TARGETING WEE1 IN STANDARD THERAPY RESISTANT ESTROGEN RECEPTOR POSITIVE BREAST CANCER A Dissertation submitted to the Faculty of the Graduate School of Arts and Sciences of Georgetown University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Tumor Biology By Yassi Fallah, M.S. Washington, DC March 10, 2021 Copyright 2021 by Yassi Fallah, M.S. All Rights Reserved ii TARGETING WEE1 IN STANDARD THERAPY RESISTANT ESTROGEN RECEPTOR POSITIVE BREAST CANCER Yassi Fallah, M.S. Thesis Advisor: Ayesha N. Shajahan-Haq, Ph.D. ABSTRACT Despite the success of antiestrogens in extending overall survival of patients with estrogen receptor positive (ER+) breast tumors, resistance to these therapies is prevalent. ER+ tumors that progress on antiestrogens are treated with antiestrogens and CDK4/6 inhibitors. However, 20% of these tumors never respond to CDK4/6 inhibitors (intrinsic resistance). Here, we used endocrine sensitive ER+ MCF7 and T47D breast cancer cells to generate long-term estrogen deprived (LTED) endocrine resistant cells that are intrinsically resistant to CDK4/6 inhibitors. Since treatment with antiestrogens arrests cells in the G1 phase of the cell cycle, we hypothesized that a defective G1 checkpoint allows resistant cells to escape this arrest but increases their dependency on G2 checkpoint for DNA repair and growth, and hence, targeting the G2 checkpoint will induce cell death. Indeed, inhibition of WEE1, a crucial G2 checkpoint regulator, with AZD1775 (Adavosertib), significantly decreased cell proliferation and increased G2/M arrest, apoptosis and gamma-H2AX levels (a marker for DNA double stranded breaks) in resistant cells compared with sensitive cells. Thus, targeting WEE1 is a promising anti-cancer therapeutic strategy in endocrine resistant breast cancer. iii DEDICATION I dedicate this work to my parents. To my mother, Faranak and father, Hamid, I thank you for everything that you do for me. Thank you for your unconditional love and support. This thesis is for all the sacrifices that you have made for me. I am grateful to have you as parents. To my brother, Kayvan thank you for being the most caring human I know. You always believe in me and I thank you. May one-day cancer deaths become a tragic memory. iv ACKNOWLEDGEMENTS I would like to first express my sincere gratitude to my mentor, Dr. Ayesha N. Shajahan-Haq for her guidance throughout my studies. Your encouragement and thoughtful criticisms motivated me to continue and do better as a scientist. I am grateful for your continuous support and patience. A special thank you to my great friend, Diane Demas. I had the pleasure of being trained by you and working with you throughout these past 4 years. I will deeply miss our science and non- science discussions. Without you, this journey would not have been the same. I would like to thank my incredible thesis committee members: Dr. Marc Lippman, Dr. Anton Wellstein, Dr. Filipa Lynce and Dr. Amrita Cheema for all the clinical and preclinical knowledge and supervision. A special thank you to my co-mentor and thesis committee chair, Dr. Lippman for all of our meetings in critiquing my presentations and for your helpful guidance. Also, a warm thank you to Dr. Wellstein for your advice and support. I learned the most about cancer biology from your teachings. I am truly honored to have you all throughout this journey. Thank you to all the members of the Tumor Biology Training Program, especially Dr. Anna Riegel, Dr. Rebecca Riggins and our director, Dr. Michael Johnson for your crucial roles and leaderships. I also would like to extend my gratitude to the faculty and staff of Georgetown University Lombardi Comprehensive Cancer Center. In particular, I thank Dr. Karen Creswell, the director of the Flow Cytometry and Cell Sorting Shared Resources and Dan Xun for your expertise and assistance. v TABLE OF CONTENTS CHAPTER I: INTRODUCTION.....................................................................................................1 Hormone Receptor Positive (HR+) Breast Cancer………………………………………. 