Functional Elucidation and Targeting of the Bcl-2 Pro-Survival Proteins in Various Cancers

Cancer is a heterogeneous group of diseases defined by distinct capabilities, including resistance to programmed cell death, or apoptosis. The Bcl-2 protein family governs the integrity of the mitochondria, which acts as a central nexus of the apoptotic cascade and is commonly dysregulated in cancers. The Bcl-2 protein family consists of a delicate balance of pro- and anti- apoptotic members that interact with one another via a selective network of protein-protein interactions. In many cancers, the anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1, and Bfl-1) are overexpressed and contribute to aggressive malignancy and therapeutic resistance. Selective small molecule inhibitors of these proteins (BH3 mimetics) are therapeutic modalities with promise in diverse cancers. Potent inhibitors of Bcl-2, Bcl-xL, and Mcl-1 are being clinically evaluated, but small molecule Bfl-1 inhibitors have remained elusive. In this body of work, we utilized and developed selective chemical tools to probe the Bcl-2 proteins in a wide spectrum of cancers. Using BH3 profiling we revealed distinct functional pro-survival trends in various hematologic malignancies. These findings were highlighted by the preferential survival dependence in lymphoma based on cell of origin, where B-cell and T-cell lymphomas predominantly display sole dependence on Bcl-2 and Mcl-1, respectively. These functional discoveries were accompanied by selective sensitivities to the respective BH3 mimetics. The translational relevance of these findings was substantiated by testing primary patient samples. In a genetically diverse panel of solid tumor cell lines we discovered profound sensitivity to a combination of Bcl-xL and Mcl-1 inhibitors, with some responding to single agent BH3 mimetic treatment. Functional analysis of Bcl-2 family proteins in ovarian cancer identified a highly Mcl-1 dependent patient derived xenograft cell line. In melanoma we discovered that BRAF/MEK inhibitors selectively prime cells for Mcl-1 dependence and led to increased sensitivity to Mcl-1 inhibitors, especially in the context of acquired resistance. Additionally, we explored the role of Bfl-1 in melanoma, which is the cancer where it is most abundantly expressed and highlight an unmet need to develop Bfl-1 inhibitors. We undertook medicinal chemistry efforts to discover first-in-class small molecule dual inhibitors of the Mcl-1 and Bfl-1 proteins. Building from our previous work on Mcl-1 inhibitors, we designed and developed a class of 2,5-substituted benzoic acid compounds selective for both Mcl-1 and Bfl-1. The most potent inhibitor in this series, compound 24, displayed equipotent binding (Ki = 100 nM) to both Mcl-1 and Bfl-1, with 200-fold selectivity over Bcl-2 and Bcl-xL. Compound 24 selectively disrupts protein-protein interactions between endogenous Mcl-1/Bfl-1 and pro-apoptotic peptides and selectively kills model Eμ-Myc lymphoma cell lines that depend on Mcl-1 and Bfl-1 for survival. From optimizing functional diagnostic strategies, to the development of novel small molecule inhibitors, we have outlined a body of work that contributes to the therapeutic targeting of the Bcl-2 protein family. These findings highlight the use of chemical biology to interrogate various cancers with the translational goal of improving patient outcomes in a precision manner.PhDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163306/1/kjkump_1.pd