Investigation of the role of autocrine and paracrine growth factors in the survival and proliferation of chronic myeloid leukaemia stem and progenitor cells

Gallipoli, Paolo (2013) Investigation of the role of autocrine and paracrine growth factors in the survival and proliferation of chronic myeloid leukaemia stem and progenitor cells. PhD thesis, University of Glasgow.

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Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder arising in a haemopoietic stem cell (HSC) as a result of the reciprocal translocation between the long arms of chromosomes 9 and 22 (t9;22), leading to the formation of the fusion oncogene BCR-ABL. BCR-ABL has constitutive tyrosine kinase (TK) activity which drives, at least during the chronic phase (CP) of the disease, myeloid progenitor cells expansion through terminally differentiated cells and is necessary for the transformed phenotype. The introduction at the end of the last century of BCR-ABL TK inhibitors (TKI) has dramatically changed the management of newly diagnosed CP CML patients as the vast majority achieve deep molecular responses while enjoying good quality of life when treated with TKI. However about 20% of patients still show various degree of resistance to all currently available TKI while in those achieving deep responses, there is compelling evidence of persistent minimal residual disease demanding lifelong treatment which has obvious implications in terms of compliance, adverse events and costs.
It is now known that the main reason for disease persistence in CML patients treated with TKI lies in the insensitivity of the most primitive CML leukaemia stem cell (LSC). More recent evidence has demonstrated that, in contrast to more mature leukaemic progenitor cells, CML LSC are not addicted to BCR-ABL kinase activity but rather rely on other stem cell intrinsic pathways for their survival. The main focus in the CML field is therefore to identify these pathways while also trying to exploit them therapeutically to achieve CML LSC eradication and as a result disease cure.
Growth factor (GF) signals are known to provide survival cues to CML stem and progenitor cells (SPC) and potentially support their survival even in the presence of TKI. Moreover CML SPC are also known to produce higher levels of some GFs via an autocrine loop and support their survival and proliferation through this mechanism. In this thesis, the characterisation of the autocrine GF production by CML SPC was extended while also investigating the role of several GFs and downstream signals in survival, proliferation and self-renewal of CML SPC. Whenever possible, the consequences of therapeutic targeting of these signals on CML SPC survival and proliferation were also assessed in vitro.
In particular the role of the intracellular janus kinase (JAK) 2, which relays several myeloid GF signals, such as those from interleukin (IL)-3 and granulocyte macrophage colony-stimulating factor (GM-CSF), in CML SPC survival and proliferation was investigated mainly because higher levels of autocrine expression of GM-CSF by CML SPC relative to normal were demonstrated, while autocrine IL-3 production by CML SPC had already been shown. Moreover the cognate receptor of both GM-CSF and IL-3 (CSF2RB) was also shown to be expressed at higher levels in CML SPC relative to their normal counterparts, further supporting investigations on the role of JAK2 in CML SPC biology. Indeed targeting JAK2 with small molecule inhibitors in CML SPC in vitro, particularly in the presence of maximal BCR-ABL TK inhibition, resulted in increased apoptosis, reduced proliferation and colony output of CML SPC. The JAK2 inhibitor plus TKI combination treatment, compared to either single agent, further reduced survival of the more primitive quiescent LSCs in vitro, while also reducing engraftment of primary CML CD34+ cells in vivo in immunocompromised hosts. Although a degree of toxicity to normal haemopoietic stem and progenitor cells (HSPC) was observed, this was not as great as seen in CML SPC, thus suggesting that a therapeutic window for using JAK2 inhibitors in CML patients might be present when a carefully selected concentration of these compounds is chosen.
Tumour necrosis factor (TNF)-α was another GF shown to be produced in an autocrine fashion at higher levels by CML SPC relative to normal HSPC. Moreover its levels of production by CML SPC were not modulated by BCR-ABL TK activity. Using a small molecule TNF-α inhibitor and exogenous TNF-α, it was shown that autocrine TNF-α acts as a survival and proliferative signal in CML SPC. Moreover its role became even more important in the presence of TKI, as combining TNF-α inhibition with TKI led to high levels of apoptosis in CML CD34+ cells, including the more primitive quiescent population, while also causing increased apoptosis in a population enriched for CML LSCs based on its surface marker expression (CD34+ CD38-).
Finally given the known importance of quiescence and self-renewal pathways in CML LSC persistence following TKI treatment, the role of transforming growth factor (TGF)-β1 and novel neurotransmitter mediated pathways in CML LSC quiescence and self-renewal was investigated based on the findings of a genome and epigenome-wide screen of primary CML LSCs and normal HSCs carried out in our laboratory. Using in vitro assays the putative role of the neuromediators norepinephrine and acethylcoline in CML LSC self-renewal was demonstrated. Moreover the role of TGF-β1 in inducing primary CML LSC quiescence mainly by modulating the AKT pathway was also demonstrated.
Overall the work presented in this thesis has furthered our understanding of the role of both autocrine and paracrine known and novel regulators of haemopoiesis in several aspects of CML SPC biology such as their survival, proliferation and self-renewal. Furthermore the efficacy in eradicating CML SPC of therapeutic strategies targeting some of these GF signals has been explored in vitro, thus providing evidence supporting their subsequent testing in in vivo assays and in due course in clinical studies. It is hoped therefore that the work presented will contribute to devise novel therapeutic strategies to eradicate CML LSC and in turn lead to a cure for CML patients.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Chronic myeloid leukaemia, leukaemia stem cell, growth factors
Subjects: R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing
Supervisor's Name: Holyoake, Prof. Tessa
Date of Award: 2013
Depositing User: Dr Paolo Gallipoli
Unique ID: glathesis:2013-4172
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 26 Apr 2013 11:32
Last Modified: 26 Apr 2013 11:32

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