Severe functional alterations in vitro in CD34(+) cell subpopulations from patients with chronic myeloid leukemia

Chronic myeloid leukemia (CML) arises from the malignant transformation of a hematopoietic stem cell (HSC) that gives rise to functionally defective progeny, including primitive and relatively mature progenitor cells (HPC). Both HSC and HPC are comprised within the population of CD34(+) cells, normally present in bone marrow (BM). In the present study, we have separated two different subpopulations of CD34(+) cells from CML marrow: Population I, enriched for CD34(+) Lin(-) cells; and Population II, enriched for CD34(+) CD36(-) CD38(-) CD45RA(-) Lin(-) cells, and assessed their progenitor cell content as well as their capacity to proliferate and expand in response to a combination of hematopoietic cytokines in serum- and stroma-free long-term liquid cultures. The absolute cell numbers recovered in Population I from normal and CML samples were similar; in contrast, we found that Population II from CML was amplified four-fold, as compared to normal. In spite of this latter observation, no significant differences were observed in terms of the absolute number of CFC when comparing Populations I and II from CML patients and normal subjects. Interestingly, the proliferation and expansion potentials of CML cells were clearly deficient as compared to their normal counterparts. Indeed, in cultures of Population I cells the maximum fold increase in total and progenitor cell numbers corresponded to 30 and 8%, respectively, of those observed in cultures of normal marrow-derived Population I cells. Such functional deficiencies were even more evident in Population II cells in which the maximum fold increase in total and progenitor cell numbers corresponded to 3 and 0.5%, respectively, of the levels found in cultures of Population II cells from normal marrow. The present study demonstrates that bone marrow-derived CD34(+) cells from CML patients possess functional abnormalities, clearly evident in the in vitro system used by us. Among the two CML subpopulations studied here, the more immature one (Population II; enriched for CD34(+) CD36(-) CD38(-) CD45RA(-) Lin(-) cells) was the one that showed the most severe abnormalities, as compared to its relatively more mature counterpart (Population I; enriched for CD34(+) Lin(-) cells).     

Coculture and transplant of purified CD34(+)Lin(-) and CD34(-)Lin(-) cells reveals functional interaction between repopulating hematopoietic stem cells.

The human hematopoietic stem cell compartment is comprised of repopulating CD34(+) and CD34(-) cells. The interaction between these subsets with respect to their reconstitution capacity in vivo remains to be characterized. Here, lineage-depleted (Lin(-)) human CD34(+) and CD34(-) hematopoietic cells were isolated from human male and female umbilical cord blood (CB) and transplanted into immune-deficient NOD/SCID EMV(null) mice, thereby allowing the use of human and Y-chromosome-specific DNA sequences to discriminate human reconstitution contributed by CD34(+) vs CD34(-) repopulating stem cells. Although cultured human CB CD34(-)Lin(-) cells transplanted alone possessed only minimal repopulating capacity, with 15% of mice achieving low levels of engraftment, transplantation of cocultured male CD34(-)Lin(-) cells with female CD34(+)Lin(-) cells demonstrated human repopulation with a contribution from CD34(-)Lin(-)-derived progeny in 80% of the recipients. After coculture and transplantation, male CD34(-)Lin(-) cells gave rise to primitive CD34(+)CD38(-) cells isolated in vivo, which demonstrated clonogenic progenitor function into multiple lineages. Taken together, our study indicates that the presence of CD34(+)Lin(-) cells in coculture enhanced the low repopulating function of human CD34(-)Lin(-) cells in vivo. We propose that CD34(+)Lin and CD34(-)Lin cells represent phenotypically distinct, but related cell types that exhibit unique and previously unappreciated functional interaction.     

Apoptosis induction in peripheral leukemia cells by remission induction treatment in vivo: selective depletion and apoptosis in a CD34+ subpopulation of leukemia cells.

