Characterization of Signal Transduction Abnormalities Revealed Spleen Tyrosine Kinase as a Therapeutic Target in High-Risk Precursor B Cell Acute Lymphoblastic Leukemia by Tatiana Perova A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Medical Biophysics University of Toronto © Copyright by Tatiana Perova 2013 Characterization of signal transduction abnormalities revealed spleen tyrosine kinase as a therapeutic target in high-risk precursor B cell acute lymphoblastic leukemia Tatiana Perova Doctor of Philosophy Department of Medical Biophysics University of Toronto 2013 ABSTRACT Currently, the intensive chemotherapy remains the first line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although these regimens have significantly improved patient outcomes, their use is associated with debilitating morbidities and fatal relapses, highlighting the great need in new agents that target essential survival signals in leukemia. Thus, the overall goal of my project was to gain insights into the signaling abnormalities that regulate aberrant proliferation and survival of B-ALL cells in an effort to identify novel targets in this malignancy. This study demonstrated that pre-B cell receptor (pre-BCR)-independent spleen tyrosine kinase (SYK) activity was required for the survival and proliferation of a p53-/-PrkdcSCID/SCID mouse model of B-ALL. I extended this discovery to human disease, demonstrating that SYK was activated in primary B-ALL, independent of the pre-BCR expression. The small molecule SYK inhibitor fostamatinib (fosta) significantly attenuated proliferation of 79 primary diagnostic B-ALL samples at clinically achievable concentrations. Importantly, fosta treatment reduced dissemination of engrafting B-ALL cells into the spleen, liver, kidney and central nervous system (CNS) in a NOD.Prkdcscid/scidIl2rgtm1Wjl/SzJ xenotransplant model of B-ALL. Analysis of signaling abnormalities using a high-throughput phospho-flow cytometry platform demonstrated that pediatric and adult B-ALL samples exhibit variable basal activation of BCR, i i PI3K/AKT/mTOR, MAPK and JAK/STAT pathways. Importantly, we identified that fosta- mediated inhibition of SYK, PLC 2, CRKL and EIF4E phosphorylation in B-ALL was predictive of its anti-leukemic activity, and was distinct from the cellular actions of other small molecule inhibitors of key nodal signaling pathways. Examination of molecular mechanism of fosta action by gene expression profiling revealed transcriptional effects of fosta treatment that included, most notably, potent inhibition of pathways involved in lymphocyte activation and inflammation. In conclusion, this study demonstrates that SYK signaling is crucial for B-ALL survival and provides detailed characterization of cellular and molecular mechanisms of fosta action in B-ALL. These data argue in favor of testing small molecule SYK inhibitors in pediatric and adult B-ALL. ii i ACKNOWLEDGEMENTS The famous adage says “It takes a village to raise a child”. My personal experience in graduate school fits well with this proverb, since my personal and professional growth took contributions from an inspiring group of people that made my PhD a productive and exciting experience. To my mentors, Jayne Danska and Cynthia Guidos, thank you for giving me a unique opportunity to work on an exciting project that allowed me to explore in depth the world of translational cancer research, and providing me with an exceptional academic environment and inspiring guidance. This experience helped me to build essential skills required for successful research career and fully realize my scientific passion. My committee members Dr. Dwayne Barber, Dr. Jane McGlade and Dr. Ben Neel have been exceptional mentors, providing me with valuable advise and ideas that helped to structure this PhD thesis. I am extremely lucky to have had a privilege to collaborate with Dr. Johann Hitzler and Dr. Mark Minden, who made this project possible by providing access to valuable patient samples. Their perspectives and scientific knowledge were essential for the identification of the clinical contribution of my project. I am extremely humbled to have an opportunity to work with all of these inspiring individuals. I am grateful to share the many years of PhD with a fantastic group of people. To my “bestie” Eniko Papp, I don‟t think it would be possible to endure graduate school without your friendship. Your passion for science, intelligence and constant encouragement have motivated me to persevere, even in the hardest of times. I want to thank Ildiko Grandal, who welcomed and supported me in the lab from the day 1, and provided much-needed help in lengthy experiments. Phil Kousis, Andrea Wong, Sara Suliman and Peggy Wong have all been great friends and colleagues that provided support on a daily basis. I want to thank all the remarkable members of the lab, past and present, for providing a great working environment. Most importantly, I would like to acknowledge the limitless support of my family. My parents have given me an opportunity to pursue my dreams and ambitions, even if they took me thousands of miles away from home. You have given me love, encouragement and support. I am forever in your debt. I am lucky to have an incredible sister and a loving spouse, who both have unwearyingly provided tremendous daily motivation and personal support. I love you both and I am grateful to have you in my life. Thank you all for patiently waiting for me to finally finish graduate school. To your relief, I will not be an eternal student or “вечный студент”, as my dad once called me! iv TABLE OF CONTENTS ABSTRACT ................................................................................................................................................ ii ACKNOWLEDGEMENTS ....................................................................................................................... iv TABLE OF CONTENTS ............................................................................................................................ v LIST OF TABLES .................................................................................................................................. viii LIST OF FIGURES ................................................................................................................................... ix LIST OF ABBREVIATIONS .................................................................................................................. xii CHAPTER I: INTRODUCTION .............................................................................................................. 