University of Tennessee Health Science Center UTHSC Digital Commons Theses and Dissertations (ETD) College of Graduate Health Sciences 4-2020 Integrated Multimodal Genomic Analyses Reveal Novel Mechanisms of Glucocorticoid Resistance in Acute Lymphoblastic Leukemia Robert J. Autry University of Tennessee Health Science Center Follow this and additional works at: https://dc.uthsc.edu/dissertations Part of the Medical Pharmacology Commons Recommended Citation Autry, Robert J. (https://orcid.org/0000-0002-6965-2942), "Integrated Multimodal Genomic Analyses Reveal Novel Mechanisms of Glucocorticoid Resistance in Acute Lymphoblastic Leukemia" (2020). Theses and Dissertations (ETD). Paper 516. http://dx.doi.org/10.21007/etd.cghs.2020.0501. This Dissertation is brought to you for free and open access by the College of Graduate Health Sciences at UTHSC Digital Commons. It has been accepted for inclusion in Theses and Dissertations (ETD) by an authorized administrator of UTHSC Digital Commons. For more information, please contact [email protected]. Integrated Multimodal Genomic Analyses Reveal Novel Mechanisms of Glucocorticoid Resistance in Acute Lymphoblastic Leukemia Abstract Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Much has been discovered in recent decades regarding ALL biology, and the outcome of patients with ALL has vastly improved, especially in pediatric ALL patients. Despite very promising overall cure rates, patients who relapse have a greatly decreased prognosis with survival rates ranging from 30-60%. These numbers stand to improve even further with new targeted therapies that seek to improve or maintain cure rates while reducing treatment related toxicities which affect patients both acutely and chronically. Glucocorticoids (GCs) are essential components of modern chemotherapeutic intervention for ALL. Resistance to glucocorticoids is an important factor in determining early treatment response and overall patient survival. Reduction of glucocorticoid induced toxicities, such as osteonecrosis, can significantly affect patient quality of life and are associated with high dose glucocorticoid treatment in pediatric patients. Both endogenous and exogenous glucocorticoids exert their mechanism of action through various pleiotropic effects that regulate numerous cellular functions and can cause selective cytotoxicity in lymphoid malignancies. The complex mechanism of action of glucocorticoids is evident in the number of diverse clinically relevant molecular pathways that have been previously associated with resistance to glucocorticoids in ALL.The identification of genomic and epigenomic mechanisms of glucocorticoid resistance are important for improving ALL treatment outcomes. We used an agnostic genome-wide method to interrogate multiple types of genomic information (mRNA and miRNA expression, DNA methylation, SNPs, CNAs and SNVs/ Indels) in primary human acute lymphoblastic leuke Document Type Dissertation Degree Name Doctor of Philosophy (PhD) Program Biomedical Sciences Research Advisor William E. Evans, PharmD Keywords Drug Resistance, Genomics, Glucocorticoids, Leukemia, Pharmacogenomics Subject Categories Medical Pharmacology | Medicine and Health Sciences This dissertation is available at UTHSC Digital Commons: https://dc.uthsc.edu/dissertations/516 UNIVERSITY OF TENNESSEE HEALTH SCIENCE CENTER DOCTOR OF PHILOSOPHY DISSERTATION Integrated Multimodal Genomic Analyses Reveal Novel Mechanisms of Glucocorticoid Resistance in Acute Lymphoblastic Leukemia Author: Advisor: Robert J. Autry William E. Evans, PharmD A Dissertation Presented for The Graduate Studies Council of The University of Tennessee Health Science Center in Partial Fulfillment of the Requirements for the Doctor of Philosophy degree from The University of Tennessee in Biomedical Sciences: Molecular and Systems Pharmacology College of Graduate Health Sciences May 2020 Portions of Chapters 3, 4 and 5 © 2020 Springer Nature Limited. All other material © 2020 by Robert J. Autry. All rights reserved. ii DEDICATION This dissertation is dedicated first and foremost to my wonderful wife, Lindsy. You have supported my path every step of the way, inspired me to work harder and challenged me to reach higher, without you this would have been impossible. To my amazing children Fletcher and Sloane, for providing me with endless joy to distract from the hard days. Never give up on your dreams no matter what stands in your way, and may you learn from failure so that someday you can make a difference. To my parents Don and Lorie, who have pushed me and supported me to achieve my potential from childhood. To my Nana and Papa, who taught me to work hard, to take chances and to always remember importance of family. Finally, to my grandmother