In vitro studies demonstrating the induction of programmed cell death by cytotoxic drugs used in anticancer chemotherapy suggested that antileukemic treatment eliminates leukemia cells by apoptosis. We therefore analyzed apoptosis induction and activation of apoptosis signaling molecules in patients receiving remission induction treatment for AML and ALL during the initial phase of leukemia cell reduction. A coexistence of distinct populations of CD34(+) and CD34(-) leukemia cells could be identified. During chemotherapy, CD34(+) leukemia cells were more rapidly depleted than CD34(-) cells. Furthermore, a significant increase in leukemia cell apoptosis ex vivo was detected in CD34(+) cells, while no such increase was observed in the CD34(-) subpopulation, suggesting that CD34(+) leukemia cells are the main targets for apoptosis induction through antileukemic treatment. No alterations in Bax and Bcl-2 expression were found during in vivo chemotherapy, and CD95 expression and sensitivity remained low, indicating the induction of apoptosis independent of the CD95 system or regulation of protein levels of Bax and Bcl-2. The data suggest that analysis of leukemia cell subpopulations is required for further identification of apoptosis signaling molecules relevant for response to treatment and assessment of drug efficacy in vivo and in vitro.     

Human acute myeloid leukemia CD34+/CD38- progenitor cells have decreased sensitivity to chemotherapy and Fas-induced apoptosis, reduced immunogenicity, and impaired dendritic cell transformation capacities

The destruction of cells capable of initiating and maintaining leukemia challenges the treatment of human acute myeloid leukemia. Recently, CD34+/CD38- leukemia progenitors have been defined as new leukemia-initiating cells less mature than colony-forming cells. Here we show that CD34+/CD38- leukemia precursors have reduced in vitro sensitivity to daunorubicin, a major drug used in leukemia treatment, in comparison with the CD34+/CD38+ counterpart, and increased expression of multidrug resistance genes (mrp/lrp). These precursors show lower expression of Fas/Fas-L and Fas-induced apoptosis than CD34+/CD38+ blasts. Moreover, the CD34+/CD38- leukemic subpopulation induces a weaker mixed leukocyte reaction of responding T-lymphocytes than the CD34+/CD38+ leukemic counterpart, either in a MHC-unmatched or MHC-matched settings. This weaker immunogenicity could be linked to lower expression on CD34+/CD38- leukemia precursors of major immune response molecules (MHC-DR, LFA-3, B7-1, or B7-2) than CD34+/CD38+ leukemic cells. Nonetheless, the susceptibility of the immature CD38- precursors to cytotoxicity was not different from the sensitivity of the CD38+ counterpart. Finally, CD34+/CD38- leukemia precursors, in contrast with CD38+ precursors, failed, under appropriate conditions, to differentiate into dendritic cells, a central step for antigen recognition. This is to our knowledge the first demonstration that the very immature phenotype of CD34+/CD38- leukemic progenitors confers both chemotherapy resistance and decreased capacities to induce an immune response. Because the susceptibility of the immature leukemia cells as cytotoxic targets is maintained, our data underline the importance of improving the initial steps of leukemia recognition, more particularly by defining optimal conditions of dendritic cell transformation of the very immature hematopoietic precursors.     

Retinoic acid induction of CD38 antigen expression on normal and leukemic human myeloid cells: relationship with cell differentiation

Differentiation in the hematopoietic system involves, among other changes, altered expression of antigens, including the CD34 and CD38 surface antigens. In normal hematopoiesis, the most immature stem cells have the CD34+CD34 -phenotype. In acute myeloid leukemia (AML), although blasts from most patients are CD38+, some are CD38 -. AML blasts are blocked at early stages of differentiation; in some leukemic cells this block can be overcome by a variety of agents, including retinoids, that induce maturation into macrophages and granulocytes both in vitro and in vivo . Retinoids can also induce CD38 expression. In the present study, we investigated the relationship between induction of CD38 expression and induction of myeloid differentiation by retinoic acid (RA) in normal and leukemic human hematopoietic cells. In the promyelocytic (PML) CD34 -cell lines, HL60 and CB-1, as well as in normal CD34+CD34 -hematopietic progenitor cells RA induced both CD38 expression as well as morphological and functional myeloid differentiation that resulted in loss of self-renewal. In contrast, in the myeloblastic CD34+ leukemic cell lines, ML-1 and KG-1a, as well as in primary cultures of cells derived from CD34+-AML (M 0 and M 1 ) patients, RA caused an increase in CD38+ that was not associated with significant differentiation. Yet, long exposure of ML-1, but not KG-1, cells to RA resulted in loss of self-renewal. The results suggest that while in normal hematopoietic cells and in PML CD34 -cells induction of CD38 antigen expression by RA results in terminal differentiation along the myeloid lineage, in early myeloblastic leukemic CD34+ cells, induction of CD38 and differentiation are not functionally related. Since, several lines of evidence suggest that the CD38 -cells are the targets of leukemic transformation, transition of these cells into CD38+ phenotype by RA or other drugs may have therapeutic effect, either alone or in conjunction with cytotoxic drugs, regardless the ability of the cells to undergo differentiation.     

