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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 expansion of a subset of AML CD34+ cells in long-term cultures. Acute myeloid leukemia CD34+ cells (4 × 104) were sorted and plated in 12-well plates precoated with MS5 stromal cells. Cells were expanded in LTC medium supplemented with 20 ng/ml IL-3, G-CSF, and TPO. Dasatinib was added as indicated concentrations. Cultures were demi-depopulated weekly for analysis. The responses to dasatinib at 0.5 nM (n = 15) (a) and 5 nM (n = 14) (b) of all AMLs capable of long-term proliferation on stroma are shown, as compared to the growth of control group (% growth of control). The time points for cell counts indicated were at week 4/5. Cell counts indicated suspension and adherent hematopoietic cells that were separated by sorting CD45+ (human) cells. c–e Growth curves of three AMLs with growth reduction by dasatinib treatment are shown. Weekly cumulative cell counts represented cells in suspension except at time point of replating, where cell counts reflected suspension and adherent hematopoietic cells. The leukemic cells both in suspension and adherent layer were harvested from the coculture at weeks 4 and 7 to initiate the second and third cocultures on new MS5 stroma (AML no. 1)
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Fig2: Dasatinib impairs expansion of a subset of AML CD34+ cells in long-term cultures. Acute myeloid leukemia CD34+ cells (4 × 104) were sorted and plated in 12-well plates precoated with MS5 stromal cells. Cells were expanded in LTC medium supplemented with 20 ng/ml IL-3, G-CSF, and TPO. Dasatinib was added as indicated concentrations. Cultures were demi-depopulated weekly for analysis. The responses to dasatinib at 0.5 nM (n = 15) (a) and 5 nM (n = 14) (b) of all AMLs capable of long-term proliferation on stroma are shown, as compared to the growth of control group (% growth of control). The time points for cell counts indicated were at week 4/5. Cell counts indicated suspension and adherent hematopoietic cells that were separated by sorting CD45+ (human) cells. c–e Growth curves of three AMLs with growth reduction by dasatinib treatment are shown. Weekly cumulative cell counts represented cells in suspension except at time point of replating, where cell counts reflected suspension and adherent hematopoietic cells. The leukemic cells both in suspension and adherent layer were harvested from the coculture at weeks 4 and 7 to initiate the second and third cocultures on new MS5 stroma (AML no. 1)

Mentions: It has been shown previously that the propagation of AML cells partially depends on constitutively activation of receptor kinases including FLT3 and KIT, and the autocrine and paracrine production of growth factors that make use of nonreceptor protein TKs [28]. Therefore, AML cells (n = 19) were studied in long-term stromal culture assays by using exclusively the sorted CD34+ cell fraction that is enriched for leukemic stem cells, as has been described [17, 18]. The clinical characteristics of the studied patients, including FAB classification, cytogenetics, and defined mutations, are summarized in Table 1. In 79% (15/19) of the tested AML cases, long-term expanding cocultures could be generated (Fig. 2a, b). Variability in responsiveness of the different AMLs for dasatinib was observed. In 20% of the cases (3/15), a distinct decrease in long-term cell expansion of AML CD34+ cells was already observed at a dose of 0.5 nM dasatinib, ranging from 48% to 91% inhibition as compared to the untreated group. This concentration of dasatinib showed less than 15% growth inhibition in normal CD34+ cells on stroma (Fig. 1a, b). The growth curves of the three AML cases are shown in Fig. 2c–e. To demonstrate whether dasatinib also inhibited the self-renewal potential of the AML CD34+ cells, we performed replating experiments by harvesting the cells from L-CAs after 3 to 5 weeks of coculture. These cells were subsequently studied for their capacity to initiate second (and third) cocultures on new MS5 stroma (Fig. 2c). The results demonstrated that AML leukemic cells still expanded on new MS5 stroma, and that the inhibitory effect of dasatinib was less pronounced after replating. The AML cells that responded to dasatinib were characterized by chromosomal translocations or mutations in BCR–ABL (n = 1), KIT (n = 1), or undetermined (n = 1) mutations. In six FLT3–ITD-positive AMLs, no suppressive effects of dasatinib on cell expansion were noticed. Moreover, it appeared that the responsiveness to dasatinib was independent of the level of SRC mRNA expression (data not shown). A remarkable finding was the fact that in the additional AMLs (11/15), a stimulatory effect on cell proliferation was observed at low-dose (0.5 nM) dasatinib when the AML CD34+ cells were cultured in long-term stromal assay (Fig. 2a). An increase of 474% (range, 17–2417%) was observed in time. This effect of dasatinib was noticed after a period of 2 to 3 weeks of culture. At a higher concentration (5 nM), this stimulatory effect disappeared in most of the cases (Fig. 2b). Taken together, dasatinib shows pronounced inhibitory effects on proliferation in a subset of AML CD34+ progenitor cells, whereas in the additional cases, a stimulatory effect might be shown at low dose.Table 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 