Abstract 1382: The discovery of a novel antibody, 14G6, as a tool for understanding BAK conformational dynamics and their relationship to venetoclax response in leukemia

Abstract Venetoclax, a BCL2-specific inhibitor, is FDA-approved for the treatment of acute myeloid leukemia and chronic lymphocytic leukemia with varying patient responses. Conformation specific antibodies can help unravel the regulation of BCL2 family proteins and its relation to venetoclax response. We used mouse B cell cloning to identify 14G6, the first antibody that is specific for the non-activated, monomeric BAK conformation. The crystal structure of the 14G6 Fab bound to BAK revealed a binding site encompassing both the α1 helix and α5-α6 hinge regions of BAK, the two sites involved in the transition to the active, dimerisation-ready, conformation of BAK. This makes it a useful tool for determining the levels of non-activated, monomeric BAK in cancer cells by blue native PAGE. We found that 14G6 bound the majority of BAK in several leukemia cell lines, and binding decreased following treatment with venetoclax, indicating only minor levels of activated or sequestered BAK in those cells. 14G6 is thus an important new tool to assess BAK status in response to anti-cancer treatments and understand mechanisms of resistance to venetoclax, potentially aiding in the development of more reliable biomarkers to predict patient response. Citation Format: Michelle S. Miller, Hema P. Subas-Satish, Sweta Iyer, Melissa X. Shi, Agnes W. Wong, Karla C. Fischer, Ahmad Z. Wardak, Daisy Lio, Rachel T. Uren, Peter E. Czabotar, Ruth M. Kluck. The discovery of a novel antibody, 14G6, as a tool for understanding BAK conformational dynamics and their relationship to venetoclax response in leukemia [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 1382.