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Understanding the Role of Group I Paks in Thyroid Cancer DISSERTATION Presented in Partial Fulfillment of the Requirements for T

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Understanding the Role of Group I Paks in Thyroid Cancer DISSERTATION Presented in Partial Fulfillment of the Requirements for T Understanding the Role of Group I PAKs in Thyroid Cancer DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Christina Michelle Knippler, BA Biomedical Sciences Graduate Program The Ohio State University 2019 Dissertation Committee: Matthew Ringel, MD, Advisor David Carbone, MD, PhD Joanna Groden, PhD Michael Ostrowski, PhD Mark Parthun, PhD Copyrighted by Christina Michelle Knippler 2019 Abstract Thyroid cancer incidence has been increasing over the last several decades. Most thyroid cancers are curable, however, aggressive tumors do not respond to standard therapy and have only limited responses to recently-approved targeted therapies, especially when metastatic cancer is present. Many “driver” mutations of thyroid cancer have been determined, but it is becoming increasingly clear that these oncogenic pathways do not act as singular entities to “drive” malignancy. It is, therefore, pivotal to understand the complexity of thyroid cancer signaling in order to determine the best therapeutic approach. The MAPK pathway is overactivated in the majority of thyroid cancers, with the most common mutation causing the valine to glutamic acid point mutation at amino acid 600 in the kinase BRAF (i.e. BRAFV600E). Members of the MAPK pathway, and in particular BRAFV600E, are therapeutic targets of high interest, with ongoing clinical trials using pathway inhibitors. Preliminarily, these drugs result in only partial responses and resistance. Research is ongoing to determine the mechanisms of innate and acquired resistance to these drugs. Our prior research has identified a family of serine/threonine kinases, the group I p21-activated kinases (PAKs), as a novel indicator of aggressive characteristics in thyroid cancer. PAK1 protein levels and activation are highly expressed in the invasive edges of thyroid cancers, compared to the center of the tumor or normal thyroid tissue. BRAF and PAK1 signaling are interconnected, both necessary for thyroid cancer migration, with ii PAK activation in vitro and in vivo regulated by BRAF expression. Further, BRAF and PAK1 physically interact. Therefore, the connection between BRAF and PAK1 are significant for thyroid cancer progression. The objective of the current work was to characterize the BRAF-PAK1 interaction in order to develop novel therapeutic approaches. This research determined the ability of PAK1 to be pharmacologically inhibited and tested the hypothesis that group I PAK inhibitors would reduce thyroid cancer growth and motility – two important hallmarks of cancer progression. Biochemical approaches tested how PAK1 physically interacts with BRAF, and its constitutively active mutated form, BRAFV600E. We hypothesized that PAK1 and BRAF physically interact as a complex with other mediating proteins and that these proteins provide additional targets for better therapeutic efficacy. This work utilized both in vitro and in vivo models to provide a comprehensive understanding of the BRAF and PAK1 complex. In vitro studies using several different thyroid cancer cell lines determined that group I PAKs could be pharmacologically inhibited and this reduced thyroid cancer cell growth, induced cell cycle arrest, and reduced cell invasion. Further, PAK inhibition could be combined with BRAFV600E and AKT inhibition for further synergistic reductions on thyroid cancer cell growth, useful for future clinical application. Importantly, group I PAK inhibition was effective in vivo in a mouse model of thyroid cancer where BRAFV600E was overexpressed in the thyroid. PAK inhibition restricted thyroid size and also carcinoma formation. iii In vitro biochemical approaches determined that both wild-type BRAF and BRAFV600E can physically interact with PAK1 in precise cell contexts, most often during mitosis. The interaction is unlikely to be direct, but may be facilitated through part of the PAK1 regulatory domain and does not require PAK1 kinase function. Proteomic evidence suggests that this interaction is mediated by chaperone proteins, allowing BRAF and PAK1 to interact transiently. These studies expand our understanding of the signaling crosstalk between BRAF and PAK1 and identify the group I PAKs as therapeutically targetable kinases in thyroid cancer. This provides further evidence to continue efforts in drug development targeting the group I PAKs, as well as potential combination strategies with BRAF, AKT, and possibly HSP90 inhibitors, for patients with aggressive thyroid cancers. iv Dedication To my parents, sister, and fiancé for their constant love, support, and encouragement. To Grammy