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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 inhibits Mer and Flt3 dependent signaling in AML patient samples(A) Immunoblot analysis of Mer and Flt3 expression in lysates prepared from AML patient samples. (B) Flt3 mutation status of patient samples determined by molecular profiling. (C, D) Dose-dependent inhibition of Mer and Flt3 phosphorylation in response to treatment with UNC1666. AML blasts from patient sample #10510 (Mer positive, Flt3-ITD high allelic ratio) were treated with UNC1666 or vehicle for two hours. (C) Mer and Flt3 were immunoprecipitated from cell lysates and phosphorylated Mer (p-Mer), total Mer (~180 kDa), phosphorylated Flt3 (p-Flt3) and total Flt3 (130/160 kDa) were detected by immunoblot. (D) Phosphorylation of downstream signaling molecules was assessed by immunoblot after treatment with UNC1666 or vehicle.
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Figure 6: UNC1666 inhibits Mer and Flt3 dependent signaling in AML patient samples(A) Immunoblot analysis of Mer and Flt3 expression in lysates prepared from AML patient samples. (B) Flt3 mutation status of patient samples determined by molecular profiling. (C, D) Dose-dependent inhibition of Mer and Flt3 phosphorylation in response to treatment with UNC1666. AML blasts from patient sample #10510 (Mer positive, Flt3-ITD high allelic ratio) were treated with UNC1666 or vehicle for two hours. (C) Mer and Flt3 were immunoprecipitated from cell lysates and phosphorylated Mer (p-Mer), total Mer (~180 kDa), phosphorylated Flt3 (p-Flt3) and total Flt3 (130/160 kDa) were detected by immunoblot. (D) Phosphorylation of downstream signaling molecules was assessed by immunoblot after treatment with UNC1666 or vehicle.

Mentions: Mer and wild-type Flt3 expression levels were assessed in blasts obtained from six patients diagnosed with acute myeloid leukemia using immunoblot analysis (Figure 6A), and FLT3-ITD alleles were detected using standard clinical molecular techniques (Figure 6B). Additional clinical information relevant to each patient samples is described in Figure 6B. In our ongoing studies, we have not identified any cell line other than MV4;11 (which expresses very little Mer) that jointly expresses Mer and a Flt3-ITD mutation (data not shown), however five out of six of our randomly obtained patient samples did co-express these molecules. All samples expressed Mer (consistent with our previously published results), but demonstrated a range of Mer expression levels (Figure 6A). Since sensitivity of Mer expressing malignant cell lines does not appear to directly correlate with the degree of Mer expression (data not shown) and Mer dependence may instead be related to autocrine ligand expression or some other property of the cell, we opted to score all patient samples that express Mer as Mer positive (Merpos). All samples except #123009 expressed Flt3-ITD mutations, though #11612 and #41206 were noted to have a low allelic ratio. Since there is a correlation between high allelic ratio of the Flt3-ITD (> 0.4) and poor prognosis in patients [16], we accordingly denoted when a patient sample was of high (“Flt3-ITDhighAR”) or low (“Flt3-ITDlowAR”) Flt3 allelic ratio. None of the patient samples demonstrate detectable levels of Trk proteins (Supplemental Figure 4A). Sample #10510, which was Merpos and Flt3-ITDhighAR, was analyzed to determine intracellular signaling alterations after treatment with UNC1666. Mer and Flt3 were immunoprecipitated from whole cell lysates and immunoblot analysis confirmed inhibition of both kinases in response to UNC1666 (Figure 6C). Phosphorylation of both Mer and Flt3 was inhibited by greater that 50% after treatment with UNC1666 at concentrations as low as 50 nM, though it is notable that UNC1666 appears to decrease Flt3 phosphorylation at a lower dose compared to its effect on Mer, similar to what was observed in cell lines. Immunoblot analysis of Erk1/2, Akt and Stat5 phospho-proteins demonstrated marked decreases in response to UNC1666 treatment (Figure 6D), which correlated the decrease in Mer and Flt3 phosphorylation status.