C-type lectin-like molecule-1: a novel myeloid cell surface marker associated with acute myeloid leukemia

Acute myeloid leukemia (AML) has a poor prognosis due to treatment-resistant relapses. A humanized anti-CD33 antibody (Mylotarg) showed a limited response rate in relapsed AML. To discover novel AML antibody targets, we selected a panel of single chain Fv fragments using phage display technology combined with flow cytometry on AML tumor samples. One selected single chain Fv fragment broadly reacted with AML samples and with myeloid cell lineages within peripheral blood. Expression cloning identified the antigen recognized as C-type lectin-like molecule-1 (CLL-1), a previously undescribed transmembrane glycoprotein. CLL-1 expression was analyzed with a human anti-CLL-1 antibody that was generated from the single chain Fv fragment. CLL-1 is restricted to the hematopoietic lineage, in particular to myeloid cells present in peripheral blood and bone marrow. CLL-1 is absent on uncommitted CD34(+)/CD38(-) or CD34(+)/CD33(-) stem cells and present on subsets of CD34(+)/CD38(+) or CD34(+)/CD33(+) progenitor cells. CLL-1 is not expressed in any other tissue. In contrast, analysis of primary AMLs demonstrated CLL-1 expression in 92% (68 of 74) of the samples. As an AML marker, CLL-1 was able to complement CD33, because 67% (8 of 12) of the CD33(-) AMLs expressed CLL-1. CLL-1 showed variable expression (10-60%) in CD34(+) cells in chronic myelogenous leukemia and myelodysplastic syndrome but was absent in 12 of 13 cases of acute lymphoblastic leukemia. The AML reactivity combined with the restricted expression on normal cells identifies CLL-1 as a novel potential target for AML treatment.     

Gene expression profiles of AML derived stem cells; similarity to hematopoietic stem cells.

Tumors contain a fraction of cancer stem cells that maintain the propagation of the disease. The CD34(+)CD38(-) cells, isolated from acute myeloid leukemia (AML), were shown to be enriched leukemic stem cells (LSC). We isolated the CD34(+)CD38(-) cell fraction from AML and compared their gene expression profiles to the CD34(+)CD38(+) cell fraction, using microarrays. We found 409 genes that were at least twofold over- or underexpressed between the two cell populations. These include underexpression of DNA repair, signal transduction and cell cycle genes, consistent with the relative quiescence of stem cells, and chromosomal aberrations and mutations of leukemic cells. Comparison of the LSC expression data to that of normal hematopoietic stem cells (HSC) revealed that 34% of the modulated genes are shared by both LSC and HSC, supporting the suggestion that the LSC originated within the HSC progenitors. We focused on the Notch pathway since Jagged-2, a Notch ligand was found to be overexpressed in the LSC samples. We show that DAPT, an inhibitor of gamma-secretase, a protease that is involved in Jagged and Notch signaling, inhibits LSC growth in colony formation assays. Identification of additional genes that regulate LSC self-renewal may provide new targets for therapy.     

