Date of Award

2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Ben Z. Stanger

Abstract

During cancer progression, tumor cells undergo various molecular and phenotypic changes collectively referred to as “plasticity,” leading to both cell autonomous and non-cell autonomous consequences. There is increasing evidence that epigenetic modifiers – enzymes and complexes involved in the removal, deposition, and molecular interpretation of chromatin post-translational modifications – play an important role in regulating tumor plasticity. Still, our understanding of how or even the extent to which chromatin-based mechanisms contribute to tumor biology remains limited. Here we employed CRISPR-based forward genetic screening to unbiasedly address the epigenetic basis of tumor plasticity. Through an in vitro screen, we first identified two histone-modifying enzymes involved in the writing and erasing of H3K36me2 that act reciprocally to regulate epithelial plasticity, a particular form of cellular plasticity whereby cells transition between epithelial and mesenchymal states, as well as tumor differentiation and metastasis. Mechanistically, we found that global changes in H3K36me2 reprogram enhancers associated with master transcriptional regulators of the mesenchymal state. While these results indicate that a large-scale, epigenome-wide mechanism underlies epithelial plasticity in cancer, we subsequently identified focal epigenetic alterations as the basis for microenvironmental plasticity. Specifically, using an in vivo CRISPR-Cas9 screen, we identified the histone demethylase KDM3A as a potent tumor cell-intrinsic epigenetic regulator of the immune microenvironment. Through transcriptomic and epigenomic analyses, we found that KDM3A and its two downstream mediators, KLF5 and SMAD4, transcriptionally converge on EGFR to suppress T cell infiltration, and that loss of any of the components of this molecular axis is sufficient to convert the immune microenvironment from non-T cell-inflamed to T cell-inflamed, thereby sensitizing tumors to combination immunotherapy. Thus, using the power of CRISPR genetic screening, we identified key epigenetic determinants of cellular plasticity, which lead to both cell intrinsic and extrinsic changes. These epigenetic modifiers also represent a promising class of therapeutic targets for some of the most lethal aspects of tumor progression, including metastasis and therapy resistance.

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