SRC is a signaling mediator in FLT3-ITD– but not in FLT3-TKD–positive AML

Mutations of the Fms-like tyrosine kinase 3 (FLT3) are among the most frequently detected molecular abnormalities in AML patients. Internal tandem duplications (ITD) are found in approximately 25% and point mutations within the second tyrosine kinase domain (TKD) in about 7% of AML patients. Patients carrying the FLT3-ITD but not FLT3-TKD mutation have a significantly worse prognosis. Thus, both FLT3 mutations seem to exert different biological functions. FLT3-ITD but not FLT3-TKD has been shown to induce robust activation of the STAT5 signaling pathway. Here we investigated the mechanisms leading to differential STAT5 activation and show that FLT3-ITD but not FLT3-TKD utilizes SRC to activate STAT5. Co-immunoprecipitation and pulldown experiments revealed an exclusive interaction between SRC but not other Src family kinases and FLT3-ITD, which is mediated by the SRC SH2 domain. We identified tyrosines 589 and 591 of FLT3-ITD to be essential for SRC binding and subsequent STAT5 activation. By using site-specific antibodies we found both residues significantly stronger phosphorylated in FLT3-ITD compared to FLT3-TKD. SRC inhibition and knockdown blocked STAT5 activation and proliferation induced by FLT3-ITD but not by FLT3-TKD. Thus, SRC might be a therapeutic target in FLT3-ITD positive AML.