Abstract
Tumor cells adapt to the inflammatory tumor microenvironment (TME) and develop resistance to immunotherapy, with ferroptosis being a major form of tumor cell death. However, the mechanisms by which tumor cells coordinate TME stimuli and their unique metabolic traits to evade ferroptosis and develop resistance to immunotherapy remain unclear. Here we showed that interferon-γ (IFNγ)-activated calcium/calmodulin-dependent protein kinase II phosphorylates phosphoserine aminotransferase 1 (PSAT1) at serine 337 (S337), allowing it to interact with glutathione peroxidase 4 (GPX4) and stabilize the protein, counteracting ferroptosis. PSAT1 elevates GPX4 stability by promoting α-ketoglutarate-dependent PHD3-mediated GPX4 proline 159 (P159) hydroxylation, disrupting its binding to HSC70 and inhibiting autophagy-mediated degradation. In mice, reconstitution of PSAT1 S337A or GPX4 P159A promotes ferroptosis and suppresses triple-negative breast cancer (TNBC) progression. Blocking PSAT1 pS337 with CPP elevates IFNγ-induced ferroptosis and enhances the efficacy of programmed cell death protein 1 (PD-1) antibodies in TNBC. Additionally, PSAT1-mediated GPX4 hydroxylation correlates with poor immunotherapy outcomes in patients with TNBC, highlighting PSAT1’s noncanonical role in suppressing ferroptosis and immunotherapy sensitivity.
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Data availability
Primary research data underlying this study are documented in the article and Supplementary Information. Extended datasets are available from the corresponding researcher through appropriate academic protocols. The UniProt protein database (EMBL-EBI) was used for protein identification. Gene IDs were determined from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/gene). Mass spectrometry raw data have been archived in ProteomeXchange under dataset identifier PXD061373. All source files are included as supplemental materials accompanying this publication. Source data are provided with this paper.
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Acknowledgements
This study was supported by grants from the Ministry of Science and Technology of the People’s Republic of China (2021YFA0805600 to D.X. and 2020YFA0803300 to Z.L.), the National Natural Science Foundation of China (82188102 and 82030074 to Z.L.; 32470815, 92157113 and 82072630 to D.X.; 82273955 and 82473949 to Z.T.; 82372814 and 82173114 to Z.W.; 82402048 to Z.H. and 82103658 to Q.Z.), the Zhejiang Natural Science Foundation Key Project (LD22H160002 to D.X.), Zhejiang Natural Science Foundation Discovery Project (LQ22H160023 to Z.W.), Zhejiang Leading Innovation and Entrepreneurship Team (2022R01006 to X.Q.), Zhejiang University Research Fund (188020*194221901/029 to Z.L.), Postdoctoral Fellowship Program of Chinese Postdoctoral Science Foundation (GZC20241481 to Z.H.) and Shanghai Pujiang Program (2022PJD040 to Q.Z. and 2023PJD049 to Y.O.). Z.L. is the Kuancheng Wang Distinguished Chair. The author gratefully acknowledges the support of the K.C. Wong Education Foundation.
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D.X. conceived and designed the study. D.X., Z.T. and X.W. supervised the study. P.Z., Z.H., Y.S. and L.G. designed most of the experiments, performed most of the experiments (including cell biology and biochemical experiments and animal study) and analyzed most of the data. Y.O., Y.D., G.J. and B.D. performed the experiments and analyzed western blot and qPCR data. Y.L., T.W., Q.T. and Y.H. performed the experiments and conducted FACS analysis. Q.Z., X.S., X.C., K.W., S.L., S.W., Y.S. and Y.B. performed the experiments and conducted immunohistochemical analysis. M.L., L.X., Q.W., Y.M., G.L. and Z.W. performed statistical analysis and interpretation of the data. D.X., P.Z., Z.H., Y.S. and L.G. wrote the manuscript. Y.D., Y.W., G.L., Z.L. and X.L. reviewed and edited the manuscript. X.B. and S.D. provided technical support.
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Extended data
Extended Data Fig. 1 CAMK2 phosphorylates PSAT1 at S337 upon IFNγ stimulation.
a–d,f–h, Immunoprecipitation and immunoblotting were performed with the indicated antibodies. All experiments were repeated three times independently with consistent results, and representative data are shown. a,f, MDA-MB-231 cells transfected with or without CAMK2 shRNA were treated with or without IFNγ for 2 h. b, MDA-MB-231 and BT549 cells were treated with or without IFNγ (25 ng ml−1) for 2 h. c, A GST pull-down assay was performed as indicated. d, Recombinant Flag-CAMK2 WT or Flag-CAMK2-KD purified from 293T cells (100 ng) was incubated with bacterially purified His-PSAT1 (1000 ng) in 40 μl of kinase buffer (10 mM HEPES (pH 7.2), 1 mM EGTA, 5 mM MgCl2 and 2 mM CaCl2) and then incubated with 0.5 mM normal ATP at 30 °C for 30 min and then subjected to SDS–PAGE and immunoblotting. Immunoblotting analyses were performed with the indicated antibodies. e, Flag-CAMK2 WT and KD purified from 293T cells were analyzed by SDS–PAGE. g, MDA-MB-231 cells with expression of the indicated CAMK2 proteins were treated with or without IFNγ (25 ng ml−1) for 2 h. h, The indicated cells expressing PSAT1 shRNA with reconstituted expression of the indicated PSAT1 proteins were collected.
