Investigation into the relevance of BCR-ABL forthe survival of cancer stem cells in chronicmyeloid leukaemia

R Medicine (General);RM Therapeutics. Pharmacology

Hamilton, Ashley ; Holyoake, Tessa

University:University of Glasgow

Department:Institute of Cancer Sciences

关键词: CML, stem cells, BCR-ABL, dasatinib, drug resistance;   

Others  :  http://theses.gla.ac.uk/2208/1/2010hamiltonphd.pdf

来源: University of Glasgow

【 摘 要 】

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder of thehaemopoietic stem cell, defined by the Philadelphia chromosome (Ph) - theoutcome of a balanced, reciprocal translocation between the long arms ofchromosomes 9 and 22. The novel fusion oncogene generated on chromosome22 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 thedisease. The introduction of the orally available, tyrosine kinase inhibitor (TKI) -imatinib mesylate (IM) - marked a major advance in CML treatment in terms ofefficacy and tolerability and has now become the first line of therapy. IM acts bycompeting with ATP to block ABL-kinase activity, resulting in the selectiveapoptosis induction of BCR-ABL+ cells. However, despite the success of IM asstandard therapy for CML, only a small proportion of patients obtain a completemolecular response, where they become negative for BCR-ABL transcripts by RTPCR.It is hypothesised that this minimal residual disease is the result of aprimitive quiescent subpopulation of leukaemic cells, which may be the cause forrelapse at a later date. Another major clinical concern is the observation ofmolecular resistance in IM-treated patients. Proposed mechanisms of resistanceinclude BCR-ABL amplification, decreased intracellular IM concentrations causedby drug efflux proteins such as multi drug resistance-1 and the development ofpoint 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 resistantor -intolerant patients with Ph+ leukaemias, which has a 325-fold greaterpotency 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 impressiveclearance of Ph+ cells, however, the primitive quiescent cell population did notappear to undergo apoptosis even after several days TKI exposure. Therefore, itwas still not clear whether early CML progenitor cells depend on BCR-ABL fortheir growth and survival. In this study we have attempted to determine whetherCML stem cells are dependent on BCR-ABL TK activity for their survival and/orproliferation using dasatinib treatment and aimed to characterise the cells whichsurvived drug exposure. We found that 10% of the CML cells were able to survivethe dasatinib treatment. We also showed that maximal BCR-ABL TK inhibition wasachieved in the surviving CML cells, both in the bulk population of cells and themore problematic primitive stem cell population. Those cells which survived thedasatinib treatment were found to be primitive, residing mainly in the undivided cellfraction 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 longtermculture-initiating cell (LTC-IC) colonies; these data suggested that ~10% ofprimitive CD34+ CML cells are not addicted to BCR-ABL TK activity for theirsurvival. This also suggested that these primitive, resistant CML cells appeared tosurvive and proliferate by BCR-ABL-independent mechanisms. Therefore, the nextexperiments were then designed to investigate the cellular process of autophagyas a potential means of primitive CML cell survival. Analysis of the properties ofCD34+ CML cells which remained viable following dasatinib treatment, revealedthe existence of cytoplasmic autophagic structures determined by electronmicroscopy and significantly increased autophagosome-asociated LC3-II,particularly in the cells cultured without growth factors (GF)s. This suggested thatautophagy is induced following GF deprivation of CML cells and is significantlyincreased within these cells, upon BCR-ABL inhibition following dasatinib treatment. Furthermore, we also found that the inhibition of autophagy greatlypotentiated the effect of TKI treatment on the reduction of primitive CML progenitorcells, in terms of the effective eradication of functionally defined colony formingcells and LTC-ICs.Overall, this thesis has shown for the first time that the most TKI-resistant primitiveCML cells are likely to be independent of BCR-ABL TK activity for theirproliferation and/or survival. Furthermore, we have shown that these resistantCML stem cells rely on the BCR-ABL independent autophagy process for survivalin response to stressful conditions, such as, GF deprivation and TKI treatment.