TWIST-1 promotes cell growth,drug resistance and progenitor clonogenic capacities in myeloid leukemia and is a novel poor prognostic factor in acute myeloid leukemia

Alterations of TWIST-1 expression are often seen in solid tumors and contribute to tumorigenesis and cancer progression. However, studies concerning its pathogenic role in leukemia are scarce. Our study shows that TWIST-1 is overexpressed in bone marrow mononuclear cells of patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Gain-of-function and loss-of-function analyses demonstrate that TWIST-1 promotes cell growth, colony formation and drug resistance of AML and CML cell lines. Furthermore, TWIST-1 is aberrantly highly expressed in CD34⁺CD38⁻ leukemia stem cell candidates and its expression declines with differentiation. Down-modulation of TWIST-1 in myeloid leukemia CD34⁺ cells impairs their colony-forming capacity. Mechanistically, c-MPL, which is highly expressed in myeloid leukemia cells and associated with poor prognosis, is identified as a TWIST-1 coexpressed gene in myeloid leukemia patients and partially contributes to TWIST-1-mediated leukemogenic effects. Moreover, patients with higher TWIST-1 expression have shorter overall and event-free survival (OS and EFS) in AML. Multivariate analysis further demonstrates that TWIST-1 overexpression is a novel independent unfavourable predictor for both OS and EFS in AML. These data highlight TWIST-1 as a new candidate gene contributing to leukemogenesis of myeloid leukemia, and propose possible new avenues for improving risk and treatment stratification in AML.     

Flow cytometric characterization of acute myeloid leukemia: IV. Comparison to the differentiation pathway of normal hematopoietic progenitor cells.

Gradual increase of CD38 on cells expressing CD34 characterizes the early cell differentiation pathway of normal human hematopoietic progenitors. In this study the coordinated expression pattern of CD34 and CD38 was assessed on leukemic blasts from bone marrow aspirates of 95 patients with newly diagnosed acute myeloid leukemia (AML). Expression was divided into six categories analogous to the differentiation pathway of normal bone marrow. The CD38 antigen was expressed on the leukemic cells of all patients and CD34+ leukemic cells were found in 79 patients (83%). In 93 patients, the leukemic cells were found along the differentiation pathway defined by CD34 and CD38. In 33 of the 93 patients, a part of the CD34+ cells did not express the CD38 antigen (categories 1 and 2). In another 33 patients, all CD34+ cells expressed CD38 (categories 3 and 4). In the remaining 27 patients, only cells were found which dimly expressed CD34 or did not express CD34 (categories 5 and 6). Of the 93 patients, 88 were treated with intensive chemotherapy according to the protocol of the German AML Cooperative Group. Of these, 21 died early and were not evaluable for treatment response. Complete remission was achieved in 14 of 22 patients (64%) in categories 1 and 2, in 19 of 26 patients (73%) in categories 3 and 4, and in 18 of 19 patients (95%) in categories 5 and 6. The event-free survival was significantly longer in patients of categories 5 and 6 compared to patients in categories 1 and 2 (p less than 0.01) and categories 3 and 4 (p less than 0.05), respectively. We conclude that in the majority of AML patients the immunophenotype of leukemic cells follows the early cell differentiation pathways defined by coordinated expression of CD34 and CD38 similar to that of normal hematopoietic progenitors. The presence of cells in the late cell differentiation stages (CD34+/-, CD38 /+) identifies patients with a higher complete remission rate and longer complete remission duration.     

High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival.

