TARGETING AN NRF2/G6PDH PATHWAY TO REVERSE MULTI-DRUG RESISTANCE IN DIFFUSE LARGE B-CELL LYMPHOMA A Dissertation by SEYEDHOSSEIN MOUSAVIFARD Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chair of Committee, Steve Maxwell Committee Members, James C. Sacchettini Raquel Sitcheran David W. Threadgill Warren Zimmer Head of Program, Warren Zimmer May 2017 Major Subject: Medical Sciences Copyright 2017 Seyed Hossein Mousavi-Fard ABSTRACT A leading cause of mortality in diffuse large B-cell lymphoma (DLBCL) patients is the development of resistance to the CHOP regimen, the anthracycline-based chemotherapy consisting of cyclophosphamide, doxorubicin, vincristine, and prednisone. Our first objective of this work was to investigate the impact of Nuclear factor erythroid–related factor 2 (Nrf2)/ glucose-6-phosphate dehydrogenase (G6PDH) pathway on CHOP-resistance in DLBCL cell lines. We provide evidence here that a Nrf2/G6PDH pathway plays a role in mediating CHOP resistance in DLBCL. We found that CHOP-resistant DLBCL cells expressed both higher Nrf2 and G6PDH activities and lower reactive oxygen (predominantly superoxide) levels than CHOP- sensitive cells. We hypothesized that increased activity of the Nrf2/G6PDH pathway leads to higher GSH production, a more reduced state (lower ROS), and CHOP-resistance. In support of our hypothesis, direct inhibition of G6PDH or knockdown of Nrf2/G6PDH lowered both NADPH and GSH levels, increased ROS, and reduced tolerance or CHOP-resistant cells to CHOP. We also present evidence that repeated cycles of CHOP treatment select for a small population of Nrf2High/G6PDHHigh/ROSLow cells that are more tolerant of CHOP and might be responsible for the emergence of chemoresistant tumors. We propose that sensitive Nrf2Low /G6PDHLow /ROSHigh cells are essentially killed off by CHOP allowing for the selective propagation of the small population of CHOP-resistant Nrf2High/G6PDHHigh/ROSLow cells, thereby resulting in relapse of lymphoma. Our second objective was to study rifamycins’ potency at sensitizing drug resistant cancer cells to chemotherapeutics. We have discovered a novel chemosensitizer (RTI-79, a rifamycin- derivative) with a broad spectrum of action that includes ovarian cancer and double and triple hit non-Hodgkin’s lymphoma. RTI-79 is relatively non-toxic and has favorable in vivo safety and ii pharmacokinetic (PK) profiles. RTI-79 in combination therapies is effective in multiple drug resistant cancers in mouse models. RTI-79 works by dramatically increasing intracellular reactive oxygen species (ROS), primarily superoxide, through redox cycling. The level of ROS induction is directly correlated with drug sensitivity. Importantly, RTI-79 also triggers the unfolded protein response (UPR) that results in increased ubiquitination and loss of Nrf2, the primary sensor for intracellular ROS. Thus, RTI-79 both increases ROS and squelches Nrf2’s ability to respond to ROS. This unique mechanism provides a broad and novel approach for the very safe application of RTI-79, and other rifamycins, in treating drug resistant cancers. We also showed that RTI-79 acts to increase the oxidative state in chemoresistant cancer cells by inducing superoxide (O2-) and downregulating the Nrf2/G6PDH/NADPH/GSH pathway. RTI-79 also increased the ubiquitination state of several mitochondrial chaperone proteins (mtDnaJ [HSP40]), HSP60, HSP70mt [mortalin]) and decreased activated CREB, which are known to play roles in mitochondrial unfolded protein response (UPRmt) retrograde signaling. In summary, this research explores a new way to overcome the chemoresistance particularly in DLBCL and ovarian carcinoma. The new findings of this study will significantly impact our understanding of the role of Nrf2/G6PDH signaling pathway in DLBCL, and provide hope for the development of rational therapies for both newly diagnosed patients with DLBCL and also patients with relapsing DLBCL. iii ACKNOWLEDGEMENTS I would like to thank my committee chair, Dr. Steve Maxwell, and my committee members, Dr. Sacchettini, Dr. Sitcheran and Dr. Threadgill, and Dr. Zimmer for their invaluable guidance, support, encouragement, and knowledge throughout the course of this research. I would like to express my profound gratitude to Dr. Sacchettini for the financial support and scientific collaborations. I also thank the department of Molecular and Cellular Medicine at Texas A&M Health Science Center for providing funds for this research. Thanks also go to my friends and colleagues and the department of Molecular and Cellular Medicine faculty and staff for making my time at Texas A&M University a great experience. Finally, thanks to my family for their encouragement and support over the years