p110δ activity is required for Akt phosphorylation and proliferation of AML blasts. (A) IC87114 dose-response relationship and specificity. BMMCs from patient no. 89 (90% leukemic blasts) or OPM2 cells were incubated for 30 minutes with the indicated concentrations of IC87114 or 25 μM LY294002. Cell extracts from 10⁶ cells were analyzed by Western blot using antibodies against phosphoSer473 of Akt (Cell Signaling Technology). After stripping, the blots were reprobed with antiactin antibodies to ascertain equal loading of the samples. (B) IC87114 inhibits Flt-3 ligand–stimulated PI3K activity. BMMCs from patient no. 102 were incubated for 30 minutes with or without 10 μM IC87114 or 25 μM LY294002, followed by stimulation for 15 minutes with 50 ng/mL FLT-3 ligand, and Ser473 phosphorylation of Akt was analyzed by Western blotting. (C) IC87114 inhibits constitutive PI3K activity. BMMCs were incubated for 30 minutes with 10 μM IC87114 or 25 μM LY294002 or with solvent (dimethylsulfoxide [DMSO]) alone. Cell extracts from 10⁶ cells were analyzed by Western blot using anti–p-Akt (Ser473) and antiactin antibodies. Inset blots show the results of a typical experiment using blasts from patient no. 51. Similar experiments were performed with the blasts of each patient presented in Table 1 and Figure 1. The blots were scanned and analyzed using ImageJ software. For each sample, Akt phosphorylation observed in the absence of inhibitor was set at 100% and Akt phosphorylation in the presence of inhibitors was expressed relative to this value (main graph). Error bars represent standard deviations of the 10 patient samples. Significance determined by Student t test: control/LY294002, P < .001; control/IC87114, P < .001; LY294002/IC87114, P > .2 (not significant). (D) IC87114 inhibits AML cell proliferation. BMMCs from each patient presented in Table 1 were incubated for 48 hours with or without 10 μM IC87114 and pulsed for 6 hours with [³H]-thymidine. Moreover, BMMCs from patient nos. 89, 102, and 110 were also incubated for 48 hours with 10 ng/mL FLT-3 ligand in the presence or absence of IC87114. For each BMMC sample, [³H]-thymidine incorporation in the absence of FLT-3 ligand and IC87114 (control) was set at 100% and [³H]-thymidine incorporation in the presence of FLT-3 ligand and/or IC87114 was expressed relative to this value. Significance determined by Student t test: control/IC87114, P < .001; FLT-3L/FLT-3L + IC87114, P < .01. Error bars indicate standard deviation.