Abstract 2524: Critical interactions and tumor-specific mutations of Bcl-2 transmembrane domains revealed by a novel split luciferase assay

Abstract In intrinsic apoptosis, the interaction network of the Bcl-2 protein family controls the decision over life and death. Cells are sentenced to death when pro-apoptotic multidomain effector proteins BAX, BAK or BOK oligomerize and form pores in the mitochondrial outer membrane. This releases cytochrome c, which induces the activation of cell-wrecking proteases, the caspases. Interactions of Bcl-2 proteins with other family members essentially regulate cell death. The interaction via the BH3 region was intensively studied in the last decades. As a result, small-molecule drugs, BH3-mimetics, were developed which bind and inhibit anti-apoptotic Bcl-2 proteins. Since anti-apoptotic Bcl-2 proteins are often overexpressed in hematopoietic malignancies, BH3-mimetics e.g. Venetoclax are approved for anti-cancer therapy. Unlike the BH3 interaction site, the c-terminal α9-helix referred to as the transmembrane domain (TMD) is mostly neglected in Bcl-2 interaction studies. However, TMDs not only dictate subcellular localization, but also substantially influence protein-protein interactions. Interestingly, Bcl-2 TMDs can harbor several tumor-specific mutations. The functional basis for TMD interaction as well as the resulting functional relevance for apoptosis signaling, however, remains poorly understood. To unravel the Bcl-2 TMD interaction network, we developed a split luciferase assay system enabling us to detect Bcl-2 TMD interactions in living cells. Simultaneously encoding for the expression of a fluorophore and TMD fusion peptides this system was used to generate fluorescence-normalized luminescence-based interaction data. Here, we identified a homotypic interaction pattern among effector TMDs of BAX, BAK and BOK. Molecular modelling of effector TMD interaction in mimics of cellular membranes also supports these findings. TMD swap experiments show significant influence of TMD sequence on subcellular localization and cell death signaling as assessed via confocal laser scanning microscopy and flow cytometry-based cell death assays. Moreover, we tested previously described mutations of the BAX-TMD (S184A, S184D) as well as a tumor-specific mutation (V180G) in the novel interaction assay. S184 (de-)phosphorylation as mimicked with S184A/S184D affects subcellular localization. In accordance, we find that S184A enhanced and S184D abolished interaction with wildtype BAX-TMD. Intriguingly, V180G not only modulates BAX subcellular localization but also prevents interaction with wildtype BAX-TMD. These findings verify a crucial role of Bcl-2 TMDs in subcellular localization and furthermore strongly support a function in interaction and cell death regulation. Further efforts to explore the Bcl-2 TMD interaction network as well as functional analysis of tumor-specific TMD mutations could pave the way to establish TMDs as a target of cancer therapy. Citation Format: Tobias B. Beigl, Alexander Paul, Sandra Weller, Benjamin Schäfer, Walter E. Aulitzky, Hans-Georg Kopp, Thomas Fellmeth, Kristyna Pluhackova, Markus Rehm, Frank Essmann. Critical interactions and tumor-specific mutations of Bcl-2 transmembrane domains revealed by a novel split luciferase assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2524.