Characterization of CD34+ Hematopoietic Progenitor Cells in JAK2V617F and Calr-mutated Myeloproliferative Neoplasms

Abstract Introduction: The dominance of the JAK2V617F-positive clone at the CD34+ compartment is an important modifier of the disease phenotype in myeloproliferative neoplasms (MPN). Recently, mutations in the calreticulin gene (CALR) have been described in around 40-70% of JAK2V617F and MPL wild-type essential thrombocythemia (ET) and myelofibrosis (MF) patients. However, there is limited information regarding the role of CALR mutant clone in hematopoietic progenitor cells. Objective: To study the mutant allele burden at progenitor level in JAK2V617F-positive and CALR-mutated MPN. Methods: Sixty-five patients with MPN including 36 with polycythemia vera (PV) all JAK2V617F-positive, 13 with ET (7 JAK2V617F-positive and 6 CALR-mutated) and 16 with MF (9 JAK2V617F-positive post-PV MF, 4 CALR-mutated primary MF and 3 CALR-mutated post-ET MF) were included in the study. Granulocytes were isolated from peripheral blood by density gradient, whereas CD34+ cells were purified by immunomagnetic positive selection. Stem cells (CD34+CD38-) and progenitors (CD34+CD38+) populations were further separated by fluorescence-activated cell sorting. JAK2V617F and CALR allele burden was measured by quantitative PCR and PCR followed by fragment analysis, respectively, in stem cells, progenitor cells and granulocytes. The study was approved by the local Ethics Committee and informed consent was obtained according to the Declaration of Helsinki. Results: CALR-mutated ET patients harbored a higher mutant load in CD34+CD38- than JAK2V617F-positive ET patients (30.6 vs 6.3%, p=0.01), whereas no significant differences were observed in CD34+CD38+ and in granulocytes allele burdens. Moreover, CALR-mutated ET patients showed a higher mutational load in CD34+CD38- than JAK2V617F-positive PV (30.6% vs 15.7%, p=0.04) but the mutant load in granulocytes was lower (29.6% vs 63.3%, p<0.001). The mutant allele burden in granulocytes and CD34+ cells was higher in patients with JAK2V617F-positive MF than in those with CALR-mutated MF (CD34+CD38-: 71% vs 47.2% p=0.05, CD34+CD38+: 68.4% vs 40.6% p=0.018, granulocytes: 76.9% vs 53.7% p=0.05). Finally, we could demonstrate that the mutant load was lower in CALR-mutated ET patients than in CALR-mutated MF at progenitor level and in granulocytes (CD34+CD38-: 30.6% vs 47.1% p=0.08, CD34+CD38+: 17.8% vs 40.6% p=0.03, granulocytes: 29.6% vs 53.7% p=0.004). Conclusion: CALR-mutated ET patients have a higher mutant load in CD34+CD38- than JAK2V617F-positive ET and PV patients, whereas the JAK2V617F-positive hematopoietic progenitor cells have more differentiation potential than those CALR-mutated. Moreover, in the MF phase of MPN, the expansion of the mutated clone at the progenitor level is greater in JAK2V617F-positive than in CALR-mutated patients. Disclosures No relevant conflicts of interest to declare.

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Sociology of Normal Stem and Progenitor Cells in CML Niche

Blood ◽

10.1182/blood.v120.21.1234.1234 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1234-1234

Author(s):

Robert S Welner ◽

Giovanni Amabile ◽

Deepak Bararia ◽

Philipp B. Staber ◽

Akos G. Czibere ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mouse Model ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Hematopoietic Progenitor Cells ◽

