Figure 5: Pharmacological inhibition of p110β suppresses leukaemia in vitro and in Pten-deficient mice.
(a) Colony-forming assays were performed with 1 × 104 (BM) or 5 × 104 (spleen) cells obtained from 7 DPI PtenΔ/Δ animals. Cells were cultured with added DMSO, BYL719, TGX221,GS1101, NVSPI35, GDC0941 or RAD001 with the following concentrations based on published data: 1, 0.5, 0.1 μM (BYL719, TGX221 and GDC0941); 5, 1, 0.5 μM (GS1101); 0.1, 0.05, 0.01 μM (NVSPI35 and RAD001). Colonies were counted 7 days after incubation. Data are represented as mean±s.e.m. (n=3, measured in duplicates) and all data are compared with the DMSO control for statistical analysis. *P<0.05; **P<0.001. Two-way ANOVA test was applied to compare the colony formation. (b) Schematic representation of drug treatment. (c) Survival of PtenΔ/Δ animals treated with vehicle (blue; n=7; median survival=23 days), BYL719 (green; n=8; median survival=32 days), KIN193 (red; n=7; median survival=50 days), IC87114 (orange; n=7; median survival=28 days) and AS605240 (brown; n=6; median survival=23 days). *P<0.05; **P<0.001; ***P<0.0001. The log-rank test was applied to compare survival. (d) Spleen weight of vehicle-treated (n=4) and KIN193-treated animals (n=3) at the moribund stage. *P<0.05. Student’s t-test was applied. (e) Western blot analysis of Akt signalling in whole BM cells at 7 DPI. Freshly isolated BM cells were treated with DMSO, BYL719, KIN193, GS1101 or NVSPI35 at the 1-μM dose for 2 h (n=6) for each treatment. (f) Phospho-flow analysis of p-Akt on isoform-selective inhibitor-treated animals. Freshly isolated lineage-negative BM cells were treated with inhibitors as in (e) and subjected to flow cytometry for LSK staining and intracellular P-Akt staining (n=3) for each, and median fluorescence intensities were normalized to control.
