The Role of Cytokines in governing the Expansion of the T315I mutation in Chronic Myeloid ...

239 Pages·2014·5.58 MB·English

The Role of Cytokines in governing the
Expansion of the T315I mutation in Chronic
Myeloid Leukaemia

Oi-Lin Lee

The Melissa White Laboratory
Cancer Theme, South Australian Heath and Medical Research Institute (SAHMRI)
Department of Haematology, SA Pathology
Centre for Personalised Cancer Medicine (CPCM), University of Adelaide
Cancer for Cancer Biology, University of Adelaide
Adelaide, South Australia
&
Faculty of Health Sciences
School of Medicine
Discipline of Paediatrics
The University of Adelaide
Adelaide, South Australia

A thesis submitted to the University of Adelaide in candidature for the degree of Master of
Philosophy
February 2014
Table of Contents
Abbreviations ............................................................................................................ 6
Abstract ................................................................................................................... 14
Thesis Declaration .................................................................................................. 16
Acknowledgements……………………………………………………………………… 17
Chapter 1 Introduction ........................................................................................... 18
1.1 Biology of CML .......................................................................................................... 19
1.1.1 Philadelphia Chromosome (Ph) ........................................................................ 19
1.1.2 Pathophysiology of CML ................................................................................... 24
1.2 Clinical and laboratory features of CML .................................................................. 31
1.2.1. Clinical features ................................................................................................. 31
1.2.2. Laboratory features........................................................................................... 32
1.3 Treatment ................................................................................................................. 32
1.3.1. Chemotherapy ................................................................................................... 32
1.3.2 Interferon-α ....................................................................................................... 33
1.3.3 Tyrosine kinase inhibitors (TKIs) ....................................................................... 33
1.3.4 Allogeneic Haematopoeitic Stem Cell Transplant (Allogeneic HSCT) ............. 39
1.4 Mechanisms of Resistance ....................................................................................... 40
1.4.1 Mutations in the Bcr-Abl kinase domain .......................................................... 41
1.4.2. Increased expression of BCR-ABL1 ................................................................... 45
1.4.3. Cytokines mediated resistance ......................................................................... 45
1.4.4 Activation of Bcr-Abl Independent Signalling Pathways ................................. 46
1.4.5 Alteration in the expression of drug transporters ........................................... 48
1.4.6. Clonal Evolution ................................................................................................ 48
1.5. Summary, Research hypothesis and Aims ............................................................... 49

Chapter 2 Materials and Methods.......................................................................... 52
2.1 Commonly used reagents and their suppliers ......................................................... 52
Table 2.1 .......................................................................................................................... 52
2.2 Solutions, buffers and media ................................................................................... 55
2.5.1 Cell culture media ............................................................................................. 55
2.2.2 Serum deprived medium (SDM) ....................................................................... 55
2.2.3 Freezing medium ............................................................................................... 55
2.2.4 Thaw medium for cell lines ............................................................................... 56
2.2.5 Flow cytometry fixative (FACS Fixative) ........................................................... 56
2.2.6 Reagents required for assessment of cell death by flow cytometry .............. 56
2.2.7 Reagents required for Western Blotting .......................................................... 56
2.2.8 Reagents needed for Phospho-Tyrosine Flow Cytometry of cell lines ........... 58
2.2.9 Tyrosine kinase inhibitors (TKI) ........................................................................ 59
2.3 Cell lines .................................................................................................................... 59
2.3.1 K562 and K562-T315I ........................................................................................ 59
2.3.2 HL60 parental, HL60 BCR-ABL1p210 and HL60 BCR-ABL1T315I ............................ 60
2.3.3 KU812 ................................................................................................................. 60
2.4 General techniques used in the laboratory ............................................................. 60
2.4.1 Maintenance of cell lines .................................................................................. 60
2.4.2 Cell counts and viability assessment by trypan blue exclusion dye................ 60
2
2.4.3 Cryopreservation of cells .................................................................................. 61
2.4.4 Thawing of cell lines .......................................................................................... 61
2.4.5 Lymphoprep density gradient centrifugation (Ficoll) of a cell line culture for
live cell enrichment ......................................................................................................... 61
2.4.6 Optimal washing of K562-T315I cell line .......................................................... 62
2.5 Special techniques .................................................................................................... 62
2.5.1 Flow cytometry to assess cell death by Annexin V and 7AAD ........................ 62
2.5.2 Flow cytometry to assess growth factor and cytokine cell surface receptors 63
2.5.3 Flow cytometry to assess the presence and proportion of intracellular
proteins ........................................................................................................................... 63
2.5.4 Carboxyfluorescein diacetate succinimidyl ester (CFSE) labelling of cells ...... 64
2.5.5 Co-culturing BCR-ABL1WT cells with BCR-ABL1T315I cells ................................... 65
2.5.6 Western blotting analyses ................................................................................ 66
2.5.7 mRNA isolation .................................................................................................. 67
2.5.8 cDNA synthesis .................................................................................................. 68
2.5.9 BCR-ABL1 kinase domain Long PCR amplification and sequencing ................ 68
2.5.10 Sequencing reaction Purification ...................................................................... 70
2.5.11 Quantification of BCR-ABL1 mRNA ................................................................... 70
2.5.12 MTS assay .......................................................................................................... 71
2.5.13 Cytokine profiling of cell culture supernatants ................................................ 72
2.6 Statistical analysis .................................................................................................... 73

