Figure 1

mTOR signaling pathway is activated and contributes to the survival of AML cells harboring mutated FLT-3. A. Western blot analysis of primary AML cells with mutated FLT3- ITD showed activation (phosphorylation) of mTOR kinase and downstream mTOR mediators (left panel). These data were confirmed also with immunohistochemistry on FLT3-mutated AML bone marrow samples (right panel). In positive cases, the majority of tumor cells show expression of phosphorylated (activated) mTOR, p70S6K, rpS6 and 4EBP1 with a cytoplasmic staining pattern (DAB chromogen, hematoxylin & eosin [H&E] counterstain, original magnification ×400). B. A causal association between activation status of mTOR signaling pathway and activating mutations of FLT3 was further supported by the upregulation of mTOR, Raptor as well as phosphorylation (activation) of downstream effectors such as p70S6K and rpS6 proteins in BaF3 cells stably transfected with mutated FLT3 compared with BaF3 cells transfected with Wt-FLT3, as shown by Western blot analysis (left panel), or immunohistochemistry performed in cell blocks (right panel). C. Western blot analysis of AML cell lines showed that pharmacologic inhibition of PI3K and mTOR kinases by LY294002 resulted in downregulation of p-AKT and downstream mediators of the mTOR pathway. This effect was more pronounced in the MV4-11 and MOLM13 cell lines harboring mutated FLT3 comparing with the U937 cell line with Wt-FLT3. D. Silencing of the mTOR gene by transient transfection of mTOR-specific siRNA resulted in downregulation of mTOR and p-AKT signaling (left panel), associated with decreased survival of FLT3- mutated MOLM13 and MV4-11 cells by 30% and 62%, respectively, as compared with a small decrease by 13% of the Wt-FLT3 U937 cells (p < 0.05), 48 hours after treatment (right panel).