UNIVERSITY OF CALIFORNIA IRVINE Targeting eIF4F Translation Initiation Complex in order to Sensitize Blood Malignancies to Targeted Agents Submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY In Biological Sciences By Lee-or Herzog Dissertation Committee: Professor David A. Fruman Professor Craig M. Walsh Associate professor Mei Kong Associate Professor Olga V. Razorenova 2020 © 2020 Lee-or Herzog DEDICATION To Eitan Herzog - My dearest son. You are my soul. You mean everything to me. To my dear parents – For always believing in me, supporting me and guiding me. To Ella, May and Ilan - For your constant encouragement, laughter and help. To my grandparents - Who are always on my mind. I keep my promises every day. To Dr. David Fruman – For your outstanding mentorship, for your help and for helping me become a better scientist each day. To my committee members – for your encouragement, useful suggestions and for helping me improve my scientific skills. To my mentees and team – I could not have done this without you. Thank you very much for all your help, your dedication and for making an impact on translational science. To my lab mates – Thank you for your help, for making the lab such a great place to work in, for your support and the good time we shared together. ii TABLE OF CONTENTS LIST OF FIGURES………………………………………………………………….. iv LIST OF TABLES…………………………………………………………………… vi ACKNOWLEDGEMENTS………………………………………………………...... vii CURRICULUM VITAE……………………………………………………………... iix ABSTRACT OF THE DISSERTATION…………………………………………….. x CHAPTER 1: Introduction….……………………………………………………….. 1 CHAPTER 2: Targeting eIF4F Translation Initiation Complex with SBI-756 Sensitizes B Lymphoma Cells to Venetoclax ……………………………………….. 97 CHAPTER 3: Targeting eIF4F translation complex sensitizes B-ALL cells to tyrosine kinase inhibition…….……………………………………………………….. 165 CHAPTER 4: Significance, discussion and future directions……………………….. 200 iii LIST OF FIGURES Figure Title Page number Figure 1.1 The hallmarks of cancer and therapeutic targeting of them 3 Figure 1.2 Overview of mTORC1 and mTORC2 Complexes, Key Substrates, and Inhibitors 4 Figure 1.3 mTOR acts as a nutrient sensor coordinating cellular functions 6 Figure 1.4 Mechanisms of oncogenic activation of PI3K/AKT or loss of PTEN 7 Figure 1.5 The Philadelphia Chromosome 10 Figure 1.6 Overview of BCL-2 family protein interactions 20 Figure 1.7 Extrinsic and intrinsic apoptotic signaling pathways 22 Figure 1.8 Targeting mTOR Pathway as a Therapeutic Strategy 44 Figure 1.9 Convergence of major signaling pathways involved in cancer toward the eIF4F 47 complex Figure 1.10 Components of eIF4F translation initiation machinery complex, and inhibitors 49 Figure 2.1 mTORC1 regulates eIF4E dependent translation via phosphorylation of 4E-BP1 112 Figure 2.2 Constitutively active 4E-BP1 mutant sensitizes lymphoma cells to venetoclax 114 (ABT-199) and navitoclax (ABT-263) similar to TOR-KI combination Figure 2.3 SBI-756 prevents eIF4E-eIF4G interaction 115 Figure 2.4 SBI-756 treatment suppresses cap-dependent translation 116 Figure 2.5 SBI-756 does not change mTOR substrate phosphorylation 117 Figure 2.6 Treatment with SBI-756 sensitizes DLBCL and MCL cells to venetoclax 119 combinational treatment Figure 2.7 Treatment with SBI-756 sensitizes DLBCL and MCL cells to venetoclax 120 combinational treatment Figure 2.8 SBI-756 in combination with venetoclax induces apoptosis 121 Figure 2.9 4E-BP1 KO clones are insensitive to TOR-KI treatment, yet remain sensitive to 123 SBI-756 treatment Figure 2.10 Lymphoma cell lacking 4EBP1 are resistant to TOR-KI yet are sensitive to SBI- 124 756 treatment Figure 2.11 SBI-756 sensitizes to venetoclax treatment in vivo 125 Figure 2.12 Venetoclax and SBI-756 combination is tolerable and retains mTOR 127 functionality, while reduces tumor volume in vivo iv Figure 2.13 SBI-756 induces a decrease in MCL-1, BCL-xL, survivin, eIF4E and eIF4G 129 Figure 2.14 Selective reduction of mRNA translation following 4 hours SBI-756 treatment 131 Figure 2.15 SBI-756 treatment reduces protein synthesis following 16 hours of treatment 133 Figure 2.16 SBI-756 is not cytotoxic to human CD4+T cells, CD8+ T cells, NK cells or 135 monocytes Figure 2.17 B lymphocytes are more sensitive to eIF4F targeting than other PBMCs 137 Figure 2.18 Components of eIF4F translation initiation machinery complex, and inhibitors 140 Figure 2.19 Model for mechanism of venetoclax sensitization by SBI-756 142 Figure 3.1 Schematic diagram of the PI3K/ mTOR/ eIF4E signaling pathway and inhibitors 167 used in this study to target the pathway Figure 3.2 4E-BP mediates cytotoxicity of TORC1 inhibition 170 Figure 3.3 Reduced Eif4e gene dosage sensitizes to dasatinib 172 Figure 3.4 Targeting the eIF4F complex sensitizes