FLT3ITD and FLTD835Y have transforming potential in Ba/F3 cells and activate STAT5 and MAPK.
(A) FLT3ITD mutants confer a competitive growth advantage to IL-3–dependent Ba/F3 cells. EGFP expression was determined by flow cytometry in Ba/F3 cells transduced with pMSCV-EGFP-IRES-FLT3ITD-NPOS retrovirus 4 days and 13 days after transduction and IL-3 withdrawal. (B) FLT3ITD-NPOS or FLT3ITD-W51, but not FLT3WT constructs, confer IL-3–independent growth to Ba/F3 cells. Ba/F3 cells stably expressing FLT3ITD-NPOS, FLT3ITD-W51, and FLT3WT were seeded at a density of 4 × 104 cells/mL in the absence or presence of IL-3 (10 ng/mL). Viable cells were counted for up to 3 days by trypan blue exclusion. (C) FLT3ITD and FLT3D835Y activate STAT5 and MAPK1/2. Protein lysates from Ba/F3 cells transformed either by FLT3ITD-NPOS and FLT3D835Y or vector-transduced cells (mock) grown in the presence of IL-3 were analyzed by Western blot. No activation of AKT and only weak activation of STAT3 could be detected.