Chapter 3 The BCR-ABL1T315I cells protect BCR-ABL1WT cells from TKI-induced
cell death through a paracrine cytokine mechanism ........................................... 74
3.1. Introduction .............................................................................................................. 74
3.2. Approach ................................................................................................................... 75
3.2.1. Characterising the K562 naïve and K562-T315I cell lines ................................ 75
3.2.2. Cytokine profiling of cell culture supernatants ................................................ 75
3.2.3. Proliferation and viability of BCR-ABL1T315I cells compared to BCR-ABL1WT cells
in SDM ........................................................................................................................... 76
3.2.4. Immunophenotyping for growth factor and cytokine receptor expression ... 78
3.2.5. Co-culturing BCR-ABL1WT cells with BCR-ABL1T315I cells ................................... 78
3.2.6. Cell rescue by exogeneous cytokines ............................................................... 79
3.2.7. FGF-2 blocking experiment ............................................................................... 79
3.2.8. Intracellular flow cytometry ............................................................................. 80
3.2.9. Phopho-protein detection by Western blotting analyses ............................... 80
3.3. Results ....................................................................................................................... 81
3.3.1. Characterisation of the K562 naïve, K562-T315I, HL60 parental, HL60 BCR-
ABL1p210 and HL60 BCR-ABL1T315I cell lines ..................................................................... 81
3.3.2. Differential cytokine profiling evident in BCR-ABL1 positive cells harbouring
the T315I mutation.......................................................................................................... 88
3.3.3 Immunophenotyping of the cytokine and growth factor receptors revealed no
significant difference between the cell lines ................................................................. 91
3.3.4. Cells harbouring the T315I mutation do not have a proliferative or survival
advantage in SDM ......................................................................................................... 102
3.3.5. Culturing BCR-ABL1WT cells together wth BCR-ABL1T315I cells reduced TKI-
induced cell death in BCR-ABL1WT cells ........................................................................ 110
3
3.3.6. Exogenous FGF-2 was able to rescue K562 naïve cells from TKI induced cell
death ......................................................................................................................... 115
3.3.7. Inhibiting FGF-2 by bFM-1, a FGF-2 neutralizing antibody ............................ 119
3.3.8. K562-T315I cells protect K562 naïve cells from imatinib-induced cell death by
secretion of FGF-2 when cultured together in SDM .................................................... 123
3.3.9. FGF-2 rescues K562 naïve cells from cell death via reactivation of pErk and
pSTAT5 ......................................................................................................................... 126
3.3.10. FGF-2 does not confer a survival advantage to K562-T315I cells through an
autocrine action ............................................................................................................ 133
3.4. Discussion ............................................................................................................... 138