Quiescent State ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Abstract Abstract 1234 Specialized bone marrow (BM) microenvironment niches are essential for hematopoietic stem and progenitor cell maintenance, and recent publications have focused on the leukemic stem cells interaction and placement within those sites. Surprisingly, little is known about how the integrity of this leukemic niche changes the normal stem and progenitor cells behavior and functionality. To address this issue, we started by studying the kinetics and differentiation of normal hematopoietic stem and progenitor cells in mice with Chronic Myeloid Leukemia (CML). CML accounts for ∼15% of all adult leukemias and is characterized by the BCR-ABL t(9;22) translocation. Therefore, we used a novel SCL-tTA BCR/ABL inducible mouse model of CML-chronic phase to investigate these issues. To this end, BM from leukemic and normal mice were mixed and co-transplanted into hosts. Although normal hematopoiesis was increasingly suppressed during the disease progression, the leukemic microenvironment imposed distinct effects on hematopoietic progenitor cells predisposing them toward the myeloid lineage. Indeed, normal hematopoietic progenitor cells from this leukemic environment demonstrated accelerated proliferation with a lack of lymphoid potential, similar to that of the companion leukemic population. Meanwhile, the leukemic-exposed normal hematopoietic stem cells were kept in a more quiescent state, but remained functional on transplantation with only modest changes in both engraftment and homing. Further analysis of the microenvironment identified several cytokines that were found to be dysregulated in the leukemia and potentially responsible for these bystander responses. We investigated a few of these cytokines and found IL-6 to play a crucial role in the perturbation of normal stem and progenitor cells observed in the leukemic environment. Interestingly, mice treated with anti-IL-6 monoclonal antibody reduced both the myeloid bias and proliferation defects of normal stem and progenitor cells. Results obtained with this mouse model were similarly validated using specimens obtained from CML patients. Co-culture of primary CML patient samples and GFP labeled human CD34+CD38- adult stem cells resulted in selective proliferation of the normal primitive progenitors compared to mixed cultures containing unlabeled normal bone marrow. Proliferation was blocked by adding anti-IL-6 neutralizing antibody to these co-cultures. Therefore, our current study provides definitive support and an underlying crucial mechanism for the hematopoietic perturbation of normal stem and progenitor cells during leukemogenesis. We believe our study to have important implications for cancer prevention and novel therapeutic approach for leukemia patients. We conclude that changes in cytokine levels and in particular those of IL-6 in the CML microenvironment are responsible for altered differentiation and functionality of normal stem cells. Disclosures: No relevant conflicts of interest to declare.

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Neutophil Gelatinase –Associated Lipocalin (LCN2) Affects Primary Melofibrosis Hematopoietic Progenitor Cells As Well As Microenvironmental Cells

Blood ◽

10.1182/blood.v122.21.2839.2839 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2839-2839

Author(s):

Min Lu ◽

Lijuan Xia ◽

Rona Singer Weinberg ◽

Ronald Hoffman

Keyword(s):

Progenitor Cells ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Cxcr4 Expression ◽

