INTERFERON-GAMMA-MEDIATED IMMUNOEVASIVE STATEGIES IN MULTIPLE MYELOMA DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Paul David Ciarlariello Graduate Program in Molecular Cellular and Developmental Biology The Ohio State University 2016 Dissertation Committee: Don M. Benson, Jr., MD, PhD -Advisor Michael A. Caligiuri, MD Gregory Lesinski, PhD Natarajan Muthusamy, DVM, PhD -Advisor Flavia Pichiorri, PhD Copyrighted by Paul David Ciarlariello 2016 Abstract Natural killer (NK) cells are a major source of interferon-γ (IFN-γ) and may play a key role in innate immunity against multiple myeloma (MM). IFN-γ abrogates MM tumor cell in vitro proliferation, however, clinical trials of recombinant IFN-γ in patients with MM showed no benefit. MM cells exhibit strategies designed to evade NK cell surveillance and lysis. Herein, we provide evidence of a novel means of MM immune evasion in which IFN-γ appears to play a reciprocal relationship between MM and NK cells. Patients with MM exhibit higher levels of serum IFN-γ levels than in the healthy setting. NK cells produce IFN-γ in response to MM cells which express functional IFN-γ receptors. Stimulation with IFN-γ leads to increased transcription and expression of the inhibitory ligands HLA-E and PD-L1 by MM cells. This effect may be overcome by interruption of the NKG2A / HLA-E interaction. Intriguingly, MM cells release extracellular vesicles (EV) which are capable of enhancing IFN-γ production of NK cell, thus describing a potential cyclic mechanism of perpetual immune detriment. Taken in total, these results suggest that MM cells subvert the favorable effects of IFN-γ to attenuate NK cell tumor-directed cytotoxicity. ii Dedication To my wife, Nicole, and my baby, Gianluca. To my parents, Paul and Sue. I am a scientist - I seek to understand me all of my impurities and evils yet unknown I am a scientist - I seek to understand me I am an incurable, and nothing else behaves like me I am a scientist- I seek to understand me [...] the hole I dig is bottomless, but nothing else will set me free -Guided By Voices iii Acknowledgments I would like to thank my advisor Dr. Don M. Benson, Jr. for taking the plunge and adopting me as his first and only graduate research student. Don, you have created a supporting environment in which I could grow as a scientist. If even an ounce of your knowledge, generosity, hospitality, and disposition have rubbed off on me, I will no doubt be a better researcher, father, and overall human being. Thank you, for lending your expertise and shepherding my training. I am forever grateful. Of course, I would like to acknowledge my co-mentor Dr. Raj Muthusamy for the many constructive conversations which kept me on track to graduate. Raj, curiosities pour out of you and I have had such a rewarding experience discussing my data and its implications. I will always admire your method and approach to properly designed and controlled experiments. A special thanks to Dr. Michael A. Caligiuri who brought me on to his team in 2007. Mike, you dispense positivity and influence with every word and action to those around you, and I am grateful for your support and guidance throughout the years. To Dr. Flavia Pichiorri, thank you for the many collaborative efforts and willingness to meet and discuss our mutual findings. I have enjoyed our colorful conversations and your constant excitement for decoding life’s secrets. To Dr. Greg Lesinski, thank you for your constructive and honest feedback throughout the years. I have always felt at ease in our exchanges, knowing that you were fully present and engaged in my scientific stories. iv I extend my endless respect and gratitude to my science “mommy”, Dr. Rossana Trotta. Rox, you make hypothesis-driven methods look deceivingly easy. I did not have a full appreciation of your expertise in experimental design until I was on my own. I think of you when considering experimental controls and especially when I am qualifying the conditions of my results. You have left such an impression on the scientist I have become. Thank you. I must honor and recognize my lab mate, Dr. Tiffany Hughes for her many supportive roles during my tenure. You have offered your help every day for the past six years and have asked nearly nothing in return. We as a lab were truly lucky to recruit you, and I am confident your many contributions will help rid the world of myeloma, once and for all. Some of the most rewarding experiences in the lab were moments while mentoring my undergraduates D. Maxwell Banaszak and Rohit Menon. I am glad to have been a part of your experience here at OSU. I know you both are ready to take on the world. My parents Paul and Sue Ciarlariello have selflessly supported me my entire life. Through their efforts, I have been afforded the free reign of pursuing my passions. Mom and Dad, I am without words as to how incredible my life experience has been. Let this accomplishment symbolize your success as parents. I have been and will always be proud to be your son. To my love, Nicole Danielle Ciarlariello. You are the most intuitive, thoughtful, and gentle soul I have ever come across and I am lucky to have you by my side. We now embark on new beginnings with our baby boy, Gianluca. I don’t care where we end up, as long as we are together. Thank you for believing in me, and trusting me with your heart. v Vita November 4, 1983 ............................................ Born, Dayton, Ohio June 2002 .......................................................... West Carrollton High School June 2006 .......................................................... B.S. Biochemistry, The Ohio State University September 2010 - Present ................................. Molecular, Cellular, and Developmental Biology Graduate School Program, The Ohio State University Publications Harshman, S. W., A. Canella, P. D. Ciarlariello, K. Agarwal, O. E. Branson, A. Rocci, H. Cordero, M. A. Phelps, E. M. Hade, J. A. Dubovsky, A. Palumbo, A. Rosko, J. C. Byrd, C. C. Hofmeister, D. M. Benson, Jr., M. E. Paulaitis, M. A. Freitas and F. Pichiorri (2016). "Proteomic characterization of circulating extracellular vesicles identifies novel serum myeloma associated markers." J Proteomics. Briercheck EL, R. Trotta ,L. Chen , A. S. Hartlage, J. P. Cole, T. D. Cole, C. Mao , P. P. Banerjee, H. T. Hsu , E. M. Mace, D. Ciarlariello, B. L. Mundy-Bosse, I. Garcia-Cao, S. D. Scoville, L. Yu, R. Pilarski, W. E. Carson, G. Leone, P. P. Pandolfi, J.Yu, J. S. Orange and M. A. Caligiuri (2015). “PTEN Is a Negative Regulator of NK Cell Cytolytic Function” J Immunol 194(4):1832-40. doi: 10.4049/jimmunol.1401224. Epub 2015 Jan 16. Collins, S. M., C. E. Bakan, G. D. Swartzel, C. C. Hofmeister, Y. A. Efebera, H. Kwon, G. C. Starling, D. Ciarlariello, S. Bhaskar, E. L. Briercheck, T. Hughes, J. Yu, A. Rice and D. M. Benson, Jr. (2013). "Elotuzumab directly enhances NK cell cytotoxicity against myeloma via CS1 ligation: evidence for augmented NK cell function complementing ADCC." Cancer Immunol Immunother 62(12): 1841-1849. vi Harshman, S. W., A. Canella, P. D. Ciarlariello, A. Rocci, K. Agarwal, E. M. Smith, T. Talabere, Y. A. Efebera, C. C. Hofmeister, D. M. Benson, Jr., M. E. Paulaitis, M. A. Freitas and F. Pichiorri (2013). "Characterization of multiple myeloma vesicles by label-free relative quantitation." Proteomics 13(20): 3013-3029. Trotta, R., L. Chen, S. Costinean, S. Josyula, B. L. Mundy-Bosse, D. Ciarlariello, C. Mao, E. L. Briercheck, K. K. McConnell, A. Mishra, L. Yu, C. M. Croce and M. A. Caligiuri (2013). "Overexpression of miR-155 causes expansion, arrest in terminal differentiation and functional activation of mouse natural killer cells." Blood 121(16): 3126-3134. Trotta, R., L. Chen, D. Ciarlariello, S. Josyula, C. Mao, S. Costinean, L. Yu, J. P. Butchar, S. Tridandapani, C. M. Croce and M. A. Caligiuri (2012). "miR-155 regulates IFN-gamma production in natural killer cells." Blood 119(15): 3478-3485. Trotta, R., D. Ciarlariello, J. Dal Col, H. Mao, L. Chen, E. Briercheck, J. Yu, J. Zhang, D. Perrotti and M. A. Caligiuri (2011). "The PP2A inhibitor SET regulates granzyme B expression in human natural killer cells." Blood 117(8): 2378-2384. Costinean, S., S. K. Sandhu, I. M. Pedersen, E. Tili, R. Trotta, D. Perrotti, D. Ciarlariello, P. Neviani, J. Harb, L. R. Kauffman, A. Shidham and C. M. Croce (2009). "Src homology 2 domain-containing inositol-5-phosphatase and CCAAT enhancer-binding protein beta are targeted by miR-155 in B cells of Emicro-MiR-155 transgenic mice." Blood 114(7): 1374- 1382. Trotta, R., J. Dal Col, J. Yu, D. Ciarlariello, B. Thomas, X. Zhang, J. Allard, 2nd, M. Wei, H. Mao, J. C. Byrd, D. Perrotti and M. A. Caligiuri (2008). "TGF-beta utilizes SMAD3 to inhibit CD16-mediated IFN-gamma production and antibody-dependent cellular cytotoxicity in human NK cells." J Immunol 181(6): 3784-3792. Trotta, R., D. Ciarlariello, J. Dal Col, J. Allard, 2nd, P. Neviani, R. Santhanam, H. Mao, B. Becknell, J. Yu, A. K. Ferketich, B. Thomas, A. Modi, B. W. Blaser, D. Perrotti and M. A. Caligiuri (2007). "The PP2A inhibitor SET regulates natural killer cell IFN-gamma production." J Exp Med 204(10): 2397-2405. Fields of Study Major Field: Molecular, Cellular, and Developmental Biology vii Table of Contents Abstract ..................................................................................................................................... ii Dedication.................................................................................................................................iii