p190 cells to dasatinib 173 Figure 3.5 Targeting the eIF4F complex suppresses colony formation to similar extent as 173 targeting mTORC1 Figure 3.6 SBI-576 sensitizes similar to mTOR inhibition but does not inhibit mTOR 174 Figure 3.7 Reduced eIF4E availability leads to reduction in protein synthesis 175 Figure 3.8 SBI sensitizes human Ph+ and Ph-like B-ALL cell lines and stromal-dependent 176 lines to dasatinib treatment Figure 3.9 SBI-756 is selectively toxic to B cells 177 Figure 3.10 SBI-576 reduces eIF4E:eIF4G1 interaction in vivo in a dose-dependent manner 178 Figure 3.11 Combination of SBI-756 and dasatinib reduces B-ALL leukemic burden while 180 suppress eIF4E:eIF4G interaction in vivo Figure 3.12 MLN0128 or SBI-756 treatment prime cells towards apoptosis 181 Figure 4.1 Strategies for evading apoptosis 206 Figure 4.2 TOR-KI and SBI-756 treatments sensitize AML cells to venetoclax combination 211 Figure 4.3 eIF4F variants reprogram the cellular translatome in response to distinct stimuli 217 Figure 4.4 Schematic diagram of the genetic model that enables conditional reduction in 219 eIF4E expression v LIST OF TABLES Table Title Page number Table 2.1 The sequences used for guiding Cas9 to 4EBP1 108 Table 2.2 The sequences used for guiding Cas9 to 4EBP2 108 Table 2.3 Genes found to be translationally down-regulated following eIF4F 150 suppression Table 2.4 Negative regulators of apoptosis genes that were found to be 164 translationally downregulated following eIF4F inhibition vi ACKNOWLEDGMENTS • This study was supported by: National Institutes of Health grants R01-CA158383 (to Dr. Fruman), UC Irvine Cancer Research Institute pilot grant from the 2018 Anti-Cancer Challenge (to Dr. Fruman), Bridge Grant from the American Society of Hematology (to Dr. Fruman), and by Cancer Center Support Grant P30-CA062203 to UC Irvine. • I was supported by National Institutes of Health grant T32 AI 060573. I want to thank Dr. Eric Pearlman, Dr. Amanda Burkhardt and Ms. Rebecca Taylor for believing in me, giving me a chance to do the research that I wanted, and for their constant support. • I thank Dr. Selina Chen-Kiang and Dr. Maurizio Di Liberto for providing MCL cell lines. • I want to thank Dr. Bert Semler for providing us with the dual-luciferase reporter construct, and for Dr. Grant MacGregor for his assistance with performance of dual-luciferase assays and establishment of mouse models. • I thank Dr. Adeela Syed for her great assistance with microscopy imaging, her patience and for her training. vii CURRICULUM VITAE EDUCATION • University of California, Irvine (UCI), California 09/2015 Ph.D. in Biological sciences - Molecular Biology & Biochemistry • Tel Aviv University, Tel Aviv, Israel 06/2012 M.Sc. in Medical Sciences (Magna cum Laude) • The Hebrew University of Jerusalem, Jerusalem, Israel 06/2005 B.Sc. in Biological Sciences RESEARCH EXPERIENCE • Oncology & Immunology, University of California, Irvine (UCI) 08/2015 - Present Graduate Student Researcher Project manager - Managing 5 research projects and 12 students to achieve goals within budget. Scientific writer - Published 3 peer-reviewed scientific publications (5 more in process) in international journals. Scientific communicator - 14 scientific presentations that received 10 awards. Collaborator - Identified and supported 5 research opportunities, that led to 3 publications. • Weizmann Institute of Science, Rehovot, Israel 06/2014 – 05/2015 Senior Research Assistant and Lab Technician Conducted diverse biological methods at the Antibodies unit and the Microbiology unit, such as: full production of monoclonal and polyclonal antibodies, tissue culture, protein purification, antibodies labeling (CyTOF), plasmid production, directed fermentations, DASGIP controlled fermentations, microscopy and diverse microbiology methods. Managed databases constant update and drafting of lab protocols, reports and SOPs. • CorAssist Cardiovascular, Herzliya, Israel 01/2014 – 04/2014 Preclinical and Clinical Research Assistant As research and development coordinator, oversaw the entire research process, development of medical device, ethics committees, practical work, operations and follow ups. Wrote research and quality assurance documentation (all inspected and approved during a successful ISO audit). • HY Laboratories, Rehovot, Israel 10/2012 – 11/2013 Technical Writer and Quality Assurance Associate Researched at GMP and ISO 17025 conditions, authored all laboratory documents (e.g. protocols, reports, SOPs) for medical devices and pharmaceutical customers for submission to regulatory authorities (FDA and CE). Conducted and certified to perform all the tests offered by the lab (Sterility, LAL, Bioburden etc.). Supervised research, development and production