Hematopoietic Progenitor Cells ◽

Adherent Cell ◽

Hematopoietic Progenitor ◽

Cd34 Cells ◽

Cell Layers

Abstract Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm (MPN) characterized by preferential proliferation of malignant hematopoietic progenitor cells which leads to excessive proliferation of marrow microenvironmental cells which are not involved by the malignant process. These events result in a clinical disorder characterized by anemia, a leukoerythroblastic blood picture, constitutive mobilization of CD34+ cells, extramedullary hematopoiesis, dacrocytosis, marrow megakaryocytic hyperplasia, progressive splenomegaly and reticulin and collagen marrow fibrosis. Several cytokines elaborated by PMF hematopoietic cells including TGF-b, vascular endothelial growth factor and tumor necrosis factor a have been implicated as playing a role in creation of the MF clinical phenotype. Neutrophil gelatinase associated Lipocalin-2 (LCN2) has been reported to have two distinct roles in the pathobiology of chronic myeloid leukemia (CML), suppressing residual normal HPC development and promoting CML proliferation (Devireddy LR et al, 2005, Cell). We, therefore hypothesized that LCN2 might also play a role in the development of the phenotypic features of PMF. Plasma LCN2 levels were measured by ELISA in 77 patients with PMF and were shown to be elevated as compared to 16 normal plasmas (P<0.001) Media conditioned by PMF MNC contained higher levels of LCN2 than media conditioned by normal MNC (p=0.03). The LCN2 receptor level was flow cytometrically analyzed and a significantly greater percentage of normal MNC and CD34+ cells than PMF MNC and CD34+ cells expressed the LCN2 receptor. The effect of increasing concentrations of recombinant LCN2 on CFU-GM and BFU-E derived colony by normal BM CD34+ cells as well as the PMF CD34+ cells was evaluated. The addition of LCN2 inhibited CFU-GM and BFU-E derived colony formation by normal CD34+ cells at a dose of 100 ng/ml (p=0.048 and p=0.043, respectively), while a similar dose of LCN2 did not influence the number of colonies cloned from PMF CD34+ cells. Previously our laboratory has reported that the expression of the chemokine receptor CXCR4 was decreased in PMF CD34+ cells which we hypothesized contributed to abnormal trafficking of CD34+ cells. LCN2 has been reported to affect CXCR4 expression by marrow CD34+ cells (Costa D, et al. 2010, Cytokine). CXCR4 expression by PMF CD34+ cells was lower than that of normal BM CD34+ cells. Incubation with LCN2, however, further reduced the expression of CXCR4 of PMF CD34+ cells by 10 to 50 % (p=0.012). By contrast, LCN2 increased CXCR4+ expression by normal CD34+ cells. We next evaluated the effects of LCN2 on the BM microenvironment. Normal BM mononuclear cells were plated in dishes exposed to the vary doses of LCN2 (10, 50, 100, 200 ng/ml) and the formation of adherent cell layers was monitored. Low doses of LCN2 (10 and 50 ng/ml) promoted the formation and proliferation of adherent cell layers composed of fibroblast-like cells after 1-3 weeks of culture. The fibroblast-like cells expressed vimentin and von Willebrand factor, indicating that they resembled mesenchymal stem cells, fibroblast and endothelial cells. We then examined the formation of adherent cell layers by normal BM MNC co-cultured with PMF, PV or normal MNCs separated by a 0.4 um trans-well for three weeks. The proliferation of confluent fibroblast-like cells was observed solely in BM MNC co-cultured with PMF MNC. These data indicate that LCN2 is generated in increased amounts by PMF mononuclear cells and likely plays a role in PMF biology by promoting malignant hematopoiesis but suppressing normal hemaopoiesis, suppressing CXCR4 expression by PMF CD34+ cells and promoting marrow fibroblast proliferation. Disclosures: No relevant conflicts of interest to declare.

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3,2′-Dihydroxyflavone Improves the Proliferation and Survival of Human Pluripotent Stem Cells and Their Differentiation into Hematopoietic Progenitor Cells

Journal of Clinical Medicine ◽

10.3390/jcm9030669 ◽

2020 ◽

Vol 9 (3) ◽

pp. 669 ◽

Cited By ~ 1

Author(s):

Kyeongseok Kim ◽

Ahmed Abdal Dayem ◽

Minchan Gil ◽

Gwang-Mo Yang ◽

Soo Bin Lee ◽

...

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Pluripotent Stem Cells ◽

Mononuclear Cells ◽

Nk Cell ◽

Human Pluripotent Stem Cells ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Differentiation Potential ◽

Proliferation And Differentiation

Efficient maintenance of the undifferentiated status of human pluripotent stem cells (hiPSCs) is crucial for producing cells with improved proliferation, survival and differentiation, which can be successfully used for stem cell research and therapy. Here, we generated iPSCs from healthy donor peripheral blood mononuclear cells (PBMCs) and analyzed the proliferation and differentiation capacities of the generated iPSCs using single cell NGS-based 24-chromosome aneuploidy screening and RNA sequencing. In addition, we screened various natural compounds for molecules that could enhance the proliferation and differentiation potential of hiPSCs. Among the tested compounds, 3,2′-dihydroxyflavone (3,2′-DHF) significantly increased cell proliferation and expression of naïve stemness markers and decreased the dissociation-induced apoptosis of hiPSCs. Of note, 3,2′-DHF-treated hiPSCs showed upregulation of intracellular glutathione (GSH) and an increase in the percentage of GSH-high cells in an analysis with a FreSHtracer system. Interestingly, culture of the 3,2′-DHF-treated hiPSCs in differentiation media enhanced their mesodermal differentiation and differentiation into CD34+ CD45+ hematopoietic progenitor cells (HPC) and natural killer cells (NK) cells. Taken together, our results demonstrate that the natural compound 3,2′-DHF can improve the proliferation and differentiation capacities of hiPSCs and increase the efficiency of HPC and NK cell production from hiPSCs.

