We tested our model by treating BaF3 cells transfected with the gain of function FLT3 D835V muta tion with either NVP BGT226 or NVP BEZ235 and probed for T308 or S473 phosphorylated AKT isoforms in a western immunoblot using whole cell lysates. Both inhibitors potently and globally suppressed AKT phosphorylation of initially maximally activated AKT. Jurkat they cells treated with well established PI3K inhibitors served as controls. NVP BGT226 displays antiproliferative and proapoptotic activity in mutant tyrosine kinase mediated AKT activated BaF3 isogenic cells We next utilized our BaF3 model to evaluate the mutant TK specific antiproliferative effect of either NVP BGT226 or NVP BEZ235 in an isogenic cellular background.

Both agents revealed compound specific but also distinct mu tation Inhibitors,Modulators,Libraries specific Inhibitors,Modulators,Libraries activity, with the parental cell line being the least sensitive for both tested agents. BCR ABL1, FLT3 D835V and KIT D816Y transfectants displayed an intermediate sensitivity pattern whereas FLT3 ITD demonstrated high sensitivity for both agents with IC50s below 10 nM. Repre sentative dose vs. effect graphs are shown in Figure 7AB. A summary of achieved IC50s is provided in Table 1 to gether with additional TK isoforms tested. When testing for induction of apoptosis, NVP BGT226 proved to be Inhibitors,Modulators,Libraries highly potent in virtually all tested cell lines, with transfectant specific IC50s raging from 120 1800 nM. In contrast, the high capacity to inhibit cellular proliferation for NVP BEZ235 did not similarly translate into potency to induce apoptosis for all tested transfectant cell lines.

Importantly, BaF3 FLT3 ITD cells, which were highly inhibited Inhibitors,Modulators,Libraries with regard to cellular pro liferation, did only show moderate induction of apoptosis towards NVP BEZ235. In analogy, BCR ABL1 transfected cells failed to achieve IC50 as well, with a proportion of 39% apop totic cells at 5000 nM. These findings are in line with Inhibitors,Modulators,Libraries our results for the corresponding tested human leukemia cell lines. Notably, other transfectants retained some level of sensitivity with regard to induction of apoptosis. Representative dose vs. effect graphs are shown in Figure 7CD. A full list of IC50s for both agents and additionally tested mutant TK BaF3 cells are provided with Table 1. We confirmed our observations Ganetespib Phase 3 at the protein level and treated BaF3 parental, FLT3 ITD, FLT3 D835V, KIT D816Y or BCR ABL1 transfected cells with NVP BGT226 or NVP BEZ235 to probe whole cell ly sates for AKT phosphorylation in an immunoblot. Dual inhibition of PI3Kinases and MTOR12 lead to potent AKT dephosphorylation of initially activated AKT in IL3 stimulated or mutant TK activated cells in the low nanomolar range. This went along with the observed antiproliferative effects for both agents on the cellular level.