1 I. 1 Thesis Overview ......................................................................................................................................... 2 I. 2 Overview of B cell development ........................................................................................................... 2 I.2.1 Regulatory networks promoting B lineage specification and commitment ............................... 3 I.2.2 Assembly of immunoglobulin genes through V(D)J recombination .............................................. 3 I.2.3 Expression of pre-BCR and BCR controls B cell differentiation program .................................... 4 I.3 Initiation of signaling through pre-BCR and BCR complexes ..................................................... 5 I.3.1 Contribution of Ig and Ig to pre-BCR and BCR signaling ............................................................... 5 I.3.2 Src family kinases are early effectors of pre-BCR and BCR signaling ............................................ 6 I.3.3 SYK is an obligatory component of pre-BCR and BCR signaling ...................................................... 7 I.4 Complexity of SYK-dependent signaling pathways in B cells ..................................................... 9 I.4.1 Essential role of BLNK and BTK in SYK signaling .................................................................................. 9 I.4.2 SYK and PLC 2-mediated activation of NF B ........................................................................................ 10 I.4.3 SYK and activation of MAPK signaling ...................................................................................................... 11 I.4.4 SYK and activation of survival PI3K/AKT/mTOR pathway ............................................................. 12 I.5 Targeting protein kinases in hematologic malignancies ........................................................... 14 I.5.1. Protein kinases as therapeutic targets .................................................................................................... 14 I.5.2 Discovery of imatinib for the treatment of BCR-ABL1 leukemia................................................... 15 I.5.3 Targeting aberrant BCR signaling in mature B cell malignancies ................................................. 15 I.6 Use of phospho-flow cytometry to study signaling perturbations ......................................... 18 I.7 B-ALL: clinical presentation and current treatment options ................................................... 18 I.7.1 Incidence and clinical presentation of B-ALL ........................................................................................ 19 I.7.2 Prognostic factors ............................................................................................................................................. 19 I.7.3 Current treatment options and challenges ............................................................................................. 20 I.8 Discovery of novel therapeutic targets in B-ALL ........................................................................... 21 I.8.1 Beginning of the genomics era in B-ALL .................................................................................................. 21 I.8.2 Identification of a novel subgroup of high-risk B-ALL ....................................................................... 21 1.8.3 Genetic alterations reveal novel therapeutic targets in B-ALL ..................................................... 22 1.8.4 It’s a long way to go: the search for personalized medicine in B-ALL continues................... 22 I.9 Project goals ............................................................................................................................................... 23 I.10 FIGURES ..................................................................................................................................................... 24 CHAPTER II: Therapeutic potential of spleen tyrosine kinase inhibition for the treatment of high-risk precursor B-cell acute lymphoblastic leukemia ......................... 32 II.1 ABSTRACT .................................................................................................................................................. 33 II.2 INTRODUCTION ....................................................................................................................................... 33 II.3 METHODS ................................................................................................................................................... 35 II.3.1 Human B-ALL Samples .................................................................................................................................. 35 v II.3.2 Cell lines............................................................................................................................................................... 35 II.3.3 Kinase Inhibitors .............................................................................................................................................. 36 II.3.4 Proliferation Assay .......................................................................................................................................... 36 II.3.5 siRNA Transfection ......................................................................................................................................... 36 II.3.6 Mice ....................................................................................................................................................................... 36 II.3.7 Lymphocyte isolation ..................................................................................................................................... 37 II.3.8 Lymphoblast Isolation from CNS, Liver and Kidneys ....................................................................... 37 II.3.9 Histology ............................................................................................................................................................. 38 II.3.10 Flow Cytometry and adoptive transfer of murine cells ................................................................ 38 II.3.11 Flow cytometric analyses of human samples .................................................................................... 39 II.3.12 Apoptosis assay ............................................................................................................................................. 39 II.3.13 Magnetic beads depletions ........................................................................................................................ 