THE ROLE OF NPM-ALK SIGNALING IN TUMOR CELL METABOLISM by Scott Robert Paul McDonnell A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular and Cellular Pathology) in the University of Michigan 2013 Doctoral Committee: Professor Megan S. Lim, Chair Professor Kathleen Cho Research Associate Professor Nicholas Donato Associate Professor Peter C. Lucas Assistant Professor Ivan Maillard Assistant Professor Zaneta Nikolovska-Coleska © Scott Robert Paul McDonnell 2013 to children with cancer ii ACKNOWLEDGEMENTS Graduate school has been an amazing time in my life, and many people throughout my tenure at Michigan have played vital roles in my thesis work. Whether through mentorship, collaborative research or emotional support, everyone in my life has contributed to this body of work presented here. My success is yours and I can’t thank you enough. My mentors Dr. Megan Lim and Dr. Kojo Elenitoba-Johnson have been immensely critical to this work. I thank you both for your guidance, advice and support during my graduate career. I have been very lucky to be mentored by two incredibly intelligent individuals who constantly put my needs as a graduate student above their own, as this is a quality not easy to come by. I would also like to acknowledge my thesis committee members (Dr. Lucas, Dr. Cho, Dr. Donato, Dr. Maillard and Dr. Nikolovska-Coleska) for their support and guidance throughout my graduate career. I also truly appreciate the support from Dr. Nick Lukacs and the Pathology graduate program, including Laura Hessler and Laura Labut. I would also like to thank the members (past and present) of the Lim and Elenitoba-Johnson labs. Carlos Murga-Zamalloa, Steven Hwang, Delphine Rolland, Fuzon Chung, Anagh Sahasrabuddhe, Kaiyu Ma, Joon Ahn, Thiru Velusamy, Mark Kiel, Johnvesley Basappa, Xiaofei Chen, Zilin Nie, Elena Ivan, Veronica Mendoza, Carla McNeil, Adam Kronk, and Jean-Marc Fontaine. Each and every one of you has helped me in my research, whether through technical advice, hands-on help, or casual conversation around coffee and I greatly appreciate it. This work has succeeded only because of the important collaborations we fostered. Thank you to Venky Basrur, Kevin Conlon and Damian Fermin for help with the proteomic work. Thank you to Charles Burant, Chunhai Ruan, and Sasha Raskind for help with the metabolomic studies. Finally, thank you to Craig Thomas iii for providing us the PKM2 activating compounds as well as your intellectual collaboration. My work in the lab couldn’t have progressed as well as it did without the love and support from the people in my life outside of the lab. I would like to thank all of my extended family in Michigan that welcomed us and made Michigan a home. I have met some amazing, life-long friends in Ann Arbor. Kreg, David, Seyth, Molly, Bernadette, Liz, Ian, Simon, River, Laura; thank you for everything and remember that there will always be a place for you in San Diego to come visit. To my parents, thank you for all of your love and support. You have opened so many doors for me and this thesis is very much a product of that. To my brother, Andrew, you have always set the example of which I strive to reach every day. To my grandparents, you have been a constant inspiration in my life and provided me with diverse life experiences that have influenced who I have become. And to my parents-in-law, thank you for your love and support and for allowing me to drag your daughter across country to expand my education. Finally, and most importantly, thank you Davina. You are the one who has provided me love and support day-in and day-out for the last five years. I couldn’t image coming out to Michigan and enduring though graduate school without you. I love you. This dissertation is the culmination of influences from everyone mentioned here. I hope I made you all proud. iv PREFACE Anaplastic large cell lymphoma represents about 10-15% of pediatric lymphomas and has an aggressive clinical course with frequent relapse. The majority of these tumors are characterized by translocations involving the ALK gene. The most common is the chromosomal translocation t(2;5)(p23;q35), which is the driving oncogenic mutation in these tumors. The consequence of the translocation is the in the expression of a constitutively active tyrosine kinase, the NPM-ALK fusion protein. While significant progress has been made in deciphering the signaling mechanisms induced by NPM-ALK, a comprehensive understanding of how NPM-ALK induces cell proliferation and survival is lacking. This level of understanding is essential for the development of novel therapeutics to treat patients with ALCL and other ALK driven neoplasms. The aim of this study is to utilize unbiased screening approaches to identify novel mediators of NPM-ALK signaling, with the goal of uncovering potential therapeutic targets. v TABLE OF CONTENTS Dedication..................................................................................................... ii Acknowledgements...................................................................................... iii Preface......................................................................................................... v List of Figures............................................................................................... viii List of Tables................................................................................................ xi List of Abbreviations..................................................................................... xii Abstract........................................................................................................ xiv Chapter 1: Introduction I. Anaplastic Lymphoma Kinase.................................................... 1 II. Cancer Metabolism...................................................................... 32 III. Summary and Rationale.............................................................. 44 Chapter 2: Integrated Phosphoproteomics and Metabolomics Identifies NPM-ALK Metabolic Signature I. Abstract........................................................................................ 45 II. Introduction.................................................................................. 46 III. Materials and Methods................................................................ 48 IV. Results......................................................................................... 55 V. Discussion.................................................................................... 93 VI. Future Directions.......................................................................... 95 Chapter 3: NPM-ALK Phosphorylates PKM2 to Drive Metabolic Reprogramming and Oncogenesis I. Abstract........................................................................................ 97 II. Introduction.................................................................................. 98 III. Materials and Methods................................................................ 99 IV. Results......................................................................................... 102 V. Discussion................................................................................... 122 vi VI. Future Directions......................................................................... 125 Chapter 4: NPM-ALK Signals Through GSK3β to Promote Oncogenesis I. Abstract........................................................................................ 126 II. Introduction.................................................................................. 127 III. Materials and Methods................................................................ 128 IV. Results......................................................................................... 131 V. Discussion................................................................................... 153 VI. Future Directions......................................................................... 156 Chapter 5: Concluding Remarks................................................................ 157 References.................................................................................................. 159 vii LIST OF FIGURES Figure 1.1: Anaplastic Lymphoma Kinase domain structure................................. 2 Figure 1.2: NPM-ALK induced STAT3 signaling................................................... 21 Figure 1.3: NPM-ALK induced PI3K/AKT signaling.............................................. 24 Figure 1.4: NPM-ALK induced MAPK signaling.................................................... 27 Figure 1.5: Metabolic programing in normal and proliferating tissues................. 34 Figure 1.6: Glycolysis provides precursors for biomass production.................... 35 Figure 1.7: Sequence alignment of PKM1 and PKM2.......................................... 41 Figure 1.8: Regulation of PKM2 activity............................................................... 42 Figure 2.1: Integrated phosphoproteomic and metabolomic experimental design.................................................................................................................... 55 Figure 2.2: Phosphoproteome signature for lymphoma cell lines....................... 57 Figure 2.3: Validation of conditions for phosphoproteomic studies..................... 59 Figure 2.4: Phosphoproteome changes in response to ALK inhibition............... 60 Figure 2.5: Biological process enrichment in phosphoproteomic