Abstract B032: USP1 inhibition induces single strand DNA gap accumulation and overcomes PARP inhibitor resistance in BRCA1 deficient cancer cells

Abstract

Abstract Targeting BRCA-deficient tumors with PARP inhibitors has improved the progression-free survival of ovarian and breast cancer patients in the clinic. However, the majority of patients develop acquired resistance. Recent studies suggest that PARP and POLQ inhibitors confer synthetic lethality in BRCA1-deficient tumors by accumulating single-stranded DNA (ssDNA) gaps at replication forks. We have previously shown that USP1 deficiency is synthetic lethal with BRCA1 deficiency, suggesting USP1 is a potential therapeutic target for overcoming PARPi resistance (Lim et al., Mol Cell, 2018). Accordingly, USP1 inhibitors (USPi) are now undergoing clinical development. Here, we show that USP1i also promote ssDNA gap accumulation during replication in BRCA1-deficient cells. USP1 inhibition results in the accumulation of monoubiquitinated PCNA at the replication fork and increased levels of non-cleaved USP1 and RAD18 at the replication fork. Accumulation of ssDNA gaps is dependent on RAD18 activity and monoubiquitinated PCNA levels. Knockdown of RAD18, the E3 ubiquitin ligase known to ubiquitinate PCNA, resulted in reduced PCNA-Ub levels, resistance to the USP1i, and suppression of ssDNA gaps. Moreover, the accumulation of ssDNA gaps is associated with increased S-phase PAR levels and is independent of PRIMPOL, suggesting defects on the lagging strand of the replication fork. Interestingly, the induction of ssDNA gaps was detected in USP1i-sensitive but not USP1i-resistant BRCA1-deficient cells, suggesting this is the mechanism of cytotoxicity by USP1 inhibition. Indeed, USP1i also sensitized BRCA1-deficient cancer cells to other ssDNA gap-inducing agents such as POLQ and PARP inhibitors. Notably, USP1i exhibited monotherapy activity in ovarian and breast cancer cell lines with acquired PARPi resistance and re-sensitized them to PARP inhibition. PARPi-resistant BRCA1-mutated patient-derived ovarian cancer organoids were also sensitized by USP1 inhibition. Similar to the cell lines, the ssDNA gap accumulation induced by USP1i in organoids strongly correlated with the drug sensitivity of both PARPi-sensitive and PARPi-resistant models. Finally, USPi overcame PARPi resistance in a BRCA1-mutant PARPi-resistant PDX model. In conclusion, USPi exhibits monotherapy activity in both PARPi-sensitive. Citation Format: Alexandre A. B. A. da Costa, Ozge Somuncu, Ramya Ravindranathan, Sirisha Mukkavalli, David B. Martignetti, Huy Nguyen, Yuqing Jiao, Benjamin Lamarre, Lisa Moreau, Joyce Liu, Divya Iyer, Jean-Bernard Lazaro, Geoffrey I. Shapiro, Kalindi Parmar, Alan D. D’Andrea. USP1 inhibition induces single strand DNA gap accumulation and overcomes PARP inhibitor resistance in BRCA1 deficient cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B032.