FLT3 Ligand Is Required for Full Activation of FLT3 Mutants.

FLT3 is a member of the class III receptor tyrosine kinase family and is primarily expressed on hematopoietic stem/progenitor cells. It is expressed in most cases of acute myeloid leukemia (AML) and acute B-lineage leukemia. FLT3 ligand (FL) binds to wild-type FLT3 (wtFLT3) receptors and is required for the activation of wtFLT3 signaling. 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, the data upon which these conclusions were based was 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 the mutant forms of FLT3. In order to determine whether or not FLT3 mutants are completely or somewhat dependent on FL for their activation, a FL deficient murine embryo fibroblast cell line (MEF) was derived from FL deficient (FL−/−) mice by SV40 large T antigen transformation. This cell line 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 exogenous FL. Retroviral introduction of FL in FL−/−MEF cells expressing FLT3 mutants led to more than 3 fold increases of FLT3 phosphorylation by FL. Further activation of FLT3 mutant receptors by FL was also observed in BaF3 and TF1 cells expressing FLT3/ITD or TKD mutants and in MV411 cells, a cell line expressing FLT3/ITD alone without expression of wtFLT3. Phosphorylation of downstream targets of FLT3 signaling including STAT5, AKT, or MAPK was also enhanced after FL stimulation. Biological function studies revealed that exogenous FL promoted the colony forming abilities of FLT3/ITD and FLT3/TKD mutant transduced bone marrow cells derived from FL−/− mice. However, FL did not increase the colony numbers formed from control vector transduced FL−/− bone marrow cells. In colony replating assay, FLT3 mutant transduced FL−/− bone marrow cells maintained recloning abilities in the presence of additional FL after replating in the second, third, and fouth generation. In contrast, replating abilities of FLT3 mutant expressing bone marrow cells cultured in the absence of FL and vector control transduced bone marrow cells cultured with and without FL were gradually lost after replating from the second to the fourth generation. Addition of FL also led to enhanced proliferation and/or decreased apoptosis of BaF3 and TF1 cells expressing FLT3/ITD or FLT3/TKD, and of MV411 cells under conditions of serum starvation. These data strongly suggest that full activation of FLT3 mutants is dependent on FL and thus it is likely that FL plays a role in the leukemogenesis mediated by FLT3 mutants. Therefore, FL targeted therapy might be useful in the treatment of FLT3 mutant AML.