1 Development of Cyclin-Dependent Kinases 4 and 6 (CDK4/6) Inhibitors.........................5 Current Treatment of Advanced HR+ Breast Cancer…………………………………....11 CHAPTER II: DRUG RESISTANCE IN BREAST CANCER…………………………………15 Endocrine Therapy Resistance...........................................................................................15 Resistant Mechanisms of CDK4/6 Inhibitors in Breast Cancer………………………….17 CHAPTER III: CDC25A AND WEE1 AS POTENTIAL TARGETS IN BREAST CANCER...23 Regulation of the Cell Cycle by Cell Division Cycle 25A (CDC25A) and WEE1 in Breast Cancer …………………………………………………………………………………...23 Preclinical Use of WEE1 Inhibitor in Breast Cancer…………………………………….29 Research Goals…………………………………………………………………………...35 CHAPTER IV: TARGETING WEE1 INHIBITS GROWTH OF BREAST CANCER CELLS THAT ARE RESISTANT TO ENDOCRINE THERAPY AND CDK4/6 INHIBITOR……………………. ……………………………………………………………….37 Introduction………………………………………………………………………………37 Materials and Methods.......................................................................................................38 Results……………………………………………………………………………………42 Discussion………………………………………………………………………………..66 Conclusion……………………………………………………………………………….69 Supplementary Figures…………………………………………………………………..70 vi CHAPTER V: CDC25A AS A TARGET IN ENDOCRINE THERAPY AND CDK4/6 INHIBITOR RESISTANT BREAST CANCER CELLS………………………………………..76 CHAPTER VI: CLOSING REMARKS AND FUTURE DIRECTIONS………………….........82 Closing Remarks…………………………………………………………………………82 Future Directions………………………………………………………………………...88 APPENDIX: TABLES...…………………………………………………………………….…..91 REFERENCES…………………………………………………………………………………..92 vii LIST OF FIGURES Figure 1. Intrinsic subtypes of breast cancer ...................................................................................2 Figure 2. Timeline of approval of antiestrogen agents by the FDA ................................................4 Figure 3. Chemical structures of CDK4/6 inhibitors .....................................................................11 Figure 4. Four main clinical trials in HR+ metastatic patients who have progressed on endocrine therapy ............................................................................................................................................13 Figure 5. Mechanisms of resistance to CDK4/6 inhibitors ............................................................19 Figure 6. Activity of CDC25A throughout the cell cycle ..............................................................24 Figure 7. Checkpoint control by WEE1 upon DNA damage .........................................................29 Figure 8. AZD1775 (WEE1 inhibitor) clinical trials .....................................................................33 Figure 9. LTED cells are resistant to antiestrogens .......................................................................45 Figure 10. LTED cells are resistant to CDK4/6 inhibitors ............................................................46 Figure 11. LTED cells are resistant to the combination of antiestrogens and CDK4/6 inhibitors ........................................................................................................................................48 Figure 12. Differential levels of CyclinD1 and pRB(780) proteins in paternal and LTED breast cancer cells treated with combination of E2 deprivation and ribociclib ........................................51 Figure 13. Increased G1 cell cycle arrest in LTED derivative cells ..............................................52 Figure 14. AZD1775 monotherapy suppressed growth of LTED cells .........................................55 Figure 15. Inhibition of WEE1 with AZD1775 increased G2/M cell cycle arrest in LTED cells…. ...........................................................................................................................................57 Figure 16. AZD1775 induced marked increase in apoptosis in LTED cells .................................60 Figure 17. Knockdown of p53 in combination with AZD1775 enhances inhibition of growth in MCF7 and T47D cells ....................................................................................................................63 viii Figure 18. Increased WEE1 gene expression correlate with survival in LTED cells ....................65 Figure 19. Basal growth rate of LTED cells ..................................................................................70 Figure 20. LTED cells are resistant to the combination of antiestrogens and CDK4/6 inhibitors. .......................................................................................................................................71 Figure 21. AZD1775 monotherapy suppressed growth of LTED cells .........................................72 Figure 22. Increased inhibition of cell proliferation in LTED cells following siRNA mediated WEE1 knockdown .........................................................................................................................73 Figure 23. Treatment with AZD1775 induced PARP cleavage .....................................................75 Figure 24. Inhibition of CDC25 suppressed growth of LTED cells ..............................................78 Figure 25. Anti-proliferative effect of BN82002 in combination with antiestrogen and CDK4/6 inhibitor was enhanced in LTED cells ...........................................................................................79 Figure 26. Interaction