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Dasatinib impairs long-term expansion of leukemic progenitors in a subset of acute myeloid leukemia cases.

Han L, Schuringa JJ, Mulder A, Vellenga E - Ann. Hematol. (2010)

Bottom Line: Moreover, the inhibitory effects of dasatinib were cytokine specific.Stem cell factor-mediated proliferation was significantly impaired, associated with a reduced phosphorylation of ERK1/2 and STAT5, whereas no effect was observed on interleukin-3 and thrombopoietin-mediated signaling despite SRC activation.In conclusion, this study demonstrates that dasatinib is a potential inhibitor in a subgroup of AML, especially those that express BCR-ABL or KIT mutations.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, University of Groningen and University Medical Center Groningen, Groningen, the Netherlands.

ABSTRACT
A number of signaling pathways might be frequently disrupted in acute myeloid leukemia (AML). We questioned whether the dual SRC/ABL kinase inhibitor dasatinib can affect AML cells and whether differences can be observed with normal CD34(+) cells. First, we demonstrated that normal cord blood (CB) CD34(+) cells were unaffected by dasatinib at a low concentration (0.5 nM) in the long-term culture on MS5 stromal cells. No changes were observed in proliferation, differentiation, and colony formation. In a subset of AML cases (3/15), a distinct reduction in cell proliferation was observed, ranging from 48% to 91% inhibition at 0.5 nM of dasatinib, in particular, those characterized by BCR-ABL or KIT mutations. Moreover, the inhibitory effects of dasatinib were cytokine specific. Stem cell factor-mediated proliferation was significantly impaired, associated with a reduced phosphorylation of ERK1/2 and STAT5, whereas no effect was observed on interleukin-3 and thrombopoietin-mediated signaling despite SRC activation. In conclusion, this study demonstrates that dasatinib is a potential inhibitor in a subgroup of AML, especially those that express BCR-ABL or KIT mutations.

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Dasatinib impairs proliferation, but not colony formation and differentiation of human CB CD34+ progenitor cells. Cord blood CD34+ cells (3 × 104) were plated in T25 flask precoated with MS5 stromal cells. Cells were expanded in LTC medium with dasatinib added weekly as indicated. Cultures were demi-depopulated weekly for counting and CFC assay. a Representative figure of weekly cumulative cell counts is shown. b Percentages of cell growth as compared to the control group (% growth of control) on stroma by adding dasatinib are shown as mean value from three independent coculture experiments. c The total number of CFCs generated per T25 flask was calculated by the number of CFCs/104 plated cells multiplied by the cell counts weekly. Percentages of colonies as compared to the control group (% colonies of control) after dasatinib treatment are shown. Data were from three independent coculture experiments. d Cells (104) in suspension were plated at weeks 2 and 3 for CFC assay. Percentages of colonies as compared to the control group (% colonies of control) after dasatinib treatment are shown. e One thousand freshly isolated CB CD34+ cells were analyzed for CFC assay. Percentages of colonies as compared to the control group (% colonies of control) after dasatinib treatment are shown. Data were from three independent experiments. f The myeloid differentiation of cells in suspension from the coculture system was monitored by FACS with antibodies against CD11b, CD14, and CD15 at weeks 2 and 4. Representative figures out of three independent experiments are shown. Columns, means of three independent experiments; bars, SE (**p < 0.05)
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Fig1: Dasatinib impairs proliferation, but not colony formation and differentiation of human CB CD34+ progenitor cells. Cord blood CD34+ cells (3 × 104) were plated in T25 flask precoated with MS5 stromal cells. Cells were expanded in LTC medium with dasatinib added weekly as indicated. Cultures were demi-depopulated weekly for counting and CFC assay. a Representative figure of weekly cumulative cell counts is shown. b Percentages of cell growth as compared to the control group (% growth of control) on stroma by adding dasatinib are shown as mean value from three independent coculture experiments. c The total number of CFCs generated per T25 flask was calculated by the number of CFCs/104 plated cells multiplied by the cell counts weekly. Percentages of colonies as compared to the control group (% colonies of control) after dasatinib treatment are shown. Data were from three independent coculture experiments. d Cells (104) in suspension were plated at weeks 2 and 3 for CFC assay. Percentages of colonies as compared to the control group (% colonies of control) after dasatinib treatment are shown. e One thousand freshly isolated CB CD34+ cells were analyzed for CFC assay. Percentages of colonies as compared to the control group (% colonies of control) after dasatinib treatment are shown. Data were from three independent experiments. f The myeloid differentiation of cells in suspension from the coculture system was monitored by FACS with antibodies against CD11b, CD14, and CD15 at weeks 2 and 4. Representative figures out of three independent experiments are shown. Columns, means of three independent experiments; bars, SE (**p < 0.05)