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Treatment with Pegylated Interferon Alpha 2a in Combination with the Bcl-Xl Inhibitor ABT-737 Specifically Targets JAK2V617F Positive Hematopoietic Progenitor Cells From Patients with Polycythemia Vera.

Blood ◽

10.1182/blood.v114.22.3916.3916 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3916-3916 ◽

Cited By ~ 1

Author(s):

Min Lu ◽

Wei Zhang ◽

Daniel Yoo ◽

Dmitriy Berenzon ◽

Yan Li ◽

...

Keyword(s):

Polycythemia Vera ◽

Progenitor Cells ◽

Interferon Alpha ◽

Colony Formation ◽

Conflicts Of Interest ◽

Philadelphia Chromosome ◽

Hematopoietic Progenitor Cells ◽

Low Doses ◽

Hematopoietic Progenitor ◽

Cd34 Cells

Abstract Abstract 3916 Poster Board III-852 Polycythemia vera (PV) is a Philadelphia chromosome negative chronic myeloproliferative neoplasm (MPN) which is characterized by acquisition of a mutation in JAK2 (JAK2V617F). The administration of a pegylated form of interferon-alpha-2a (Peg IFNa-2a) to patients with PV has recently been reported to lead to hematological remissions and a reduction of the JAK2V617F allele burden in most patients receiving this modality of therapy. The mechanism underlying this profound clinical response of PV patients to Peg IFNa-2a has been the subject of a great deal of speculation. In order to evaluate the mechanism by which Peg IFNa-2a affects hematopoiesis in PV patients, CD34+ cells isolated from cord blood and the peripheral blood of patients with PV were cultured in semisolid media in the presence and absence of 200 and 500 U of Peg IFNa-2a. These relatively low doses of Peg IFNa-2a did not alter hematopoietic colony formation by CB CD34+ cells but inhibited PV CFU-GM colony formation by 35% and 50%, and BFU-E colony formation by 60% and 80%, respectively. Furthermore, the hematopietic colonies that formed in the presence of Peg IFNa-2a were composed of far fewer cells than those cultured in the presence of cytokines alone. In addition, individual hematopoietic colonies were plucked and the JAK2 genotype was assessed by nested allele-specific PCR assay. Exposure of PV CD34+ cells to Peg-IFNa-2a (500U) resulted in a reduction in the proportion of JAK2V617F-positive hematopoietic progenitor cells from 81.7±16.3% to 50.3±27.6% (p=0.004). Samples from 81.9% of the PV patients (9 of 11 samples) responded in this fashions to Peg IFNa 2a treatment. We then showed that incubation of PV CD34+ cells but not CB CD 34+ cells with 200 and 500U of Peg IFNa-2a resulted in increased rates of apoptosis by 4.3% and 15.3%, respectively. Erythroblasts and megakayoctes from patients with PV have been previously shown to be characterized by over-expression of the anti-apoptotic proteins Bcl-xL. We then examined if the effects of IFNa-2a could be enhanced by addition of the Bcl-xL inhibitor-ABT-737. After 2 days of treatment, Peg IFNa 2a plus ABT-737 induced significantly greater degree of apoptosis (∼50%) of a JAK2V617F positive erythroleukemia cell line (HEL cells) as compared to treatment with each agent alone, (Peg-IFNa-2a, <5%; ABT-737, 20%). PV CD34+ cells were incubated with Peg IFNa 2a (500 U) alone, ABT-737 (0.25 uM) alone or ABT-737 plus Peg IFNa 2a for 4 days and the numbers of cells were decreased by 35%, 40% and 65 %, respectively; and the corresponding percentage of apoptotic cells was 20%, 15% and 60%, respectively. Western blot analysis showed that the Bcl-xL protein level in PV but not CB mononuclear cells was reduced by treatments with ABT-737 alone or in combination with Peg IFNa 2a. Furthermore, treatment of PV CD34+ cells with ABT-737 plus Peg IFNa 2a (200U) lead to the appearance of a smaller proportion of JAK2 V617F-positive (46.7±26%) hematopoietic progenitor cells as compared to cells incubated with cytokines alone (81.7±17%) or cytokines plus Peg IFNa 2a (69±20%). These data suggest that low doses of Peg IFNa 2a selectively and directly eliminate Jak2V617F hematopietic progenitor cells which likely accounts for the therapeutic responses that have been observed with the use of this agent in the clinic. The enhanced elimination of JAK2V617F hematopoietic progenitor cells observed with the combination of ABT-737 and Peg-IFNa-2a suggests that this strategy might be an even more optimal approach for the treatment of JAK2V617F positive MPN which merits further testing in the clinic. Disclosures: No relevant conflicts of interest to declare.

