Abstract 5725: The USP1 inhibitor I-138 kills BRCA1-deficient tumor cells and overcomes PARP inhibitor resistance
Authors: Da Costa Alexandre Andre B. A. , Bose Arindam , Martignetti David , Ayala-Zambrano Cecilia , Ravindranathan Ramya , Sadatrezaei Golbahar , Jiao Yuqing , Kochupurakkal Bose , Nguyen Huy , Lazaro Jean-Bernard , Parmar Kalindi , Shapiro Geoffrey I. , D’Andrea Alan D. 
Published: April 2023

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. PARP inhibitor (PARPi) resistance in tumors can occur by restoration of homologous recombination (HR) or replication fork stability and/or by suppression of single-stranded DNA (ssDNA) gaps at replication forks. USP1 is a ubiquitin specific protease which deubiquitinates PCNA and FANCD2. We have previously shown that USP1 deficiency causes replication fork instability and is synthetic lethal with BRCA1 deficiency, suggesting USP1 as a potential therapeutic target for overcoming PARPi resistance (Lim et al, Mol Cell, 2018). Accordingly, USP1 inhibitors are now undergoing preclinical and clinical development. Here, we evaluated the activity of the novel USP1 inhibitors such as I-138 (Simoneau et al, Mol Cancer Ther, 2022) and TNG-6132 in preclinical models of BRCA1-deficient tumors. I-138 preferentially killed BRCA1-deficient ovarian and breast cancer cells and increased the cellular level of ubiquitinated PCNA. An aniPOND analysis demonstrated an accumulation of ubiquitinated PCNA specifically at the replication fork in I-138-treated cells. Consistent with our previous study (Lim et al, Mol Cell, 2018), I-138 treatment led to replication fork instability and DNA damage in BRCA1-deficient cancer cells. I-138 exposure also resulted in the accumulation of ssDNA gaps, as detected by an S1 nuclease assay. Interestingly, the induction of ssDNA gaps was detected in I-138-sensitive but not in I-138-resistant BRCA1-deficient cells, suggesting a mechanism of cytotoxicity by USP1 inhibition. Persistent monoubiquitinated PCNA accumulation was the cause of cell killing after USP1 inhibition. Knockdown of RAD18, the E3 ubiquitin ligase known to ubiquitinate PCNA, in BRCA1-deficient ovarian cancer cells resulted in reduced PCNA-Ub levels, resistance to I-138, and suppression of ssDNA gaps. Notably, I-138 exhibited monotherapy activity in ovarian and breast cancer cell lines with acquired PARPi resistance and re-sensitized the cells to PARP inhibition. Similarly, PARPi-resistant BRCA1-mutated organoids, derived from patient-derived xenograft (PDX)-derived models of ovarian cancer, were also sensitized by USP1 inhibition by TNG-6132. Interestingly, the ssDNA gap accumulation induced by I-138 strongly correlated with the drug sensitivity of both PARPi-sensitive and PARPi-resistant cells. In conclusion, I-138 exhibits monotherapy activity in both PARPi-sensitive and -resistant preclinical models of BRCA1-deficient cancer, induces ssDNA gaps and overcomes PARP inhibitor resistance. Taken together, these data support use of a USP1 inhibitor, with or without a PARP inhibitor, as a therapeutic strategy for a subset of PARP-inhibitor-resistant BRCA1-deficient tumors.
Citation Format: Alexandre Andre B. A. Da Costa, Arindam Bose, David Martignetti, Cecilia Ayala-Zambrano, Ramya Ravindranathan, Golbahar Sadatrezaei, Yuqing Jiao, Bose Kochupurakkal, Huy Nguyen, Jean-Bernard Lazaro, Kalindi Parmar, Geoffrey I. Shapiro, Alan D. D’Andrea. The USP1 inhibitor I-138 kills BRCA1-deficient tumor cells and overcomes PARP inhibitor resistance. [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 5725.