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Downloaded Feb 2017, Containing 20,162 Sequences) Using the Integrated Andromeda Search Engine (229) IDENTIFYING THERAPEUTIC AGENTS FOR THE TREATMENT OF DIFFUSE LARGE B-CELL LYMPHOMA Marissa Lynn Cann A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology in the School of Medicine. Chapel Hill 2017 Approved by: David S. Lawrence Lee M. Graves Gary L. Johnson Kristy L. Richards John E. Sondek © 2017 Marissa Lynn Cann ALL RIGHTS RESERVED ii ABSTRACT Marissa Lynn Cann: Identifying Therapeutic Agents for the Treatment of Diffuse Large B-cell Lymphoma (Under the direction of David S. Lawrence) Diffuse large B-cell lymphoma (DLBCL) can be categorized into two clinically relevant subtypes: activated B-cell (ABC) DLBCL and germinal center B-cell (GCB). Patients with ABC DLBCL have a worse prognosis, and are defined by chronic, overactive signaling through the B- cell receptor and NF-κB pathways. Signaling through the B-cell receptor is heavily dependent on the Src family kinases (SFKs), and NF-κB signaling is dependent on the proteasome. In Chapters 2 and 3, we examined the effects of the SFK inhibitor dasatinib in a panel of ABC and GCB DLBCL cell lines, with a focus on changes in the kinome (Chapter 2) or the proteasome (Chapter 3). In Chapter 2, we found that the ABC DLBCL cell lines are much more sensitive to dasatinib than the GCB DLBCL cell lines. However, both subtypes display inhibition of the SFKs in response to dasatinib treatment. Subsequent analyses revealed several cell cycle kinases to be inhibited by dasatinib treatment in the ABC DLBCL subtype, but not in the GCB DLBCL subtype. In Chapter 3, we found that treatment with dasatinib results in a decrease in the proteasome’s chymotrypsin-like (ChL), trypsin-like (TL), and caspase-like (CaL) activities in the ABC but not GCB DLBCL cell lines. Furthermore, it appears that association between the proteasome 20S core particle and 19S regulatory particle is disrupted in the ABC DLBCL cell lines after dasatinib treatment. iii In Chapter 4, we explored the utility of site-specific inhibitors of the proteasome’s TL and CaL activities in a variety of cancer cell lines. We found that the TL/ChL and CaL/ChL ratios of proteasome activity correlate with sensitivity to TL and CaL inhibitors, respectively. In addition, the two hematological malignancy cell lines studied are most sensitive to the site- specific inhibitors. Furthermore, the combination of the TL or CaL site-specific inhibitors with bortezomib or carfilzomib is synergistic in all cell lines examined. The studies presented in this thesis have important implications for the clinical use of dasatinib and site-specific proteasome inhibitors for the treatment of DLBCL, either alone or in combination with other agents. These inhibitors show promise and warrant further exploration. iv To my fiancé, Garrett Berry, who keeps me on my toes & To parents, James and Cori Cann, who never stopped believing in me v ACKNOWLEDGMENTS First and foremost, I would like to thank the members of my thesis committee, starting with my thesis mentor, David Lawrence. David has been a wonderful mentor to me during my time at UNC. He has pushed me to think about my research from different perspectives, encouraged all my grant writing adventures, and his snarky sense of humor has always helped make the lab a brighter place. I would not be the scientist I am today without his guidance and support. I would also like to thank Kristy Richards, who served as my clinical co-mentor as part of UNC’s Training Program in Translational Medicine. Her input and advice has been critical for the development of my thesis project. I would also like to thank Lee Graves, my committee chair. He has been like a second mentor to me the past few years, and I am grateful for his support and advice regarding my endeavors both in and outside the lab. I would also like to thank the other members of my thesis committee, Gary Johnson and John Sondek, whose questions and critiques have been invaluable in carrying out my thesis work. I would like to thank my labmates, both current and former members of the Lawrence lab. Melanie Priestman played an essential role in my development as a scientist, and I am grateful for her wisdom and advice. My work with Weichen Xu when I first joined the Lawrence lab is what kickstarted my thesis project, and I am grateful to her for my beginnings. It has been a pleasure working with Robert Hughes, Qunzhao Wang, Nate Oien, Luong Nguyen, Weston Smith, Zach Rodgers, Colin O’Banion, Song Ding, Anwesha Goswami, Christina vi Marvin, Emilia Zwyot, Amber Lee, Brianna Vickerman, and Josh Welfare. I wish you all the best of luck in your lives and careers. I would also like to thank