Elucidation of the Mechanism by which Phosphatase and Tensin Homologue Deleted on Chromosome Ten (PTEN) Regulates Natural Killer Cell Function DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Edward Lloyd Briercheck Graduate Program in Integrated Biomedical Science Program The Ohio State University 2013 Dissertation Committee: Professor Michael A. Caligiuri, MD (Advisor) Professor William E. Carson, III, MD Professor Gregory B. Lesinski, Ph.D. Professor Guido Marcucci, MD Copyright by Edward Lloyd Briercheck 2013 Abstract Human natural killer (NK) cells are CD56+CD3- large granular lymphocytes of the innate immune system which are characterized by the ability to both directly kill and initiate an immune response to virally infected or malignantly transformed cells. Human NK cells in peripheral blood can be divided into two developmentally and functionally distinct subsets based upon surface expression of CD56. In contrast to the more mature CD56dim NK cell, the less mature CD56bright NK cell is unable to kill malignant cells at rest. We sought to determine the mechanism of this difference in cytolytic activity by exploring changes in gene expression between CD56bright NK cells and CD56dim NK cells. We observed that CD56bright NK cells showed a ~5 fold increase in PTEN protein expression over CD56dim NK cells. Human and murine NK cells overexpressing PTEN demonstrated decreased cytolytic activity and IFN-γ secretion, with concurrent decreases in their downstream (MAPK and AKT) targets that are critical for cytolysis. Paradoxically, human NK cells with near complete PTEN knockdown also showed decreased cytolytic activity despite elevations in AKT and MAPK. Confocal microscopy revealed that near complete PTEN knockdown results in a disruption of the NK cell’s ability to organize immunological synapse components including decreased adhesion, decreased polarization of the microtubule organizing center toward the target cell and decrease in coalescence of cytolytic granules. Thus, PTEN is differentially expressed in mature human NK cell subsets and our studies suggest it must be expressed at an optimum level to maximize NK cytolytic activity. ii Dedication This document is dedicated to Ani and my family. iii Acknowledgments I would like to thank my advisor Dr. Michael A. Caligiuri for his mentorship both in and outside the laboratory throughout my training. I have learned an incredible amount and am continually inspired by your passion, drive and belief in those around you. I have been truly lucky to have a mentor whose door, text messaging, email, phone is always open. Thank you for this opportunity, and your continued support. To my friends and colleagues in the Caligiuri lab. I have learned so much from all of you these past five years. I simply could not have completed this journey without you. I would especially like to thank Dr. Rosanna Trotta for her scientific expertise, willingness to teach, and providing me with a foundation of learning to do truly well designed science. I would also like to thank my students Jordan Cole, Tyler Cole, and Alex Hartlage for their contributions to this work. You all have tremendous potential and I look forward to having you as colleagues. I would like to thank my committee members Dr. Guido Marcucci, Dr. William E. Carson III, and Dr. Gregory B. Lesinski. I am very fortunate to have such an accomplished and willing committee during my graduate studies. Thank you all once again. I would also like to extend my appreciation the Medical Scientist Training Program (M.S.T.P.) and the Biomedical Sciences Graduate program at The Ohio State University iv for the opportunity to be a part of such a special group of faculty and students. I would like to thank Dr. Larry Schlesinger, Dr. Lawrence Kirschner, and Ashley Bertran for their tireless work in creating a world-class training program. I would also like to express my gratitude to the late Dr. Alan Yates who first welcomed me to the program and laid the foundation for the M.S.T.P. I would like to acknowledge the Pelotonia Graduate Fellowship program, the Medical Scientist Training Program and the National Cancer Institute for their support of the research presented in this thesis. I would like to thank my mother for her commitment to creativity and education and my father who has never wavered in his belief in hard work and his son. Thank you both and thank you to the rest of my family including Brennen Cocklin for their tremendous support. Finally, Ani there are no words to describe what you mean to me and how you have supported me through this process, most often from 800 miles away. I am constantly amazed at your talent, determination, and grace. You make me want to be a better person. With love, thank you. v Vita May 2002 .......................................................Jackson High School May 2006 .......................................................B.S. Biology, University of Toledo 2006 to present ..............................................MSTP Program The Ohio State University Publications Trotta R, Chen L, Costinean S, Josyula S, Mundy-Bosse BL, Ciarlariello D, Mao C, Briercheck EL, McConnell KK, Mishra A, Yu L, Croce CM, Caligiuri MA. Blood. 