Abstract Acute myeloid leukemia (AML) is the most common leukemia in adult patients, with a 5-year survival rate of less than 30 percent. Therefore, more effective therapeutic strategies are required to prolong the survival of AML patients. Importantly, anti-apoptotic proteins, especially B-cell lymphoma 2 (Bcl-2), overexpression in AML is associated with uncontrolled growth as well as chemoresistance. Unsurprisingly, Bruton’s tyrosine kinase (BTK) overexpresses in AML and associated with poor prognosis and chemoresistance. The FDA-approved BTK inhibitor, ibrutinib, has been successful in treating other hematologic malignancies, but a proportion of patients relapse mainly because of acquired mutations at Cys481Ser (C481S) in the kinase domain of BTK. If we are to develop effective BTK-based therapeutic regimens, it is critical that we account for these likely mutations and avoid agents that target the kinase domain. To develop a more selective and effective BTK inhibitor, we modified our first-generation BTK inhibitor to avoid the C481S mutation in the BTK kinase domain. This advance allowed us to develop a second-generation BTK inhibitor, KS151, that shows in vitro potency against C481S mutants. Because BTK and Bcl-2 are critical in chemoresistance and poor prognosis, combination therapy targeting BTK and Bcl-2 is a more viable option for AML patients to achieve better therapeutic outcomes. We propose the combination of KS151 with venetoclax (ABT-199) or ABT-737 in resistant AML cells. We employed a panel of three AML cell lines: Kusami-1, U937, and THP-1. Remarkably, KS151 was able to induce AML cell death with an average IC50 of 4 μM. Furthermore, KS151 showed a significant increase in both venetoclax and ABT-737 efficacy against AML cells. Western blot analysis demonstrated that Kusami-1 cells have the highest expression of both BTK and Bcl-2 proteins. Taken further, flow cytometry (Annexin V staining) demonstrated that KS151 combined with inhibition of Bcl-2 by venetoclax synergistically induced apoptosis (approximately 95%) of Kusami-1 cells. Collectively, the combination therapy showed promising and synergistic in vitro anti-leukemic activity against AML cell lines and represents a new viable treatment for AML patients. Future studies will apply the combination strategies in AML-resistant cell lines and include assessment in patient samples and in vivo studies. Citation Format: Nicole K. Schmalbach, Omar S. Al-Odat, Weam Othman Elbezanti, Gabriella Yao, Daniel A. Guirguis, Krishne Gowda, Shantu G Amin, Tulin Budak-Alpdogan, Subash C. Jonnalagadda, Manoj K. Pandey. Development of a novel BTK inhibitor against resistant acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A01.