Abstract Mitotic kinesins are essential regulators of cancer cell replication and migration. The microtubule associated motor protein KIF20A (also called MKlp2), a member of the kinesin-6 family, plays an essential role during cytokinesis and is also involved in the fission of RAB6-positive vesicles from Golgi membrane, therefore contributing to intracellular vesicular trafficking (1). KIF20A plays a critical role in the development and progression of many cancers, and its high expression is associated with disease progression and poor survival outcome (2). More specifically, immature hematopoietic cells, exhibit high KIF20A expression, whereas mature peripheral blood cells do not (3). Herein we describe the preclinical anti-leukemic efficacy of DIACC2010, sole-in-class selective KIF20A inhibitor. DIACC2010 demonstrated potent and consistent cytotoxic activity in-vitro against a panel of 9 human AML cell lines, with median IC50 of 40 nM (range 10-77 nM). In the same experimental conditions, cytarabine (CYTA) had median IC50 of 207 nM (range 4-1580 nM). In parallel, no in-vitro toxicity of DIACC2010 was observed on human normal cells such as peripheral blood mononuclear cells (PBMC) and primary hepatocytes (IC50 >50 µM). DIACC2010 was subsequently evaluated in-vivo in xenograft models of CYTA-sensitive and CYTA-resistant AML cell lines: DIACC2010 significantly improved overall survival as compared to CYTA in all models, with dose-dependent efficacy. On a mechanistic standpoint, DIACC2010-exposed AML cells displayed characteristic Golgi scattering leading to cell death, as hallmarks of DIACC2010 mode of action and target engagement. Altogether, these results confirm the relevance of KIF20A-directed therapeutic approaches and support the development of DIACC2010 for the treatment of AML. (1) Coupling fission and exit of RAB6 vesicles at Golgi hotspots though kinesin-myosin interactions, S. Miserey-Lenkei et al, Nature com, 2017, 104:300 (2) Prognostic significance of KIF2A and KIF20A expression in human cancer. X. Li et al, Medicine, 2019, 98:46 (3) KIF20A, highly expressed in immature hematopoietic cells, supports the growth of HL60 cell line, H. Morita et al, Int J Immunol, 2018,108:607 Citation Format: Yves COLLETTE, Rémy CASTELLANO, Michel AURRAND-LIONS, Hélène SICARD, Norbert VEY, Cécile BOUGERET. DIACC2010, a selective inhibitor of KIF20A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1813.