39 II.3.14 Phospho-specific Flow Cytometry ......................................................................................................... 39 II.3.15 Fluorescence Compensation .................................................................................................................... 40 II.3.16 B-ALL Xenograft Assays ............................................................................................................................. 40 II.3.17 Western Blot Analysis ................................................................................................................................. 41 II.3.18 Gene Expression Microarrays .................................................................................................................. 42 II.3.19 Statistical Analysis ........................................................................................................................................ 42 II.4 RESULTS ..................................................................................................................................................... 43 II.4.1 pre-BCR-independent SYK activation in a p53-/-; Prkdcscid/scid model of early B-ALL .......... 43 II.4.2 Pre-BCR-independent SYK activation in human B-ALL ................................................................... 44 II.4.3 SYK activation promotes B-ALL survival and proliferation ........................................................... 45 II.4.4 Kinase specificity of SYK inhibitor effects ............................................................................................. 45 II.4.5 Fosta limits B-ALL growth after xenotransplantation ..................................................................... 46 II.5 DISCUSSION ............................................................................................................................................... 49 II.6 TABLES ........................................................................................................................................................ 52 II.7 FIGURES ...................................................................................................................................................... 57 CHAPTER III: Phospho-flow cytometric profiling of SYK-dependent signaling networks in high-risk precursor B-cell acute lymphoblastic leukemia .......................... 81 III.1 ABSTRACT ................................................................................................................................................ 82 III.2 INTRODUCTION ...................................................................................................................................... 83 III.3 METHODS ................................................................................................................................................. 86 III.3.1 Patient samples ............................................................................................................................................... 86 III.3.2 Cell lines ............................................................................................................................................................. 86 III.3.3 Small molecule kinase inhibitors ............................................................................................................. 86 III.3.4 Proliferation assay ......................................................................................................................................... 87 III.3.5 Antibodies ......................................................................................................................................................... 87 III.3.6 Phospho-flow analysis in cell lines ......................................................................................................... 87 III.3.7 Phospho-flow analysis in B-ALL patient samples ............................................................................. 88 III.3.8 Data normalization and visualization .................................................................................................... 89 III.3.9 Mutation analysis ........................................................................................................................................... 90 III.3.9 Statistical analysis .......................................................................................................................................... 90 III.4 RESULTS .................................................................................................................................................... 90 III.4.1 Optimization and validation of phospho-flow platform for detection of basal signaling 90 III.4.2 Dissection of pre-BCR-independent SYK signaling in high-risk B-ALL ................................... 93 III.4.3 Aberrant SYK-independent MAPK signaling networks in B-ALL ............................................... 95 III.4.4 Role of basally active BCR-related proteins in proliferation of B-ALL ..................................... 96 III.4.5 AKT-independent inhibition of pS6 and pEIF4E by fosta ............................................................. 97 III.4.6 Aberrant activation of pSTAT5 in high-risk BCR-ABL- B-ALL ..................................................... 99 III.4.7 B-ALL patients organized by similarities in basal phosphorylation signature display similar clinical characteristics ............................................................................................................................ 101 v i III.5 DISCUSSION ........................................................................................................................................... 102 III.6 TABLES ................................................................................................................................................... 107 III.7 FIGURES .................................................................................................................................................. 111 CHAPTER IV: Identification of transcriptional effects of fostamatinib in high-risk precursor B-cell acute lymphoblastic leukemia .................................................................... 139 IV.I ABSTRACT ............................................................................................................................................... 140 IV.II INTRODUCTION ................................................................................................................................... 