BMI1 has emerged as a key oncogenic factor in many cancers, associated with unregulated cellular proliferation, tumor metastasis and cancer-initiating cell self-renewal. BMI1 is best characterized as a component of the canonical vertebrate polycomb repression complex 1 (PRC1) which negatively regulate transcription of hundreds of genes through ubiquitination of histone H2A. Previous work suggested that BMI1 has multiple protein binding partners within the PRC1 complex and we were motivated by the prospects to target these protein-protein interactions (PPIs) with small molecule inhibitors. This dissertation describes a multi-pronged campaign to: 1) characterize BMI1 PPIs at the molecular level and 2) develop novel chemical tools to explore BMI1 function in both normal and cancer biology.
Using X-ray crystallography and solution NMR approaches we solved the 3D structure of BMI1 in complex with its PRC1 binding partner protein PHC2. Supporting biochemical and biophysical characterization of the BMI1 PPI domain demonstrated a novel mode of self-association of this domain. Mutagenic disruption of both BMI1-PHC2 and BMI1-BMI1 interactions blocks cellular proliferation demonstrating that multiple PPIs are critical for BMI1 function.
To identify small molecule inhibitors of BMI1 we designed two biochemical assays to quantify the BMI1-PHC2 interaction and these assays were used as a platform for high-throughput screening. Through this screen we identified three classes of small molecule inhibitors that bind directly to BMI1 to disrupt the BMI1-PHC2 interaction, representing three different strategies for BMI1 inhibitor development.
As a complementary approach to inhibit BMI1 we developed a specific inhibitor of Ring1B/BMI1- mediated H2A ubiquitination with potent inhibitory activity both in vitro and in cells. Mechanistic characterization demonstrates that Ring1B/BMI1 inhibitors induce significant protein conformational change and the inhibitor-bound conformation is incompatible with nucleosome binding by Ring1B. These molecules represent the first direct-binding inhibitors of Ring1B/BMI1 and have a novel mechanism of action to block direct protein-nucleosome interaction.
Overall, this work contributes to the understanding of BMI1 function through characterization of its multiple PPIs and demonstrates that these interactions can be inhibited by small molecules representing novel strategies to target this protein for development of new chemical tools or potential therapeutics for cancer.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113363/1/flvgray_1.pd
