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DEK Is a Homologous Recombination DNA Repair Protein and Prognostic Marker for a Subset of Oropharyngeal Carcinomas

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DEK Is a Homologous Recombination DNA Repair Protein and Prognostic Marker for a Subset of Oropharyngeal Carcinomas DEK is a homologous recombination DNA repair protein and prognostic marker for a subset of oropharyngeal carcinomas A dissertation submitted to the Graduate School Of the University of Cincinnati In partial fulfillment of the requirements to the degree of Doctor of Philosophy (Ph.D.) in the Department of Cancer and Cell Biology of the College of Medicine March 29, 2017 by ERIC ALAN SMITH B.S Rose-Hulman Institute of Technology, 2010 Dissertation Committee: Susanne I. Wells, Ph.D. (Chair) Paul R. Andreassen, Ph.D. Nancy Ratner, Ph.D. Peter J. Stambrook , Ph.D. Kathryn A. Wikenheiser-Brokamp, M.D., Ph.D. Abstract The DEK oncogene is currently under investigation as a therapeutic target and clinical biomarker for tumor progression, chemotherapy resistance and poor outcomes for multiple types of malignancies. With regard to most cancer types, the degree of DEK overexpression correlates with higher stage tumors and worse patient survival, marking this molecule as a promising prognostic factor. Prior to this work, the utility of DEK as a biomarker had not been assessed in oropharyngeal squamous cell carcinoma (OPSCC), an aggressive disease characterized by poor survival and high rates of treatment comorbidities. As discussed in chapter 1, OPSCC is comprised of two subtypes based on the presence or absence of human papillomavirus (HPV) infection. In general, HPV+ OPSCCs have improved therapy response and an overall better prognosis than their HPV- counterparts. This treatment sensitivity may be due in part to the HPV oncogenes, which inactivate and degrade tumor suppressors as discussed in chapter 2, removing the need for the development of mutations that promote radiation and chemotherapy resistance. Due to improved therapy response, clinical trials are now testing de- intensified regimens for the HPV+ OPSCC subset to prevent unnecessary morbidity from aggressive treatment. However, there is a population of HPV+ tumors that have poor outcomes from standard treatment and may not respond to de-intensified therapy. Since current staging tools do not fully account for therapy response, it is unclear which HPV+ OPSCC patients will not benefit from de-intensified treatment, but biomarkers, such as DEK, may play a critical role in identifying such patients. ii Chapter 3 describes the biochemical and cellular functions of the DEK oncogene and focuses on how DEK can promote chemotherapy/radiation resistance through DNA repair. Chapter 4 provides new experimental evidence demonstrating that DEK is strictly required for homologous recombination (HR) repair of DNA breaks. DEK was found to be required for γH2AX activation after treatment with ionizing radiation and formed a complex with RAD51, the essential HR recombinase. Based on the lack of association with BRCA1, it is hypothesized that DEK may assist RAD51 in strand invasion. While a previous study has linked the chemotherapy resistance of DEK to NHEJ function, it was instead determined that the loss of DEK is synthetic lethal with NHEJ inactivation via DNA-PK inhibitors. This suggests that cells rely on remaining NHEJ for survival in the absence of DEK-mediated HR repair. In Chapter 5, the prognostic value of DEK in predicting outcomes in OPSCC was determined using a retrospective case study with tumor resections from 194 patients. DEK expression was correlated with higher volume tumors and an increased risk of death in HPV+ OPSCC, but no association was found in HPV- disease. These findings indicate that DEK can be further developed as a biomarker for poor survival in HPV+ OPSCC and mark HPV- OPSCC as one of the few solid tumors where DEK is not predictive of survival outcomes. In summary, this dissertation identified that DEK is required for HR repair of DSBs and may be clinically useful as a prognostic marker for HPV+ OPSCC. iii iv Preface The following work in this dissertation has been published in the following peer- reviewed journals: Smith E.A., Kumar, B., Kakajan, K., Smith, S.M., Brown N.V., Zhao, S., Kumar, P., Teknos, T.N., Wells, S.I. “DEK associates with tumor stage and outcome in HPV16 positive oropharyngeal squamous cell carcinoma.” Published in Oncotarget Smith E.A., E.A., Gole, G., Willis, N.A., Soria, R., Starnes L.M., Krumpelbeck, E.F., Jegga, A.G., Abdullah M.A,, Guo, H., Meetei, A.R., Andreassen, P.R., Kappes, F., Privette Vinnedge, L.M., Daniel, J.A., Scully, R., Wiesmüller, L. Wells, S.I. “DEK is required for homologous recombination repair of DNA breaks.” Published in Scientific Reports Smith