Primitive subsets of leukemic cells isolated by using fluorescence-activated cell sorting from patients with newly diagnosed Ph⁺/BCR–ABL⁺chronic myeloid leukemia display an abnormal ability to proliferatein vitroin the absence of added growth factors. We now show from analyses of growth-factor gene expression, protein production, and antibody inhibition studies that this deregulated growth can be explained, at least in part, by a novel differentiation-controlled autocrine mechanism. This mechanism involves the consistent and selective activation of IL-3 and granulocyte colony-stimulating factor (G-CSF) production and a stimulation of STAT5 phosphorylation in CD34⁺leukemic cells. When these cells differentiate into CD34⁻cellsin vivo, IL-3 and G-CSF production declines, and the cells concomitantly lose their capacity for autonomous growthin vitrodespite their continued expression of BCR–ABL. Based on previous studies of normal cells, excessive exposure of the most primitive chronic myeloid leukemia cells to IL-3 and G-CSF through an autocrine mechanism could explain their paradoxically decreased self-renewalin vitroand slow accumulationin vivo, in spite of an increased cycling activity and selective expansion of later compartments.

Xenograft models of chronic phase human chronic myeloid leukemia (CML) have been difficult to develop because of the persistence of normal hematopoietic stem cells in most chronic phase CML patients and the lack of methods to selectively isolate the rarer CML stem cells. To circumvent this problem, we first identified nine patients' samples in which the long-term culture-initiating cells were predominantly leukemic and then transplanted cells from these samples into sublethally irradiated NOD/SCID and NOD/SCID-β2microglobulin^−/− mice. This resulted in the consistent and durable (>5 months) repopulation of both host genotypes with similar numbers of BCR-ABL⁺/Ph⁺ cells. The regenerated leukemic cells included an initial, transient population derived from CD34⁺CD38⁺ cells as well as more sustained populations derived from CD34⁺CD38⁻ progenitors, indicative of a hierarchy of transplantable leukemic cells. Analysis of the phenotypes produced revealed a reduced output of B-lineage cells, enhanced myelopoiesis with excessive production of erythroid and megakaropoietic cells and the generation of primitive (CD34⁺) leukemic cells displaying an autocrine IL-3 and G-CSF phenotype, all characteristics of primary CML cells. These findings demonstrate the validity of this xenograft model of chronic phase human CML, which should enable future investigation of disease pathogenesis and new approaches to therapy.

Imatinib mesylate treatment causes remissions in a majority of patients with chronic myeloid leukemia (CML), but relapses are an increasing problem. We hypothesized that imatinib-resistant leukemic cells emerge from CML stem cells that acquire BCR–ABL gene mutations even before exposure to BCR–ABL–targeted agents such as imatinib. Lineage-negative (i.e., immature) CD34 ⁺ CD38 ⁻ CML stem cell–enriched populations were isolated from five patients with chronic phase CML samples by fluorescence-activated cell sorting. To identify BCR–ABL gene mutations, complementary DNAs (cDNAs) prepared from purified CML stem cells were subjected to allele-specific amplification using primers corresponding to 16 kinase domain mutations, with normal bone marrow cells serving as negative controls. We also cloned and directly sequenced BCR–ABL cDNAs prepared from freshly isolated CML stem cells and from their progeny generated after 3–5 weeks of culture. In 20%–33% of cDNA preparations from freshly isolated CML stem cell–enriched populations, both allele-specific amplification and direct sequencing methods revealed mutations in sequences corresponding to the BCR–ABL kinase domain. Mutations were not observed in cDNA sequences encoding the c-ABL kinase domain that were obtained from similar types of primitive normal cells. More than 70 different BCR–ABL mutations (including frameshift mutations and premature stop codons) were identified in the progeny of cultured CML stem cells. Analysis of individual clones derived from the cultured cells demonstrated that new BCR–ABL mutations were produced. Primary CML stem cells display instability of the BCR–ABL fusion gene both in vivo and in vitro. Thus, patients may possess leukemic stem cells with BCR–ABL kinase mutations before initiation of BCR–ABL–targeted therapies and would likely be predisposed to develop resistance to these agents.