Mitchell, Rebecca (2017) Exploring BCR-ABL-independent mechanisms of TKI-resistance in ...

336 Pages·2017·7.31 MB·English

Mitchell, Rebecca (2017) Exploring BCR-ABL-independent mechanisms of
TKI-resistance in chronic myeloid leukaemia. PhD thesis.

https://theses.gla.ac.uk/7977/

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Enlighten: Theses
https://theses.gla.ac.uk/
[email protected]
Exploring BCR-ABL-independent mechanisms of
TKI-Resistance in Chronic Myeloid Leukaemia

By
Rebecca Mitchell
BSc (Hons), MRes

Submitted in the fulfilment of the requirements for the degree of
Doctor of Philosophy
September 2016

Section of Experimental Haematology
Institute of Cancer Sciences
College of Medical, Veterinary and Life Science
University of Glasgow
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Abstract
As the prevalence of Chronic Myeloid Leukaemia (CML) grows, due to the therapeutic
success of tyrosine kinase inhibitors (TKI), we are witnessing increased incidences of drug
resistance. Some of these patients have failed all currently licensed TKIs and have no
mutational changes in the kinase domain that may explain the cause of TKI resistance.
This poses a major clinical challenge as there are currently no other drug treatment
options available for these patients. Therefore, our aim was to identify and target
alternative survival pathways against BCR-ABL in order to eradicate TKI-resistant cells.
To investigate alternative survival mechanisms in TKI-resistant CML cells, ponatinib-
resistant cell line models were generated, which show resistance to all current TKIs,
despite complete inhibition of BCR-ABL activity. Additionally, DNA sequencing revealed
no mutational changes within the BCR-ABL kinase domain, which may explain TKI
resistance and RNA-sequencing showed an impaired transcriptional response following
ponatinib treatment when compared with parental TKI-sensitive cells.
Using these models, we demonstrated that the TKI-resistant cells acquired alternative
activation of mTOR. Using clinically relevant dual PI3K and mTOR inhibitors; NVP-Bez235,
VS-5584, apitolisib and gedatolisib, we validated the PI3K-AKT-mTOR pathway as a
therapeutic target in vitro in TKI-resistant CML cell lines and more importantly in bone
marrow derived mononuclear cells from CML patients resistant to TKIs and with no
known kinase domain mutational changes.
We demonstrated in vitro that TKI-resistant cell lines are highly sensitive to PI3K and
mTOR inhibitors, with EC50 values less than 30 nM compared to ponatinib, 647.3 nM.
These inhibitors reduced cell viability by causing a significant induction of apoptosis and
significant decrease in the clonogenic growth of primary TKI-resistant CML patient
samples.
Furthermore, we showed that NVP-Bez235 induced autophagy as a protective mechanism
following PI3K/mTOR inhibition. The combination of NVP-Bez235 with pharmacological
(Hydroxychloroquine (HCQ)) or specific autophagy inhibition, via ATG7 knockdown, the
efficacy of NVP-Bez235 was enhanced shown by the dramatic reduction in clonogenic
growth of TKI-resistant CML patient cells. In addition, we validated this in vivo using a
murine model by transplanting luciferase tagged TKI-resistant cells, treated with NVP-
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Bez235 in combination with HCQ, which significantly reduced tumour burden and
increased survival rates compared to controls.
These data suggested that the PI3K-AKT-mTOR pathway may be a key player responsible
for TKI-resistance and that pharmacological inhibition of this pathway, with the additional
inhibition of autophagy, may represent a potential new treatment option for TKI-resistant
CML patients, when resistance is driven by a BCR-ABL-independent mechanism.
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Table of contents
Table of Contents
Abstract .................................................................................................................................. 2
List of Tables........................................................................................................................ 11
List of Figures ...................................................................................................................... 12
Publications .......................................................................................................................... 16
Publications in Preparation .................................................................................................. 16
Acknowledgement................................................................................................................ 17
Author’s declaration ............................................................................................................. 18
Abbreviations ....................................................................................................................... 19
1 Introduction ................................................................................................................... 23
1.1 History of haemopoietic stem cells .............................................................................. 23
1.1.1 Development of the haemopoietic system ......................................................... 24
1.1.2 Haemopoietic hierarchy ..................................................................................... 24
1.1.3 HSC identification and isolation ........................................................................ 25
1.1.4 Haemopoietic stem cell characteristics and regulation ...................................... 27
1.1.5 CML is a stem cell disease ................................................................................ 28
