Mitchell, Rebecca (2017) Exploring BCR-ABL-independent mechanisms of TKI-resistance in chronic myeloid leukaemia. PhD thesis. https://theses.gla.ac.uk/7977/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given 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 2 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- 3 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. 4 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 5 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