Extended Data Fig. 2 PSAT1 pS337 increases GPX4 stability.
a,d,e, Immunoprecipitation and immunoblotting were performed with the specified antibodies. All experiments were repeated three times independently with consistent results, and representative data are shown. a–c, The indicated cells were treated with IFNγ (25 ng ml−1) for the indicated periods and collected. Relative intensity of GPX4 to β-tubulin was analyzed (b). Data are the mean ± s.d., n = 6 independent experiments (two-tailed Student’s t test). The mRNA expression levels of GPX4 gene were measured using qPCR (c). Data are the mean ± s.d., n = 6 independent experiments (two-tailed Student’s t test). d,e, MDA-MB-231 and BT549 cells expressing PSAT1 shRNA with reconstituted expression of PSAT1 proteins were stably transfected with active CAMK2-CA. The indicated cells were treated with CHX for the indicated time and collected for immunoblotting. Relative intensity of GPX4 to β-tubulin was analyzed. Data are the mean ± s.d., n = 6 independent experiments, two-tailed Student’s t test.
Extended Data Fig. 3 GPX4 P159–OH by PHD3 prevents HSC70–GPX4 interaction.
a,b, MDA-MB-231 and BT549 cells expressing GPX4 shRNA with reconstituted expression of the indicated GPX4 proteins were collected for immunoblotting and mRNA detection using quantitative PCR as indicated. Data are the mean ± s.d., n = 6 independent experiments, two-tailed Student’s t test. c, MDA-MB-231 and BT549 cells transfected with the indicated plasmids were collected. In a,c, immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. Representative results from three independent experiments were shown.
Extended Data Fig. 4 GPX4 P159–OH inhibits CMA-mediated GPX4 degradation.
a,b, MDA-MB-231 cells transfected with the indicated shRNA were treated with IFNγ (25 ng ml−1) for 2 h and collected. c,d, MDA-MB-231 and BT549 cells expressing PHD3 shRNA with reconstituted expression of the indicated PHD3 proteins were collected for immunoblotting or were treated with or without IFNγ (25 ng ml−1) for 2 h. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. e, MDA-MB-231 and BT549 cells expressing PHD3 shRNA with reconstituted expression of the indicated PHD3 proteins were treated with CHX for the indicated time in the absence or presence of IFNγ and collected for immunoblotting. Data are the mean ± s.d., n = 6 independent experiments, two-tailed Student’s t test. f, MDA-MB-231 and BT549 cells expressing PHD3 shRNA with reconstituted expression of the indicated PHD3 proteins were treated with or without IFNγ (25 ng ml−1) for 9 h. Immunoblotting analyses were performed with the indicated antibodies. For all experiments, representative results from at least three independent experiments were shown.
Extended Data Fig. 5 PSAT1–GPX4 axis suppresses ferroptosis.
a–f, MDA-MB-231 and BT549 cells with reconstituted expression of the indicated proteins were treated with erastin (20 μM) in the absence or presence of Fer1 (10 μM) for 24 h. Lipid ROS-positive cells (a,b), 4-HNE level (c,d) and cell death (e,f) were measured. Data are the mean ± s.d., two-tailed Student’s t test. Data are the mean ± s.d. All experiments were repeated three times independently with similar results.
Extended Data Fig. 6 PSAT1 suppresses ferroptosis in a GPX4-dependent manner.
a,b,d,e, MDA-MB-231 cells expressing PSAT1 and GPX4 shRNA with reconstituted expression of the indicated PSAT1 and GPX4 proteins were treated with 20 μM erastin, cysteine deprivation or RSL3 (0.5 μM) in the absence or presence of Fer1 (10 μM) for 24 h. Lipid ROS-positive cells and cell death were measured. c,f, MDA-MB-231 and BT549 cells with reconstituted expression of the indicated proteins were treated with different doses of erastin for 40 h or RSL3 for 24 h, and cell viability was measured. Data are the mean ± s.d., two-tailed Student’s t test. All experiments were repeated three times independently with consistent results.