PURPOSE: In CD34-positive acute myeloid leukemia (AML), the leukemia-initiating event originates from the CD34(+)CD38(-) stem cell compartment. Survival of these cells after chemotherapy may lead to minimal residual disease (MRD) and subsequently to relapse. Therefore, the prognostic impact of stem cell frequency in CD34-positive AML was investigated. EXPERIMENTAL DESIGN: First, the leukemogenic potential of unpurified CD34(+)CD38(-) cells, present among other cells, was investigated in vivo using nonobese diabetic/severe combined immunodeficient mice transplantation experiments. Second, we analyzed whether the CD34(+)CD38(-) compartment at diagnosis correlates with MRD frequency after chemotherapy and clinical outcome in 92 AML patients. RESULTS: In vivo data showed that engraftment of AML blasts in nonobese diabetic/severe combined immunodeficient mice directly correlated with stem cell frequency of the graft. In patients, a high percentage of CD34(+)CD38(-) stem cells at diagnosis significantly correlated with a high MRD frequency, especially after the third course of chemotherapy. Also, it directly correlated with poor survival. In contrast, total CD34(+) percentage showed no such correlations. CONCLUSIONS: Both in vivo data, as well as the correlation studies, show that AML stem cell frequency at diagnosis offers a new prognostic factor. From our data, it is tempting to hypothesize that a large CD34(+)CD38(-) population at diagnosis reflects a higher percentage of chemotherapy-resistant cells that will lead to the outgrowth of MRD, thereby affecting clinical outcome. Ultimately, future therapies should be directed toward malignant stem cells.     

Proliferation and apoptosis in acute and chronic leukemias and myelodysplastic syndrome

Clonal expansion of leukemic cells is thought to be due to proliferation in excess of apoptosis. To define and compare proliferation and apoptosis between various leukemias and myelodysplastic syndrome (MDS), we measured proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) incorporation as surrogate markers for proliferation and caspase 3 activity and annexin V surface binding as surrogate markers for activation of the apoptotic cascade in patients with MDS, chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML). We found high proliferation in bone marrow cells from MDS and CMML as measured by PCNA and BrdU incorporation. The lowest level of proliferation was found in CLL. Apoptosis was also highest in MDS and CMML as measured by annexin V and caspase 3 activity. Unexpectedly, we found no significant difference in proliferation in bone marrow CD34+ cells from various leukemias or MDS. Apoptosis was significantly higher in bone marrow CD34+ cells from MDS and CML in chronic phase as compared to CD34+ cells from AML patients. Our results illustrate differences in proliferation and apoptosis between acute and chronic leukemias and MDS. These differences may have diagnostic and therapeutic implications.     

Transplantation of human acute myeloid leukemia (AML) cells in immunodeficient mice reveals altered cell surface phenotypes and expression of human endothelial markers

To better characterize acute myeloid leukemia (AML) development in non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice, we transplanted samples from patients with AML or KG-1 and EOL-1 cell lines. We found 9/12 primary AML samples and both cell lines to engraft within 2-8 weeks, with 5-80% human cells in bone marrow. Compared with freshly isolated AML cells, percentages of human CD33+, CD38+, CD31+ CD13+ or CD15+ subpopulations increased after transplantation, whereas percentages of CD34+ cells decreased. Engrafted mice frequently showed expression of human endothelial cell markers. Thus, transplantation of human AML into NOD/SCID mice reveals expression of hematopoietic and endothelial differentiation markers.     

急性髓系白血病患者来源白血病干细胞的分选与鉴定

 目的　分选急性髓系白血病患者白血病干细胞并进行鉴定,为白血病干细胞的进一步研究奠定基础。方法　采用Ficoll密度梯度离心法从患者骨髓中分离单个核细胞。采用流式细胞术从单个核细胞中分选CD+34 CD+123白血病干细胞,通过集落形成能力和鹅卵石形成能力检测分选后白血病干细胞的自我更新与分化能力,同时检测CD+34 CD+123 细胞纯度,观察其细胞形态。结果　分选后的CD+34 CD+123 白血病干细胞比例占总分选细胞的10.7 %,具有集落形成能力和鹅卵石形成能力,CD+34 CD+123 细胞纯度为62.1 %。结论　成功分选与鉴定了白血病干细胞,可用于其后的进一步研究工作。     

Notch ligand Delta-1 differentially modulates the effects of gp130 activation on interleukin-6 receptor alpha-positive and -negative human hematopoietic progenitors

