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Vempati, Sridhar (2007): The Role of the Juxtamembrane Domain of FLT3-ITDs in Acute Myeloid Leukemia. Dissertation, LMU München: Faculty of Medicine
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Abstract

Activating mutations in the juxtamembrane domain of FLT3 (FLT3-internal tandem duplications, FLT3-ITDs) represent the most frequent genetic alterations in acute myeloid leukemia (AML). FLT3-internal tandem duplications (FLT3-ITDs) are a heterogenous group of mutations in patients with acute leukemias that are prognostically important. To characterize the mechanism of transformation by FLT3-ITDs, we sequenced the juxtamembrane region (JM) of FLT3 from 284 patients with acute leukemias. The length of FLT3-ITDs varied from 2 to 42 amino acids (AA) with a median of 17 AA. The analysis of duplicated AAs showed that in the majority of patients, the duplications localize between AA 591 to 599 (YVDFREYEY). Arginine 595 (R595) within this region is duplicated in 77% of patients. Single duplication of R595 in FLT3 conferred factor-independent growth to Ba/F3 cells and activated STAT5. Moreover, deletion or substitution of the duplicated R595 in two FLT3-ITD constructs as well as the deletion of wildtype-R595 in FLT3-ITD substantially reduced the transforming potential, pointing to a critical role of the positive charge of R595 in stabilizing the active confirmation of FLT3-ITDs. Deletion of R595 in the FLT3-WT inhibited the growth of cells upon FL stimulation and the STAT5 activation. In this study we could also show that the tyrosine residues 589 and 591 of the FLT3-ITDs could be important phosphorylation sites and are very crucial for the activation of FLT3- ITDs. Simultaneous substitution of these two tyrosine residues with phenyalanine showed complete inhibition of the transforming potential of FLT3-ITDs and STAT5 activation. The substitution of tyrosine residues 597 and 599 did not show any effect on the transforming potential of FLT3-ITDs, supporting the previous hypothesis that these tyrosines may be only important to maintain the integrity of FLT3-WT in its inactive state. Our data provide important insights into the role of the juxtamembrane domain in the mechanism of transformation by FLT3-ITDs.