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Tumors with defects in DNA double strand break (DSB) repair pathways, including those with mutations in BRCA1/2, are often treated with either poly (ADP-ribose) polymerase-1 (PARP1) inhibitors or platinum-based chemotherapy. Despite their clinical benefits, many patients develop resistance leading to the need for novel therapeutic approaches. To address this unmet clinical need, we applied our CRISPRomics® platform to identify novel therapeutic targets for DNA damage and repair deficient cancers. One of the top ranked targets was the deubiquitinating enzyme, USP1, with known functions in DNA damage repair pathways of translesion synthesis (TLS) and fanconi anemia (FA). We developed a novel small molecule inhibitor, KSQ-4279, that selectively inhibits USP1 and leads to accumulation of mono-ubiquitinated USP1 substrates such as proliferating nuclear antigen (PCNA). In addition to KSQ-4279’s activity in BRCA mutant tumors with known homologous recombination deficiencies (HRD), KSQ-4279 also exhibits synergistic activity in combination with PARP inhibitors and re-sensitizes PARP refractory tumors. Mechanistically, we show that in sensitive cell lines, treatment with KSQ-4279 alters the replication fork dynamics resulting in replication stress via induction of replication associated single stranded DNA gaps and subsequent fork degradation. Additionally, KSQ-4279 leads to loss of the essential DNA replication and repair protein, PCNA, which can be rescued by blocking PCNA ubiquitination either through Rad18 knockout or by ectopic expression of a PCNA K164R mutant. This loss of PCNA and sensitizing ssDNA lesions eventually lead to DNA double strand breaks (DSBs) and wide-spread DNA damage accumulation that contributes to cell death. This mechanistic insight supports the ongoing clinical trial of KSQ-4279 in patients with tumors harboring BRCA1/2 or other HRD mutations, both as a single agent and in combination with PARP inhibitors. Citation Format: Sumeet Nayak, Pamela Sullivan, Yuji Mishina, Anne Dodson, Pei-Lun Kao, Hugh Gannon, Chris Middleton, Sol Shenker, Morgan Murray, Michael Schlabach, Frank Stegmeier, Erica Tobin, Louise Cadzow, Andrew Wylie. KSQ-4279 mediated USP1 inhibition induces replication associated DNA gaps that contribute to cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2771. |
