Abstract 429: Targeting translesion synthesis to sensitize ovarian cancer cells to PARP inhibitor treatment

Abstract BRCA-deficient tumor cells, characterized by defective homologous recombination (HR) repair, show high sensitivity to poly (ADP-ribose) polymerase inhibitors (PARPi). The mechanism is that PARPi blocks PARP and traps them at DNA damage sites (PARP-DNA complex), inducing replication fork stalling, leading to cancer cell death with BRCA mutation due to the accumulation of unrepaired DNA double-strand breaks. At present, PARP inhibitors have been widely used to treat ovarian cancer patients with deficient HR. However, like other chemotherapy agents, many patients acquire PARPi resistance. Replication fork protection is a key mechanism driving resistance to PARPi. Translesion synthesis (TLS) is an error-prone DNA damage tolerance pathway that can help cancer cells bypass DNA lesions and rescue replication fork stalling with Y-family DNA polymerases. Thus, we hypothesized that enhanced TLS enables BRCA-mutated ovarian cancer cells to overcome PARPi treatment via bypassing PARP-DNA complexes, and rescuing stalled replication forks, eventually leading to BRCA secondary mutations and partial restoration of HR function. To test this hypothesis, we first confirmed that PARPi can promote TLS activity by increasing the expression of monoubiquitinated PCNA in BRCA-mutated ovarian cancer cells. In addition, replication fork stalling has been observed following treatment with PARP inhibitors, as demonstrated by DNA fiber assays. Blocking TLS by knocking down REV1 exacerbates replication fork stalling, highlighting the interplay between PARPi treatment and TLS pathways. To further investigate the role of TLS polymerases, we individually knocked down TLS polymerases and evaluated their effects. Knocking down POLH (encode Polη), exacerbated Olaparib-induced replication fork stalling, irrespective of BRCA status. Functional analyses revealed that POLH knockdown sensitized BRCA-mutated cells to Olaparib but had no significant effect on BRCA wild-type cells. Next, we utilized a newly developed ChIP-based assay to assess TLS efficiency. We demonstrated that TLS inhibition through knocking down POLH or using a TLS inhibitor JH-RE-06 impairs DNA replication across Olaparib-induced PARP1-DNA complexes. Furthermore, JH-RE-06 sensitized HR-deficient ovarian cancer cell lines and patient-derived organoids to Olaparib in vitro. It also enhanced the efficacy of Olaparib in vivo, both in NSG mice bearing human BRCA1-mutated ovarian cancer cells and C57BL/6 mice with murine ovarian cancer cells harboring P53-/- and BRCA1-/- genotypes. In summary, our findings reveal that TLS facilitates replication fork recovery by enabling DNA replication across PARPi-induced PARP-DNA complexes, with Polη playing a key role. Importantly, JH-RE-06 represents a promising strategy to enhance the therapeutic sensitivity of HR-deficient ovarian cancer cells to PARP inhibitors like Olaparib. Citation Format: Na Li, Qianyun Ge, Yajing Yang, Linzhou Wang, Xiaoli Zhang, Junran Zhang, Zaibo Li, Floor Backes, Qi-En Wang. Targeting translesion synthesis to sensitize ovarian cancer cells to PARP inhibitor treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 429.