Janette who was the best example of hard work, the value of education and speaking truth for its own sake. iii ACKNOWLEDGEMENTS First, I would like to express my deepest gratitude and appreciation to my mentor Dr. William E. Evans. For the last five years, every day has been an adventure. He always challenged me to do more, pushed me to ask the right questions, taught me to be resilient and helped me push through when things were hardest. He provided so many opportunities for me to grow both as a scientist and as a professional. His insightful comments, guidance and commitment to maintaining the highest standard of excellence were an inspiration to me and the epitome of what a mentor should be. The Evans lab was the perfect fit for my graduate education, and I could not imagine a better place to do my Ph.D. Having Dr. Evans as a mentor was key to my success and has prepared me for a bright and successful future as an independent investigator. Secondly, I would like to thank my committee members Dr. Charles Mullighan, Dr. Taosheng Chen , Dr. Robert Williams and Dr. Mary V. Relling for supporting and guiding me through this process. You have all been so helpful and always had great feedback for me that made this project what it is today. I am especially thankful to Dr. Relling as both the Pharmaceutical Sciences department chair and as the closest collaborator with the Evans lab. She has provided exceptional insight, guidance and resources to me at every level of this project on a weekly basis. She went above and beyond what is expected to take interest in this project, provide a welcoming shared collaborative lab environment and helped me to succeed as a young scientist whenever possible. I would like to thank current and former members of the Evans lab: Dr. Steven Paugh for early development and inspiration for my project, Dr. Robert McCorkle for help with many aspects of the genomics studies, Dr. Daniel Ferguson for help with mechanisms, Dr. Jordan Beard for help with experiments and figures, Dr. Erik Bonten for numerous experiments and guidance through issues, Dr. Kristine Crews for help with all patient data, providing clinical perspective and guidance, Dr. Barthelemy Diouf for guidance and stimulating debate, Dr. Elixabeth Lopez-Lopez for help with computation and Calvin Lau who was a great POE student. Also, members of the Relling lab: Dr. Wenjian Yang for always being a resource for computation and finding data, Dr. Colton Smith who provided invaluable computational assistance, Dr. Seth Karol for providing a clinical perspective, guidance and humor, Monique Payton for expert guidance on the design of in vivo studies, IACUC protocol writing and 24/7 support and Dr. Emily Finch for guidance and moral support. Thank you all for providing an amazing shared lab environment, helping me generate ideas, supporting me when things were good or bad and riding this rollercoaster with me for the last five years. I want to express my appreciation for the technical staff (in the Evans and Yang Labs) who worked on both clinical assays and who helped with a number of other important experiments Hannah Williams, Natasha Atkinson, Dylan Maxwell, Jeremy Hunt, Brandon Smart, Yan Wang, August John, Lauren Rowland and Ting-Nien Lin. It iv has been such a pleasure working with such an amazing group of talented scientists and gaining very good friends. I have had the opportunity to work with so many wonderful people in the Pharmaceutical Sciences department who have helped guide this project. Most especially, Dr. Jun Yang for his exceptional guidance and as a pivotal part of the collaborative pharmacogenomic efforts in our department and the members of his lab, especially Dr. Yoshihiro Gocho for help with in vivo experiments. Also, I am very grateful to Dr. Daniel Savic for his guidance and collaboration on the project and to all the members of his lab, Dr. Christopher Coke and Dr. Jonathan Diedrich for their moral support, friendship and help with ATAC/CHIP-seq for the project and Brennan Bergeron for his moral support. I would like to thank other collaborators at St. Jude: Dr. Cheng Cheng , Dr. Stanley Pounds, Dr. Deqing Pei and Dr. Lei Shi for biostatistical guidance and excellent collaboration, Dr. Charles Gawad and all lab members for scRNA sequencing aspects of the project especially Dr. Robert Carter and Dr. Sivaraman Natarajan, Dr. Kathryn Roberts from the Mullighan lab for providing data and guidance in our understanding of molecular subtypes, Dr. Jiyang Yu for NetBID and his exceptional lab member Dr. Jingjing Liu who was very helpful with figures. I would like to thank Aman Seth, Rebecca Thorne and the HMP core for help and support with in vivo experiments. Dr. Tiffani Rogers the acting veterinarian and Dr.