Functional ABCG2 is overexpressed on primary CML CD34+ cells and is inhibited by imatinib mesylate

Abstract

IntroductionThe introduction of imatinib mesylate (IM) has transformed the treatment of chronic myeloid leukemia (CML), however a significant minority of patients does not respond and others lose response during treatment. Such states reflect IM resistance in the whole cell population, however suboptimal responses may represent the persistence of a subpopulation of insensitive leukemic stem cells (LSCs). [1][2][3][4] One possible mechanism of LSC persistence is the limitation of intracellular drug concentration due to the activity of ATP binding cassette (ABC) drugresistance proteins as seen previously with conventional chemotherapeutics. 5 One such transporter, ABCG2, is highly expressed on normal stem cell populations 6 and as such may also be expected to be expressed on LSCs.There have been a number of convincing but contradictory reports on the interaction of IM with ABCG2. Houghton et al 7 found IM to be a potent inhibitor of ABCG2, but not a substrate; Burger et al 8 found IM to be a substrate; and importantly, Nakanishi et al 9 showed that Bcr-Abl itself may regulate ABCG2 protein expression via AKT activity and suggest that ABCG2 may be a mediator of IM resistance. However, these studies all used cell lines that had been engineered to express ABCG2.It was therefore, critical to look at this potential transporter for IM in primary CML cells and particularly within the LSC compartment. We report here our investigation of the expression and function of ABCG2 on primary CD34 ϩ CML cells, and the effect of this transporter on cellular IM levels and their response to this drug. Study design Primary cell samples and cell linesLeukapheresis products from chronic-phase CML patients at diagnosis prior to IM treatment were obtained with informed consent in accordance with the Declaration of Helsinki, and with the approval of the Glasgow Royal Infirmary Ethics Committee, and enriched for CD34 ϩ cells using CLINIMACS (Miltenyi Biotec, Bisley, United Kingdom). CD34 ϩ cells were confirmed to be Bcr-Abl ϩ by fluorescence in situ hybridization (FISH) and cultured in growth factor medium (5GF) as previously published. 1 Normal CD34 ϩ cells were purchased from Cambrex Bioscience (Wokingham, United Kingdom). AML6.2 (ABCG2 high) and OCI-AML3 (parental ABCG2 negative) cell lines 10 were from Dr Brian Sorrentino (St Jude Children's Research Hospital, Memphis, TN). Efflux studiesThe efflux protocol was a modification of that previously described 11 using the ABCG2-specific inhibitor fumitremorgin C (FTC; Dr R Robey, Center for Cancer Research, NIH, Bethesda, MD). Cells were incubated with Ϯ 10 M FTC Ϯ 5 M IM (Novartis Pharma, Basel, Switzerland) for 15 minutes to block pump activity before the addition of 200 nM BODIPY-Prazosin (B-P; Molecular Probes, Eugene, OR) and a further 30-minute incubation. Cells were then washed and incubated Ϯ 10 M FTC/5 M IM for 60 minutes to allow efflux before being washed in ice-cold medium and analyzed. An Inside Blood analysis of this article appears at the front of this issue.Reprints: Joanne C...

Drug transporters: recent advances concerning BCRP and tyrosine kinase inhibitors

“…It has now been demonstrated that SP cells are present in several tumour samples, possess stem cell-like properties, overexpress BCRP and possess inherent drug resistance (Haraguchi et al, 2006). Consistently, BCRP overexpression was reported on primary CML CD34 þ cells (Jordanides et al, 2006). Imatinib is efficiently used in the clinical treatment of BCR-ABL-driven CML, but possible BCRP-mediated imatinib resistance would naturally affect the efficacy of the treatment.…”

“…Rather, imatinib may serve as a potent BCRP inhibitor thereby enabling reversal of BCRP-mediated resistance to topotecan and SN-38 (Houghton et al, 2004). Similarly, accumulation of mitoxantrone in primary chronic myeloid leukaemia (CML) CD34 þ cells overexpressing BCRP was significantly increased by 5 mM of imatinib, confirming the activity of this TKI as a BCRP inhibitor (Jordanides et al, 2006). The same authors postulated that imatinib is not a BCRP substrate, since BCRP inhibition in CML CD34 þ cells neither potentiated the effect of imatinib nor affected imatinib accumulation in these cells.…”

Influence of CYP3A5 and drug transporter polymorphisms on imatinib trough concentration and clinical response among patients with chronic phase chronic myeloid leukemia

“…50,51 Moreover, both transporters show elevated expression in CML stem cells, 52,53 and several known SNPs modify 56 Although they did not investigate the relationship between the tested SNPs and plasma IM concentration, the authors noted that interindividual variation in hepatic IM uptake and clearance could lead to changes in systemic IM concentration. Our results should be interpreted within the context of the study limitations, which primarily involve the sample size of the study.…”

Hematopoietic Stem Cells in Chronic Myeloid Leukemia

“…Imatinib has been variably reported to be a substrate and/or an inhibitor for the BCRP/ABCG2 drug efflux pump, which is overexpressed in many human tumors and also found to be functionally expressed in CML stem cells [111,112]. CML stem cells have been shown to express the ATP dependent transporter cassette protein ABCG2, which could decrease the intracellular accumulation of Imatinib in CML LSC [103].…”