291/10-1, 461507177), the German Federal Ministry of Education and Research (BMBF) FERROPath (01EJ2205B), and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. GA 884754) to M.C.; JSPS KAKENHI (20KK0363) to E.M.; Alexander von Humboldt Post-Doctoral Fellowship to J.Z.; and China Scholarship Council to W.Z.
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E.M., T.N., J.Z., and M.C. conceived the study and wrote the manuscript. E.M., T.N., J.Z., and W.Z. performed the experiments and analysis. A.S.D.M. expressed and purified recombinant FSP1 and DHODH. P.S. performed in silico modelling. All authors read and agreed on the content of the paper.
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M.C. and P.S. hold patents for some of the compounds described herein, and are co-founders and shareholders of ROSCUE Therapeutics.
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Extended data figures and tables
Extended Data Fig. 1 The synergistic effect of brequinar with ferroptosis inducers in a panel of cancer cell lines.
a. Known DHODH inhibitors in cancer-related clinical trials. Sourced from https://clinicaltrials.gov/, August 2022. b. Heatmaps of cell viability showing the synergistic effects of brequinar (BQR) with ML210, erastin and BSO in HT-1080 cells. Viability was measured after 48 h (ML210 and erastin) and 72 h treatment (BSO). c. Heatmaps of cell viability showing the synergistic effects of BQR with RSL3 and ML210 in 786-O, A375, MDA-MB-436 and A549 cells. Viability was measured after 48 h. d. Evaluation of cellular toxicity of brequinar. HT-1080, 786-O and MDA-MB-436 cells were treated with the indicated concentrations of BQR in the presence or absence of the ferroptosis inhibitor liproxstatin-1 (Lip1, 1 μM) for 24 h. BQR treatment alone was not sufficient to induce ferroptosis. e. Representative images of HT-1080 cells treated with or without BQR (1 μM) and uridine (100 μM) for 5 days. The cells were seeded at a density of 200 cells/well in a 96-well plate. Scale, 100 μm. Data is mean ± s.d. of n = 3 (d). Data is representative of two independent experiments (b-e).
Extended Data Fig. 2 Inhibitory effects of DHODH inhibitors against FSP1 enzyme activity.
a. (Left) Scheme of the FSP1 enzyme activity assay. Resazurin (100 μM), a substrate of FSP1, is reduced to resorufin by incubation with recombinant FSP1 protein (50 and 40 nM of human and mouse FSP1, respectively) and NADH (200 μM). The amount of resorufin evaluated by fluorescent intensity (ex 540/em 590 nm) indicates FSP1 enzymatic activity. (Right) Scheme of the DHODH enzyme activity assay. Enzyme reaction of recombinant human DHODH (25 nM), dihydroorotate (DHO, 500 μM) and CoQ0 (100 μM) reduces an electron acceptor 2, 6-dichlorophenolindophenol (DCIP, 120 μM) to DCIPH2. The change in absorbance of DCIP (at absorbance 600 nm) indicates DHODH enzyme activity. b. NADH consumption assay using recombinant human FSP1 protein (25 nM) in combination with or without brequinar (BQR, 300 μM). Menadione (50 μM) was used as a substrate of FSP1. Brequinar inhibited the FSP1-dependent NADH consumption. c. The inhibitory effect of BQR and iFSP1 on mouse FSP1 enzymatic activity. d. Heatmaps showing the viability and immunoblotting of hFSP1-overexpressed (OE) and Dhodh KO-Pfa1 cells with or without overexpression of hDHODH. Viability was measured after treatment with RSL3 for 24 h. Combination of RSL3 with BQR synergistically induced cell death in both cell lines. e. The inhibitory effect of known DHODH inhibitors on human and mouse FSP1 enzyme activity. f. Calculated IC50 values of iFSP1 and DHODH inhibitors against recombinant human and mouse FSP1. g. The inhibitory effect of DHODH inhibitors and iFSP1 against human DHODH enzymatic activity. h. Heatmaps showing the viability of HT-1080 cells (5,000 cells per well) treated with RSL3 in combination with vidofludimus or BAY-2402234 for 24 h. The values of the groups treated with zero or 0.01 μM of RSL3 are also shown in the right graphs. i. The binding prediction of iFSP1 in human FSP1 protein. Data is mean ± s.d. of n = 3 (b). Data is representative of three (b, c and e) and two independent experiments (d, g and h), respectively.
