Oncometabolite fumarate impairs ATR-CHK1 signaling by succinating RPA1 in fumarate hydratase-deficient renal cell carcinoma cells.

backgroundFumarate hydratase (FH) is a crucial enzyme that converts fumarate to malate in the tricarboxylic acid cycle. FH dysregulation results in abnormal accumulation of oncometabolite fumarate, which drives susceptibility in fumarate hydratase-deficient renal cell carcinoma (FH-dRCC), but the precise mechanisms remain not fully understood.

methodsSuccinated proteins in UOK268 cells were identified by LC–MS/MS. FH wild-type and FH mutant cells were treated with ionizing radiation or hydroxyurea, and ATR-CHK1 activation was analyzed by Western blotting. Cell cycle distribution was assessed using a cell cycle arrest assay, and homologous recombination assay was performed to evaluate the efficiency of DNA double-strand break repair. Succination sites of replication protein A1 (RPA1) were determined by LC–MS/MS. RPA1 DNA-binding activity was examined by electrophoretic mobility shift assay.

resultsHigh fumarate levels impair activation of ATR-CHK1 signaling in response to replication stress and DNA damage. Mechanistically, fumarate modifies RPA1, an essential factor for ATR-CHK1 activation through succination, a post-translational modification. Succination of RPA1 occurs mainly at cysteine residues 481 and 486, which reduces its binding affinity for single-stranded DNA (ssDNA). RPA1 succination leads to deficient recruitment of TOPBP1 to ssDNA, resulting in attenuated CHK1 activation and defective cell cycle arrest in response to DNA damage. Succinated RPA1 compromises homologous recombination-mediated DNA repair.

conclusionOur findings establish that fumarate-induced succination of RPA1 impairs DNA repair and cell cycle control, promoting genomic instability in FH-dRCC. This work reveals a novel mechanism by which oncometabolites contribute to genomic instability.