1.2 Chronic Myeloid Leukaemia ........................................................................................ 29
1.2.1 CML epidemiology and clinical characteristics ................................................ 30
1.2.2 Diagnosis and monitoring .................................................................................. 32
1.2.3 BCR-ABL structure ........................................................................................... 33
1.2.4 BCR-ABL functional domains .......................................................................... 33
1.2.5 BCR-ABL oncogenic pathway .......................................................................... 35
1.2.6 BCR-ABL mimics growth factor signalling ...................................................... 39
1.2.7 BCR-ABL alteration of the HSC niche ............................................................. 40
1.3 History of CML treatment ............................................................................................. 41
1.3.1 The development of tyrosine kinase inhibitors (TKIs) ...................................... 42
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1.3.2 Imatinib in clinical trial...................................................................................... 44
1.3.3 Treatment responses .......................................................................................... 45
1.4 Mechanisms of resistance .............................................................................................. 47
1.4.1 Intolerance and non-compliance ........................................................................ 47
1.4.2 BCR-ABL-dependent mechanisms of resistance .............................................. 48
1.4.3 BCR-ABL-independent mechanisms of resistance ........................................... 51
1.5 Strategies to combat TKI resistance ............................................................................ 55
1.5.1 Imatinib dose escalation..................................................................................... 55
1.5.2 TKIs ................................................................................................................... 55
1.5.3 Second-generation TKIs .................................................................................... 55
1.5.4 Third-generation TKIs ....................................................................................... 58
1.5.5 An alternative experimental BCR-ABL inhibitor .............................................. 60
1.5.6 Omacetaxine ...................................................................................................... 61
1.6 Autophagy ....................................................................................................................... 62
1.6.1 Autophagy initiation .......................................................................................... 63
1.6.2 Autophagosome formation ................................................................................ 63
1.6.3 Autophagosome completion .............................................................................. 64
1.6.4 Autolysosome formation.................................................................................... 65
1.6.5 Autophagy and Cancer....................................................................................... 65
1.6.6 Autophagy and CML ......................................................................................... 66
1.6.7 Autophagy inhibition in CML ........................................................................... 68
1.6.8 CHOICES clinical trial ...................................................................................... 69
1.6.9 New generation of autophagy inhibitors ............................................................ 69
2 Materials and Methods .................................................................................................. 73
2.1 Materials .......................................................................................................................... 73
2.1.1 Small molecule inhibitors .................................................................................. 73
2.1.2 Tissue culture materials ..................................................................................... 73
2.1.3 Molecular biology supplies ................................................................................ 76
2.1.4 FACS ................................................................................................................. 80
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2.1.5 Immunofluorescence .......................................................................................... 80
2.1.6 Seahorse ............................................................................................................. 81
2.1.7 Animal work ...................................................................................................... 81
2.1.8 PCR primer sequences ....................................................................................... 82
2.1.9 Equipment .......................................................................................................... 83
2.2 Preparation of medium and solutions .......................................................................... 84
2.2.1 Tissue culture mediums and solutions ............................................................... 84
2.2.2 Western blot solutions ....................................................................................... 87
2.2.3 Flow cytometry .................................................................................................. 90
