PARG Governs a PARylation-Ubiquitination Toggle that Stabilizes RAD51AP1 to Drive Homologous Recombination-Mediated Chemoresistance.

Gastric cancer is a lethal malignancy with limited targeted therapeutic options, particularly for HER2-negative disease. Through integrated bioinformatic analysis of multiple gastric cancer cohorts, we identified poly(ADP-ribose) glycohydrolase (PARG) as a DNA damage response protein that is frequently overexpressed and associated with poor prognosis. Although PARG inhibition as a monotherapy showed limited efficacy, high-throughput screening of FDA-approved drugs revealed a synthetic lethal interaction between PARG inhibition and fluoropyrimidines, including 5-fluorouracil (5-FU). Mechanistically, PARG directly bound the homologous recombination (HR) cofactor RAD51-associated protein 1 (RAD51AP1) via its N-terminal domain and removed PAR chains. The dePARylation prevented RNF169-mediated K48-linked ubiquitination of RAD51AP1, thereby protecting RAD51AP1 from proteasomal degradation. Consequently, loss of PARG destabilized RAD51AP1 and impaired HR repair, resulting in increased sensitivity to fluoropyrimidine-induced DNA lesions. In patient-derived xenograft models, PARG inhibition significantly enhanced the efficacy of both 5-FU and the oral prodrug capecitabine. Moreover, restoring RAD51AP1 expression substantially reversed the chemosensitizing effect conferred by PARG deficiency. Clinically, PARG and RAD51AP1 protein levels were strongly correlated in gastric cancer tissues, and their coexpression defined a subset of patients with the worst survival. Collectively, these findings identify a PARylation-ubiquitination switch regulated by PARG that stabilizes RAD51AP1 to enhance HR repair, unveiling the PARG-RAD51AP1 axis as a key determinant of fluoropyrimidine sensitivity and a biomarker-directed target for combination therapy in gastric cancer. SIGNIFICANCE: PARG inhibition confers synthetic lethality to fluoropyrimidines by disrupting RAD51AP1-mediated homologous recombination, supporting the potential of PARG as a functional determinant of DNA repair capacity and a predictive biomarker for chemotherapy stratification.