Characterization of Signal Transduction Abnormalities Revealed Spleen Tyrosine Kinase as a ...

255 Pages·2013·6.47 MB·English

by  Tatiana Perova

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,
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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.
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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!
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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
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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
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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

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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
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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
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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
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