for fostering my love of science. v Acknowledgments I am truly appreciative of everyone who has helped and supported me along this journey. My mentor, Dr. Matthew Ringel, has been a cornerstone in my development as a young scientist. He taught me how to think critically and creatively about my science. I am thankful for our long meetings discussing results and developing new hypotheses, and his sincere support in my personal growth. His persistent encouragement, no matter the result of an experiment, helped motivate me to push through the challenges of research and never lose sight of the overall goal and the discoveries we make along the way. I am very thankful for all I have learned from Dr. Ringel and will use my experiences from him to mentor young scientists in the future. I would also like to especially thank Dr. Motoyasu Saji from the Ringel Lab for his selfless guidance and expertise each day in the lab. My knowledge and skill of experimental techniques is largely due to his patience and passion for teaching. His constant care for the lab, organization, and management kept this lab running on all cylinders so that I could pursue my research goals. The Ringel Lab would not function smoothly without the endless help and work from our administrator, Nanci Edgington. Often behind the scenes, Nanci was the glue that kept this lab together, and I am immensely grateful for her time and effort in helping me with ordering, setting up meetings, troubleshooting problems, and much more. vi Additionally, I am thankful for opportunities to get feedback and inspiration from other members of the Ringel Lab, past and present, including Dr. Anisley Valenciaga, Dr. Chaojie Wang, Dr. Steven Justiniano, Dr. Anisha Hammer, Dr. Adlina Mohd Yusof, Dr. Kara Rossfeld, Neel Rajan, Ceimoani Bumrah, and Luis Bautista. Thank you to my committee members, Drs. David Carbone, Joanna Groden, Michael Ostrowski, and Mark Parthun, for their guidance and advice throughout the my graduate studies to help me advance my research and to critically analyze my work. This research would not be possible without the expertise from our collaborators. I’d like to thank Kyle Porter from the Department of Biomedical Informatics for his data analyses. Dr. Krista La Perle from the Department of Veterinary Biosciences was instrumental in analyzing the mouse pathology samples. I am grateful for Dr. Michael Freitas (OSU) and Dr. Salim Merali (Temple, Philadelphia, PA) for their proteomics expertise and to Dr. Mark Parthun for helping me with the chromatography experiments. Additionally, I am appreciative of the expertise of many of the OSU Shared Resources. I am very appreciative of the Pelotonia Fellowship Program for not only funding my research, but also giving me the opportunities to share my passion and research with the community. Finally, I am grateful for the support and mentorship from the Biomedical Sciences Graduate Program, in particular the past and current program directors, Drs. Joanna Groden, Jeffrey Parvin, and Michael Freitas. vii Vita 2009................................................................Chantilly High School, VA 2013................................................................B.A. Biology, B.A. Music, University of Virginia, VA 2013-Present ..................................................Biomedical Sciences Graduate Program, The Ohio State University, OH 2013-2014.......................................................University Fellowship, The Ohio State University, OH 2014-2016.......................................................T32 Systems in Integrated Biology Fellowship, The Ohio State University, OH 2016-2018.......................................................Pelotonia Graduate Fellowship, The Ohio State University, OH 2018-Present .................................................Graduate Research Associate, The Ohio State University, OH Publications Knippler CM, Saji M, Rajan N, Porter K, La Perle KMD, Ringel MD. MAPK- and AKT-activated thyroid cancers are sensitive to group I PAK inhibition. Endocr Relat Cancer. 2019 May 1; [Epub ahead of print] viii Valenciaga A, Saji M, Yu L, Zhang X, Bumrah C, Yilmaz AS, Knippler CM, Miles W, Giordano TJ, Cote GJ, Ringel MD. Transcriptional targeting of oncogene addiction in medullary thyroid cancer. JCI Insight. 2018 Aug 23; 3(16) Stellfox ME, Nardi IK, Knippler CM, Foltz DR. Differential binding partners of the Mis18α/β YIPPEE domains regulate Mis18 complex recruitment to centromeres. Cell Rep. 2016 Jun 7; 15(10): 2127-2135 Nardi IK, Zasadzińska E, Stellfox ME, Knippler CM, Foltz DR. Licensing of centromeric chromatin assembly through the Mis18α-Mis18β heterotetramer. Mol. Cell. 2016 Mar 3; 61(5): 774-787 McCarty SK, Saji M, Zhang X, Knippler CM, Kirschner LS, Fernandez S, Ringel MD. BRAF activates and physically interacts with PAK to regulate cell motility. Endocr Relat
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