Abstract Tumors with BRCA1/2 mutations demonstrate homologous recombination (HR) repair deficiencies, leading to genomic instability. Genomic instability is a defining hallmark of numerous carcinomas, making them susceptible to therapies targeting the DNA damage response (DDR) pathways. Among these, poly (ADP-ribose) polymerase (PARP) inhibitors have shown representative therapeutic options. However, incomplete disease control and the emergence of resistance remain challenges for many patients. Ubiquitin-specific protease 1 (USP1) plays a critical role in regulating DNA damage repair including the pathway of translesion synthesis (TLS) and Fanconi anemia (FA), which enables resistance bypass mechanisms against PARP inhibitors (PARPi). Therefore, USP1 has been considered as a synthetic lethal target with BRCA1/2-deficient and PARPi resistant tumors. Many investigational USP1 inhibitors have been discovered and developed, but key leading programs showed limited safety or efficacy. Of these, the phase 1/2 clinical trial of TNG348 was terminated due to toxicity observed in the initial study cohorts and KSQ-4279 showed limited efficacy and hematologic toxicity in its early clinical study. Based on these findings, it is necessary to understand if heme and liver toxicities are induced by on-target inhibition of USP1, and whether efficacy can be improved through higher exposure and tolerability. VRN19, a novel oral allosteric USP1 inhibitor, exhibited comparable efficacy, favorable safety, and pharmacological profiles, indicating that the previously identified limitations of USP1 programs may be overcome. VRN19 has been designed to increase target binding affinity, minimize toxicity, and increase solubility. Briefly, VRN19 inhibits the catalytic activity of the USP1-UAF1 complex by directly binding to USP1, confirmed by determining CryoEM complex structures. In the cellular assay, VRN19 showed double-digit nanomolar (nM) GI50, cytotoxic activity against BRCA1-mutated cancer cells, sparing BRCA WT cancer cells over 500-fold, indicating high selectivity to target cells. Moreover, unlike KSQ-4279, VRN19’s oral administration demonstrated dose-proportional drug exposure in the plasma at doses up to 300 mg/kg in mice and effective dissolution in FaSSGF (Fasted State Simulated Gastric Fluid) at 2 mg/mL. In the BRCA1-mutated CDX model (MDA-MB-436) and PDX with resistance to PARP inhibitors, VRN19 demonstrated synergistic effect with PARP inhibitors, particularly with olaparib, and showed efficacy comparable to KSQ-4279 without toxicity. In a 2-week toxicity study in rats, administration of 300 mg/kg VRN19 showed no significant changes in anemia markers, such as RBC, Hb, and reticulocytes, nor hepatotoxicity indicators like GGT and bilirubin. However, administration of 300 mg/kg KSQ-4279 significantly elevated anemia-related markers and GGT levels, consistent with the adverse events observed in clinical trials with KSQ-4279. In conclusion, VRN19 is a promising novel USP1 inhibitor with favorable safety and improved therapeutic outcomes. Citation Format: Se-Hyuk Kim, Seohyun Jo, Kibum Kim, Seonah Hwang, hayeong Kim, Donghyun Park, Soochan Kim, Youngyi Lee, Junhee Kim, Kyungah Seo, Younho Lee, Daekwon Kim, Sunghwan Kim, Jaeyoung Ahn. VRN19: A novel USP1 Inhibitor with anti-tumor efficacy and favorable safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB015.