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A Small-Molecule Inhibitor of Wee1, Azd1775, As a Chemosensitizing

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A Small-Molecule Inhibitor of Wee1, Azd1775, As a Chemosensitizing A SMALL-MOLECULE INHIBITOR OF WEE1, AZD1775, AS A CHEMOSENSITIZING AGENT IN ACUTE LEUKEMIA by TAMARA BURLESON GARCIA B.S., University of Alabama at Birmingham, 2011 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Cancer Biology Program 2017 This thesis for the Doctor of Philosophy degree by Tamara Burleson Garcia has been approved for the Cancer Biology Program by Mary E. Reyland, Chair Arthur Gutierrez-Hartmann Mingxia Huang Andrew Thorburn Jing Wang Rajeev Vibhakar, Advisor Christopher C. Porter, External Advisor Date: 08/18/2017 ii Garcia, Tamara Burleson (Ph.D., Cancer Biology) A Small-Molecule-Inhibitor of WEE1, AZD1775 as a Chemosensitizing Agent in Acute Leukemia Thesis Directed by Associate Professor Rajeev Vibhakar ABSTRACT Although some patients with acute leukemia have good prognoses, the prognosis of adult and pediatric patients who relapse or cannot tolerate standard chemotherapy is poor. Thus, novel therapies are necessary to improve outcomes in acute leukemia. Conventional chemotherapy is the mainstay of treatment for acute leukemia, and one approach to improving outcomes is to enhance the efficacy of these drugs. Inhibition of the cell cycle protein WEE1 has been shown to enhance the efficacy of a number of conventional chemotherapy agents by abrogating cell cycle arrest, increasing DNA damage accumulation, and promoting apoptosis. Increased understanding of the functions of WEE1 and the consequences of WEE1 inhibition, particularly in combination with targeted and non- targeted agents, will be useful in translating small-molecule WEE1 inhibitors to the clinic. This dissertation seeks to further the understanding of the consequences of WEE1 inhibition in acute leukemia. Chapter I provides an overview of the clinical characteristics and molecular changes in acute myeloid leukemia and acute lymphoblastic leukemia and general principles of cell cycle regulation and DNA damage repair. I also review the function of WEE1 and pre-clinical and clinical studies with a first-in-class small-molecule inhibitor of WEE1, AZD1775. Finally, I review mechanisms of resistance to kinase inhibitors with a focus on epigenetic-mediated drug resistance. Chapter II describes the materials and methods iii used to generate the data presented in this dissertation. In Chapter III, I seek to determine the relative contribution of CDK1 and CDK2 to the combinatorial activity of AZD1775 and cytarabine. In Chapter IV, I identify a novel synergistic combination in acute leukemia: AZD1775 and olaparib. In Chapter V, I characterize three acute leukemia cell lines with acquired resistance to AZD1775 and demonstrate that the resistance is mediated by an altered epigenetic landscape leading to elevated MYC expression. In Chapter VI, I summarize these findings and discuss future directions for this work. Collectively, this work enhances the understanding of mechanisms of combinatorial activity of WEE1 inhibition and targeted and non-targeted agents and describes a potential mechanism of resistance which can be informative in clinical translation. The form and content of this abstract are approved. I recommend its publication. Approved: Rajeev Vibhakar iv To my husband, Jesse, and my parents, Keith and Danita. I am forever grateful for your love, encouragement, and prayers. v ACKNOWLEDGMENTS I would like to thank Chris Porter for his guidance and mentorship over the past four years. It was an honor and a pleasure to work in the Porter lab, and this experience has been invaluable in my career development. I would also like to acknowledge the rest of the former Porter lab members for their support, advice, and friendship. I would like to thank Lori Gardner and Christy Gearheart for their support and encouragement during this journey and Courtney Jones for being a wonderful scientific role model and source of scientific and career advice. Thank you to Cathy Lee-Miller who allowed me to shadow her in the clinic and has been a great clinical role model. Thank you to Susan Fosmire for being a great lab mate in both the Porter and Vibhakar labs and for cloning the CDK1/2-AF constructs. Dmitry Baturin trained me to work with mice and assisted in all in vivo studies. Annemie van Linden developed the AZD1775-resistant cell lines and performed the AZD1775 and Ara-C combination studies in sensitive and resistant cells. Jonathan Snedeker helped with olaparib and AZD1775 combination studies. I am greatly indebted to Rajeev Vibhakar for allowing me to work in his lab for the past year. It has been a great experience, and my interactions with him and the rest of the neuro-oncology team, both in the lab and in the clinic, have solidified my desire to pursue a career in pediatric neuro-oncology. Many thanks to rest of the Vibhakar lab for welcoming me this past year. Thank you to Sujatha Venkataraman for all the scientific advice she has given me and for patiently answering my many technical questions. Many thanks to my thesis committee for their insight and advice and for helping me develop as a researcher. vi I would like to thank the MSTP administration for their support and encouragement. I would also like to thank my MSTP classmates for their friendship and advice. It is an honor to be a part of such a smart, talented, and kind group of people. Many thanks to my classmate Sally Peach for her encouragement, advice, and friendship, and for entertaining me with stories of her many world travels. I would like to thank my undergraduate research mentors, Christopher Willey and Andrei Stanishevsky, who introduced me to research and inspired me to pursue a career as a physician scientist. I would like to thank my husband Jesse for his love and encouragement. Thank you for helping me maintain a good work-life balance throughout this journey and for making me laugh every day, even when my experiments were not working. Lastly, I cannot thank my parents enough for their support and prayers. I am thankful for the sacrifices they have made that have enabled me to pursue my dream of being a physician scientist and for instilling in me their work ethic that has made this work possible. Soli Deo Gloria vii TABLE OF CONTENTS CHAPTER I. INTRODUCTION .....................................................................................................1 Acute Myeloid Leukemia ..............................................................................1 Acute Lymphoblastic Leukemia ....................................................................6 General Principles of Cell Cycle Regulation and DNA Damage Repair .......11 WEE1 Tyrosine Kinase..................................................................................21 Resistance to Kinase Inhibitors ......................................................................37 II. MATERIALS AND METHODS ...............................................................................42 Cell Culture ....................................................................................................42 Lentivirus Preparation ....................................................................................42 Chemotherapies, Antibodies, and Reagents ...................................................43 Comet Assays.................................................................................................43 Immunofluorescence ......................................................................................44 Flow Cytometry .............................................................................................44 Beta-Galactosidase Staining ..........................................................................44 Animal Experiments ......................................................................................45 RNAseq ..........................................................................................................45 viii Statistical Analysis .........................................................................................46 III. INCREASED ACTIVITY OF WEE1 TARGETS CDK1 AND CDK2 IS NECESSARY FOR THE COMBINATORIAL ACTIVITY OF AZD1775 AND CYTARABINE ......................................................................................................................47 Introduction ....................................................................................................47 Results ............................................................................................................48 Summary of Findings .....................................................................................52 IV. A SMALL-MOLECULE INHIBITOR OF WEE1, AZD1775, SYNERGIZES WITH OLAPARIB BY IMPAIRING HOMOLGOUS RECOMBINATION AND ENHANCING DNA DAMAGE AND APOPTOSIS IN ACUTE LEUKEMIA ..................56 Introduction ....................................................................................................56 Results ............................................................................................................58 Summary of Findings .....................................................................................78 V. ACQUIRED RESISTANCE TO AZD1775 IS MEDIATED BY HDAC REGULATED EXPRESSION OF C-MYC ..........................................................................80 Introduction ....................................................................................................80 Results ............................................................................................................81
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