PI3K upregulation in acquired resistance is sensitive to idelalisib and AKT or PDK1 inhibitor combinations.

<p>(A) Protein lysates were generated from TMD8^IDELA-S and TMD8^IDELA-R cells, and analyzed by western blot (p-AKT S473, p-AKT T308, AKT, p-S6RP S235/236, S6RP, p-GSK3β S9, GSK3β) and Simple Western (PTEN). (B) Cell viability of TMD8^IDELA-S (left) and TMD8^IDELA-R (right) cells treated with idelalisib, MK-2206 or combination of idelalisib and MK-2206 (1 μM), 96 hours CellTiterGlo assay, mean ± SEM, n = 4. Combination curve is normalized to the single agent alone. (C) Cell viability of TMD8^IDELA-S (left) and TMD8^IDELA-R (right) cells treated with idelalisib, GSK2334470 or combination of idelalisib and GSK2334470 (3 μM), 96 hours CellTiterGlo assay, mean ± SEM, n = 4. Combination curve is normalized to the single agent alone. (D) TMD8^IDELA-R cells were treated with vehicle, idelalisib (1 μM), MK-2206 (1 μM), and idelalisib plus MK-2206, or (E) Vehicle, idelalisib (1 μM), GSK2334470 (1 μM), and idelalisib plus GSK2334470 for 48 hours. Apoptosis was assessed by 7AAD and PE annexin V staining, and analyzed by flow cytometry, mean ± SD, n = 3. (F) TMD8^IDELA-R cells were treated with vehicle, idelalisib (1 μM), MK-2206 (1 μM), or idelalisib plus MK-2206 and (G) Vehicle, idelalisib (1 μM), GSK2334470 (1 μM), or idelalisib plus GSK2334470 for 2 hours. Protein lysates were generated and analyzed by western blot, representative experiment of n = 3. TMD8^IDELA-S vehicle control was included as a reference on the same membrane.