Extended Data Fig. 7 CAMK2–PSAT1–GPX4 axis attenuates ICB therapeutic efficacy.
a–c, 4T-1 cells with expression of indicated proteins were subcutaneously (a,b) or mammary pad-orthotopically (c) injected into BALB/c female mice (n = 6 per group). These mice were given anti-mPD-1 antibody (100 μg/100 µL) or control hamster IgG (100 μg/100 µL) by i.p. injection starting on the seventh day after tumor inoculation and treated every 3 days a time for a total of five treatments. Liproxstatin-1 (Lip-1) was intraperitoneally injected daily from the 4th day at a dose of 10 mg/kg until the end point at day 28 (a). Tumor volumes (top) and tumor weight (bottom) were calculated. Bioluminescence imaging (BLI) images (c, left) and quantification of BLI signals on day 28 (c, right) were shown. Data are presented as the mean ± s.d., two-tailed Student’s t test.
Extended Data Fig. 8 Manipulating PSAT1–GPX4 boosts antitumor immune response.
a–d, 4T-1 cells with expression of the indicated proteins were mammary pad-orthotopically injected into BALB/c female mice (n = 6 per group). These mice were given anti-mPD-1 antibody (100 µg/100 µl) or control hamster IgG (100 µg/100 µl) by i.p. injection starting on the seventh day after tumor inoculation and treated every 3 days a time for a total of five treatments. Intratumoral cells were collected for flow cytometry (FACS) analyses. Quantification for IFNγ+ CD8+ T cells, GzmB+ CD8+ T cells and perforin+ CD8+ T cells (a,b) and IFNγ+ CD4+ T cells, GzmB+ CD4+ T cells and perforin+ CD4+ T cells (c,d) were shown. Data are presented as the mean ± s.d., two-tailed Student’s t test.
Extended Data Fig. 9 Improved ICB efficacy by PSAT1 or GPX4 mutant requires IFNγ.
a–d, 4T-1 cells with expression of the indicated proteins were mammary pad-orthotopically injected into BALB/c female mice (n = 6 per group). These mice were given anti-mPD-1 antibody (100 µg/100 µl) or control hamster IgG (100 µg/100 µl) by i.p. injection starting on the seventh day after tumor inoculation and treated every 3 days a time for a total of five treatments. Mice are pretreated with or without IFNγ antibodies before and during therapy with anti-PD-1 antibodies. Bioluminescence imaging (BLI) images (a,b, top) and quantification of BLI signals on day 28 (a,b, bottom) were shown. Data are the mean ± s.d., two-tailed Student’s t test. IHC analyses of the indicated tumors from BABL/c mice were performed with the indicated antibodies. The indicated staining scores for the indicated tumor samples were compared using two-tailed Mann–Whitney U test (c,d).
Extended Data Fig. 10 PSAT1 pS337 blockade improves ICB therapeutic efficacy.
a–c, Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. Representative results from at least three independent experiments were shown. a,b, The indicated MDA-MB-231 were treated with the indicated peptides with or without IFNγ (25 ng ml−1) for 2 h. d, MDA-MB-231 cells were treated with CHX for the indicated time together with IFNγ (25 ng ml−1) and the indicated peptides for 9 h. Relative intensity of GPX4 to β-tubulin was analyzed. Data are the mean ± s.d., n = 6 independent experiments, two-tailed Student’s t test. d,e, MDA-MB-231 and BT549 cells were treated with IFNγ (100 ng ml−1) and the indicated peptides in the absence or presence of Fer1 (10 μM) for 24 h. Lipid ROS-positive cells (d) and cell death (e) were measured. Data are the mean ± s.d., two-tailed Student’s t test. All experiments were repeated three times independently with consistent results. f–i, 4T-1 cells were mammary pad-orthotopically injected into BALB/c female mice (n = 6 per group). These mice were given anti-mPD-1 antibody (100 μg/100 µl) or control hamster IgG (100 μg/100 µl) by i.p. injection starting on the seventh day after tumor inoculation and treated every 3 days a time for a total of five treatments. The resulting tumors were resected 28 days after injection (f), and tumor weight (g) was calculated. Data are presented as the mean ± s.d., two-tailed Student’s t test. IHC analyses of the indicated mammary pad-orthotopic tumors from BALB/c mice were performed with the indicated antibodies. Representative staining images are shown (h). The indicated staining scores for the indicated tumor samples were compared using two-tailed Mann–Whitney U test (i). Data are the mean ± s.d.
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Zheng, P., Hu, Z., Shen, Y. et al. PSAT1 impairs ferroptosis and reduces immunotherapy efficacy via GPX4 hydroxylation. Nat Chem Biol 21, 1420–1432 (2025). https://doi.org/10.1038/s41589-025-01887-3
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DOI: https://doi.org/10.1038/s41589-025-01887-3
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