Figure 5
Figure 5. FLT3-ITD activates MCL-1 expression via STAT5 signaling. (A) FLT3-WT, FLT3-ITD, and FLT3-ITD-Y589/591F mutants were transduced into purified murine HSCs and GMPs to test changes in the amount of MCL-1 transcripts. During the transduction, cells were cultured for 48 hours in the presence of FLT3 and SCF. In this condition, the amount of MCL-1 transcripts in HSCs transfected with FLT3-WT was almost equal to HSCs with an empty vector. Significant up-regulation of MCL-1 (∼ 9-fold higher) was seen when HSCs were transfected with FLT3-ITD. This effect was more pronounced in HSCs than GMPs. The up-regulation of MCL-1 was completely inhibited in HSCs transduced with FLT3-ITD-Y589/591F mutant, indicating that FLT3-ITD–specific STAT5-docking phosphorylation sites are necessary to stimulate MCL-1 transcription. (B) Schematic presentation of signaling pathways of FLT3-ITD to induce MCL-1 expression. In FLT3-WT, FLT3 ligation triggers both PI3K/Akt and RAS/MAPK pathways. In contrast, FLT3-ITD induces STAT5-dependent MCL-1 expression. STAT5 might induce MCL-1 expression by PI3K/Akt activation presumably through posttranscriptional mechanism (1), and by directly stimulating MCL-1 transcription (2). These 2 distinct FLT3-ITD signaling pathways for MCL-1 expression contribute to the development of AML. (C) STAT5 siRNA was transduced into MV4-11 cell lines to suppress STAT5 expression. The sorted live cells were analyzed at 48 hours after STAT5 siRNA transduction. STAT5 siRNA suppressed the expression of MCL-1 transcript and protein. STAT5 siRNA also suppressed phospho-Akt, but not phospho-MEK, pathway. Vertical lines have been inserted to indicate a repositioned gel lane. Results are shown as mean ± SD (*P < .05) (D) The expression of MCL-1 in MV4-11 24 hours after treatment with pharmacologic inhibitors of PKC (PKC-412), PI3K (Ly294002), JAK2 (AG490), or MEK (U0126). PKC-412 inhibited MCL-1 mRNA expression. PKC-412 and Ly294002 but not U0126 or AG490 inhibited the MCL-1 protein expression. RE indicates relative expression levels. Results are shown as mean ± SD (*P < .05).

FLT3-ITD activates MCL-1 expression via STAT5 signaling. (A) FLT3-WT, FLT3-ITD, and FLT3-ITD-Y589/591F mutants were transduced into purified murine HSCs and GMPs to test changes in the amount of MCL-1 transcripts. During the transduction, cells were cultured for 48 hours in the presence of FLT3 and SCF. In this condition, the amount of MCL-1 transcripts in HSCs transfected with FLT3-WT was almost equal to HSCs with an empty vector. Significant up-regulation of MCL-1 (∼ 9-fold higher) was seen when HSCs were transfected with FLT3-ITD. This effect was more pronounced in HSCs than GMPs. The up-regulation of MCL-1 was completely inhibited in HSCs transduced with FLT3-ITD-Y589/591F mutant, indicating that FLT3-ITD–specific STAT5-docking phosphorylation sites are necessary to stimulate MCL-1 transcription. (B) Schematic presentation of signaling pathways of FLT3-ITD to induce MCL-1 expression. In FLT3-WT, FLT3 ligation triggers both PI3K/Akt and RAS/MAPK pathways. In contrast, FLT3-ITD induces STAT5-dependent MCL-1 expression. STAT5 might induce MCL-1 expression by PI3K/Akt activation presumably through posttranscriptional mechanism (1), and by directly stimulating MCL-1 transcription (2). These 2 distinct FLT3-ITD signaling pathways for MCL-1 expression contribute to the development of AML. (C) STAT5 siRNA was transduced into MV4-11 cell lines to suppress STAT5 expression. The sorted live cells were analyzed at 48 hours after STAT5 siRNA transduction. STAT5 siRNA suppressed the expression of MCL-1 transcript and protein. STAT5 siRNA also suppressed phospho-Akt, but not phospho-MEK, pathway. Vertical lines have been inserted to indicate a repositioned gel lane. Results are shown as mean ± SD (*P < .05) (D) The expression of MCL-1 in MV4-11 24 hours after treatment with pharmacologic inhibitors of PKC (PKC-412), PI3K (Ly294002), JAK2 (AG490), or MEK (U0126). PKC-412 inhibited MCL-1 mRNA expression. PKC-412 and Ly294002 but not U0126 or AG490 inhibited the MCL-1 protein expression. RE indicates relative expression levels. Results are shown as mean ± SD (*P < .05).

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