PROJECT SUMMARY/ABSTRACT BCL-2 proteins participate in a dynamic interaction network that determines whether a cell will live or die. Deregulation of this essential signaling pathway underlies the pathogenesis of human cancer and resistance to treatment. The goal of this R35 research program is to elucidate the fundamental protein interaction mechanisms that drive the apoptotic program and harness these insights to develop next-generation cancer treatments. Over the last five years of R35 support, we applied novel chemical tools and a host of analytical technologies to achieve mechanistic discoveries that revealed new druggable binding sites and compounds to target them. We found that covalent modification of distinct cysteines in pro-apoptotic BAX and anti-apoptotic MCL-1 and BFL-1 differentially regulate their apoptotic functions. Our pursuit of covalent ligands that mimic these post-translational modifications are yielding prototype BAX activators and MCL-1 and BFL-1 inhibitors for cancer therapy. Deciphering how BAX and BAK are directly activated, and the conformational mechanisms that underlie their conversion from latent monomers into toxic mitochondrial oligomers, has also been a major focus of our work. Indeed, the elusive structures of the BAX and BAK death channels represent the “holy grail” of apoptosis research. We recently generated the first full-length homogeneous BAX oligomer (BAXO) amenable to structure- function characterizations, providing a glimpse into the macromolecular organization of a functional BAXO species. BAXO and its mutants are enabling us to pinpoint the structural determinants for each step of the BAX- activation pathway and thus inform new control points for pharmacologic activation of apoptosis. In addition to dissecting these high-priority, canonical BCL-2 protein interactions, we have developed proteomic tools to identify non-canonical targets and recently found that MCL-1 directly interacts with the fatty acid oxidation enzyme VLCAD, revealing a dual role for MCL-1 at the intersection of apoptosis and metabolic regulation. We hypothesize that MCL-1-driven cancers rely on both apoptotic suppression and fatty acid metabolism to maximize pathologic survival, potentially explaining why MCL-1 is the most widely expressed anti-apoptotic protein across human cancers. Here, we build on our newest mechanistic insights to interrogate a spectrum of BCL-2 family interactions that drive human cancer and mine each opportunity to pharmacologically subvert them. Specifically, our next set of R35 goals are: (1) identify the structural and functional determinants that mediate the “execution phase” of mitochondrial apoptosis; (2) solve the structure of a BAX oligomer; (3) characterize the non-canonical role of MCL-1 at the intersection of apoptosis and cancer metabolism; and (4) advance the development and in vivo testing of BCL-2 family molecular modulators as next-generation therapies for human cancer. We tackle these goals using multidisciplinary approaches that span chemistry, structural biology, proteomics, biochemistry, cell biology, and in vivo testing. As a chemical biologist and pediatric oncologist, I am committed to transforming our fresh mechanistic insights into new therapies for relapsed and refractory cancers.

PROJECT NARRATIVE BCL-2 family proteins are master arbiters of mitochondrial apoptosis and deregulation of their protein interaction mechanisms contributes to the development, maintenance, and chemoresistance of human cancer. The overarching goal of this R35 research program is to develop unique chemical probes and apply the latest analytical technologies to tackle longstanding enigmas of the apoptotic pathway, including how death proteins self-associate to permeabilize the mitochondria, novel modes of post-translational BCL-2 family regulation, and unanticipated interaction partners that integrate apoptosis with critical cellular signaling pathways. By revealing new apoptotic mechanisms, targets, and binding surfaces, our studies not only inform fundamental cancer biology but also provide next-generation treatments for reactivating apoptosis in human cancer.