FLT3 Mutant Activation Is Still Dependent on FLT3 Ligand.

FLT3 is a member of the class III receptor tyrosine kinase family and is primarily expressed on hematopoietic stem/progenitor cells. FLT3 ligand (FL) interacts with wild-type FLT3 (wtFLT3) receptors leading to the activation of FLT3 signaling. Constitutive activation of wt FLT3 through autocrine activation by FL is also observed in a number of cell lines and primary AML samples. Incubation of leukemic blasts expressing FLT3 with FL results in enhanced proliferation and decreased spontaneous apoptosis in many cases of acute leukemia. Somatic mutations of FLT3 involving internal tandem duplication (ITD) of the juxtamembrane domain or point mutations in the tyrosine kinase domain (TKD) have been identified in approximately 17–34% and 7–9% of acute myeloid leukemia (AML) patients, respectively. The ITD and TKD mutations appear to activate the tyrosine kinase domain of FLT3 in a FL-independent manner. However, these data were all obtained in human and murine cell lines that express FL. Thus, it is not clear whether FL also plays a role in the activation of FLT3 mutants. In order to determine whether or not FLT3 mutants are completely or somewhat dependent on FL, a FL deficient murine embryo fibroblast cell line (MEF) was derived from FL deficient (FL−/−) mice by SV40 large T antigen transformation. This eliminates the possibility of autocrine stimulation of transfected FLT3 receptor by FL. Expression of FLT3/ITD and FLT3/TKD mutations in FL−/−MEF cells resulted in some constitutive phosphorylation of FLT3 receptor. However, a more than 4 fold increase of FLT3 activation was induced by addition of FL. Retroviral introduction of FL in FL−/− MEF cells expressing FLT3 mutants led to more than 3 fold and 2 fold increase of tyrosine phosphorylation of FLT3 and AKT, respectively. In addition, FL expression resulted in a 3 fold increase of phosphorylation of STAT5 in FL−/−MEF cells expressing FLT3/ITD. In these FL expressing cells, addition of exogenous FL showed no further stimulation of FLT3, AKT and STAT5. These data strongly suggest that the level of FLT3 activation mediated by FLT3 mutants is still largely dependent on FL.