Application of a674563 in acute leukemia carrying flt3 mutant gene
A kind of FLT3-D835Y, mutant technology, applied in the field of medicine
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Embodiment 1
[0045] Example 1: Effect of A674563 on proliferation of cancer cells
[0046] The selectivity of A674563 to inhibit the proliferation of cancer cells was evaluated by testing the effect of A674563 on the growth of cancer cells.
[0047] In this embodiment, acute myeloid leukemia cells HL-60 (expressing wild-type FLT3 gene), acute promyelocytic leukemia cell line NB-4 (Lu+) (expressing wild-type FLT3 gene), human acute myeloid leukemia cells OCI-AML-3 (expressing FLT3 A680V mutant gene), human acute myeloid leukemia cell line MOLM-14 (expressing FLT3-ITD mutant gene and wild-type FLT3 gene), human acute myeloid leukemia cell line MOLM-13 (express FLT3-ITD mutant gene and wild-type FLT3 gene), human acute myeloid leukemia cell line MV-4-11 (express FLT3-ITD mutant gene), MDS-RAEB (myelodysplastic syndrome-increased blast type) cell line SKM-1 (expressing wild-type FLT3 gene), human acute myeloid leukemia cell line U-937 (expressing wild-type FLT3 gene), mouse cell BaF3. The ...
Embodiment 2
[0060] Example 2: Effects of A674563 on Upstream and Downstream Signaling Pathways in Cells
[0061] In human acute myeloid leukemia cell line MV-4-11 (expressing FLT3-ITD mutant gene), human acute myeloid leukemia cell line MOLM-13 (expressing FLT3-ITD mutant gene and wild-type FLT3 gene) and human In the acute myeloid leukemia cell line MOLM-14 (expressing FLT3-ITD mutant gene and wild-type FLT3 gene), the effects of A674563 on FLT3 protein kinase, PKB / AKT kinase and the phosphorylation of STAT5, ErK, GSK3β, FOX01, PRAS40, p70S6K, and 4EBP1 proteins closely related to FLT3 kinase and PKB / AKT kinase. At the same time, we also detected the effect of A674563 on the phosphorylation of protein C-Myc and transcription factor NF-κB subunit p65 ( figure 1 a). With different concentrations of 0 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM (in DMSO) of A674563 and 1 μM (in DMSO) of PKB / AKT inhibitor GSK690693, PKB / AKT inhibitor MK2206, FLT3 inhibitor TCS359 Treat MOLM-13, MOLM-14, MV...
Embodiment 3
[0065] Example 3: Effect of A674563 on FLT3 autophosphorylation in the BaF3 cell line at the isogenic locus
[0066] Mouse TEL-FLT3-BaF3 (activating kinase stably expressing wild-type FLT3) cells, mouse BaF3-FLT3-ITD (activating kinase stably expressing FLT3 ITD mutation) cells, mouse BaF3-FLT3-D835Y (stably expressing FLT3D835Y mutant activated kinase) cells, mouse BaF3-FLT3-ITD-D835Y (activated kinase stably expressing FLT3-ITD+D835Y mutation) cells, mouse BaF3-FLT3-ITD-F691L (stable activated kinase) cells (see Example 1 for the construction method of these cells). The effect of A674563 on the autophosphorylation of FLT3 and / or FLT3-ITD mutant protein kinases in cells was tested in the above cell lines ( figure 2 ). With different concentrations of 0 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM (in DMSO) A674563, 0.05 μM (in DMSO) FLT3 kinase inhibitor AC220 (purchased from Hao Yuan Chemexpress Company (Shanghai)) respectively BaF3 cell lines treated with different isogeni...
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