In clinical trials, the tyrosine kinase inhibitor STI571 has proven highly effective in reducing leukemic cell burden in chronic myeloid leukemia (CML). The overall sensitivity of CML CD34 ؉ progenitor cells to STI571 and the degree to which cell death was dependent on cell cycle status were determined. Stem cells (Lin ؊ CD34 ؉ ) from the peripheral blood of patients with CML in chronic phase and from granulocyte-colony-stimulating factor-mobilized healthy donors were labeled with carboxy-fluorescein diacetate succinimidyl diester dye to enable highresolution tracking of cell division. Then they were cultured for 3 days with and without growth factors ؎ STI571. After culture, the cells were separated by fluorescence-activated cell sorting into populations of viable quiescent versus cycling cells for genotyping. For healthy controls, in the presence of growth factors, STI571 affected neither cell cycle kinetics nor recovery of viable cells. In the absence of growth factors, normal cells were unable to divide. For CML samples, in the presence or absence of growth factors, the response to STI571 was variable. In the most sensitive cases, STI571 killed almost all dividing cells; however, a significant population of viable CD34 ؉ cells was recovered in the undivided peak and confirmed to be part of the leukemic clone. STI571 also appeared to exhibit antiproliferative activity on the quiescent population. These studies confirm that CML stem cells remain viable in a quiescent state even in the presence of growth factors and STI571. Despite dramatic short-term responses in vivo, such in vitro insensitivity to STI571, in combination with its demonstrated antiproliferative activity, could translate into disease relapse after prolonged therapy. IntroductionChronic myeloid leukemia (CML) is a clonal myeloproliferative disease characterized by the t(9;22) chromosome translocation that, in turn, creates the BCR-ABL oncogene. [1][2][3] The fusion gene product is a p210 oncoprotein containing a constitutively active tyrosine kinase that confers certain growth advantages to the Philadelphia-positive (Ph ϩ ) clone compared with normal hematopoietic cells. 4 We have demonstrated recently the existence of a population of rare, primitive, quiescent stem cells in all chronic-phase CML patient samples, whether derived from peripheral blood or bone marrow. These stem cells are predominantly Ph ϩ , express high levels of CD34 ϩ but lack the markers CD38, CD45RA, or CD71, and can spontaneously exit G 0 to enter a continuously proliferating state, either in vitro or to produce Ph ϩ progeny in immunocompromised mice in vivo. 5,6 Many cancers are treated with relatively nonselective cytotoxic drugs that affect normal and malignant cells. Because most available chemotherapeutic agents show some degree of S-phase specificity, cells that are not actively dividing may prove resistant to such drugs. This raises the possibility that the quiescent leukemic cells we have identified in patients with CML are likely to survive standard chemotherapy r...

Chronic myeloid leukemia (CML) stem and progenitor cells overexpress BcrAbl and are insensitive to imatinib mesylate (IM). We therefore investigated whether these cells were efficiently targeted by nilotinib. In K562, the inhibitory concentration (IC50) of nilotinib was 30 nM versus 600 nM for IM, consistent with its reported 20-fold-higher potency. However, in primary CD34(+) CML cells, nilotinib and IM were equipotent for inhibition of BcrAbl activity, producing equivalent but incomplete reduction in CrkL phosphorylation at 5 microM. CML CD34(+) cells were still able to expand over 72 hours with 5 microM of either drug, although there was a concentration-dependent restriction of amplification. As for IM, the most primitive cells (CFSE(max)) persisted and accumulated over 72 hours with nilotinib and remained caspase-3 negative. Furthermore, nilotinib with IM led to further accumulation of this population, suggesting at least additive antiproliferative effects. These results confirmed that, like IM, the predominant effect of nilotinib is antiproliferative rather than proapoptotic.

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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...