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Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia.

Lee-Sherick AB, Zhang W, Menachof KK, Hill AA, Rinella S, Kirkpatrick G, Page LS, Stashko MA, Jordan CT, Wei Q, Liu J, Zhang D, DeRyckere D, Wang X, Frye S, Earp HS, Graham DK - Oncotarget (2015)

Bottom Line: Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle.These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition.In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML.

View Article: PubMed Central - PubMed

Affiliation: University of Colorado, Department of Pediatrics, Aurora, CO, USA.

ABSTRACT
Mer and Flt3 receptor tyrosine kinases have been implicated as therapeutic targets in acute myeloid leukemia (AML). In this manuscript we describe UNC1666, a novel ATP-competitive small molecule tyrosine kinase inhibitor, which potently diminishes Mer and Flt3 phosphorylation in AML. Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle. These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition. Treatment of primary AML patient samples expressing Mer and/or Flt3-ITD with UNC1666 also inhibited Mer and Flt3 intracellular signaling, induced apoptosis, and inhibited colony formation. In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML.

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UNC1666 induces apoptosis and decreases colony formation in AML patient samples(A) Graphic representation of apoptosis and cell death in AML patient samples after treatment with UNC1666 or vehicle for 72 hours. Apoptotic and dead cells were determined by flow cytometry after staining with YO-PRO-1 iodide and propidium iodide. Values derived from each sample are shown. (B) Colony-forming assays were performed in methylcellulose with the indicated treatments. Graphic representation of reduced colony number after treatment with UNC1666, compared to vehicle. Mean values and standard errors derived from triplicate samples are shown. (C) Graphic representation of the effect of UNC1666 on normal cord blood colony forming potential. Mean values and standard errors were derived from 3 independent experiments. **p < 0.01, NS = not significant.
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Figure 7: UNC1666 induces apoptosis and decreases colony formation in AML patient samples(A) Graphic representation of apoptosis and cell death in AML patient samples after treatment with UNC1666 or vehicle for 72 hours. Apoptotic and dead cells were determined by flow cytometry after staining with YO-PRO-1 iodide and propidium iodide. Values derived from each sample are shown. (B) Colony-forming assays were performed in methylcellulose with the indicated treatments. Graphic representation of reduced colony number after treatment with UNC1666, compared to vehicle. Mean values and standard errors derived from triplicate samples are shown. (C) Graphic representation of the effect of UNC1666 on normal cord blood colony forming potential. Mean values and standard errors were derived from 3 independent experiments. **p < 0.01, NS = not significant.

Mentions: To replicate the conditions utilized with AML cell lines, patient samples were treated with UNC1666 or vehicle and apoptosis was determined using flow cytometry as described above. Patient samples were co-cultured with HS27 stromal cells, which provide essential factors to support myeloid cells and allowed us to assess response to UNC1666. Under the stromal cell enhanced growth conditions, treatment with UNC1666 was sufficient to induce apoptosis in all patient samples, thereby demonstrating efficacy on AML blasts with a wide range of Mer expression levels or Flt3-ITD status (Figure 7A & Supplemental Figure 4B). Induction of apoptosis in response to UNC1666 was independent of position in the cell cycle, though at high doses may correlate with accumulation in G2/M phase as previously noted in AML cell lines (Supplemental Figure 4C).


Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia.

Lee-Sherick AB, Zhang W, Menachof KK, Hill AA, Rinella S, Kirkpatrick G, Page LS, Stashko MA, Jordan CT, Wei Q, Liu J, Zhang D, DeRyckere D, Wang X, Frye S, Earp HS, Graham DK - Oncotarget (2015)

UNC1666 induces apoptosis and decreases colony formation in AML patient samples(A) Graphic representation of apoptosis and cell death in AML patient samples after treatment with UNC1666 or vehicle for 72 hours. Apoptotic and dead cells were determined by flow cytometry after staining with YO-PRO-1 iodide and propidium iodide. Values derived from each sample are shown. (B) Colony-forming assays were performed in methylcellulose with the indicated treatments. Graphic representation of reduced colony number after treatment with UNC1666, compared to vehicle. Mean values and standard errors derived from triplicate samples are shown. (C) Graphic representation of the effect of UNC1666 on normal cord blood colony forming potential. Mean values and standard errors were derived from 3 independent experiments. **p < 0.01, NS = not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4466645&req=5

Figure 7: UNC1666 induces apoptosis and decreases colony formation in AML patient samples(A) Graphic representation of apoptosis and cell death in AML patient samples after treatment with UNC1666 or vehicle for 72 hours. Apoptotic and dead cells were determined by flow cytometry after staining with YO-PRO-1 iodide and propidium iodide. Values derived from each sample are shown. (B) Colony-forming assays were performed in methylcellulose with the indicated treatments. Graphic representation of reduced colony number after treatment with UNC1666, compared to vehicle. Mean values and standard errors derived from triplicate samples are shown. (C) Graphic representation of the effect of UNC1666 on normal cord blood colony forming potential. Mean values and standard errors were derived from 3 independent experiments. **p < 0.01, NS = not significant.
Mentions: To replicate the conditions utilized with AML cell lines, patient samples were treated with UNC1666 or vehicle and apoptosis was determined using flow cytometry as described above. Patient samples were co-cultured with HS27 stromal cells, which provide essential factors to support myeloid cells and allowed us to assess response to UNC1666. Under the stromal cell enhanced growth conditions, treatment with UNC1666 was sufficient to induce apoptosis in all patient samples, thereby demonstrating efficacy on AML blasts with a wide range of Mer expression levels or Flt3-ITD status (Figure 7A & Supplemental Figure 4B). Induction of apoptosis in response to UNC1666 was independent of position in the cell cycle, though at high doses may correlate with accumulation in G2/M phase as previously noted in AML cell lines (Supplemental Figure 4C).

Bottom Line: Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle.These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition.In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML.

View Article: PubMed Central - PubMed

Affiliation: University of Colorado, Department of Pediatrics, Aurora, CO, USA.

ABSTRACT
Mer and Flt3 receptor tyrosine kinases have been implicated as therapeutic targets in acute myeloid leukemia (AML). In this manuscript we describe UNC1666, a novel ATP-competitive small molecule tyrosine kinase inhibitor, which potently diminishes Mer and Flt3 phosphorylation in AML. Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle. These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition. Treatment of primary AML patient samples expressing Mer and/or Flt3-ITD with UNC1666 also inhibited Mer and Flt3 intracellular signaling, induced apoptosis, and inhibited colony formation. In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML.

Show MeSH
Related in: MedlinePlus