Expression and function of drug transporters in primitive CML cells

[摘要] Chronic myeloid leukaemia (CML) is a stem cell (SC) disorder initiated by thereciprocal translocation between chromosome 9 and 22, giving rise to thePhiladelphia (Ph) chromosome and the resulting expression of the oncogenicfusion protein BCR-ABL. The current first line of treatment is imatinib mesylate(IM), a tyrosine kinase inhibitor (TKI) that competes with ATP to block ABL kinaseactivity, which in turn prevents tyrosine phosphorylation of downstream moleculesand selectively induces apoptosis of BCR-ABL cells. However, despite excellentcytogenetic responses, only a minority of patients achieve complete molecularresponse (CMR). We have previously identified a population of quiescent (q) Ph+SC found in chronic phase (CP) CML that are relatively insensitive to IM and otherTKIs and which may be responsible for the molecular persistence of this disease.This population may be insensitive because TKIs do not reach therapeuticconcentrations within the cell. Such resistance to classical chemotherapeuticdrugs, the phenomenon of multidrug resistance (MDR), is mediated by ABCtransporters. In this study we have investigated whether CML SC express theclinically relevant ABC transporters and determine their interaction with TKIs. Inaddition, we determined whether the inhibition of these transporters increased theefficacy of TKI against CML SC.Using CML CD34+ cells isolated from newly diagnosed patients, normal CD34+cells and cell lines transduced with specific transporters as controls, the relativeexpression of drug transporters were determined in CML CD34+ cells andintracellular staining confirmed protein expression. The interaction of drugtransporters with TKIs was assessed using a combination of substratedisplacement assays and radiolabelled assays. The effect of transporter inhibitors3with TKIs on the growth and differentiation of q34+ and more mature CD34+ cellsfrom CML patients in CP were assessed with regard to cell division, apoptosis andBCR-ABL kinase activity.When compared to normal CD34+ cells, CML CD34+ cells over-expressed ABCG2mRNA. In contrast MDR1 expression was reduced in CML CD34+ cells and MRP1was detected at similar expression levels in both populations. All three drugtransporters were expressed at the protein levels in CML CD34+ cells. It wasdetermined that at therapeutic concentrations (5μM) IM and nilotinib both inhibitedABCG2 and MDR1 and nilotinib also inhibited MRP1. Neither drug was a substratefor any of the transporters. In contrast, dasatinib was shown to be a substrate forABCG2 and MRP1, but had no effect on MDR1. Therefore activity andconcentration of dasatinib but not IM or nilotinib may be altered by the activity ofthese proteins. In keeping with their inhibitory activity, neither IM nor nilotinibdemonstrated significantly increased efficacy when combined with specific ABCtransporter inhibitors (FTC or PSC 833). Surprisingly, although dasatinib was asubstrate for ABCG2 and MRP1, dasatinib did not further increase apoptosis, orreduce the qSC population.Therefore, although MDR1, MRP1 and ABCG2 were found to be expressed andfunctional in CML CD34+ cells and to interact with TKI, the co-treatment of TKIswith drug transporter inhibitors did not further increase apoptosis, reduce BCRABLkinase activity or reduce the qSC population. Therefore, modulation ofindividual transporter activity is unlikely to reverse the resistance of this populationof cells to TKI and will not improve the clinical response to these drugs.

[发布机构] University:University of Glasgow;Department:Institute of Cancer Sciences

[关键词] QP Physiology