Greatwall in replication stress/DNA damage responses and oral cancer resistance

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Oral cancer, including cancers of the mouth and the back of the throat, is the sixth most common cancer worldwide. In the United States, approximately 50,000 new oral cancer cases are diagnosed each year. First- line treatments for oral cancer typically include surgery and radiation, with chemotherapy added to decrease the possibility of metastasis, to eliminate residual tumor cells after surgery, to enhance the efficacy of radiation (chemoradiation), and for patients with confirmed distant metastasis. Radiation and oral cancer chemotherapeutics confer cytotoxicity largely by disrupting DNA replication to induce DNA damage. Unfortunately, the prognosis of oral cancer, particularly HPV(-) cases, remains relatively poor, calling for a better understanding of how cells respond to replication stress and DNA damage, and accordingly, developing more effective treatment options and combinations to overcome drug resistance. In the current project, we characterize a new role of Greatwall (Gwl) kinase in the replication stress and DNA damage responses. Gwl was frequently upregulated in HPV(-) oral cancer, in correlation with cancer progression, tumor recurrence, and poor patient survival. Gwl promoted the recovery and resistance of oral cancer cells to drugs that induce replication stress and DNA damage. Gwl depletion or inhibition sensitized the drug responses in oral cancer cells and mouse tumor models. Building on these findings, we hypothesize that Gwl mediates the cellular responses to replication stress and DNA damage, and is therefore a potent target for oral cancer therapy. We will uncover detailed mechanisms underlying the function and regulation of Gwl in the replication stress and DNA damage responses; we will also establish the crucial proof-of-principle for the development of Gwl targeting in cancer treatment. In Aim 1, we will delineate how Gwl is recruited to stalled replication forks via its interaction with replication protein A (RPA) to regulate a phosphatase-mediated response to replication stress. This study will shed new light on cancer progression and treatment, given that replication stress is a hallmark of cancer, and that anti-replication drugs are commonly used in cancer therapy. In Aim 2, we will reveal a new mechanism that leads to Gwl stabilization and accumulation after replication stress and DNA damage, potentially as a key event that initiates cell recovery and confers tumor resistance. Upregulation of Gwl is mediated directly by DNA damage signaling, suggesting a self-engaged “timer” mechanism that initiates cell recovery and treatment resistance. Finally, guided by our mechanistic investigations, we will explore in Aim 3 therapeutic targeting of Gwl, using unique small molecule inhibitors which interfere with either Gwl kinase activation or its interaction with RPA. Both patient-derived oral tumor xenograft and orthotopic syngeneic oral tumor models will be utilized to comprehensively evaluate the therapeutic potential of Gwl inhibition. Together, this project will lead to a deeper understanding of the cellular responses to replication stress and DNA damage, and characterize a new drug target to improve oral cancer therapy.