Kuntz, Elodie Marie (2017) an Investigation of Metabolic Vulnerabilities in Chronic Myeloid Leukaemic Stem Cells

Kuntz, Elodie Marie (2017) An investigation of metabolic vulnerabilities in chronic myeloid leukaemic stem cells. PhD thesis. http://theses.gla.ac.uk/8615/ 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 http://theses.gla.ac.uk/ [email protected] An Investigation of Metabolic Vulnerabilities in Chronic Myeloid Leukaemic Stem Cells Elodie Marie Kuntz Thesis submitted to the University of Glasgow in accordance with the requirements for the degree of Doctor of Philosophy Institute of Cancer Science College of Medical, Veterinary and Life Sciences University of Glasgow October 2017 CRUK Beatson Institute Garscube Estate Switchback Road Bearsden, Glasgow 1 Abstract Chronic myeloid leukaemia (CML) is a myeloproliferative disorder that originates at the haematopoietic stem cell (HSC) level. CML is driven by BCR-ABL, a fusion oncoprotein with a constitutive tyrosine kinase activity. The discovery of imatinib, a c-Abl specific tyrosine kinase inhibitor (TKI), revolutionised the treatment of CML by inducing cytogenetic and molecular responses in the majority of CML patients in chronic phase. However, imatinib and second/third generation TKIs do not eradicate leukaemic stem cells (LSCs), leading to disease persistence with associated risk of toxicity, drug resistance and relapse. This suggests that effective eradication of CML LSCs requires identification of novel target(s) that can be exploited therapeutically in combination with TKI treatment. In recent years, a plethora of studies have demonstrated that cancer cells rewire their metabolism to fuel their high energy demands and targeting these metabolic alterations can be of therapeutic benefit. Thus far, investigation of CML LSCs metabolism has been restricted by technical limitations. In this study, we aimed to identify and target the metabolic dependencies in CML LSCs using stem cell enriched (CD34+) primary cells isolated from CML patients and healthy donors. We initially investigated the metabolism of differentiated CD34- and primitive CD34+ cells and demonstrated that glucose and fatty acid oxidation was elevated in CD34+ CML cells. We as well demonstrated that CML CD34+ cells displayed an increase in their mitochondrial oxygen consumption rate (OCR). Next, we compared the metabolism of CD34+ and CD34+CD38- CML cells to their respective normal counterparts, which revealed that stem cell-enriched CML cells possess increased mitochondrial functions in comparison to normal cells. Of clinical significance, we show that the antibiotic tigecycline, an inhibitor of mitochondrial translation, reduced this aberrant oxidative metabolism. The combination of imatinib and tigecycline targeted primitive CML cells at a clinically achievable concentration while having minimal effect on colony formation potential of CD34+ cells derived from healthy donors. To validate these findings in vivo, human CML CD34+ cells were injected into irradiated immune-deficient mice. Remarkably, four-week combination treatment with tigecycline and imatinib in vivo eliminated the majority of CML LSCs, targeting 95% of the cells. Moreover, 2 mice maintained low levels of CML LSCs upon discontinuation of the combination treatment whereas imatinib-treated mice showed signs of relapse. These results indicate that oxidative phosphorylation is crucial for the survival of CML LSCs and inhibition of mitochondrial metabolism with tigecycline, in combination with imatinib treatment, might be a suitable therapeutic strategy to selectively target these cells and improve cure rates. 3 Contents Abstract ............................................................................................................................... 2 Author’s declaration ............................................................................................................. 9 Acknowledgments ............................................................................................................. 10 Abbreviations ..................................................................................................................... 12 Introduction ........................................................................................................................ 17 1.1 Haematopoiesis and haematopoietic stem cells (HSCs) .................................... 17 1.1.1 Ontogeny of haematopoiesis ....................................................................... 17 1.1.2 Hematopoietic cells hierarchy ...................................................................... 17 1.1.3 Regulation of HSCs ..................................................................................... 18 1.1.4 Identification and isolation of HSCs ............................................................. 20 1.2 The cancer stem cell (CSC) model ..................................................................... 21 1.2.1 Introduction .................................................................................................. 21 1.2.2 Historical overview ....................................................................................... 21 1.2.3 Others characteristics of CSCs .................................................................... 22 1.2.4 Cell of origin ................................................................................................. 22 1.2.5 Controversies of the CSC model ................................................................. 23 1.3 CML .................................................................................................................... 24 1.3.1 Introduction .................................................................................................. 24 1.3.2 Epidemiology ............................................................................................... 25 1.3.3 Disease progression .................................................................................... 25 1.3.4 Molecular pathophysiology of CML .............................................................. 26 1.3.5 CML diagnosis and monitoring .................................................................... 27 1.3.6 BCR-ABL signalling pathway ....................................................................... 28 1.3.7 CML Treatment ............................................................................................ 29 1.3.8 CML LSCs persistence ................................................................................ 35 1.4 Metabolism and HSC fate ................................................................................... 38 1.4.1 Metabolism at a glance ................................................................................ 38 1.4.2 Use of stable isotopes as tracers ................................................................. 40 1.4.3 Metabolic regulation of HSCs ...................................................................... 40 1.5 Cancer Metabolism ............................................................................................. 42 1.5.1 Metabolic reprogramming of cancer cells .................................................... 42 1.5.2 The Warburg effect and glycolysis in cancer ............................................... 43 1.5.3 Mitochondrial metabolism in cancer............................................................. 44 1.5.4 Targeting metabolic vulnerabilities of cancer cells ....................................... 46 1.6 Aims .................................................................................................................... 50 4 Chapter 2 Material and Methods ...................................................................................... 51 2.1 Material ............................................................................................................... 51 2.1.1 General reagents ......................................................................................... 51 2.1.2 Flow cytometry reagents .............................................................................. 53 2.1.3 Western blot antibodies ............................................................................... 54 2.1.4 Primers ........................................................................................................ 54 2.1.5 Primary cells ................................................................................................ 54 2.1.6 Cell lines ...................................................................................................... 55 2.1.7 Equipment .................................................................................................... 55 2.1.8 Composition of tissue culture media, solutions and buffers ......................... 55 2.2 Methods .............................................................................................................. 59 2.2.1 Primary samples .......................................................................................... 59 2.2.2 Tissue Culture .............................................................................................

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