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Microarray and Proteomic Analysis of Myeloproliferative Neoplasms,

Blood ◽

10.1182/blood.v118.21.3856.3856 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3856-3856

Author(s):

Vladan P Cokic ◽

Pascal Mossuz ◽

Jing Han ◽

Milos Diklic ◽

Mirela Budec ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Myeloproliferative Neoplasms ◽

Response To Therapy ◽

Hematopoietic Progenitor Cells ◽

Healthy Individuals ◽

Hematopoietic Progenitor ◽

Jak2 Mutation ◽

Allele Burden ◽

Protein Levels

Abstract Abstract 3856 The gene and protein expression profiles in myeloproliferative neoplasms (MPN) may reveal gene markers of a potential clinical function in diagnosis and prediction of response to therapy. Using cDNA microarray analysis, involving 25,100 unique genes, we studied the gene expression profile of hematopoietic CD34+ progenitor cells and granulocytes obtained from peripheral blood of patients with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) compared with healthy individuals. The microarray analyses of the hematopoietic progenitor cells and granulocytes have been performed on 9 patients with ET, 8 patients with PV, 4 patients with PMF and 8 healthy donors. The granulocytes for proteomic studies have been pooled in 4 groups: PV with JAK2 mutant allele burden above 80%, ET with JAK2 mutation, PMF with JAK2 mutation and ET/PMF with no JAK2 mutation. We focused our analysis to hematopoiesis related genes. In the collected patient samples, the increased number of granulocytes allowed for further validation by protein analysis of microarray gene expression suggested from less differentiated hematopoietic progenitor cells. Folate receptor 3 (FOLR3), constitutively secreted in hematopoietic tissues, has increased protein levels in granulocytes of JAK2V617F homozygous PV as well as mRNA levels in hematopoietic progenitor cells of patients with PV. The enzyme matrix metallopeptidase 9 (MMP9), involved in IL-8-induced mobilization of hematopoietic progenitor cells from bone marrow, also has significantly increased protein levels in granulocytes of PV patients with increased JAK2 mutation allele burden. In addition, Ras-related C3 botulinum toxin substrate 2 (RAC2) protein level, essential for erythropoiesis, is increased specifically in PV granulocytes with JAK2V617F homozygosity. Moreover, RAC2 gene expression is significantly increased in hematopoietic progenitor cells of PV, with no changes in its granulocytes. Although, like PV, RAC2 gene expression was also increased in ET and PMF hematopoietic progenitor cells compared to healthy individuals, in granulocytes of ET and PMF patients with JAK2 mutation RAC2 protein levels were decreased, contrary to the elevated level in PV. Furthermore, inconsistent with JAK2V617F homozygous PV patients, granulocytes of ET and PMF with the JAK2 mutation exhibit FOLR3 protein at levels lower than the ET and PMF with no JAK2 mutation. Investigating the extent to which these genes participate in the complex molecular and cellular mechanisms of MPN will likely lead to new insights of malignancy development. In conclusion, molecular profiling of hematopoietic progenitor cells and granulocytes of MPN patients revealed gene expression patterns that are beyond their recognized function in disease pathogenesis and can be related to patients' clinical characteristics with imminent prognostic relevance. Disclosures: No relevant conflicts of interest to declare.