expansion of a subset of AML CD34+ cells in long-term cultures. Acute myeloid leukemia CD34+ cells (4 × 104) were sorted and plated in 12-well plates precoated with MS5 stromal cells. Cells were expanded in LTC medium supplemented with 20 ng/ml IL-3, G-CSF, and TPO. Dasatinib was added as indicated concentrations. Cultures were demi-depopulated weekly for analysis. The responses to dasatinib at 0.5 nM (n = 15) (a) and 5 nM (n = 14) (b) of all AMLs capable of long-term proliferation on stroma are shown, as compared to the growth of control group (% growth of control). The time points for cell counts indicated were at week 4/5. Cell counts indicated suspension and adherent hematopoietic cells that were separated by sorting CD45+ (human) cells. c–e Growth curves of three AMLs with growth reduction by dasatinib treatment are shown. Weekly cumulative cell counts represented cells in suspension except at time point of replating, where cell counts reflected suspension and adherent hematopoietic cells. The leukemic cells both in suspension and adherent layer were harvested from the coculture at weeks 4 and 7 to initiate the second and third cocultures on new MS5 stroma (AML no. 1)
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Related In: Results  -  Collection

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Fig2: Dasatinib impairs expansion of a subset of AML CD34+ cells in long-term cultures. Acute myeloid leukemia CD34+ cells (4 × 104) were sorted and plated in 12-well plates precoated with MS5 stromal cells. Cells were expanded in LTC medium supplemented with 20 ng/ml IL-3, G-CSF, and TPO. Dasatinib was added as indicated concentrations. Cultures were demi-depopulated weekly for analysis. The responses to dasatinib at 0.5 nM (n = 15) (a) and 5 nM (n = 14) (b) of all AMLs capable of long-term proliferation on stroma are shown, as compared to the growth of control group (% growth of control). The time points for cell counts indicated were at week 4/5. Cell counts indicated suspension and adherent hematopoietic cells that were separated by sorting CD45+ (human) cells. c–e Growth curves of three AMLs with growth reduction by dasatinib treatment are shown. Weekly cumulative cell counts represented cells in suspension except at time point of replating, where cell counts reflected suspension and adherent hematopoietic cells. The leukemic cells both in suspension and adherent layer were harvested from the coculture at weeks 4 and 7 to initiate the second and third cocultures on new MS5 stroma (AML no. 1)
Mentions: It has been shown previously that the propagation of AML cells partially depends on constitutively activation of receptor kinases including FLT3 and KIT, and the autocrine and paracrine production of growth factors that make use of nonreceptor protein TKs [28]. Therefore, AML cells (n = 19) were studied in long-term stromal culture assays by using exclusively the sorted CD34+ cell fraction that is enriched for leukemic stem cells, as has been described [17, 18]. The clinical characteristics of the studied patients, including FAB classification, cytogenetics, and defined mutations, are summarized in Table 1. In 79% (15/19) of the tested AML cases, long-term expanding cocultures could be generated (Fig. 2a, b). Variability in responsiveness of the different AMLs for dasatinib was observed. In 20% of the cases (3/15), a distinct decrease in long-term cell expansion of AML CD34+ cells was already observed at a dose of 0.5 nM dasatinib, ranging from 48% to 91% inhibition as compared to the untreated group. This concentration of dasatinib showed less than 15% growth inhibition in normal CD34+ cells on stroma (Fig. 1a, b). The growth curves of the three AML cases are shown in Fig. 2c–e. To demonstrate whether dasatinib also inhibited the self-renewal potential of the AML CD34+ cells, we performed replating experiments by harvesting the cells from L-CAs after 3 to 5 weeks of coculture. These cells were subsequently studied for their capacity to initiate second (and third) cocultures on new MS5 stroma (Fig. 2c). The results demonstrated that AML leukemic cells still expanded on new MS5 stroma, and that the inhibitory effect of dasatinib was less pronounced after replating. The AML cells that responded to dasatinib were characterized by chromosomal translocations or mutations in BCR–ABL (n = 1), KIT (n = 1), or undetermined (n = 1) mutations. In six FLT3–ITD-positive AMLs, no suppressive effects of dasatinib on cell expansion were noticed. Moreover, it appeared that the responsiveness to dasatinib was independent of the level of SRC mRNA expression (data not shown). A remarkable finding was the fact that in the additional AMLs (11/15), a stimulatory effect on cell proliferation was observed at low-dose (0.5 nM) dasatinib when the AML CD34+ cells were cultured in long-term stromal assay (Fig. 2a). An increase of 474% (range, 17–2417%) was observed in time. This effect of dasatinib was noticed after a period of 2 to 3 weeks of culture. At a higher concentration (5 nM), this stimulatory effect disappeared in most of the cases (Fig. 2b). Taken together, dasatinib shows pronounced inhibitory effects on proliferation in a subset of AML CD34+ progenitor cells, whereas in the additional cases, a stimulatory effect might be shown at low dose.Table 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