Abstract A010: Synergistic ovarian cancer killing by combination of PARG and USP1 inhibitors: Role of single stranded DNA gaps as a predictive biomarker

Abstract Poly(ADP-ribose) glycohydrolase (PARG), the primary de-PARylating enzyme, plays a critical role in resolving DNA damage. PARG inhibitors (PARGi) are currently in clinical development for patients with tumors exhibiting homologous recombination deficiency (HRD). PARGi induces toxic single-stranded DNA gaps (ssGAPs) in ovarian cancer models by exacerbating S-phase PARylation and causing Okazaki fragment processing (OFP) defects (https://www.pnas.org/doi/10.1073/pnas.2413954121). Similarly, inhibition of USP1, a deubiquitinating enzyme, promotes the accumulation of ssGAPs during replication in BRCA1-deficient cells. Notably, cellular sensitivity to either PARGi or USP1 inhibitor (USP1i) correlates strongly with ssGAP formation, identifying ssGAPs as a predictive biomarker of drug response. Recently CRISPR screens with USP1 inhibitors have identified OFP genes (e.g., PARG, PARP1, FEN1) as USP1i sensitizers. Given the clinical challenges associated with USP1i monotherapy, there is increasing interest in rational combination strategies that maintain efficacy while potentially improving tolerability. Here we show that co-targeting PARG and USP1 induces a novel synergistic interaction in ovarian cancer models by amplifying replication stress and DNA damage. USP1i augmented the PARGi-induced PARylation at DNA replication forks, leading to a greater increase in ssGAPs gaps, compared to treatment with either single agent alone. This dual inhibition led to accumulation of unresolved replication intermediates, culminating in mitotic catastrophe, as demonstrated by increased micronuclei formation and chromosomal aberrations. Importantly, the PARGi and USP1i combination also enhanced tumor cell killing in a subset of PARP-inhibitor-resistant BRCA1-deficient tumors. Mechanistically we identified RAD18-mediated PCNA ubiquitination as a key driver of synergistic lethality. Loss of RAD18 rescued fork-associated PARylation, reduced replication stress, and suppressed ssGAPs thereby mitigating the combined effects of USP1 and PARG inhibition and causing drug resistance. Consistently, USP1 knockout cells exhibited increased sensitivity to PARGi , which was reversed by RAD18 depletion, highlighting RAD18’s essential role in translesion synthesis-mediated ssGAP filling and combination-induced lethality. Notably, USP1i and PARGi also synergized with other ssGAP-inducing agents (e.g., FEN1 inhibitors), suggesting broader combinatorial potential. Finally, we observed synergistic activity in specific HRD patient derived ovarian cancer organoids (PDOs). Our data provides novel mechanistic insights into the therapeutic potential of combining these inhibitors, offering preclinical evidence to support its advancement for treating tumors with and without HRD and addressing PARPi resistance. Ovarian cancer PDOs enable rapid sensitivity testing, with ssGAP accumulation serving as a potential predictive biomarker. This combination strategy holds promise for enhancing treatment efficacy while reducing toxicity, paving the way for more targeted and effective therapeutic options. Citation Format: Ramya Ravindranathan, Nicholas William. Ashton, Alexandre Andre. Da Costa, Ozge Somuncu, Ben Lamarre, Sirisha Mukkavalli, Kalindi Parmar, Geoffrey Shapiro, Alan D'Andrea. Synergistic ovarian cancer killing by combination of PARG and USP1 inhibitors: Role of single stranded DNA gaps as a predictive biomarker [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A010.