Targeting the Cell Cycle in Oral, Head, and Neck Squamous Cell Carcinoma

Thesis (Ph.D.)--University of Washington, 2022A fundamental hallmark of cancer is the cell’s ability to sustain proliferative signaling thereby deregulating the cell cycle. Uncovering the molecular changes that inappropriately drive proliferation has allowed researchers to specifically target cancer cell vulnerabilities related to cell cycle control. An important class of proteins that contribute to this control are the cyclins and cyclin-dependent kinases (CDKs). Cancer cell mutations that lead to aberrant activation of cyclin-CDKs are common in many cancers including oral, head, and neck squamous cell carcinomas (OHNSCCs). As the sixth most common cancer worldwide, OHNSCC represents a key opportunity for researchers to study the application of targeted inhibitors to cell cycle control. In my dissertation, I explore the diverse roles that cyclin-CDKs play not only in driving cancer cell proliferation, but also in how they might contribute to therapeutic resistance of targeted inhibitors. Specifically, we employed a genome-wide CRISPR-Cas9 screen to reveal mediators of replication stress failure in cells with constitutively active CDK2 complexes. Data from this screen implicated CDK2 in activating apoptotic signaling. However, rather than contributing to apoptosis, CDK2 may instead activate non-canonical functions of apoptotic signaling molecules to initiate senescence. While our screen level data strongly suggested the involvement of apoptotic signaling in constitutively active CDK2 cells experiencing replication stress, validation of individual hits were unable to confirm this finding. We also investigated the role of cyclin E in conferring CDK4/6 inhibitor resistance in OHNSCC. Our data show that contrary to the established view that high cyclin E activity serves as a bypass mechanism to this class of drug, it is actually insufficient on its own phosphorylate pRb in lieu of CDK4/6 activity. Finally, we applied a chemical genetic approach refined in our lab to conduct a proteomic screen for the discovery of novel CDK6 substrates. With this technique, we found a host of novel candidate CDK substrates and validated one of these candidates, CDC45, in-vitro. Together, these studies have expanded our knowledge of cyclin-CDK biology while also providing an essential clarification in how these complexes contribute to therapeutic resistance. These insights will serve as the basis for future investigations on cell cycle deregulation and the appropriate application of targeted therapies