AbstractBackground:KIF18A, a molecular motor protein, is crucial for regulating microtubule length, essential for maintaining proper cell function and division. Chromosomal instability (CIN) significantly contributes to cancer progression and is targeted by emerging therapies such as PARP inhibitors, which exploit synthetic lethality in cancer cells. CIN resulting from whole genome duplication (WGD) is associated with poor outcomes and metastatic disease, with cancers demonstrating sensitivity to spindle assembly checkpoint disruption. This makes KIF18A a promising target in this drug class.Methods and Results:We have discovered HW221043, a highly potent and orally bioavailable small molecule inhibitor of KIF18A. Biochemical assays revealed that HW221043 potently inhibits the ATPase activity of KIF18A (IC50, 6 nM) without affecting other kinesin family members, even at the highest tested concentration of 10 μM. In cell-based assays, HW221043 markedly inhibited the viability of CIN-positive tumor cell lines at nanomolar concentrations, with no effect on CIN-negative cells even at 10 μM. The compound demonstrated excellent pharmacokinetic (PK) profiles in mice, rats, dogs, and monkeys. In two cell-derived xenograft (CDX) models (OVCAR3 and BT549), oral administration of HW221043 dose-dependently inhibited tumor growth, with nearly complete inhibition observed at 15 mg/kg once daily. Higher doses led to tumor regression. All in vitro and in vivo PK and safety tests yielded favorable results, supporting the preclinical development of HW221043.Conclusion:These findings provide pharmacological proof-of-concept for the synthetic lethal effect of HW221043 and support KIF18A inhibition as a novel therapeutic strategy in oncology. HW221043 is currently in the Investigational New Drug (IND) enabling stage of development.Citation Format:Qun Li, yang zang, xin zhao, qian wu, tiejun bing, lifei liu, xuejun zhang. Discovery of HW221043, a potent and orally bioavailable KIF18A inhibitor for treating chromosomal instability in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1482.