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A chemoproteomic portrait of the oncometabolite fumarate

Abstract

Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth. Understanding these mechanisms thus represents a vital goal. Toward this goal, here we report a chemoproteomic map of fumarate, a covalent oncometabolite whose accumulation marks the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). We applied a fumarate-competitive chemoproteomic probe in concert with LC–MS/MS to discover new cysteines sensitive to fumarate hydratase (FH) mutation in HLRCC cell models. Analysis of this dataset revealed an unexpected influence of local environment and pH on fumarate reactivity, and enabled the characterization of a novel FH-regulated cysteine residue that lies at a key protein–protein interface in the SWI-SNF tumor-suppressor complex. Our studies provide a powerful resource for understanding the covalent imprint of fumarate on the proteome and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.

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Fig. 1: Fumarate is a covalent oncometabolite.
Fig. 2: Global chemoproteomic profiling of FH-regulated cysteine residues
Fig. 3: Analyzing the reactivity and abundance of FH-regulated cysteines.
Fig. 4: Establishing the molecular determinants of fumarate–protein interactions.
Fig. 5: Functional analyses of FH-regulated Cys residues.
Fig. 6: Chemoproteomic discovery of ligandable cysteines upregulated by FH mutation.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its supplementary information files. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the identifiers PXD009378 (Supplementary Datasets 14) and PXD009202 (Supplementary Dataset 5). All of the data are accessible in the supplemental data sets (Supplementary Datasets 17) and can further be explored using our web-based resource (https://ccr2.cancer.gov/resources/Cbl/proteomics/fumarate).

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Acknowledgements

The authors thank C. Grose (Protein Expression Laboratory) for cloning and preparation of plasmid DNA, T. Archer (NIEHS) for the gift of the SMARCC1 and SNF5 plasmids, B. Weinberg (MIT) for the gift of the pLKO.1 puro plasmid (Addgene plasmid # 8453), A. Roberts, J. Garlick, and T. Zengeya (NCI) for assisting with preliminary studies. This work was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research (ZIA BC011488-05, ZIA BC011038-10) and the CCR FLEX Program. Support for E.W. was provided by the NIH (1R01GM117004 and 1R01GM118431-01A1).

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Authors

Contributions

R.A.K., D.W.B., E.W. and J.L.M. designed experiments. R.A.K. and D.W.B. performed all chemical proteomic labeling and enrichment experiments. D.W.B. and E.W. performed all LC–MS/MS studies and relative stoichiometry analyses of IA-alkyne enrichment experiments. R.A.K. and A.E.B. synthesized compounds. R.A.K., S.E.B. and J.H.S. performed all cell-based analyses and co-immunoprecipitation experiments. C.A.B. assisted with cell-based analyses and performed S-succination reversibility studies. J.L.M., D.W.B., A.L.T. and R.A.K. performed bioinformatics analyses. D.W. and W.M.L. performed HLRCC spheroid growth inhibition studies and assisted with SWI–SNF analyses. A.A. and E.C.D. performed glycerol gradient fractionation analysis of the SWI–SNF complex and SNF5-dependent gene expression in HLRCC cell lines. N.F. provided the S-succination antibody and literature analysis. W.M.L. and D.R.C. provided HLRCC cell lines and advised experimental design. M.P.W., L.F. and M.L. performed whole-proteome MudPIT S-succination analyses of HLRCC cells. R.A.K. and J.L.M. analyzed data and wrote the manuscript with input from all authors.

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Correspondence to Jordan L. Meier.

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Supplementary information

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Supplementary Figures 1–10

Reporting Summary

Supplementary Note 1

Synthetic Procedures

Supplementary Dataset 1

FH-regulated cysteines identified by comparative profiling of FH−/− HLRCC cell line (UOK262) and a FH+/+ rescue HLRCC cell line (UOK262WT); n = 3 independent experiments.

Supplementary Dataset 2

Compiled list of S-succinated cysteine residues previously characterized in the literature, and annotation with chemoproteomic data (if available).

Supplementary Dataset 3

Sequences used for motif analysis, as well as results for analyses of conservation based functional impact (FI), gene ontology (GO), and genomic lesions found in covalent fumarate targets in kidney cancer.

Supplementary Dataset 4

Fumarate-sensitive cysteines identified by competitive profiling of HEK-293 cells treated and untreated with fumarate (1 mM); n = 4 independent experiments.

Supplementary Dataset 5

Peptides identified as targets of S-succination in MudPIT LC–MS/MS analyses of HLRCC cell (UOK262 and UOK268) proteomes.

Supplementary Dataset 6

Analysis of transcripts co-regulated by FH and SNF5 in publicly accessible RNA-seq datasets.

Supplementary Dataset 7

Solvent-exposed surface area analysis of FH-regulated (Supplementary Dataset 1), exogenous fumarate sensitive (Supplementary Dataset 4), and hyperreactive cysteines.

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Kulkarni, R.A., Bak, D.W., Wei, D. et al. A chemoproteomic portrait of the oncometabolite fumarate. Nat Chem Biol 15, 391–400 (2019). https://doi.org/10.1038/s41589-018-0217-y

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