Abstract 6910: Synergistic tumor cell killing with dual inhibition of PARG and USP1

Abstract Poly (ADP-ribose) glycohydrolase (PARG) is an enzyme that aid in DNA repair primarily through the removal of poly (ADP-ribose) (PAR) chains from PARylated proteins. PARG inhibitors are currently in clinical development for patients with tumors exhibiting homologous recombination deficiency (HRD). Recently, we have demonstrated that similar to PARP inhibitors, PARG inhibitors also promote the formation of single stranded DNA gaps (ssGAPs) in preclinical ovarian cancer models. PARGi treatment increases the levels of toxic S phase PARylation resulting from Okazaki fragment processing (OFP) defects. More importantly the cellular sensitivity to PARGi directly correlates with the formation of ssGAPs. Additionally, our recent findings show that inhibitor of USP1, a deubiquitinating enzyme, also promotes the accumulation of ssGAPs during replication in BRCA1-deficient cells, and this phenotype correlates with the drug sensitivity. Interestingly, a CRISPR screen with USP1 inhibitor (USP1i) KSQ-4279 has identified gRNAs against genes encoding proteins in the OFP and base excision repair (BER) pathways, particularly PARG and PARP1, as key sensitizers. Combining PARP inhibitor (PARPi) with USP1i has shown promise in preclinical setting, extending PARPi benefits to tumors without homologous recombination defects and addressing PARPi resistance. In this study, we provide the evidence that PARGi synergizes with USP1i to maximize vulnerability to drug-induced replication stress in a panel of ovarian cancer and breast cancer cells. The combination treatment exacerbated DNA damage and replication stress as detected by an increase in markers such as pKAP1, pRPA, pCHK1 and γH2AX. Notably, USP1i augmented the PARGi-induced S phase PARylation and specifically PARylation at the replication forks. As predicted, the combination treatment induced ssGAP formation in the sensitive cells. Dual inhibition of PARG and USP1 caused an accumulation of unresolved replication intermediates, resulting in mitotic catastrophe as evidenced by a marked increase in micronuclei formation and chromosomal aberrations. Importantly, PARGi and USP1i combination enhanced tumor cell killing in a subset of PARP-inhibitor-resistant BRCA1-deficient tumors. Overall, these results indicate that the combination treatment with a PARGi and a USP1i causes accumulation of toxic PARylation at the replication fork leading to ssGAPs and replication catastrophe. Both PARG inhibitors and USP1 inhibitors are orally available drugs currently in clinical trials. 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 HRD and those resistant to PARP inhibitors. 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, Alexandre A. da Costa, Benjamin P. Lamarre, Sirisha Mukkavalli, Ozge Somuncu, Kalindi Parmar, Geoffrey I. Shapiro, Alan D. D’Andrea. Synergistic tumor cell killing with dual inhibition of PARG and USP1 [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 6910.