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Fbxw11 Variants Control the Quiescence of HSCs

Blood ◽

10.1182/blood.v128.22.5052.5052 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5052-5052

Author(s):

Lina Wang ◽

Wenli Feng ◽

Xiao Yang ◽

Feifei Yang ◽

Rong Wang ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Hematopoietic Progenitor Cells ◽

Control Vector ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

The Impact

Abstract Ubiquitination is a posttranslational mechanism that controls diverse cellular processes. Fbxw11, a constituent of the SCF (Skp1-Cul1-F-box) ubiquitin ligase complex, targets for degradation of several important transcription factors, including NF-κB, β-cantenin and ATF4. Fbxw11 may play pivotal roles in many aspects of hematopoiesis through regulating various signal transduction pathways. However, the role of Fbxw11 on HSCs quiescence remains largely unknown. In this study, we cloned three transcript variants (Fbxw11a, Fbxw11c and Fbxw11d) to study the biological function of Fbxw11 in hematopoiesis. To elucidate Fbxw11 distribution in hematopoietic system, hematopoietic cell populations at different stages of differentiation were isolated from bone marrow of 8 week-old mice and Fbxw11 expression was studied by real-time PCR. Expression of Fbxw11 were lower in purified long term hematopoietic stem cells (LT-HSC, LSK CD34- Flt3-), but higher in short term hematopoietic stem cells (ST-HSC, LSK CD34+ Flt3-), hematopoietic stem and progenitor cells (LSK), and various hematopoietic progenitor cells. The results reveal that Fbxw11 is preferentially expressed in more mature progenitor cells. The expression of Fbxw11 in mature blood cells was also studied showing that Fbxw11 was expressed at lower level in neutrophils, higher level in B and T lymphocytes, and moderate level in monocytes. To assess the impact of Fbxw11 on reconstitution capacity of LT-HSCs, we cloned Fbxw11a, Fbxw11c and Fbxw11d into retrovirus system, respectively. LSK cells were infected with MSCV-Fbxw11a/Fbxw11c/Fbxw11d-IRES-GFP or the blank control vector MSCV-GFP. Competitive repopulation assays we performed 48h later after infection, and reconstitution in peripheral blood (PB) was analyzed every 4 weeks. Repopulation of donor cells expressing high level of Fbxw11 variants was significantly lower than those infected with control vector at 1 and 4 months in PB and at 4 months in BM after transplantation. These data indicate that Fbxw11 is negative for the long-term repopulating capacity of HSCs. To further confirm the effects of Fbxw11 variants in hematopoiesis, the effect of Fbxw11 variants on the growth and enumeration of hematopoietic progenitor cells was detected by colony-forming cell assay (CFC). The number of CFU-G, CFU-GM, CFU-GEMM and the total number of CFU were lower in LSK over-expressing Fbxw11 variants when compared with LSK control. To determine the cell-cycle distribution of HSC cells, Hoechst 33342 and Ki67 staining were performed showing that G0 phase LSK cells were decreased when they over-expressing Fbxw11 variants. In conclusion, our data reveal unrecognized roles for Fbxw11 in the regulation of HSPCs. Our findings suggest that Fbxw11 variants have negative effect on reconstitution capacity of LT-HSCs. Fbxw11 variants decrease the reconstitution capacity through promoting cell proliferation, which results in loss of hematopoietic stem cell quiescence. We anticipate that our experiments will facilitate the understanding of hematopoiesis through which Fbxw11-mediated signals control HSC quiescence and functions. The work was supported by the Grants 81300376, 81370634, 81570153 from the National Natural Science Foundation of China (NSFC); 14JCQNJC10600 from the Tianjin Science and Technology Programs; Disclosures No relevant conflicts of interest to declare.

