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Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dick, Frederick A.

2nd Supervisor

DiMattia, Gabriel E.

Abstract

High-grade serous ovarian cancer (HGSOC) is the most common form of ovarian cancer. The majority of women are disproportionately diagnosed at an advanced stage (stage III-IV) of the disease when tumours have progressed beyond the ovaries or fallopian tubes and into the peritoneal cavity. Survival rates at late-stage are as low as 25% and chemoresistant disease recurrence is common, affecting up to 90% of patients. Multicellular clusters called spheroids contribute to dormancy, chemoresistance, and metastases and are a major challenge to treatment of HGSOC. Spheroid cells undergo reversible quiescence to evade chemotherapy in a process mediated by the mammalian DREAM complex and its initiating kinase, DYRK1A. Depletion of DYRK1A reduces spheroid cell survival and increases sensitivity to chemotherapy, highlighting it as an attractive therapeutic target. Herein we demonstrate the long-term consequences of DREAM loss in adult mice. DREAM deficient mice do not have proliferative control defects but develop systemic amyloidosis as a result of overexpression of apolipoproteins Apoa1 and Apoa4. Overexpression of Apoa1 and Apo4 were marked with increased B-MYB-MuvB (MMB) and decreased H2AZ deposition within gene bodies. The prolonged latency before developing amyloidosis suggests depriving cells of quiescence is tolerable for short periods of time. To broadly identify genetic vulnerabilities in spheroid cells, we employed an integrated strategy in which we investigated the transcriptional programming and also performed a loss-of-function genome-wide CRISPR screen in HGSOC spheroid cells. Towards this aim, we developed novel bioinformatic tools and methodology to facilitate high-throughput discovery of essential genes and pathways and anticipate these tools will have broad usability in transcriptional and loss-of-function studies. Using these tools, we identified the netrin signaling pathway as an essential mediator of HGSOC spheroid cell survival. Specifically, components of netrin signaling are upregulated in spheroid cells and depletion of netrin ligands or receptors was sufficient to reduce spheroid cell viability. Our work highlights netrin signaling as a potential target for new metastatic ovarian cancer therapies. Taken together, the work presented herein provide more insight into the roles of DREAM and DYRK1A in HGSOC spheroid survival as well as implications of therapeutically targeting this pathway. HGSOC is a very deadly disease and there is an urgent need to develop new therapeutic strategies that can specifically target dormant chemoresistant spheroids in patients to treat or prevent relapse.

Summary for Lay Audience

The majority of ovarian cancer cases are diagnosed at an advanced stage when the disease has spread into the abdominal cavity. Survival rates in these women are extremely low. Treatment in these women is made difficult due to the presence of spheroids, which are clusters of cancer cells that have dislodged from tumours and float within the abdominal fluid. Spheroids are resistant to drugs and persist in a “sleeping” state called dormancy. These cells travel through the abdominal fluid and reawaken to spread the disease to new sites in the body. We previously identified the protein complex known as DREAM and the protein known as DYRK1A as essential factors that promote the survival of spheroids. DYRK1A is required to mobilize DREAM. Loss of DREAM or DYRK1A in spheroids reduces survival and enhances sensitivity to drugs, suggesting these are attractive targets for therapies. However, the mechanisms by which DREAM and DYRK1A promote spheroid survival and dormancy are not fully understood and the long-term consequences of DREAM loss in adults is not known. Herein we demonstrate the side-effects of prolonged DREAM loss in adult mice to investigate what would happen if DREAM is inhibited. We found that prolonged DREAM loss leads to the development of amyloidosis indicating this must be an import consideration for anti-DREAM drug therapies in ovarian cancer. Next, to identify new drug targets in dormant ovarian cancer, we screened spheroids and identified a family of genes that were previously uncharacterized in ovarian cancer and we show that they are regulated by DYRK1A. We show that disabling these genes reduces spheroid survival, indicating their potential as drug targets. We also describe new computational tools that facilitated this discovery and anticipate these tools will have broad usability in other studies. In summary, the work presented here add to our understanding of the roles of DREAM and DYRK1A in ovarian cancer and inform of the implications of targeting this pathway with drugs. Ovarian cancer is a very deadly disease and there is an urgent need to develop new treatment strategies to improve patient survival.

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

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