140 IV.3 METHODS ............................................................................................................................................... 142 IV.3.1 Patient samples ............................................................................................................................................ 142 IV.3.2 Reagents .......................................................................................................................................................... 142 IV.3.3 Proliferation Assay ..................................................................................................................................... 142 IV.3.4 Flow Cytometry ............................................................................................................................................ 143 IV.3.5 Apoptosis Assay ........................................................................................................................................... 143 IV.3.6 Treatment and RNA extraction .............................................................................................................. 143 IV.3.7 Microarray experiments ........................................................................................................................... 144 IV.3.8 Statistical analyses of microarray data .............................................................................................. 144 IV.3.9 Gene set enrichment analyses of expression data ......................................................................... 145 IV.3.10 Validation by quantitative real-time PCR (qRT-PCR) ................................................................ 145 IV.4 RESULTS ................................................................................................................................................. 146 IV.4.1 Optimization of treatment conditions to profile expression signature of fosta effects . 146 IV.4.2 Analysis of fosta effects in high-risk adult and pediatric B-ALL .............................................. 148 IV.4.3 Distinct inhibition signatures of fosta and dexamethasone ...................................................... 150 IV.5 DISCUSSION ........................................................................................................................................... 151 IV.6 TABLES .................................................................................................................................................... 155 IV.7 FIGURES .................................................................................................................................................. 174 Chapter V: CONCLUSIONS AND FUTURE DIRECTIONS .......................................................... 197 V.1 Thesis overview .................................................................................................................................... 198 V.2 Clinical relevance of this study ........................................................................................................ 199 V.3 Phospho-flow: a move towards personalized medicine ........................................................ 200 V.4 Cellular effects of fosta in B-ALL: SYK inhibition or off-target effects? ............................. 201 V.5 Identification of transcriptional consequences of fosta treatment in B-ALL ................. 202 V.6 Mechanisms of pre-BCR-independent SYK signaling in B-ALL ............................................ 203 V.7 Targeting of leukemia initiating cells (LICs) by fosta ............................................................. 204 V.8 Signal transduction therapies in B-ALL: the road ahead ....................................................... 205 CHAPTER VI: REFERENCES ............................................................................................................ 207 vi i LIST OF TABLES Table II.1 Summary of B-ALL patient samples .......................................................................... 52 Table II.2 List of antibodies used for compensation in flow cytometry experiments ................. 53 Table II.3 Clinical characteristics of B-ALL patient samples used for in vivo experiments with fosta ............................................................................................................................................... 54 Table II.4 Summary of regimen 1 effects on the weight organs in a xenotransplant model of human B-ALL ............................................................................................................................... 55 Table II.5 Summary of regimen 2 effects on the weight of organs in a xenotransplant model of early human B-ALL ...................................................................................................................... 56 Table III.1 List of small molecule inhibitors used in phospho-flow profiling of early B-ALL samples ........................................................................................................................................ 107 Table III.2 List of phospho-specific and intracellular antibodies for profiling of early B-ALL samples ........................................................................................................................................ 108 Table III.3 List of antibodies used for compensation in flow cytometry experiments .............. 109 Table III.4 Clinical characteristics of B-ALL patient samples .................................................. 110 Table IV.1 Sequences of primers used for quantitative real-time PCR ..................................... 155 Table IV.2 Plasmids used to prepared standard curved for quantitative real-time PCR ........... 156 Table IV.3 A list of top 60 differentially expressed gene at 4 h fosta treatment ....................... 157 Table IV.4 A list of top 60 differentially expressed genes at 8 h fosta treatment ...................... 160 Table IV.5 Top 60 gene sets enriched vehicle-treated pediatric B-ALL samples ..................... 163 Table IV.6 Top 60 gene sets enriched vehicle- versus fosta-treated adult B-ALL .................... 165 Table IV.7 Top 60 gene sets enriched vehicle- versus DEX-treated adult B-ALL samples ..... 167 Table IV.8 A list of unique fosta-sensitive genes in adult B-ALL ............................................ 169 Table IV.9 A list of unique DEX-sensitive genes in adult B-ALL ............................................ 171 vi ii LIST OF FIGURES Figure I.1 Simplified overview of transcriptional and signaling networks that control generation of B-cell progenitors ..................................................................................................................... 