and to my wife for her patience and love. iv CONTRIBUTORS AND FUNDING SOURCES Contributors This work was supported by a dissertation committee consisting of Dr. Steve Maxwell, Dr. Raquel Sitcheran and Dr. David W. Threadgill of the Department of Molecular and Cellular Medicine, Dr. James C. Sacchettini of the Department of Biochemistry and Biophysics, and Dr. Warren E. Zimmer of the Department of Medical Physiology. Steve Maxwell and Seyed Hossein Mousavi-Fard researched and prepared the manuscript for chapter I. Seyed Hossein Mousavi-Fard and Steve Maxwell designed experiments and analyzed the data for Chapter II. Seyed Hossein Mousavi-Fard carried out the transductions, western blots, GSH, and NADPH experiments. Seyed Hossein Mousavi-Fard and Timothy Davis conducted experiments on G6PDH activities and effects on cell growth. Seyed Hossein Mousavi-Fard and Steve Maxwell carried out the reactive oxygen species analyses. Steve Maxwell conceived the idea for the project and generated the cell lines. Deeann Wallis and Jim Sacchettini commented on the data. Deeann Wallis involved in much of the conception and experimental design, data generation, analysis, writing and editing the manuscript for Chapter III. Nian Zhou leads medicinal chemist determined SAR, conceived, designed and generated all analogs. Dwight Baker designed and implemented initial HTS and validated hits and determined synergy, edited manuscript. Seyed Hossein Mousavi-Fard, Kimberly Loesch, Stacy Galaviz, Qingan Sun, Thomas Ioerger, Michael DeJesus, Wen Dong, and Gwen Seemann involved in experimental design, data generation, and analysis. Carolina Mantilla Rojas and David W. Threadgill generated, analyzed, and interpreted v mouse echocardiogram data. Maureen T. O'Brien and Theresa Fossum generated, analyzed, and interpreted histology on mouse hearts. Theresa Fossum, Steve Maxwell, and James C. Sacchettini conceived the initial idea, involved in experimental design, data generation, and analysis, writing and editing manuscript. All other work conducted for the dissertation was completed by Seyed Hossein Mousavi-Fard independently. Funding Sources The preparation of this dissertation was supported by institutional funds provided to Dr. Steve Maxwell from the Texas A&M Health Science Center of the Department of Molecular and Cellular Medicine. In addition, James C. Sacchettini provided funding for data generation and analysis for chapter III. vi TABLE OF CONTENTS Page ABSTRACT .…………………………………………………………………………………. ii ACKNOWLEDGEMENTS ……………..…………………………………………………… iv CONTRIBUTORS AND FUNDING SOURCES……………………………………………… v TABLE OF CONTENTS …………………………………………………………………….. vii LIST OF FIGURES ………………………………………………………………………….. xi LIST OF TABLES …………………………………………………………………………… xiii CHAPTER I GENERAL INTRODUCTION AND LITERATURE REVIEW: NON-HODGKIN’S B CELL LYMPHOMA: ADVANCES IN MOLECULAR STRATEGIES TARGETING DRUG RESISTANCE……………………………………...... 1 1.1 Introduction…………………………………………………………………....…. 1 1.1.1 The problem of multidrug resistance in non-Hodgkins lymphoma ..…. 1 1.1.2 Innate and acquired drug resistance varies among subtypes of NHL…. 2 1.1.3 Drug-resistant DLBCL….………………………………….…….…… 3 1.1.4 Mantle-cell lymphoma ...……………………………………………… 4 1.2 Drug-resistance pathways in B-cell NHL…………………………………....….. 5 1.2.1 PI3K/Akt pathway in B-cell NHL drug resistance ..………………….. 5 1.2.2 Dysregulated apoptotic pathways ……..……………………………… 6 1.2.2.1 Extrinsic (death receptor) pathway ..……………………….. 6 1.2.2.2 Intrinsic pathway ..…………………………………………. 7 1.2.3 IAP proteins ..…………………………………………………………. 9 1.2.4 DNA damage response ..……………………………………………… 9 1.2.5 NFκB pathway ..………………………………………………………. 10 1.2.6 microRNAs ..………………………………………………………….. 10 1.2.7 TGFβ pathway ...………………………………………………………. 11 1.2.8 Oxidative stress pathway ..……………………………………………. 11 1.2.9 Reactive oxygen species ..…………………………………………….. 12 1.2.10 Proton pumps ..………………………………………………………. 12 1.2.11 GSH .………………………………………………………………… 13 1.2.12 CD antigens ………………………………………………………… 13 1.2.12.1 CD20-rituximab ..…………………………………………. 13 1.2.12.2 CD147 ..…………………………………………………… 14 1.2.13 MDR proteins ..………………………………………………………. 15 1.2.14 Drug metabolizing pathways .……………………………………….. 16 1.2.15 MAP kinase pathway .……………………………………………….. 17 1.2.16 Tyrosine protein kinases ..…………………………………………… 17 1.2.17 p53 tumor suppressor gene ..………………………………………… 18 1.2.18 Proteasome ...………………………………………………………… 19 vii 1.2.19 Epigenetics…………………………………………………………… 20 1.2.20 MSI instability ...….………………………………………………….. 20 1.2.21 Stromal influences ...…………………………………………………. 21 1.2.22 Other ...……………………………………………………………….. 23 1.2.22.1 Sphingosine kinase...……………………………………… 23 1.2.22.2 Geranylation ……………………………………………… 23 1.2.22.3 Zinc transporter....………………………………………….