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Autopalmitoylation of IDH1-R132H regulates its neomorphic activity in cancer cells

Nature Chemical Biology (2026)Cite this article

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Abstract

Gain-of-function mutations of isocitrate dehydrogenase 1 (IDH1) lead to oncometabolite (R)-2-hydroxyglutarate production, contributing to the tumorigenesis of multiple human cancers. While fatty acid biosynthesis is critical for IDH1-mutant tumor growth, the underlying mechanisms remain unclear. Here, leveraging chemical probes and chemoproteomic profiling, we identified that oncogenic IDH1-R132H is uniquely autopalmitoylated at C269, which is not observed in wild-type IDH1. This modification responds to fatty acids and regulates R132H enzymatic activity by enhancing substrate and cofactor binding, as well as dimerization. Loss of C269 palmitoylation reverses IDH1-R132H-induced metabolic reprogramming and hypermethylation phenotypes and impairs cell transformation. Interestingly, C269 autopalmitoylation occurs within a hydrophobic pocket, targeted by a clinical IDH1-mutant inhibitor (LY3410738). Our study reveals that autopalmitoylation, conferred by the IDH1R132H mutation, links fatty acid metabolism to the regulation of IDH1 mutant activity and represents a druggable vulnerability in IDH1-mutant cancers.

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Fig. 1: Chemoproteomic profiling reveals that IDH1 has reactive cysteine residues near a hydrophobic pocket.
Fig. 2: Cys269 is specifically autopalmitoylated in mutant IDH1.
Fig. 3: Cys269 autopalmitoylation enhances the enzymatic activity of IDH1mt.
Fig. 4: IDH1mt autopalmitoylation regulates the binding of cofactor and substrate and promotes dimerization.
Fig. 5: IDH1mt C269 autopalmitoylation regulates 2-HG production and induces metabolic alterations in IDH1-mut cells.
Fig. 6: IDH1mt C269 autopalmitoylation induces epigenetic alterations and drives cell transformations.

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Data availability

The raw RNA-seq data were deposited to the National Center Biotechnology Information Gene Expression Omnibus under accession number GSE260937. The raw proteomics data were deposited to the ProteomeXchange Consortium through the PRIDE partner repository with the dataset identifier PXD070770. The atomic models and cryo-EM density maps were deposited to the Protein Data Bank and EM Data Bank under the following accession numbers: IDH1-R132H, PDB 9YHA and EMD-72964; mtC269S, PDB 9YHB and EMD-72965. Statistical source data for the supplementary figures are available in Supplementary Data 3. Initial and final configurations of all the structures generated in the MD simulations are available in Supplementary Data 4 and 5, respectively. All other data are available in the main text or Supplementary Information. Source data are provided with this paper.

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  • 16 January 2026

    In the version of the article initially published, the author of ref. 30 was given as “Dixon, S. J.” but should have been “Kahlson, M. A. et al”. This correction has been made to the HTML and PDF versions of the article.

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Acknowledgements

We thank MGH Executive Committee on Research (ECOR) Interim Support Funding to X.W. for supporting the work. L.H. is partly supported by a postdoctoral fellowship from the Antidote Health Foundation for Cure of Cancer and the MGH ECOR Fund for Medical Discovery Fundamental Research Fellowship Award. Y.S. is partly supported by Natural Science Foundation of China grant 8251101517. The work in the S.S. lab was supported by an award from the Canada Excellence Research Chair program. We thank Q. Xu, C. Braithwaite and V. Alvarado for technical assistance, the MGH confocal image core for fluorescence imaging and the Taplin mass spec core at Harvard Medical School for chemoproteomic profiling.

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Authors and Affiliations

  1. Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA

    Lu Hu, Yang Sun, Jianan Zhang & Xu Wu

  2. iHuman Institute, Shanghai Tech University, Shanghai, China

    Jinyu Lin, Liping Sun & Yibing Shan

  3. Department of Biochemistry & Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada

    Alison M. Berezuk, Katharine S. Tuttle, Xing Zhu & Sriram Subramaniam

  4. Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Hyuk-Soo Seo & Sirano Dhe-Paganon

  5. Chemical Biology Program, Dana-Farber Cancer Institute, Boston, MA, USA

    Hyuk-Soo Seo & Sirano Dhe-Paganon

  6. Cancer Institute, Xuzhou Medical University, Xuzhou, China

    Pan Li & Yang Sun

  7. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Lisheng Ni, Xiaochen Bai & Xuelian Luo

  8. Antidote Health Foundation for Cure of Cancer, Cambridge, MA, USA

    Dazhi Tan & Yibing Shan

  9. Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    Hiroaki Wakimoto & Daniel P. Cahill

  10. Mass Spectrometry Core, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    John M. Asara

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Contributions

L.H. contributed to project conceptualization, synthesis, biochemical and cellular data curation, analysis and manuscript preparation. A.M.B, K.S.T., X.Z. and S.S. contributed to cryo-EM sample preparation, data processing and analysis. H.-S.S and S.D.-P. contributed to ITC data acquisition and analysis. P.L. and Y.S. contributed to RNA-seq data analysis. L.N., X.B. and X.L. contributed to CD spectrum acquisition and analysis. L.S., J.L., D.T. and Y.S. contributed to structural analysis, MD simulations and manuscript preparation. J.Z. tested the expression levels of ZDHHC14/23 in knockdown experiments. H.W. and D.P.C. contributed to experiments using MGG152 cells and revised the manuscript. J.M.A contributed to metabolomic profiling and analysis. X.W. contributed to project conceptualization, design, analysis and manuscript preparation.