24 Figure I.2 Critical checkpoints in early B cell development ....................................................... 26 Figure I.3 Schematic representation of SRC and SYK protein structure .................................... 28 Figure I.4 SYK is a central component of pre-BCR and BCR-mediated signaling .................... 29 Figure I.5 BLNK acts as an adaptor protein linking SYK to downstream signaling molecules . 30 Figure I.6 Composite overview of signal transduction cascades activated by pre-BCR/BCR signaling ........................................................................................................................................ 31 Figure II.1 DM leukemias display a pre-BCR-independent pro-B to pre-B cell transition ........ 57 Figure II.2 pre-BCR-independent SYK activity drives proliferation and survival of DM B-ALL cells ............................................................................................................................................... 59 Figure II.3 Analysis of expression on BCR signaling components in human early B-ALL ....... 61 Figure II.4 Phospho-flow analysis of SYK-dependent signaling in B-ALL ............................... 62 Figure II.5 pre-BCR-independent SYK activity in human early B-ALL .................................... 63 Figure II.6 SYK activity is necessary for proliferation and survival of human early B-ALL ..... 64 Figure II.7 Anti-proliferative effects on SYK inhibitors in B-ALL ............................................ 66 Figure II.8 SYK inhibitors‟ effects on B-ALL proliferation are SYK-specific and are not due to off-target effects on FLT3 or SRC ................................................................................................ 67 Figure II.9 SYK knockdown reduces proliferation of early B-ALL cell lines ............................ 69 Figure II.10 In vivo inhibition of SYK activity shows therapeutic potential in a xenotransplant model of early B-ALL .................................................................................................................. 70 Figure II.11 Fostamatinib reduces tumor burden in a xenograft model of early B-ALL ............ 72 Figure II.12 Therapeutic potential of inhibition of SYK activity in a well-established xenograft model ............................................................................................................................................. 74 Figure II.13 Tumor burden in NSG mice at the start of fosta regimen 2 .................................... 76 Figure II.14 Fostamatinib reduces burden of an established leukemia. ...................................... 77 Figure II.15 Fostamatinib reduced leukemia-initiating cells in CNS and spleen ........................ 79 Figure III.1 High-throughput phospho-flow protocol for analysis of signaling networks in B- ALL ............................................................................................................................................. 111 Figure III.2 Optimization of phospho-flow antibodies to detect basal signaling responses ..... 113 ix Figure III.3 Validation of high-throughput phospho-flow protocol to detect basal signaling responses in B-ALL .................................................................................................................... 115 Figure III.4 Profiling of BCR signaling responses in B-ALL ................................................... 117 Figure III.5 Analysis of BCR signaling in B-ALL .................................................................... 119 Figure III.6 Distinct inhibition profiles of fosta and PD184352 ............................................... 121 Figure III.7 Analysis of potentiated ERK signaling in early B-ALL ........................................ 123 Figure III.8 Comparison of inhibition profiles of fosta and DAS in B-ALL ............................ 125 Figure III.9 Profiling of basal PI3K/AKT/mTOR signaling in B-ALL .................................... 127 Figure III.10 Differential in vitro sensitivity to fosta and LY ................................................... 129 Figure III.11 Validation of phospho-flow platform to detect basal pSTAT3 and pSTAT5 signaling responses ..................................................................................................................... 131 Figure III.12 Identification of aberrant pSTAT5 responses in B-ALL ..................................... 132 Figure III.13 Distinct inhibition profiles of fosta and SAR ...................................................... 134 Figure III.14 Differential inhibition of BCR signaling by fosta and SAR ................................ 136 Figure III.15 Phospho-flow profiling of B-ALL reveals five groups with distinct phosphorylation signatures that correlate with clinical outcomes .............................................. 137 Figure IV.1 Protocol for optimization of treatment conditions to detect fosta-induced changes in gene expression ........................................................................................................................... 174 Figure IV.2 Comparison of gene expression genes following fosta treatment at 4h and 8h time- points ........................................................................................................................................... 176 Figure IV.3 Gene set enrichment analysis (GSEA) of fosta effects following 4 h and 8 h treatment ..................................................................................................................................... 178 Figure IV.4 Zoom in of the lymphocyte activation and innate immune response clusters in the fosta treatment enrichment map .................................................................................................. 180 Figure IV.5 Validation of microarray results using quantitative real-time PCR ....................... 181 Figure IV.6 Analysis of fosta effects in pediatric B-ALL ......................................................... 183 Figure IV.7 Representative GSEA plots showing enrichment score for vehicle-enriched gene sets after 6h fosta treatment in pediatric B-ALL ........................................................................ 185 Figure IV.8 Representative examples of gene sets enriched in fosta-group .............................. 186 Figure IV.9 Analysis of fosta effects in high-risk adult B-ALL ................................................ 187 Figure IV.10 Enrichment map for fosta treatment in pediatric and adult B-ALL ..................... 189 x
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