Jordanides, Niove E.
Expression and function of drug transporters in primitive CML cells.
PhD thesis, University of Glasgow.
Full text available as:
Chronic myeloid leukaemia (CML) is a stem cell (SC) disorder initiated by the
reciprocal translocation between chromosome 9 and 22, giving rise to the
Philadelphia (Ph) chromosome and the resulting expression of the oncogenic
fusion 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 kinase
activity, which in turn prevents tyrosine phosphorylation of downstream molecules
and selectively induces apoptosis of BCR-ABL cells. However, despite excellent
cytogenetic responses, only a minority of patients achieve complete molecular
response (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 other
TKIs and which may be responsible for the molecular persistence of this disease.
This population may be insensitive because TKIs do not reach therapeutic
concentrations within the cell. Such resistance to classical chemotherapeutic
drugs, the phenomenon of multidrug resistance (MDR), is mediated by ABC
transporters. In this study we have investigated whether CML SC express the
clinically relevant ABC transporters and determine their interaction with TKIs. In
addition, we determined whether the inhibition of these transporters increased the
efficacy 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 relative
expression of drug transporters were determined in CML CD34+ cells and
intracellular staining confirmed protein expression. The interaction of drug
transporters with TKIs was assessed using a combination of substrate
displacement assays and radiolabelled assays. The effect of transporter inhibitors
with TKIs on the growth and differentiation of q34+ and more mature CD34+ cells
from CML patients in CP were assessed with regard to cell division, apoptosis and
BCR-ABL kinase activity.
When compared to normal CD34+ cells, CML CD34+ cells over-expressed ABCG2
mRNA. In contrast MDR1 expression was reduced in CML CD34+ cells and MRP1
was detected at similar expression levels in both populations. All three drug
transporters were expressed at the protein levels in CML CD34+ cells. It was
determined that at therapeutic concentrations (5μM) IM and nilotinib both inhibited
ABCG2 and MDR1 and nilotinib also inhibited MRP1. Neither drug was a substrate
for any of the transporters. In contrast, dasatinib was shown to be a substrate for
ABCG2 and MRP1, but had no effect on MDR1. Therefore activity and
concentration of dasatinib but not IM or nilotinib may be altered by the activity of
these proteins. In keeping with their inhibitory activity, neither IM nor nilotinib
demonstrated significantly increased efficacy when combined with specific ABC
transporter inhibitors (FTC or PSC 833). Surprisingly, although dasatinib was a
substrate for ABCG2 and MRP1, dasatinib did not further increase apoptosis, or
reduce the qSC population.
Therefore, although MDR1, MRP1 and ABCG2 were found to be expressed and
functional in CML CD34+ cells and to interact with TKI, the co-treatment of TKIs
with drug transporter inhibitors did not further increase apoptosis, reduce BCRABL
kinase activity or reduce the qSC population. Therefore, modulation of
individual transporter activity is unlikely to reverse the resistance of this population
of cells to TKI and will not improve the clinical response to these drugs.
Actions (login required)