E.A., Matrka, M.C.; Wells, S.I. “HPV Virology: Cellular Targets of HPV Oncogenes and Transformation” in “HPV Virology: Cellular Targets of HPV Oncogenes and Transformation” in Human Papillomavirus (HPV)-Associated Oropharyngeal Cancer; Miller D.L.; Stack, S.M., Eds.; Springer International Publishing AG Switzerland, 2015; pp 69-101. v Acknowledgements Out of all of the sections of this dissertation, this is probably the hardest for me to write. It is not because of lack of content, but rather there are too many people to thank, and words hardly suffice to state their importance in my PhD journey. Still, here I am giving it a go anyway! First, I must thank my labmates and all of the support they have given me through the years. We are a rather unique group: eager, rambunctious, loud, jocular, passionate, and fiercely loyal. When you join The Wells lab, it is not just a work group. You join a family. We play pranks on each other, joke alongside each other, and, like any family, argue amongst each other. Despite a whole turnover of the lab besides Susa (indispensable) and Marie (wishes she was not indispensable), I am happy to say that the spirit that first attracted me to the lab still remains, although we may actually be somewhat quieter now! One of the aspects that I will always cherish about the lab is that it is a lot like Hotel California: you can always join the lab, but you can never leave. I am always seeing and reconnecting with old alumni, even the original personnel in Susa’s laboratory. Our comradery is what defines the lab, and knowing that you always have their support is a feeling I cannot put into words. Even among a group as awesome as The Wells lab, there are special people that stand out. I want to specifically mention three here. The first is Beth Hoskins. She was the first person who truly believed in me, showing that belief by humbling me until I realized how little I knew and supporting me when I came to doubt myself. I especially want to thank Allie Adams for giving me a chance to dust myself off, train hard, and start anew. I do not know where I would be without her support and guidance. Of course, I vi should probably also thank my boss, Susa. If I am lucky enough to become a PI in the future, I sincerely hope that I am half as thoughtful and devoted to my work and my people as Susa. She embodies that type of leadership that I value most: Transformational Leadership. Where authoritarian leaders bark orders and servant leaders lead through example, transformational leaders inspire their compatriots to push themselves beyond their normal limits and accomplish great things (or if you are me, to push myself beyond my limits and end up in the ER, but that’s a story for a different time!). One of the things I admire most about Susa and may have the hardest difficulty replicating is her steadfast belief in her students and post-docs. She never gives up on people and believes in their potential, even when others would have long dismissed them. Like many of her students, I needed a mentor who defines her success by the lifelong accomplishments of her protégés, and I was fortunate to have been able to work for her. I will always be in her debt. Hopefully, I’ll get my just deserts when I also have a student who knocks on my door every day to give me a status update. One of the things I have learned from Susa is that collaboration is the life-blood of science, and that is a skill I have learned to hone during my time in her lab. From our peers at Children’s and UC, to my dear friends Ferdi, Haihong, and Chris in Aachen Uniklinic and everyone in-between, I am thankful to have been able to work with and study alongside some of the brightest minds in academia. Special thanks go to Emma Lou and Dr. Robert Cardell for their fellowship award. Without your support, I could not have travelled to study DEK biochemistry in Ferdi’s lab, attend two international meetings, or have met two wonderful and enthusiastic emeritus professors. vii To my thesis committee, I thank you for keeping me on track and my feet to the fire. Your advice has always been well-appreciated. In particular, I want to thank Paul for all of his guidance in the past several years. He was a second mentor to me, and we would spend hours after committee meetings and randomly in his office discussing hypotheses, politics, and random ideas about how in the world DEK actually functions. One day, I hope to visit Seattle long enough for me to see everything that he has recommended during our talks. Most of all, I would like to thank my wonderful family for all of their love and support. My mom was the trailblazer, obtaining the first PhD in my family’s history during my first year of graduate school, and she has served as a constant source of guidance and understanding.
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