Extended Data Fig. 3 Immunoblotting of genetic deletion or overexpression of FSP1 and DHODH, and the effect of cell confluency on ferroptosis sensitivity.
a. Immunoblotting of lysates of FSP1 KO and DHODH KO cells using HT-1080, 786-O, A375 and MDA-MB-436 cell lines. Each parental cell was used as wild type (WT). b. Immunoblotting of lysates of Pfa1 cells with stable overexpression (OE) of C-terminally HA-tagged human DHODH (hDHODH) or FSP1 (hFSP1). One experiment was performed (a, b). c. Relative cell counts of Dhodh KO Pfa1 cells with or without stable OE of hDHODH seeded 200 cells/well in a 96-well plate and incubated with or without uridine (50 μM) for 5 days. hDHODH OE rescued the suppression of cell growth in Dhodh KO Pfa1 cells without uridine supplementation. d. Immunoblotting of lysate and viability of A375 cells of WT, GPX4 KO, GPX4 KO with hFSP1 OE and GPX4/DHODH double KO with hFSP1 OE. For the measurement of the viability, 500 cells/well were seeded in a 96-well plate and incubated with or without Lip1 (1 μM) for 4 days. Viability of the cells incubated with Lip1 (1 μM) was taken as 100%. e. Immunoblotting of lysate and viability of Gpx4 and Dhodh double KO Pfa1 cells with stable OE of hFSP1. The cells were seeded at a density of 300 cells/well in a 96-well plate and incubated with or without uridine (50 μM) and Lip1 (1 μM) for 5 days. The Gpx4 and Dhodh double KO Pfa1 cells with OE of hFSP1 cells can survive without Lip1. f. The effect of cell density of HT-1080 cells on RSL3-induced cell death. The cells were seeded at densities of 3,000, 8,000 or 20,000 cells/well in a 96-well plate. On the next day, the cells were treated with RSL3 for 6 h and viability was determined. Scale, 100 μm. Data is mean ± s.d. of n = 9 (c) and n = 3 (d-f). Two-tailed t-test (c); one-way ANOVA with Dunnett’s test (d).
Extended Data Fig. 4 Expression pattern and subcellular localization of GPX4 isoforms.
a. Structural organization of the GPX4 gene, mRNA and protein of the GPX4 isoforms. Arrows indicate the transcription initiation sites. The dashed lines indicate the different splicing variants. ATG indicates the initiation codon of methionine. MTS, mitochondrial targeting sequence; NLS, nuclear localization signal. NLS also functions for stretching of DNA binding motifs. b. A scheme depicting the reported subcellular localization of each GPX4 isoform in somatic and testicular cells. The short form is abundantly expressed in the cytoplasm and mitochondrial extra-matrix space of somatic cells, while the mitochondrial matrix form is abundantly expressed in the mitochondrial matrix of testicular cells. The illustration was created using BioRender.com (a, b). c. Viability of GPX4 KO HT-1080 cells (500 cells/well) overexpressing the short or mitochondrial matrix form of GPX4 for three days after withdrawal of ferrostatin-1 (a ferroptosis inhibitor). The cells were prepared by infection with the indicated serial dilution of lentiviral particles containing the expression plasmids. Immunoblotting validated the overexpression of each form. Viability of the cells incubated with Lip1 (1 μM) was taken as 100%. d. The design of the primer pairs detecting both the short and mitochondrial matrix forms (106 bp) and specific for the mitochondrial matrix form (196 bp). Agarose gel images show the amplification of the specific single band. The ratio of the mitochondrial matrix form/short and mitochondrial matrix forms of GPX4 mRNA expression in the cancer cell lines was calculated as 2−ΔCT in quantitative RT-PCR. Data is representative of two independent experiments (c and d). Data is mean ± s.d. of n = 3 (c and d).
Supplementary information
Supplementary Information
This file contains Supplementary Table 1 and Supplementary Figure 1. Supplementary Table 1: Sequences of sgRNA guides used to generate the knockout cell lines. For making knockout cell lines, following plasmids were used: pCW-Cas9-Blast (83481, Addgene), lentiCas9-Blast (52962, Addgene), LentiGuide-Neo (139449, Addgene), pLentiCRISPRv2_Neo (127644, Addgene), pKLV-U6gRNA(BbsI) (50946, Addgene), pLentiCRISPRv2_Puro (98290, Addgene), pLentiCRISPRv2_Blast (98293, Addgene). Nucleotides in italics show the overhangs introduced into oligos that are necessary for cloning in the BbsI site of pKLV-U6gRNA(BbsI) vectors and in the BsmBI restriction sites of lentiCRISPR v2 and LentiGuide vectors. Supplementary Figure 1: Gel source images.
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Mishima, E., Nakamura, T., Zheng, J. et al. DHODH inhibitors sensitize to ferroptosis by FSP1 inhibition. Nature 619, E9–E18 (2023). https://doi.org/10.1038/s41586-023-06269-0
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DOI: https://doi.org/10.1038/s41586-023-06269-0
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