Figure 6.

Resistance to FLT3 TKIs is partially mediated through activation of Akt and/or MAPK signaling pathways. (A) Molm14, M14(R)5214, and M14(R)701 cells (10 × 10⁶ cells) were treated with 5 μM PI3K or MEK inhibitors and/or 50 nM CEP-5214 for 1 hour at 37°C. Immunoprecipitates and total protein extracts were resolved by 8% SDS-PAGE or 10% SDS-PAGE, respectively, and subjected to immunoblot analysis with the indicated phospho-specific antibodies. The same blots were then stripped and reprobed with protein-specific antibodies. (B) Molm14, (C) M14(R)5214, and (D) M14(R)701 cells (300 000 cells/mL) were incubated with 0 to 50 nM CEP-701 and/or 5 μM PI3K or MEK inhibitors for 48 hours at 37°C. Cellular activity/proliferation was determined using the MTT assay in triplicates and normalized to control cells that were not treated with CEP-701. (E) Cells (300 000 cells/mL) were incubated with 25 nM CEP-701 and/or 5 μM PI3K inhibitor and/or 5 μM MEK inhibitor for 48 hours at 37°C. Induction of apoptosis was assessed by measuring annexin V/7-AAD binding by flow cytometry. Values indicate relative fold increase in the percentage of cells that were annexin V/7-AAD positive. Results are representative of 2 independent experiments. Error bars indicate SEM.