Internal Tandem Duplication (ITD) in the Tyrosine Kinase Domain of FLT3 Displays Higher Oncogenic Potential in Acute Myeloid Leukemia

Up to 30% of patients with acute myeloid leukemia (AML) harbor a mutation in FMS like tyrosine kinase 3 (FLT3). This mutation is not only the most frequent, but also the most clinically challenging, because it is associated with increased risk of relapse and poor overall survival. The most common oncogenic FLT3 mutation is the internal tandem duplication (ITD) in the juxtamembrane domain (ITD-JM). Other less frequently occurring mutations include ITD mutations in the tyrosine kinase domain (ITD-TDK) and point mutations in the TKD (e. g. D835Y-TKD). Use of FLT3 inhibitors has shown initial promise, though an acquired resistance remains a problem. A need therefore remains for additional therapeutic targets, and in order to identify these we need to have a better understanding of the mechanisms by which the FLT3 mutations drive leukemogenesis. Although ITD-JM and D835Y-TKD mutations have been studied extensively, the role of the recently identified ITD-TKD remains poorly understood. In this study we compared the ITD-TKD domain mutations with other well-studied FLT3 mutations. We observed that transfection of cytokine-dependent Ba/F3 cells with ITD-TKD is sufficient to induce formation of colonies in semisolid medium in the absence of cytokines. Number and size of colonies were comparable to that of ITD-JM, while D835Y-TKD transfected cells failed to form colonies suggesting that the ITD-TKD mutations have stronger transforming potential than other TKD mutations. Similar to colony formation assays, proliferation and cell survival was significantly higher in ITD-TKD transfected cells compared to cells transfected with D835Y-TKD. ITD-TKD selectively enhanced STAT5 and AKT phosphorylation while ERK1/2 and p38 phosphorylation remained unchanged. Collectively our data suggest that ITD-TKD is a more potent oncogenic mutant compared to the TKD mutants.