Inhibition of Translesion DNA Synthesis Sensitizes BRCA-Deficient Ovarian Cancer to PARP Inhibitors

Abstract Poly(ADP-ribose) polymerase inhibitors (PARPi) exploit synthetic lethality to treat BRCA1/2-mutated ovarian cancer, yet clinical responses are often incomplete and resistance frequently emerges. PARPi exert cytotoxic effects by inhibiting single-strand break repair and trapping PARP on DNA, which impedes replication fork progression and generates lethal double-strand breaks. However, tumor cells can mitigate these effects through activation of alternative DNA repair mechanisms. Here, we investigated the role of translesion synthesis (TLS), a DNA damage tolerance pathway in which specialized polymerases bypass DNA lesions to sustain replication, in mediating PARPi resistance using homologous recombination (HR)-deficient epithelial ovarian cancer cell lines, patient-derived organoids (PDOs), and orthotopic xenograft models. PARPi treatment markedly slowed replication fork progression and activated TLS. Genetic depletion of the TLS scaffold protein REV1 and TLS polymerase η (Polη, encoded by POLH) exacerbated olaparib-induced replication stress, impaired fork restart, and sensitized BRCA1/2-deficient cells to PARPi. Importantly, pharmacological TLS inhibition with JH-RE-06 synergized with olaparib to enhance cytotoxicity in HR-deficient cells and PDOs, while in vivo combination therapy delayed or prevented resistance and achieved sustained tumor regression. Notably, TLS blockade also suppressed the emergence of BRCA2 reversion mutations in BRCA2-mutated ovarian cancer cells and prevented the development of olaparib resistance during chronic olaparib exposure. Collectively, these findings demonstrate that TLS, particularly Polη-dependent bypass, is a critical mechanism enabling replication fork rescue and survival under PARPi therapy. Targeting TLS provides a promising therapeutic strategy to augment PARPi efficacy and prevent resistance in BRCA-deficient ovarian cancer.