Interleukin (IL)-6 plays pleiotropic roles in human hematopoiesis and immune responses by acting on not only the IL-6 receptor-alpha subunit (IL-6Ralpha)(+) but also IL-6Ralpha(-) hematopoietic progenitors via soluble IL-6R. The Notch ligand Delta-1 has been identified as an important modulator of the differentiation and proliferation of human hematopoietic progenitors. Here, it was investigated whether these actions of IL-6 are influenced by Delta-1. When CD34(+)CD38(-) hematopoietic progenitors were cultured with stem cell factor, flt3 ligand, thrombopoietin and IL-3, Delta-1, in combination with the IL-6R/IL-6 fusion protein FP6, increased the generation of glycophorin A(+) erythroid cells but counteracted the effects of IL-6 and FP6 on the generation of CD14(+) monocytic and CD15(+) granulocytic cells. Although freshly isolated CD34(+)CD38(-) cells expressed no or only low levels of IL-6Ralpha, its expression was increased in myeloid progenitors after culture but remained negative in erythroid progenitors. It was found that Delta-1 acted in synergy with FP6 to enhance the generation of erythroid cells from the IL-6Ralpha(-) erythroid progenitors. In contrast, Delta-1 antagonized the effects of IL-6 and FP6 on the development of monocytic and granulocytic cells, as well as CD14(-)CD1a(+) dendritic cells, from the IL-6Ralpha(+) myeloid progenitors. These results indicate that Delta-1 interacts differentially with gp130 activation in IL-6Ralpha(-) erythroid and IL-6Ralpha(+) myeloid progenitors. The present data suggest a divergent interaction between Delta-1 and gp130 activation in human hematopoiesis.     

Residual normal stem cells can be detected in newly diagnosed chronic myeloid leukemia patients by a new flow cytometric approach and predict for optimal response to imatinib

Insensitivity of chronic myeloid leukemia (CML) hematopoietic stem cells to tyrosine kinase inhibitors (TKIs) prevents eradication of the disease and may be involved in clinical resistance. For improved treatment results more knowledge about CML stem cells is needed. We here present a new flow cytometric approach enabling prospective discrimination of CML stem cells from their normal counterparts within single-patient samples. In 24 of 40 newly diagnosed CML patients residual normal CD34(+)CD38(-) stem cells could be identified by lower CD34 and CD45 expression, lower forward/sideward light scatter and by differences of lineage marker expression (CD7, CD11b and CD56) and of CD90. fluorescent in situ hybridization (FISH) analysis on Fluorescence-activated cell sorting sorted cells proved that populations were BCR-ABL positive or negative and long-term liquid culture assays with subsequent colony forming unit assays and FISH analysis proved their stem cell character. Patients with residual non-leukemic stem cells had lower clinical risk scores (Sokal, Euro), lower hematological toxicity of imatinib (IM) and better molecular responses to IM than patients without. This new approach will expand our possibilities to separate CML and normal stem cells, present in a single bone marrow or peripheral blood sample, thereby offering opportunities to better identify new CML stem-cell-specific targets. Moreover, it may guide optimal clinical CML management.     

Resistance of leukemic stem-like cells in AML cell line KG1a to natural killer cell-mediated cytotoxicity

Leukemic stem cells (LSCs) play the central role in the relapse and refractory of acute myeloid leukemia (AML) and highlight the critical need for the new therapeutic strategies to directly target the LSC population. However, relatively little is known about the unique molecular mechanisms of drug and natural killer cells (NK)-killing resistance of LSCs because of very small number of LSCs in bone marrow. In this study, we investigated whether established leukemia cell line contains LSCs. We showed that KG1a leukemia cell line contained leukemic stem-like cells, which have been phenotypically restricted within the CD34(+)CD38(-) fractions. CD34(+)CD38(-) cells could generate CD34(+)CD38(+) cells in culture medium and had renewal function. Moreover, CD34(+)CD38(-) cells had self-renewal potential. We found that leukemic stem-like cells from KG1a cells were resistant to chemotherapy and NK-mediated cytotoxicity. NKG2D ligands involve in protecting LSCs from NK-mediated attack. Taken together, our studies provide a novel cell model for leukemic stem cells research. Our data also shed light on mechanism of double resistant to chemotherapy and NK cell immunotherapy, which was helpful for developing novel effective strategies for LSCs.