Abstract Background: While PARP inhibitors (PARPi) have demonstrated clinical benefit in patients with BRCA1/2-mutated ovarian cancer by exploiting synthetic lethality, only a subset of patients respond, and many eventually acquire resistance. PARPi exert cytotoxic effects by blocking single-strand break repair and trapping PARP on DNA, resulting in replication fork stalling and subsequent double-strand break formation. However, cells may circumvent these effects through translesion DNA synthesis (TLS), a DNA damage tolerance pathway that allows replication to continue past DNA lesions using specialized polymerases, potentially leading to replication fork recovery and reduced therapeutic efficacy. We hypothesized that TLS contributes to PARPi resistance in BRCA-deficient ovarian cancer by enabling cells to bypass PARP-DNA complexes and avoid lethal fork collapse. Methods: We used a panel of homologous recombination (HR) -deficient epithelial ovarian cancer (EOC) cell lines and patient-derived organoids (PDOs) to investigate the role of TLS in mediating resistance to PARPi. DNA fiber assays were performed to assess replication fork dynamics following treatment with olaparib or talazoparib. TLS activity was disrupted genetically by depleting REV1 and POLH or pharmacologically using the TLS inhibitor JH-RE-06. Synergistic effects with olaparib were evaluated in vitro. In vivo efficacy and resistance prevention were assessed using both orthotopic xenograft and syngeneic ovarian cancer mouse models. Results: PARPi treatment significantly increased replication fork stalling and the expression of monoubiquitinated PCNA, a key marker of TLS activation. Depletion of REV1, a key factor in TLS, sensitized BRCA2-deficient PEO1 cells to olaparib and intensified replication fork stalling, indicating that TLS is functionally activated to counteract PARPi-induced replication stress. Among TLS polymerases, POLη (encoded by POLH) played a predominant role in facilitating fork progression and promoting cell survival following PARPi treatment. The TLS inhibitor JH-RE-06 synergized with olaparib to induce greater cytotoxicity in HR-deficient EOC cells and PDOs. In vivo, JH-RE-06 enhanced the anti-tumor efficacy of olaparib and delayed or prevented the emergence of PARPi resistance in both BRCA2-deficient xenograft and syngeneic ovarian cancer models. Notably, long-term olaparib exposure led to secondary BRCA2 mutations and acquired resistance in PEO1 cells, while no such mutations or resistant clones emerged in POLH-depleted PEO1 cells, suggesting that TLS may promote the development of acquired PARPi resistance by facilitating secondary BRCA1/2 mutations and partial restoration of HR. Conclusion: Our findings reveal that TLS, particularly POLη-mediated bypass, plays a key role in promoting replication fork rescue and survival in BRCA-deficient ovarian cancer treated with PARPi. Inhibiting TLS with JH-RE-06 enhances PARPi sensitivity and prevents resistance, providing a strong rationale for co-targeting TLS in patients with HR-deficient ovarian cancer. Citation Format: Na Li, Qianyun Ge, Yajing Yang, Linzhou Wang, Aidan Li, Jessica Miao, Junran Zhang, Qi-En Wang. Targeting translesion synthesis to enhance PARP inhibitor efficacy and prevent resistance in BRCA-deficient ovarian cancer abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85 (18Suppl): Abstract nr A054.
Li et al. (Fri,) studied this question.