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The Tyrosine Kinase Inhibitor TK258 Differentially Targets Proliferating Human Hematopoietic Progenitor Cells but Does Not Eliminate the Quiescent Fraction.

Blood ◽

10.1182/blood.v110.11.3356.3356 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3356-3356

Author(s):

Caroline Alvares ◽

Ian Titley ◽

Mel Greaves ◽

Michael Potter ◽

Lyndal Kearney ◽

...

Keyword(s):

Stem Cells ◽

Tyrosine Kinase ◽

Progenitor Cells ◽

Kinase Inhibitor ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Quiescent Cells ◽

Cd34 Cells ◽

Normal Human

Abstract NOD-SCID mouse models have shown that, as with normal hematopoietic stem cells, leukemic stem cells (LSC) exist in a quiescent state and are thus capable of sustaining disease in vivo. Targeted therapy of LSC may therefore have a significant impact on disease eradication, relapse rates and toxicity reduction. Inhibition of FLT3 and c-kit is a current therapeutic strategy for AML but the efficacy of this approach on quiescent cells is unknown. Furthermore, although both FLT3 and c-kit play crucial roles in the development of normal human hematopoietic progenitor (HPC) and stem cells, the hematopoietic consequences of FLT3 and c-kit inhibition remain undefined. Here we show that the FLT3, c-kit and PDGF directed tyrosine kinase inhibitor TK258 specifically targets slowly dividing human HPCs, but not rapidly proliferating HPCs or quiescent cells displaying an immature phenotype. For normal CD34+ cells, treatment of short-term cultures with TK258 resulted in a dose dependent inhibition of cell expansion. Moreover, treatment of normal CD34+ cells with TK258 within the clinical dose range [0.2μM to 1.5μM] inhibited myeloid colony growth but had no effect on erythroid colony output, suggesting a differential effect on lineage-committed progenitors. To further elucidate the effect of TK258 on dividing and also quiescent CD34+ cells, normal CD34+ cells were labelled with a non-toxic fluorescent cell membrane dye, PKH26, prior to TK258 treatment in vitro. Coupled with flow cytometry, this dye enabled the discrimination of progenitor cells with different proliferative capacities. Thus, using this technique, we defined a CD34+/CD133+ quiescent population of cells that retained the same PKH fluorescence intensity as from day 0 of culture. TK258 treatment in culture generated a significant increase in the CD34+/CD133+ quiescent fraction (15.8% treated versus 5.9% untreated of total viable cells). In contrast with untreated cells this fraction maintained c-kit expression, suggesting maintenance of an immature population by TK258. Total viable cell recoveries were higher in the TK258 treated quiescent fraction (24% treated versus 13.5% untreated, p=0.037). Rapidly dividing progenitors were unaffected by TK258, while progenitors with a slower division rate showed significant sensitivity to the compound. The anti-proliferative effect of TK258 was mediated via G0/G1 cell cycle arrest, but not by apoptosis of CD34+ cells. We assessed the functional read-out of TK258 pre-treated progenitors in CFU assays, by sorting cells on the basis of their PKH26 fluorescence. These assays demonstrated a higher total colony output and diverse colony type for TK258 pre-treated ‘quiescent’ cells compared to untreated controls, in keeping with the preservation of a more immature population by TK258. Rapidly proliferating populations displayed a lower CFU potential, attributable to a reduction in myeloid output, which was not significantly altered by TK258 pre-treatment. CAFC assays and gene expression profiling of PKH26-sorted subsets are in progress. Taken together, these data demonstrate that TK258 has significant inhibitory effects on normal human hematopoietic progenitor cells in vitro and that the sensitivity of progenitor cells to the compound can be predicted from their proliferative index. Importantly, immature hematopoietic quiescent cells are preserved, which may have implications for the activity of tyrosine kinase inhibitors on non-cycling human leukemia cells.

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