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Mutations of Fms-like tyrosine kinase 3 (FLT3) are among the most frequently detected molecular abnormalities in AML patients. Internal tandem duplications (ITDs) are found in approximately 25% and point mutations within the second tyrosine kinase domain (TKD) in approximately 7% of AML patients. Patients carrying the FLT3-ITD but not the FLT3-TKD mutation have a significantly worse prognosis. Therefore, both FLT3 mutations seem to exert different biologic functions. FLT3-ITD but not FLT3-TKD has beenshown to induce robust activation of the STAT5 signaling pathway. In the present study, we investigated the mechanisms leading to differential STAT5 activation and show that FLT3-ITD but not FLT3-TKD uses SRC to activate STAT5. Coimmunoprecipitation and pull-down 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. Using sitespecific Abs, we found that both residues were significantly more strongly phosphorylated in FLT3-ITD compared with FLT3-TKD. SRC inhibition and knockdown blocked STAT5 activation and proliferation induced by FLT3-ITD but not by FLT3-TKD. We conclude that SRC might be a therapeutic target in FLT3-ITD ؉ AML. IntroductionThe Fms-like tyrosine kinase-3 (FLT3) receptor tyrosine kinase is expressed on blast cells in most patients with acute myeloid leukemia (AML). 1 Activating mutations of FLT3 have been detected in approximately 30% of AML patients. 2,3 Two distinct groups of FLT3 mutations have been described: (1) internal tandem duplications (FLT3-ITDs), which are most common in the juxtamembrane or the beta1-sheet of the tyrosine kinase domain-1 coding sequence in approximately 20%-27% of patients with AML; and (2) point mutations within the second tyrosine kinase domain (FLT3-TKDs) in approximately 7% of AML patients. 2,4,5 Both types of mutations activate the FLT3 receptor constitutively, leading to activation of downstream signaling proteins and resulting in factor-independent proliferation of murine lymphoid and myeloid cells. [6][7][8][9][10] Studies have identified FLT3-ITD as a prognostic factor because patients harboring this type of mutation have elevated peripheral blood and BM blast counts and an increased chance of relapse and inferior overall survival. 2,11,12 For FLT3-TKD mutations, a clear association with an inferior outcome could not be demonstrated in all studies and the relevance of FLT3-TKD for clinical prognosis is being investigated further. 13 Because FLT3 mutations can be found in approximately 30% of all AML patients and FLT3-ITD is associated with an inferior clinical prognosis, FLT3 is considered as an attractive therapeutic target. 14 Therefore, several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been developed and tested in AML patients. 15,16 These studies showed that a single application of the inhibitor led to a short-term clinical response in some pat...

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