localization of the receptor. In contrast, FLT3-WT receptor was mainly found on the cell surface and showed a clear response to FL. Again those effects were on a continuum within the group of FLT3 mutants representing the diverse activating potential of FLT3 mutations. FLT3 receptors in ITD expressing cells were mainly located intracellular and non-responsive to FL treatment. The localization of the FLT3-ITD in the endoplasmic reticulum seems to be a major factor in compartment-specific 1702259-66-2 activation of STAT5. The analysis of signaling revealed a distinct activation of STAT5 by FLT3-ITD mutants, as reported before. STAT5 is directly or SRC-dependently activated in FLT3-ITD expressing cells in vitro via tyrosine residues 589 and 591. Accordingly, enhanced phosphorylation of Y591 was found 
in FLT3-ITD expressing cells in contrast to FLT3-WT expressing cells. Interestingly, also FLT3-I867S, D839G and -D835Y expressing cells showed phosphorylation of Y591, indicating additional functions of this residue or undetected STAT5 activation. Y591 was identified as a docking site for suppressor of cytokine signaling 6, Lnk, an adaptor protein with negative regulator influence on FLT3 and as interaction site of c-CBL, inducing degradation of the FLT3 receptor. We used predefined STAT5 target gene sets for the evaluation of our GEP analysis to demonstrate a potential influence of FLT3 mutations on STAT5 activity in primary AML cells. We were able to show differences in STAT5 target gene expression between FLT3-ITD and -TKD with distinct characteristics. These results have to be interpreted carefully due to the highly complex role of transcription factors and many unknown variables. To account for this, we tried to adjust our analysis for additional well known mutations in AML by defining subgroups. The reproducibility of these results indicates a significant role but diverging pathways and targets of FLT3 mutations in STAT5 activity. AKT and MAPK are signaling pathways of the membranebound FLT3-WT receptor. Our Western blot analysis revealed an activation of MAPK and AKT in FLT3 point mutation expressing cells in contrast to FLT3-WT and FLT3-ITD expressing cells. Accordingly, only FLT3 point mutation but not FLT3-ITD expressing cells showed growth inhibition after treatment with the AKT pathway inhibitor MK 2206, indicating Oncogenic Potential of FLT3 Mutations aberrantly enhanced activation of this pathway. There are differing reports of AKT and MAPK activation by FLT3-ITD mutants. Consistent with our in vitro analysis we were able to detect differences in the gene expression with respect to FLT3 mutation status. Our analysis of 213 CN-AML patients was able to validate a distinct gene expression profile especially in case of FLT3-ITD, but also FLT3-TKD. Genes that were highly significantly differentially expressed in our analysis like SOCS2 and ENPP2 in the ITD subgroup have also been described by other groups recently. FLT3-TKD positive patients showed PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19630872 an association with higher PRUNE2 and ART3 expression levels which has not been described PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19632179 before. In an earlier study, gene expression levels of patients with newly diagnosed CN-AML showed distinct differences in gene expression profiles with respect to FLT3-ITD and FLT3-TKD mutation. This analysis did not exclude a potential influence of NPM1 mutations, which are highly significantly associated with FLT3 mutations, and was conducted in a relatively small patient cohort. This might account for the very