2.2.4 Immunofluorescence solutions .......................................................................... 90
2.2.5 PCR assay solutions ........................................................................................... 90
2.2.6 Transfection solutions ........................................................................................ 91
2.2.7 Microbiology solutions ...................................................................................... 92
2.3 Methods ........................................................................................................................... 93
2.3.1 Cell Culture ........................................................................................................ 93
2.3.2 Cell proliferation assays..................................................................................... 96
2.3.3 Colony Forming Cell (CFC) assay .................................................................... 97
2.3.4 Flow cytometry .................................................................................................. 98
2.3.5 Immunofluorescence microscopy .................................................................... 101
2.3.6 XTT cell viability assay ................................................................................... 102
2.3.7 Repurposing NIH approved oncology drug screen.......................................... 103
2.3.8 Western blotting ............................................................................................... 105
2.3.9 DNA sequencing .............................................................................................. 109
2.3.10 RNA-seq .......................................................................................................... 111
2.3.11 shRNA constructs ............................................................................................ 112
2.3.12 Lentivirus transfection ..................................................................................... 112
2.3.13 Transduction of CML cell lines ....................................................................... 113
2.3.14 CRISPR guide design ...................................................................................... 113
2.3.15 Target Guide Sequence Cloning Protocol ....................................................... 117
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2.3.16 Animal work .................................................................................................... 119
3 Results (I): Identification and validation of suitable TKI-resistant models for the study
of BCR-ABL-independent TKI-resistance ........................................................................ 122
3.1 Aims and objectives ..................................................................................................... 123
3.2 An assessment of current imatinib-resistant cell line models and their relevance
for the study of BCR-ABL-independent TKI-resistance ...................................................... 123
3.3 The generation of a ponatinib-resistant CML cell model ....................................... 126
3.3.1 Ponatinib resistant CML cell model is pan-TKI-resistant ............................... 131
3.3.2 Pre-clinical BCR-ABL inhibitor, ABL001, is ineffective against KCL22Pon-Res
and BaF3Pon-Res cells .................................................................................................... 132
3.4 Characterisation of the mechanism causing TKI resistance in the ponatinib-
resistant cell models .................................................................................................................. 136
3.4.1 DNA sequencing reveals no kinase domain mutation ..................................... 136
3.4.2 KCL22Pon-Res cells have an impaired transcriptional response to TKI ............. 137
3.4.3 mTOR activity is sustained in KCL22Pon-Res cells despite BCR-ABL inhibition
139
4 Results (II): Screening for potential novel drug target candidates for the treatment of
BCR-ABL-independent TKI-resistant CML...................................................................... 141
4.1 Aim and objective ........................................................................................................ 141
4.2 National Institute of Health (NIH) approved oncology drug screen ..................... 141
4.2.1 Confirmation that KCL22Pon-Res cells are pan-TKI-resistant ........................... 143
4.2.2 37 clinically approved cancer drugs have the potential to induce cell death in
TKI-resistant CML cells .............................................................................................. 143
4.2.3 KCL22Pon-Res cells are highly sensitive to mTOR inhibitors ............................ 143
4.2.4 KCL22Pon-Res cells are sensitive to HDAC inhibition ...................................... 144
4.2.5 KCL22Pon-Res cells are sensitive to MEK inhibition ......................................... 145
4.3 Validation of the primary drug screen using the BaF3Pon-Res cell model ............... 148
4.3.1 mTORC1 inhibition is insufficient to affect cell viability in BaF3Pon-Res cells 148
4.3.2 BaF3Pon-Res cells are highly sensitive to dual PI3K and mTOR inhibition ....... 150
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4.3.3 Potential selectivity using trametinib against BaF3Pon-Res cells compared to
BaF3parental cells ........................................................................................................... 153
4.4 A tertiary screen was performed to assess toxicity of the drugs to normal