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Correspondence to Lu Hu, Yibing Shan or Xu Wu.

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Competing interests

L.H. and X.W. are inventors of a patent application covering chemical probes for chemoproteomic profiling. D.P.C. has received financial compensation from Lilly (2020), Boston Pharmaceuticals (2020), GlaxoSmithKline (2021), Pyramid Biosciences (equity interest 2021), the Massachusetts Institute of Technology (2022), Incephalo (2023), Servier (2023) and Boston Scientific (2023) for advisory input. D.P.C. has also received financial compensation and travel reimbursement from Merck for invited lectures (2016) and from the US National Institutes of Health and Department of Defense for clinical trial and grant review (2023). S.S. is the founder and chief executive officer of Gandeeva Therapeutics. X.W. has financial interest in Tasca Therapeutics, which is developing small-molecule modulators of TEAD palmitoylation and transcription factors. X.W.’s interests were reviewed and are managed by MGH and Mass General Brigham in accordance with their conflict of interest policies. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 B4 is an activity-based probe for the autopalmitoylated proteome.

a-b, Comparison of labeling efficiency of different probes in HEK293A lysate (a) or live cells (b). A21, A63, B1, B4: 10 µM; alkyne 2-BP: 50 µM. c, Competition of B4 (0.1 µM) labeling of His-TEAD2 by palmitoyl-CoA. d, Inhibition of His-TEAD2 autopalmitoylation by non-tag-B4. Alkyne palmitoyl-CoA: 1 µM. e, Competition of B4 (0.1 µM) labeling of His-Bet3 by palmitoyl-CoA. f, Inhibition of His-Bet3 autopalmitoylation by non-tag-B4. Alkyne palmitoyl-CoA: 1 µM. g, Mutation of C359 blocked the labeling of Myc-TEAD1 by B4 (10 µM) in HEK293A cells. h, Labeling of endogenous ZDHHC7 by B4 (10 µM) in HEK293A cells. i, Mutation of C160 inhibited the labeling of HA-ZDHHC7 by B4 (10 µM). j-k, Gel-based profiling of autopalmitoylated proteome with B4 (10 µM) in HEK293A cells (j) or lysate (k) at different time points. The experiments in a-k were repeated independently three times with similar results and representative results are presented.

Source data

Extended Data Fig. 2 Both IDH1 WT and R132H are S-fatty acylated.

a, Streptavidin pulldown and western blot analysis showing that exogenous IDH1wt and IDH1mt were palmitoylated in HEK293A cells. Alk-16C: 50 µM. Relative palmitoylation levels of IDH1 were normalized to Input and indicated. b, Streptavidin pulldown and western blot analysis showing that endogenous IDH1wt and IDH1mt were palmitoylated in HEK293A and MGG152 cells, respectively. c, Fatty acid preference of IDH1wt/mt lipidation using chemical probes (50 µM) with different carbon-chain lengths. Relative lipidation levels of IDH1 were normalized to Input and indicated. d, Treatment of hydroxylamine (1 M) abolished palmitoylation signals of both IDH1wt and IDH1mt. e, ABE assay showing that endogenous IDH1wt and IDH1mt were palmitoylated at cysteine residues in HEK293A and MGG152 cells, respectively. f, ABE assay showing that exogenous IDH1wt and IDH1mt were palmitoylated at cysteine residues. g, Analysis of palmitoylation levels of IDH1wt and IDH1mt using various site-directed mutants. Relative palmitoylation levels of IDH1 were normalized to Input and indicated. h, Measurement of Km value of His-IDH1wt autopalmitoylation in vitro (n = 3 independent experiments). Top, representative western blot images. Bottom, the plot for the Km value calculation. Data were shown as mean ± SEM. i-j, Biotin switch to assess disulfide bond (i) and S-sulfenylation (j) of Flag-IDH1mt and Flag-IDH1mt C269S in HEK293A cells. TCEP, 10 mM; m-Arsenite, 200 mM. The experiments in a-g and i-j were repeated independently three times with similar results, and representative results are presented.