Chapter 4 In BCR-ABL1T315I expressing cells, the Mitogen activated protein
kinase pathway is hyperactivated in the presence of tyrosine kinase inhibitors
................................................................................................................................ 145
4.1. Introduction ............................................................................................................ 145
4.2. Approach ................................................................................................................. 146
4.2.1. Viability and proliferation of K562-T315I cells cultured in SDM with TKI .... 146
4.2.2. Phosphoprotein detection of pErk and pSTAT5 by western blotting ........... 147
4.2.3. Phosphoprotein detection of pAkt by intracellular flow cytometry ............. 148
4.2.4. Cytokine profile of K562-T315I cells cultured overnight with different TKI in
SDM ......................................................................................................................... 148
4.3. Results ..................................................................................................................... 149
4.3.1. K562-T315I cells are more viable in the presence of TKI ............................... 149
4.3.2. K562-T315I cells do not appear to have a greater proliferation index when
cultured with TKI when assessed with CFSE staining .................................................. 151
4.3.3. Ki-67 expression is increased in K562-T315I cells when incubated with TKI 155
4.3.4. pErk signalling is increased in K562-T315I cells when cultured with TKI
compared to untreated ................................................................................................. 160
4.3.5. pSTAT5 expression is not increased in K562-T315I compared to K562 naïve
cells when cultured with TKI ......................................................................................... 160
4.3.6. pAkt expression is decreased in K562-T315I when cultured in TKI compared to
untreated ....................................................................................................................... 163
4.3.7. Cytokine profile of the supernatant of cells harbouring the T315I mutation
when cultured in SDM with TKI .................................................................................... 163
4.4. Discussion ............................................................................................................... 169

Chapter 5 Discussion ........................................................................................... 176
5.1. Introduction ............................................................................................................ 176
5.1.1. BCR-ABL1T315I cells have a different cytokine profile compared to BCR-ABL1WT
cells ......................................................................................................................... 178
5.1.2. Cytokine receptor expression on BCR-ABL1WT and BCR-ABL1T315I cells ......... 180
5.1.3. BCR-ABL1T315I do not proliferate more rapidly compared to BCR-ABL1WT in
SDM ......................................................................................................................... 181
5.1.4. BCR-ABL1T315I cells over secrete FGF-2 which protects BCR-ABL1WT cells from
TKI-induced cytotoxicity ............................................................................................... 181
5.1.5. FGF-2 rescues BCR-ABL1WT cells from TKI-induced cell death by reactivation of
Erk and STAT5 signalling ............................................................................................... 183
4
5.1.6. K562-T315I cells are more viable and proliferate more rapidly when exposed
to TKI and this is mediated through hyperactivation of the MAP kinase pathway ... 184
5.1.7. Hyperactivation of MAP kinase signalling does not appear to be cytokine-
mediated........................................................................................................................ 185
5.2. Future directions .................................................................................................... 186
5.3. Summary ................................................................................................................. 186

Appendix Supplementary Figures....................................................................... 188
Bibliography .......................................................................................................... 219

