Chemical-induced proximity of chromatin remodelers and small-molecule inhibitors thereof

Cellular fate is hierarchically controlled by the underlying genetic code, DNA methylation, histone modifications, and chromatin remodeling, which when disrupted can lead to cancer. For this reason, chemical tools to modulate the epigenome contribute greatly to our understanding of both the fundamental mechanisms of gene regulation, and the druggability of a variety of epigenetically-disrupted diseases. This works seeks to develop and employ various chemical tools to modulate the epigenome on multiple levels— first through the temporal control, in vivo characterizations, and simulations of histone modifications and nucleosome exchange, and second through the development of inhibitors of chromatin remodeling complexes. Utilizing a combination of chemical-induced-dimerization, theoretical simulations, and epigenome meta-analysis, the unknown mechanisms governing the regulation of histone methylation through nucleosome turnover are explored. Utilizing chemical induced proximity, I selectively methylate Lysine 79 of Histone 3 (H3K79me) by recruiting the DOT1L complex to H3K79me-deficient genes in vivo, which provided a characterization of the complete system of reactions describing the transition from unmodified histones through mono-, di, and tri-methylation at distinct chromatin substrates. Despite the fact that members of the DOT1L complex are aberrantly fused with the MLL protein and result in > 80% of MLL-rearranged leukemias, the mechanisms of H3K79-mediated genetic regulation remain poorly understood. Utilizing kinetic first-principles I developed a model which predicts that nucleosome turnover can establish varied methylation states in the absence of demethylation, in agreement with my in vivo results. This model also successfully predicts the chromatin landscape in the presence of demethylation for both Histone 3 Lysine 27 methylation (regulated by polycomb repressive complexes) and the heterochromatin-inducing methylation of Histone 3 Lysine 9. I then utilized this model to successfully predict the H3K79me-addicted epigenetic landscape in Mixed Lineage Leukemias, along with genome-wide landscapes of both repressive and activating histone marks in worms, flies, and mice. In doing so, I propose a conserved general principle for the establishment of the epigenetic landscape through the concerted actions of methylation, demethylation, and nucleosome turnover. Beyond regulating histone modifications, nucleosome turnover is a critical component in maintaining a stable epigenetic-state and is mediated by chromatin remodeling complexes. A second major focus of this work is understanding the mechanisms of human SWI/SNF (BAF) complexes, which are a diverse family of ATP-dependent chromatin remodelers that exhibit combinatorial specificity to regulate specific genetic programs. Genomic studies have shown that subunits of the BAF complex are mutated in about 20% of human cancers and a large number of neurologic diseases, including autism. SWI/SNF complexes regulate transcription, replication and DNA repair through a variety of mechanisms including nucleosome mobilization, polycomb opposition, and Top2- mediated DNA decatenation. Despite the obvious need for the development of small-molecules targeting these large combinational complexes, no probes against SWI/SNF have demonstrated utility in cancer, and few exist. Through the development of a facile high-throughput drug-synergy screen, I demonstrated that a novel SWI/SNF inhibitor, BAFi-1, functions synergistically with inhibitors of the ATR/ATM kinase, which are under investigation for treatment of a broad group of human cancers. Despite the fact that this molecule was discovered in a chemical screen for the repressive function of the embryonic BAF complex, since BAFi-1is not detectably toxic, these studies suggest an avenue for therapeutic enhancement of ATR/ATM inhibition without additional toxicity. Further, this work presents a first-in-kind demonstration of chromatin remodeling inhibition as a therapeutic strategy in cancer.

Keywords: Chemical Engineering.
Advisor: Crabtree Gerald R. 
Spakowitz Andrew James 
Khosla Chaitan
Kim Peter