Source data

Extended Data Fig. 3 C269 palmitoylation was observed in other IDH1 mutants.

a, Mutation of C269 substantially decreased palmitoylation signals of IDH1 R132G and R132S. b, Mutation of C269 blocked the labeling of IDH1 R132G and R132S by B4 (10 µM). c, Mutation of C269 did not decrease palmitoylation signals of IDH1 R132C. d, Mutation of C269 decreased the labeling of IDH1 R132C by B4 (10 µM). The experiments in a-d were repeated independently three times with similar results, and representative results are presented.

Source data

Extended Data Fig. 4 C269 palmitoylation of mutant IDH1 was not mediated by ZDHHC family enzymes.

a-c, Screening of mouse Zdhhc-PATs that are responsible for IDH1mt palmitoylation. HEK293A cells were co-transfected with Flag-IDH1mt and HA-Zdhhcs. Palmitoylation signals of IDH1mt were determined by streptavidin pulldown and western blot analysis. d, Analysis of palmitoylation levels of IDH1mt 3CS (C73S/C114S/C379S) after knocking down endogenous ZDHHC14 or 23 using shRNA. Bar graph showing knockdown efficiency of ZDHHC14 or ZDHHC23 at the mRNA level (n = 3 independent experiments). Data were shown as mean ± SEM. Statistical significance was assessed using an unpaired, two-sided Student’s t-test. e-f, Analysis of palmitoylation levels of IDH1mt 3CS after knocking down endogenous ZDHHC7 (e) or 9 (f) using siRNAs. g, Analysis of palmitoylation levels of IDH1mt 3CS after individually overexpressing Zdhhc7/9/14/23. h, Co-IP assays showing there were no interactions between IDH1mt 3CS and Zdhhc7, 9, 14 or 23. The experiments in a-c and e-h were repeated independently three times with similar results, and representative results are presented.

Source data

Extended Data Fig. 5 IDH1mt autopalmitoylation induced transcriptional reprogramming in IDH-mut cells.

a, 3D PCA plot with genes plotted in three dimensions using their projections onto the first three principal components, and nine samples plotted using their weights for the components (n = 3 independent experiments). Green, control group. Red, mtC269S group. Blue, IDH1mt group. b, Heatmap analysis of global gene expression in control, IDH1mt and mtC269S U87 cells (n = 3 independent experiments). c, GO enrichment analysis with differential genes in IDH1mt and mtC269S cells showing the most significant 10 upregulated biological processes. d, DO enrichment analysis with differential genes in IDH1mt and mtC269S cells showing the most significant 20 DO Terms. Statistical significance in c-d was assessed using a hypergeometric test and adjusted for multiple comparisons using the Benjamini-Hochberg false discovery rate (FDR) method.

Source data

Supplementary information

Supplementary Information (download PDF )

Supplementary Tables 1–4, Figs. 1–16, Note and Source data for blots in supplementary figures.

Reporting Summary (download PDF )

Supplementary Data 1 (download XLSX )

List of proteins identified in the chemoproteomic profiling with B4.

Supplementary Data 2 (download XLSX )

List of metabolites identified in the metabolic profiling of U87 cells.

Supplementary Data 3 (download XLSX )

Statistical source data for supplementary figures.

Supplementary Data 4 (download TXT )

Initial configurations for all structures generated in MD simulations.

Supplementary Data 5 (download TXT )

Final configurations for all structures generated in MD simulations.

Supplementary Table 5 (download XLSX )

Sequences of primers.

Supplementary Video 1 (download MP4 )

Supplementary videos for MD simulations.

Source data

Source Data Fig. 1 (download PDF )

Unprocessed western blots and/or gels.

Source Data Fig. 2 (download PDF )

Unprocessed western blots and/or gels.

Source Data Fig. 2 (download XLSX )

Statistical source data.

Source Data Fig. 3 (download PDF )

Unprocessed western blots and/or gels.

Source Data Fig. 3 (download XLSX )

Statistical source data.

Source Data Fig. 4 (download PDF )

Unprocessed western blots and/or gels.

Source Data Fig. 4 (download XLSX )

Statistical source data.

Source Data Fig. 5 (download PDF )

Unprocessed western blots and/or gels.

Source Data Fig. 5 (download XLSX )

Statistical source data.

Source Data Fig. 6 (download PDF )

Unprocessed western blots and/or gels.

Source Data Fig. 6 (download XLSX )

Statistical source data.

Source Data Extended Data Fig. 1 (download PDF )

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Source Data Extended Data Fig. 2 (download PDF )

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 2 (download XLSX )

Statistical source data.

Source Data Extended Data Fig. 3 (download PDF )

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 4 (download PDF )

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 4 (download XLSX )

Statistical source data.

Source Data Extended Data Fig. 5 (download XLSX )

Statistical source data.

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Hu, L., Lin, J., Sun, L. et al. Autopalmitoylation of IDH1-R132H regulates its neomorphic activity in cancer cells. Nat Chem Biol (2026). https://doi.org/10.1038/s41589-025-02131-8

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