haemopoietic cells ..................................................................................................................... 155
4.4.1 Dual PI3K and mTOR inhibitors may have a therapeutic window for treatment
for BCR-ABL-independent TKI-resistant CML patients ............................................ 155
4.4.2 MEK inhibition is not toxic to non-CML haemopoietic cells ......................... 158
5 Validation of mTOR inhibitors in ponatinib-resistant cell lines ................................. 160
5.1 Ponatinib resistant CML cells are sensitive to mTOR inhibition .......................... 161
5.2 mTOR inhibitors induce apoptosis and reduce CFC output in KCL22Pon-Res cells
163
5.3 KCL22Pon-Res cells are highly sensitive to catalytic PI3K and mTOR inhibitors . 165
5.4 BaF3Pon-Res cells are highly sensitive to NVP-Bez235 ............................................ 168
5.5 KCL22Pon-Res cells transcriptional response is restored upon NVP-Bez235
treatment ..................................................................................................................................... 170
6 Validation of mTOR inhibitors in primary TKI-resistant CML cells ......................... 178
6.1 MNCs derived from TKI-resistant CML patients are sensitive to NVP-Bez235-
mediated mTOR inhibition ex vivo ......................................................................................... 178
6.2 Ponatinib is an effective treatment against BCR-ABL-dependent TKI-resistant
CML cells ................................................................................................................................... 180
6.3 Ponatinib treatment causes different transcriptional responses in TKI sensitive
CML, T315I CML and BCR-ABL-independent TKI-resistant CML ................................ 182
6.4 mTORC1 inhibition induces protective autophagy in KCL22Pon-Res cells ............ 192
6.5 NVP-Bez235 mediated mTOR inhibition induces autophagy in KCL22Pon-Res cells
193
6.6 ATG7 knock out mediated autophagy inhibition increases sensitivity of
KCL22Pon-Res cells to NVP-Bez235 ......................................................................................... 195
6.7 Pharmacological autophagy inhibition enhances the effect of NVP-Bez235
against primary TKI-resistant CML cells ............................................................................... 201
6.8 Autophagy inhibition enhances the effect of dual PI3K and mTOR inhibitors
against primary TKI-resistant CML cells ............................................................................... 203
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6.9 Pharmacological inhibition of autophagy enhances the effect of NVP-Bez235 in
vivo 205
6.10 The combination treatment of NVP-Bez235 and HCQ has minimal toxicity
compared to omacetaxine in non-CML cells ......................................................................... 207
6.11 The combination treatment of NVP-Bez235 and HCQ was well tolerated by NSG
mice and significantly extended the survival of TKI-resistant CML xenograft mice. ..... 210
7 Results: Establishing CRISPR as a genome-wide screening tool for the study of BCR-
ABL-independent TKI-resistance ...................................................................................... 213
7.1 Gene knock-down ........................................................................................................ 213
7.2 Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas system
214
7.2.1 CRISPR as a genome editing tool.................................................................... 217
7.2.2 CRISPR as a genome-wide forward screening tool......................................... 218
7.2.3 Method: Genome-wide forward CRISPR screen............................................. 221
7.2.4 CRISPR 2-vector screen .................................................................................. 226
7.2.5 Preliminary results ........................................................................................... 236
7.2.6 Future work ...................................................................................................... 239
8 Discussion ................................................................................................................... 242
8.1 mTOR and autophagy .................................................................................................. 251
8.2 NVP-Bez235 and toxicity ........................................................................................... 253
8.3 Conclusion ..................................................................................................................... 255
8.4 Future work ................................................................................................................... 256
9 Appendices .................................................................................................................. 259
9.1 Plasmid maps ................................................................................................................ 259
9.1.1 pLKO.1 puro .................................................................................................... 259
9.1.2 psPAX2 – Addgene (12260) ............................................................................ 260
9.1.3 pCMV-VSV-G – Addgene (8454) ................................................................... 261
9.1.4 LentiCRISPR v2 Addgene (52961) ................................................................. 262
9.1.5 LentiCas9-Blast – Addgene (52962) ............................................................... 263
9.1.6 LentiGuide-Puro – Addgene (52963) .............................................................. 264