Abstract A034: USP1 inhibition disrupts DNA repair and potentiates the cytotoxic effects of DNA-damaging agents via persistent DNA damage in cancer cells

Abstract Ubiquitin-specific protease 1 (USP1) is a critical regulator of DNA damage response (DDR) pathways, exerting its function via the deubiquitination of key DNA repair substrates, including FANCD2 and proliferating cell nuclear antigen (PCNA). These deubiquitination events are essential for the functional integrity of the Fanconi anemia (FA) pathway and translesion DNA synthesis (TLS), both of which are vital for resolving DNA interstrand crosslinks and replication-associated DNA lesions. DNA-damaging agents, a broad class of therapeutics exert their cytotoxic effects by inducing various forms of DNA lesions, including base modifications, single-strand breaks (SSBs), double-strand breaks (DSBs), and DNA crosslinks. These lesions disrupt DNA replication and transcription, triggering replication stress, genomic instability, and ultimately cell death if left unrepaired. However, cancer cells often activate DDR pathways that coordinate DNA repair, cell cycle checkpoints, and survival responses, which can confer resistance to DNA-damaging treatments by enhancing lesion resolution and maintaining genomic integrity. Our previous studies showed that USP1 inhibition impaired the resolution of DNA lesions, resulting in persistent activation of DNA damage markers such as γH2AX. This indicates that USP1 inhibition could deepens the genotoxic stress imposed by DNA-damaging agents and promotes apoptotic cell death by blocking effective DNA repair In this study, we investigated whether pharmacological inhibition of USP1 could enhance the cellular response to DNA-damaging agents by disrupting key DNA repair mechanisms. Human cancer cell lines were treated with our proprietary USP1 inhibitor, VRTX531, either alone or in combination with DNA-damaging agents. VRTX531 treatment alone led to the accumulation of monoubiquitinated FANCD2 and PCNA, confirming effective USP1 inhibition and disruption of FA and TLS pathway activity. Combined treatment with VRTX531 and DNA-damaging agents resulted in a synergistic increase in cytotoxicity, as reflected by significantly reduced cell viability relative to DNA damage alone. These findings underscore the broader therapeutic potential of USP1 inhibition in not only sensitizing cancer cells to DNA-damaging agents but also overcoming chemo-resistance driven by DDR pathway activation. Citation Format: Prashant K. Bhavar, Uday K. Surampudi, Partha Pratim. Sarma, Appaji Mandhare, Neetu Singh, Nandini Narayani, Anuj Kshisagar. USP1 inhibition disrupts DNA repair and potentiates the cytotoxic effects of DNA-damaging agents via persistent DNA damage in cancer cells [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 A034.