Targeting BAX Oligomerization in Hematologic Disease

DESCRIPTION (provided by applicant): The protein interactions of the BCL-2 family regulate programmed cell death or apoptosis, and thereby maintain the critical balance between cellular life and death. Pro-apoptotic BAX is a critical executioner protein that lies dormant in the cytosol until triggered by cellular stress to inflict irreversible damage on the mitochondria. Because of its role as a gatekeeper of cell death, pharmacologic modulation of BAX has the potential to alternatively block or reactivate cell death in diseases of deregulated apoptosis. BAX activation is believed to be a highly regulated, multi-step process involving an interaction-triggered conformational change, mitochondrial translocation, and homo-oligomerization to form a lethal pore within the outer mitochondrial membrane. Using Stabilized Alpha-Helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis, we recently identified by NMR analysis a novel BAX interaction site that triggers its activation. Having tackled the initial step of BAX activation, I now propose to determine the elusive molecular mechanism of BAX auto-activation that leads to homo-oligomerization, so that this critical control point of the apoptotic pathway can be exploited therapeutically to inhibit cell death in hematologic disease. Specifically, I aim to (1) synthesize structurally-reinforced alpha-helices corresponding to the BH3 death domain of BAX to identify and characterize its interaction(s) with pro-apoptotic BAX, (2) determine the solution structures of the BAX SAHB-BAX complex and an intermediate BAX conformer, and (3) investigate the mechanism of BAX propagation and the impact of its pharmacologic inhibition in hematologic cells. By operating at the interface of chemistry, biology, and hematology, I hope to contribute new insight into our understanding of the BAX auto- activation pathway, revealing new sites of BCL-2 family protein interaction and determining how they can be pharmacologically reprogrammed for the betterment of hematology patients. The multidisciplinary scope of this proposal will require advanced training and expertise. With the mentorship of Dr. Loren D. Walensky and Dr. Alan D'Andrea, I will acquire new skills and knowledge in chemical biology, apoptosis biology, and hematology, in addition to preparing for the transition to independence through training in grantsmanship, laboratory management, the job application process, and other junior faculty survival skills. The proposed training and career development program within the Department of Pediatric Oncology at the Dana-Farber Cancer Institute and Harvard Medical School offers state-of-the-art resources, world class faculty advisors and collaborators, and an outstanding environment to facilitate a successful transition to academic independence. My career goal is to become an independently funded principal investigator with a tenure-track position at a major academic research center. I am committed to a scientific career focused on the structure and function of protein interactions that regulate cell death, with direct application to the development of novel pharmacologic strategies to treat hematologic disease. PUBLIC HEALTH RELEVANCE: Programmed cell death or apoptosis regulates the critical balance between cellular life and death and, when deregulated, contributes to the pathogenesis of a wide variety of hematologic diseases characterized by too many or too few blood cells. BCL-2 family proteins regulate apoptosis and analysis of their structure and function promises to elucidate opportunities for modulating apoptosis for therapeutic benefit. Using a combination of novel chemical tools, structural biology analyses, and hematologic cell experimentation, I aim to dissect and inhibit the activation mechanism of a critical executioner protein called BAX in order to protect hematologic cells from premature or unwanted cell death.