my collaborators in the Graves lab and the UNC Proteomics Core facility. Laura Herring and Karim Gilbert were instrumental in helping me with my proteomics experiments, and I greatly appreciate all of the time Laura spent chatting with me about my data and experiments. Ian McDonald, Mike East, and Denis Okumu were always fun to chat with whenever I had to pop into the Graves lab (and their requests for cookies were always flattering!). The administrative staff in the Department of Pharmacology also deserve a shout-out. In particular, Nicole Arnold and Arlene Sandoval have been critical for keeping the Department of Pharmacology running. They are always willing to help folks in the department, and have truly helped make the Department of Pharmacology feel like a home to me. Their dedication to the department and the graduate students is felt and appreciated by all of us. One of the best parts of graduate school has been the friends I’ve made, and I am grateful for all of the wonderful people I have met during this journey. James Shellhammer has been my pharmacology buddy since day one, and I wish him the best of luck in his post-doc in Ireland. I am also thankful for the close friendships I have formed with Emily Tegowski, Cassandra Hayne, and Meagan Ryan. They have been incredible friends, and I am grateful to have enjoyed many a girls’ night with them. I look forward to our future escapades. I would also like to thank my friends from Michigan State University, especially Lauren Luethy and Chelsea Cojocari, who have always lent an understanding ear during the trials of graduate school. I would like to thank my parents, Jim and Cori Cann, and my brother, Shane, for their love and support during my time in graduate school. Their belief in me and my future success vii has meant much to me while I progressed in graduate school. I always got a kick out of my dad asking how “my little amoebas were doing”, even though they’re a completely different organism from what I’ve studied for the past 6 years. I am lucky to have such a loving and supportive family. I would also like to thank my future in-laws, Paula, Wayne, Colton, and Kira, who have been just as supportive of my journey as they have been of my fiancé’s. It was always nice to hear Paula brag about how her “babies are going to be scientists”. I am grateful to become part of their family. Most importantly, I would like to thank my fiancé, Garrett Berry. He has been with me every step of the way regarding graduate school, from studying for the GRE all the way to writing and defending my dissertation. He has kept me grounded, been a comfort during the bad days in lab (we all have them), celebrated my triumphs, and has been there to push me when my tendencies toward procrastination threatened to get the better of me. I can honestly say I could not have done this without him. Garrett, I thank you for everything you are and what you have done for me, and I am honored and thrilled to become your wife. Last, but not least (okay well maybe least), I would like to thank my handy-dandy laptop. She’s been with me since the beginning of my undergraduate career and, while she may be slow and ancient in computer years, she has been my trusty sidekick all throughout my undergraduate and graduate careers. She hasn’t given me her last blue screen of death yet, but when that time comes, may she rest in peace. viii TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................... xi LIST OF FIGURES ...................................................................................................................... xii LIST OF ABBREVIATIONS ....................................................................................................... xv CHAPTER 1: INTRODUCTION ................................................................................................... 1 1.1 Diffuse Large B-cell Lymphoma ..................................................................................... 1 1.2 The Src Family Kinases (SFKs) ....................................................................................... 3 1.3 The SFKs and B-cell Receptor (BCR) Signaling ............................................................. 5 1.4 Role of the SFKs in Cancer and Lymphoma ................................................................... 6 1.5 Measuring Kinase Activity – A Global Challenge........................................................... 8 1.6 The Proteasome .............................................................................................................. 25 1.7 Role of the Proteasome in Cancer and Lymphoma ........................................................ 26 1.8 Strategies for Measuring
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