5
Abbreviations
7-AAD 7-Aminoactinomycin D
ABCB1 ATP binding cassette, sub-family B1
ABCG2 ATP binding cassette, sub-family G2
ABL1 Abelson murine leukaemia virus human homologue 1 gene
ASK1 Apoptotic signal kinase
Akt a serine threonine kinase (also known as protein kinase B)
ALL Acute lymphoblastic leukaemia
Ann V Annexin V
AP Accelerated phase
ATP Adenosine triphosphate
Bad BCL-2 antagonist of cell death
Bak Bcl-2 homologous antagonist killer
Bax Bcl-2 associated X protein
BC Blast crisis
Bcl-2 B cell lymphoma 2
Bcl-X B-cell lymphoma-extra large
L
BCR Breakpoint cluster region
BCR-ABL1 BCR-ABL1 oncogene
BELA trial Bosutinib efficacy and safety in CML
BH Bcl-2 homology
Bim BCL-2 interacting mediator of cell death
BM Bone marrow
BMSC Bone marrow stromal cells
bp Nucleotide base pair
6
BP Blastic phase
BSA Bovine serum albumin
Btk Bruton’s tyrosine kinase
Ca2+ Calcium ion
CaCl Calcium choride
CBL Casitas B-lineage lymphoma proto-oncogene
CCyR Complete cytogenetic response
CD Cluster of differentiation
cDNA Complementary DNA
C/EBP-α CCAAT enhancer-binding protein α
CFSE Carboxyfluorescein diacetate succinimidyl ester
Chr Chromosome
CHR Complete haematological response
CML Chronic myeloid leukaemia
CMR Complete molecular response
CSL Commonwealth Serum Laboratory
CO Carbon dioxide
2
CP Chronic phase
Crk CT10 sarcoma oncogene cellular homologue
Crkl CT10 regulator of kinase-like
CXCL12 C-X-C motif chemokine 12 (also known as Stromal cell-derived factor
1)
CXCR4 C-X-C motif chemokine receptor 4
DAG Diacylglycerol
das Dasatinib
7
DASISION Dasatinib versus Imatinib Study in Treatment-Naïve CML Patients
DEPC Diethyl pyrocarbonate
DMSO Dimethyl sulphoxide
DNA Deoxyribonucleic acid
dNTP Deoxynucleotide triphosphates
DTT Dithiothreitol
ECF Enhanced chemifluorescence substrate
EDTA Ethylenediaminetetra-acetic acid
eGFP Enhanced green fluorescent protein
EGFR Epidermal growth factor receptor
ENESTnd Evaluating Nilotinib Efficacy and Safety in Clinical Trials–Newly
Diagnosed Patients
Erk Extracellular signal related kinase
et al et alia
ETV6-PDGFRβ ETS translocation variant 6- Platelet derived growth factor receptor β
gene
FACS Fluorescent activated cell sorting
FBS Foetal bovine serum
FDA Federal Drug Authority
Fgr Gardner-Rasheed feline sarcoma viral oncogene homologue
FGF Fibroblast growth factor
FGFb Fibroblast growth factor basic (also known as FGF-2)
FGFR Fibroblast growth factor receptor
FIP1L1-PDGFRα Fip 1 like 1- Platelet derived growth factor receptor α gene
FLT3-ITD FMS-like tyrosine kinase 3- internal tandem domain
8
c-FMS Cellular homologue of the feline sarcoma virus, v-FMS
FOXO Forkhead O transcription factor
FOXO 3A Forkhead O family of transcription factors 3A
g Gravitational force (Relative centrifugation force)
Gab1 GRB2-associated-binding protein 1
Gab2 GRB2-associated binding protein 2
GAP Guanosine triphosphatase-activating protein
G-CSF Granulocyte colony stimulating factor
GDP Guanosine diphosphate
GIST Gastrointerstinal stromal tumours
GM-CSF Granulocyte macrophage colony stimulating factor
Grb2 Growth factor receptor bound protein 2
GTP Guanosine triphosphate
KD Kinase domain
Hck Haematopoietic cell kinase
HGF Hepatocyte growth factor
hnRNP-E2 Heterogeneous nuclear ribonucleoprotein- E2
HSC Haematopoietic stem cell
HSCT Haematopoietic stem cell transplantation
IC50 Concentration of drug required to inhibit Bcr-Abl kinase activity by
50%
IFNα Interferon-α
IκB Inhibitor of NF-κB
IKKα Inhibitor of nuclear factor kappa B kinase subunit α
IL-1α Interleukin 1α
9
IL-1β Interleukin 1β
IL-6 Interleukin 6
IL-8 Interleukin 8
im Imatinib mesylate
IMDM Iscove’s modification of Dulbecco’s medium
IP3 Inositol triphosphate
IRIS International randomised study of interferon versus STI571
IS International standard
Jak Janus kinase
JNK c-Jun N-terminal kinase
kb Kilo base pairs
kDa kilo Dalton
Lck Lymphocyte-specific protein tyrosine kinase
LIF Leukaemia inhibitory factor
LSC Leukaemic stem cells
Lyn Lck/Yes-related novel protein
mA mili (10-3) Ampere
MAPK Mitogen activated protein kinase
M-bcr Major breakpoint cluster region
m-bcr minor breakpoint cluster region
MCyR Major cytogenetic response
MCP-1 Monocyte chemo attractant protein 1
MDC Macrophage derived chemokine
MDR1 Multiple drug resistant protein 1
MEK MAP kinase/Erk kinase
10