Investigation into the relevance of BCR-ABL for the survival of cancer stem cells in chronic myeloid leukaemia

Hamilton, Ashley (2010) Investigation into the relevance of BCR-ABL for the survival of cancer stem cells in chronic myeloid leukaemia. PhD thesis, University of Glasgow.

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Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder of the
haemopoietic stem cell, defined by the Philadelphia chromosome (Ph) - the
outcome of a balanced, reciprocal translocation between the long arms of
chromosomes 9 and 22. The novel fusion oncogene generated on chromosome
22 as a result of this translocation is called BCR-ABL. In the majority of patients,
this oncogene transcribes a 210-kDa constitutively active protein tyrosine kinase,
often referred to as p210BCR-ABL, which is necessary for the transformation of the
disease. The introduction of the orally available, tyrosine kinase inhibitor (TKI) -
imatinib mesylate (IM) - marked a major advance in CML treatment in terms of
efficacy and tolerability and has now become the first line of therapy. IM acts by
competing with ATP to block ABL-kinase activity, resulting in the selective
apoptosis induction of BCR-ABL+ cells. However, despite the success of IM as
standard therapy for CML, only a small proportion of patients obtain a complete
molecular response, where they become negative for BCR-ABL transcripts by RTPCR.
It is hypothesised that this minimal residual disease is the result of a
primitive quiescent subpopulation of leukaemic cells, which may be the cause for
relapse at a later date. Another major clinical concern is the observation of
molecular resistance in IM-treated patients. Proposed mechanisms of resistance
include BCR-ABL amplification, decreased intracellular IM concentrations caused
by drug efflux proteins such as multi drug resistance-1 and the development of
point mutations within the ABL-kinase domain. In an attempt to overcome IMresistance,
a second generation of BCR-ABL inhibitors has been developed.
Dasatinib (BMS-354825, Sprycel) is a TKI developed for the treatment of IM resistant
or -intolerant patients with Ph+ leukaemias, which has a 325-fold greater
potency than IM against cells expressing wild-type BCR-ABL, and is effective against all IM-resistant BCR-ABL mutants tested in vitro, except T315I. Previously,
we showed that dasatinib induced durable inhibition of BCR-ABL and impressive
clearance of Ph+ cells, however, the primitive quiescent cell population did not
appear to undergo apoptosis even after several days TKI exposure. Therefore, it
was still not clear whether early CML progenitor cells depend on BCR-ABL for
their growth and survival. In this study we have attempted to determine whether
CML stem cells are dependent on BCR-ABL TK activity for their survival and/or
proliferation using dasatinib treatment and aimed to characterise the cells which
survived drug exposure. We found that 10% of the CML cells were able to survive
the dasatinib treatment. We also showed that maximal BCR-ABL TK inhibition was
achieved in the surviving CML cells, both in the bulk population of cells and the
more problematic primitive stem cell population. Those cells which survived the
dasatinib treatment were found to be primitive, residing mainly in the undivided cell
fraction and the very early cell divisions. Since these BCR-ABL TK-inhibited,
resistant cells were also able to grow when re-cultured in cytokines and form longterm
culture-initiating cell (LTC-IC) colonies; these data suggested that ~10% of
primitive CD34+ CML cells are not addicted to BCR-ABL TK activity for their
survival. This also suggested that these primitive, resistant CML cells appeared to
survive and proliferate by BCR-ABL-independent mechanisms. Therefore, the next
experiments were then designed to investigate the cellular process of autophagy
as a potential means of primitive CML cell survival. Analysis of the properties of
CD34+ CML cells which remained viable following dasatinib treatment, revealed
the existence of cytoplasmic autophagic structures determined by electron
microscopy and significantly increased autophagosome-asociated LC3-II,
particularly in the cells cultured without growth factors (GF)s. This suggested that
autophagy is induced following GF deprivation of CML cells and is significantly
increased within these cells, upon BCR-ABL inhibition following dasatinib treatment. Furthermore, we also found that the inhibition of autophagy greatly
potentiated the effect of TKI treatment on the reduction of primitive CML progenitor
cells, in terms of the effective eradication of functionally defined colony forming
cells and LTC-ICs.
Overall, this thesis has shown for the first time that the most TKI-resistant primitive
CML cells are likely to be independent of BCR-ABL TK activity for their
proliferation and/or survival. Furthermore, we have shown that these resistant
CML stem cells rely on the BCR-ABL independent autophagy process for survival
in response to stressful conditions, such as, GF deprivation and TKI treatment.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: CML, stem cells, BCR-ABL, dasatinib, drug resistance
Subjects: R Medicine > R Medicine (General)
R Medicine > RM Therapeutics. Pharmacology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
Supervisor's Name: Holyoake, Professor Tessa
Date of Award: 2010
Depositing User: miss ashley hamilton
Unique ID: glathesis:2010-2208
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 28 Oct 2010
Last Modified: 10 Dec 2012 13:52

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