2013 Feb 19 Overexpression of miR-155 cause expansion, arrest in terminal differentiation and functional activation of mouse natural killer cells. Blood. 2013 Feb 19 [Epub ahead of print] McClory S, Hughes T, Freud AG, Briercheck EL, Martin C, Trimboli AJ, Yu J, Zhang X, Leone G, Nuovo G, Caligiuri MA. Evidence for a stepwise program of extrathymic T cell development within the human tonsil. J Clin Invest. 2012 Apr 2;122(4):1403-15. Trotta R, Ciarlariello D, Dal Col J, Mao H, Chen L, Briercheck EL, Yu J, Zhang J, Perrotti D, Caligiuri MA. The PP2A inhibitor SET regulates granzyme B expression in human natural killer cells. Blood. 2011 Feb 24;117(8):2378-84. doi: 10.1182/blood-2010- 05-285130. Epub 2010 Dec 14. Hughes T, Becknell B, McClory S, Briercheck EL, Yu J, Mao C, Giovenzana C, Nuovo G, Wei L, Zhang X, Gavrilin M, Wewers M, Caligiuri MA. IL-1β selectively expands and sustains IL-22(+) immature natural killer cells in secondary lymphoid tissue. Immunity. 2010 Jun 25;32(6):803-14 Briercheck EL, Freud A, Caligiuri MA. Natural Killer Cells: Basic Science and Clinical Application Nov 2009 Human Natural Killer cell development, Elsevier. Hughes T, Becknell B, McClory S, Briercheck EL, Freud AG, Zhang X, Mao C, Nuovo GJ, Yu J, Caligiuri MA. Stage 3 immature human natural killer cells found in secondary lymphoid tissue constitutively and selectively express the Th17 cytokine interleukin-22. Blood 113(17): 4008-10, 2009. vi Chang JS, Santhanam R, Trotta R, Neviani P, Eiring AM, Briercheck EL, Rochetti M, Roy DC, Calabretta B, Caligiuri MA. High levels of the BCR/ABL oncoprotein are required for the MAPK-hnRNP-E2 dependent suppression of C/EBPalpha-driven myeloid differentiation. Blood 110(2):994-1003, 2007. Fields of Study Major Field: Integrated Biomedical Science Program vii Table of Contents Abstract .............................................................................................................................. ii Dedication .......................................................................................................................... iii Acknowledgments.............................................................................................................. iv Vita ..................................................................................................................................... vi List of Figures .................................................................................................................... xi CHAPTER 1: BACKGROUND ......................................................................................... 1 1.1 NK Cell Biology........................................................................................................ 1 1.1a The role of NK cells in immunity ......................................................................... 1 2 1.1b NK cell development..................................................................................... 2 4 1.1c NK signaling through activating and inhibitory receptors .......................... 4 9 1.1d Phosphoinositide-3 kinase pathway in NK cell function ............................. 6 11 1.1e Mechanism of the NK cell immunological synapse .................................. 8 1.2 Phosphatase and tensin homologue deleted on chromosome ten (PTEN) .............. 12 1.2a PTEN as a tumor suppressor .............................................................................. 12 1.2b The regulation of PTEN .................................................................................... 14 1.2c PTEN in immunity ............................................................................................. 15 viii 1.3 Summary and Significance .................................................................................. 16 CHAPTER 2. PHOSPHATASE AND TENSIN HOMOLOGUE DELETED ON CHROMOSOME TEN (PTEN): A CRITICAL BALANCE OF EXPRESSION IS REQUIRED FOR MAXIMUM NATURAL KILLER CELL CYTOLYSIS OF TUMOR CELLS .............................................................................................................................. 17 2.1 Introduction ......................................................................................................... 17 2.2a Results