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 inhibits Mer and Flt3 dependent signaling in AML patient samples(A) Immunoblot analysis of Mer and Flt3 expression in lysates prepared from AML patient samples. (B) Flt3 mutation status of patient samples determined by molecular profiling. (C, D) Dose-dependent inhibition of Mer and Flt3 phosphorylation in response to treatment with UNC1666. AML blasts from patient sample #10510 (Mer positive, Flt3-ITD high allelic ratio) were treated with UNC1666 or vehicle for two hours. (C) Mer and Flt3 were immunoprecipitated from cell lysates and phosphorylated Mer (p-Mer), total Mer (~180 kDa), phosphorylated Flt3 (p-Flt3) and total Flt3 (130/160 kDa) were detected by immunoblot. (D) Phosphorylation of downstream signaling molecules was assessed by immunoblot after treatment with UNC1666 or vehicle.
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Figure 6: UNC1666 inhibits Mer and Flt3 dependent signaling in AML patient samples(A) Immunoblot analysis of Mer and Flt3 expression in lysates prepared from AML patient samples. (B) Flt3 mutation status of patient samples determined by molecular profiling. (C, D) Dose-dependent inhibition of Mer and Flt3 phosphorylation in response to treatment with UNC1666. AML blasts from patient sample #10510 (Mer positive, Flt3-ITD high allelic ratio) were treated with UNC1666 or vehicle for two hours. (C) Mer and Flt3 were immunoprecipitated from cell lysates and phosphorylated Mer (p-Mer), total Mer (~180 kDa), phosphorylated Flt3 (p-Flt3) and total Flt3 (130/160 kDa) were detected by immunoblot. (D) Phosphorylation of downstream signaling molecules was assessed by immunoblot after treatment with UNC1666 or vehicle.
Mentions: Mer and wild-type Flt3 expression levels were assessed in blasts obtained from six patients diagnosed with acute myeloid leukemia using immunoblot analysis (Figure 6A), and FLT3-ITD alleles were detected using standard clinical molecular techniques (Figure 6B). Additional clinical information relevant to each patient samples is described in Figure 6B. In our ongoing studies, we have not identified any cell line other than MV4;11 (which expresses very little Mer) that jointly expresses Mer and a Flt3-ITD mutation (data not shown), however five out of six of our randomly obtained patient samples did co-express these molecules. All samples expressed Mer (consistent with our previously published results), but demonstrated a range of Mer expression levels (Figure 6A). Since sensitivity of Mer expressing malignant cell lines does not appear to directly correlate with the degree of Mer expression (data not shown) and Mer dependence may instead be related to autocrine ligand expression or some other property of the cell, we opted to score all patient samples that express Mer as Mer positive (Merpos). All samples except #123009 expressed Flt3-ITD mutations, though #11612 and #41206 were noted to have a low allelic ratio. Since there is a correlation between high allelic ratio of the Flt3-ITD (> 0.4) and poor prognosis in patients [16], we accordingly denoted when a patient sample was of high (“Flt3-ITDhighAR”) or low (“Flt3-ITDlowAR”) Flt3 allelic ratio. None of the patient samples demonstrate detectable levels of Trk proteins (Supplemental Figure 4A). Sample #10510, which was Merpos and Flt3-ITDhighAR, was analyzed to determine intracellular signaling alterations after treatment with UNC1666. Mer and Flt3 were immunoprecipitated from whole cell lysates and immunoblot analysis confirmed inhibition of both kinases in response to UNC1666 (Figure 6C). Phosphorylation of both Mer and Flt3 was inhibited by greater that 50% after treatment with UNC1666 at concentrations as low as 50 nM, though it is notable that UNC1666 appears to decrease Flt3 phosphorylation at a lower dose compared to its effect on Mer, similar to what was observed in cell lines. Immunoblot analysis of Erk1/2, Akt and Stat5 phospho-proteins demonstrated marked decreases in response to UNC1666 treatment (Figure 6D), which correlated the decrease in Mer and Flt3 phosphorylation status.

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