# Dissecting the Execution Phase of BAK-Mediated Apoptosis in Cancer

> **NIH NIH F30** · HARVARD MEDICAL SCHOOL · 2022 · $38,696

## Abstract

PROJECT SUMMARY
 BCL-2 family proteins are critical regulators of apoptosis and deregulation of their protein interactions
contributes to the development and chemoresistance of cancer. The cardinal executioners of cell death, BAX
and BAK, respectively reside in the cytosol and mitochondria of the cell as monomers until activated by stress
stimuli to self-associate and porate the mitochondrial outer membrane, leading to apoptotic cell death. To thwart
chemotherapy-induced apoptosis and enforce cellular immortality, cancer usurps the cell survival arm of the
BCL-2 pathway by overexpressing anti-apoptotic members, which can bind to BAX and BAK and prevent their
transformation into toxic mitochondrial pores. The longstanding inability to generate stable and homogeneous
oligomeric forms of full-length BAX and BAK has precluded the determination of their porating structures, which
would inform both the execution phase of mitochondrial apoptosis and reveal novel surfaces for therapeutic
activation of BAX and BAK in cancer. The Walensky laboratory recently reported a novel strategy for generating
a BAX oligomer that was amenable to a battery of structure-function studies. In contrast to BAX, BAK
constitutively residues at the mitochondria, is triggered by a distinct binding site, and exhibits differential
expression patterns in mammalian tissues and in human cancers. Further, whereas full-length BAX has long
been amenable to expression in recombinant monomeric form, production of BAK has been especially
challenging. The Walensky lab developed a triple-mutant construct that allowed for the expression of full-length
monomeric BAK that exhibited physiologic activation and membrane-porating activity. In preliminary studies, I
have applied our learnings from the production of monomeric BAK and oligomeric BAX to produce a full-length
BAK oligomer for the first time. Here, I propose to optimize and validate the stability and homogeneity of this
species for rigorous biochemical and structural characterization (SA1). By mutating discrete functional regions
of BAK, I further propose to identify the structural determinants of each step of the activation pathway, and
evaluate the mechanistic findings and their functional implications in BAK-dependent leukemia cells (SA2). To
achieve my goals, I will apply multidisciplinary approaches that include protein engineering, biochemical assays
in model membranes and mitochondria, hydrogen-deuterium exchange mass spectrometry, cryo-electron
microscopy, and cellular apoptosis analyses. I am eager to embark on the rigorous training program proposed
for my graduate studies and look forward to developing as an independent and innovative physician-scientist at
the interface of chemical biology, cancer biology, and clinical oncology.

## Key facts

- **NIH application ID:** 10437624
- **Project number:** 5F30CA264846-02
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Catherine Elizabeth Newman
- **Activity code:** F30 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $38,696
- **Award type:** 5
- **Project period:** 2021-07-01 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10437624

## Citation

> US National Institutes of Health, RePORTER application 10437624, Dissecting the Execution Phase of BAK-Mediated Apoptosis in Cancer (5F30CA264846-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10437624. Licensed CC0.

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