Mentions: In contrast to BCR–ABL, which is specifically expressed in CML, in a subset of acute lymphoblastic leukemia and rarely in AML, the expression of SRC is ubiquitous throughout the normal hematopoietic system, and its activation has been associated with multiple signaling pathways [26, 27]. In order to study the effects of dasatinib treatment on normal stem/progenitor cells, CB CD34+ cells were expanded on MS5 stromal cells in the absence or presence of dasatinib. Cultures were demi-depopulated weekly for cell counting, CFC assays, and fluorescence-activated cell sorter (FACS) analysis on suspension cells. Dasatinib treatment resulted in a dose-dependent growth disadvantage of normal CD34+ progenitor cells (Fig. 1a). The growth was only significantly reduced at a higher concentration (5 nM) of dasatinib, with 77.8 ± 13.1% of control (p = 0.04) at week 2, 61.0 ± 16.5% of control (p = 0.02) at week 3, and 54.0 ± 6.3% of control (p = 0.006) at week 4 (Fig. 1b). The treatment with dasatinib (5 nM) resulted in a reduction in total progenitor (CFC) output after 3 weeks of culture (62.2 ± 10.3% of control, p = 0.01) (Fig. 1c). However, the colonies generated per 105 suspension cells were not affected by dasatinib treatment (Fig. 1d). To study whether similar results could be obtained in short-term CFC assays, we cultured 104 CD34+ cells in methylcellulose culture assay with and without dasatinib. The results demonstrated no significant suppressive effect of dasatinib on colony formation (Fig. 1e). Finally, FACS analysis of the suspension cells at weeks 2 and 4 showed no changes in the myeloid differentiation markers CD11b, CD14, and CD15, demonstrating the reduced proliferation was not associated with an impaired differentiation (Fig. 1f).Fig. 1


Dasatinib impairs long-term expansion of leukemic progenitors in a subset of acute myeloid leukemia cases.

Han L, Schuringa JJ, Mulder A, Vellenga E - Ann. Hematol. (2010)

Dasatinib impairs proliferation, but not colony formation and differentiation of human CB CD34+ progenitor cells. Cord blood CD34+ cells (3 × 104) were plated in T25 flask precoated with MS5 stromal cells. Cells were expanded in LTC medium with dasatinib added weekly as indicated. Cultures were demi-depopulated weekly for counting and CFC assay. a Representative figure of weekly cumulative cell counts is shown. b Percentages of cell growth as compared to the control group (% growth of control) on stroma by adding dasatinib are shown as mean value from three independent coculture experiments. c The total number of CFCs generated per T25 flask was calculated by the number of CFCs/104 plated cells multiplied by the cell counts weekly. Percentages of colonies as compared to the control group (% colonies of control) after dasatinib treatment are shown. Data were from three independent coculture experiments. d Cells (104) in suspension were plated at weeks 2 and 3 for CFC assay. Percentages of colonies as compared to the control group (% colonies of control) after dasatinib treatment are shown. e One thousand freshly isolated CB CD34+ cells were analyzed for CFC assay. Percentages of colonies as compared to the control group (% colonies of control) after dasatinib treatment are shown. Data were from three independent experiments. f The myeloid differentiation of cells in suspension from the coculture system was monitored by FACS with antibodies against CD11b, CD14, and CD15 at weeks 2 and 4. Representative figures out of three independent experiments are shown. Columns, means of three independent experiments; bars, SE (**p < 0.05)
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Related In: Results  -  Collection

