Author ORCID Identifier

0000-0002-0152-2666

Date of Graduation

5-2022

Document Type

Dissertation (PhD)

Program Affiliation

Cancer Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Jeffrey N. Myers, M.D., Ph.D.

Committee Member

Kunal Rai, Ph.D.

Committee Member

George Calin, M.D., Ph.D.

Committee Member

George Eisenhoffer, Ph.D.

Committee Member

Curtis Pickering, Ph.D.

Abstract

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and is associated with significant morbidity and mortality. To date, the majority of work in the field has focused on genomic alterations such as mutations and copy number alterations. However, the clinical success of targeted therapies that exploit known genomic alterations, such as EGFR mutations, has remained mixed. Over the past decade, the importance of epigenetic regulators has come to the forefront, with the realization that many of these genes are mutated in cancer. Despite this realization, the role of epigenetics in regulating tumorigenesis, progression and metastasis, and treatment response remains poorly understood. Further, outside of defined mutations in epigenetic regulators, the role of enhancer landscapes and overall chromatin dynamics have recently been recognized as important regulators of cancer cell activity and tumor microenvironment (TME) interactions. In the case of HNSCC, these facets of epigenetic regulation are understudied and present a critical opportunity to improve the understanding of HNSCC biology and design targeted therapies based on epigenetic mutations and chromatin regulation.

In this thesis, I explore the role of epigenetic regulation in HNSCC in three distinct projects. In the first project, I investigate the role of TP53, the most highly mutated gene in HNSCC, in modulating dependencies on epigenetic regulators. I explore a functional relationship between p53 and PRMT1, an arginine methyltransferase, in which mutant forms of p53 upregulate PRMT1 and increase total levels of ADMA, PRMT1’s methylation product. Further, using TCGA data, I demonstrate patients expressing high levels of PRMT1 have significantly worse survival than patients expressing low levels of PRMT1. In the second project, I investigate the enhancer landscapes of the four molecular subtypes of HNSCC. The Atypical subtype of HNSCC possesses higher enhancer activity, as measured by H3K27ac ChIP-seq and PRO-seq, than all other subtypes of HNSCC, and, interestingly, this upregulation in enhancer activity is associated with resistance to BET inhibitor treatment and regulation of MAPK, WNT, and lipid metabolism signaling pathways. In the third project, I investigate the role of KMT2D, a histone methyltransferase that is the most highly mutated epigenetic gene in HNSCC, in regulating tumorigenesis and the tumor microenvironment. KMT2D is demonstrated to function as a tumor suppressor, the loss of which is associated with 2-year recurrence in HNSCC. Consistent with this, the loss of KMT2D results in larger tumors in orthotopic models of HNSCC. Additional in vivo experiments demonstrate that the increase in tumor burden is driven by alterations in the TME. In particular, tumors with loss of KMT2D have decreased levels of anti-tumorigenic macrophages that secrete chemokines such as CXCL10 and aid in recruitment of immune cells to the tumor bed. Further, cytotoxic CD8+ T cells harvested from KMT2D knockdown tumors are found to secrete less IFNγ and possess a more exhausted phenotype than CD8+ T cells harvested from control tumors. Mechanistically, KMT2D regulates H3K4me1-marked enhancers that control the expression of genes involved in interferon signaling and antigen processing and presentation, and loss of KMT2D results in dysregulation of tumor-intrinsic immune signaling that has downstream effects on the composition of the TME.

Together, these studies serve as groundwork demonstrating the role of epigenetic regulators and chromatin dynamics in influencing tumorigenesis and treatment response in HNSCC. Additionally, I provide possible clinical applications for each of the findings, particularly in the realms of BET inhibitor resistance and immunotherapy, and, in the final chapter, I discuss future experimentation that could expand upon the work presented in this thesis.

Keywords

head and neck squamous cell carcinoma, cancer biology, bioinformatics, genomics, epigenetics, gene regulation, enhancer biology, p53, tumor microenvironment, KMT2D, chromatin

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