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Fig1: Dasatinib impairs proliferation, but not colony formation and differentiation of human CB CD34+ progenitor cells. Cord blood CD34+ cells (3 × 104) were plated in T25 flask precoated with MS5 stromal cells. Cells were expanded in LTC medium with dasatinib added weekly as indicated. Cultures were demi-depopulated weekly for counting and CFC assay. a Representative figure of weekly cumulative cell counts is shown. b Percentages of cell growth as compared to the control group (% growth of control) on stroma by adding dasatinib are shown as mean value from three independent coculture experiments. c The total number of CFCs generated per T25 flask was calculated by the number of CFCs/104 plated cells multiplied by the cell counts weekly. Percentages of colonies as compared to the control group (% colonies of control) after dasatinib treatment are shown. Data were from three independent coculture experiments. d Cells (104) in suspension were plated at weeks 2 and 3 for CFC assay. Percentages of colonies as compared to the control group (% colonies of control) after dasatinib treatment are shown. e One thousand freshly isolated CB CD34+ cells were analyzed for CFC assay. Percentages of colonies as compared to the control group (% colonies of control) after dasatinib treatment are shown. Data were from three independent experiments. f The myeloid differentiation of cells in suspension from the coculture system was monitored by FACS with antibodies against CD11b, CD14, and CD15 at weeks 2 and 4. Representative figures out of three independent experiments are shown. Columns, means of three independent experiments; bars, SE (**p < 0.05)
Mentions: In contrast to BCR–ABL, which is specifically expressed in CML, in a subset of acute lymphoblastic leukemia and rarely in AML, the expression of SRC is ubiquitous throughout the normal hematopoietic system, and its activation has been associated with multiple signaling pathways [26, 27]. In order to study the effects of dasatinib treatment on normal stem/progenitor cells, CB CD34+ cells were expanded on MS5 stromal cells in the absence or presence of dasatinib. Cultures were demi-depopulated weekly for cell counting, CFC assays, and fluorescence-activated cell sorter (FACS) analysis on suspension cells. Dasatinib treatment resulted in a dose-dependent growth disadvantage of normal CD34+ progenitor cells (Fig. 1a). The growth was only significantly reduced at a higher concentration (5 nM) of dasatinib, with 77.8 ± 13.1% of control (p = 0.04) at week 2, 61.0 ± 16.5% of control (p = 0.02) at week 3, and 54.0 ± 6.3% of control (p = 0.006) at week 4 (Fig. 1b). The treatment with dasatinib (5 nM) resulted in a reduction in total progenitor (CFC) output after 3 weeks of culture (62.2 ± 10.3% of control, p = 0.01) (Fig. 1c). However, the colonies generated per 105 suspension cells were not affected by dasatinib treatment (Fig. 1d). To study whether similar results could be obtained in short-term CFC assays, we cultured 104 CD34+ cells in methylcellulose culture assay with and without dasatinib. The results demonstrated no significant suppressive effect of dasatinib on colony formation (Fig. 1e). Finally, FACS analysis of the suspension cells at weeks 2 and 4 showed no changes in the myeloid differentiation markers CD11b, CD14, and CD15, demonstrating the reduced proliferation was not associated with an impaired differentiation (Fig. 1f).Fig. 1

Bottom Line: Moreover, the inhibitory effects of dasatinib were cytokine specific.Stem cell factor-mediated proliferation was significantly impaired, associated with a reduced phosphorylation of ERK1/2 and STAT5, whereas no effect was observed on interleukin-3 and thrombopoietin-mediated signaling despite SRC activation.In conclusion, this study demonstrates that dasatinib is a potential inhibitor in a subgroup of AML, especially those that express BCR-ABL or KIT mutations.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, University of Groningen and University Medical Center Groningen, Groningen, the Netherlands.

ABSTRACT
A number of signaling pathways might be frequently disrupted in acute myeloid leukemia (AML). We questioned whether the dual SRC/ABL kinase inhibitor dasatinib can affect AML cells and whether differences can be observed with normal CD34(+) cells. First, we demonstrated that normal cord blood (CB) CD34(+) cells were unaffected by dasatinib at a low concentration (0.5 nM) in the long-term culture on MS5 stromal cells. No changes were observed in proliferation, differentiation, and colony formation. In a subset of AML cases (3/15), a distinct reduction in cell proliferation was observed, ranging from 48% to 91% inhibition at 0.5 nM of dasatinib, in particular, those characterized by BCR-ABL or KIT mutations. Moreover, the inhibitory effects of dasatinib were cytokine specific. Stem cell factor-mediated proliferation was significantly impaired, associated with a reduced phosphorylation of ERK1/2 and STAT5, whereas no effect was observed on interleukin-3 and thrombopoietin-mediated signaling despite SRC activation. In conclusion, this study demonstrates that dasatinib is a potential inhibitor in a subgroup of AML, especially those that express BCR-ABL or KIT mutations.

Show MeSH
Related in: MedlinePlus