Treatment with PI3K/mTOR inhibitors synergizes with PTL's antileukemia activity. Primary AML samples were treated for 18 hours with either 5μM wortmannin, 2.5μM PTL, or 5μM PTL, alone or in combination. (A) Representative example of flow cytometric analysis of CD34⁺CD38⁻ populations (AML6). Dot plots show 7AAD (y-axis) vs annexin V (x-axis). (B) Percentage of viable cells normalized to untreated control for total blasts (n = 5; AML2, AML6, AML8, AML10, AML11) and (C) CD34⁺CD38⁻ populations (n = 5; AML2, AML6, AML8, AML10, AML11). Error bars represent SEM. (D) AML vs normal cells were treated with 5μM wortmannin alone, 2.5μM PTL alone, or in combination for 18 hours in suspension culture, followed by plating in methylcellulose culture. The percentage of colony-forming units (CFUs) was normalized to untreated control (n = 3; AML4, AML6, AML8). (E) Percentage of colonies relative to untreated control after treatment for 18 hours with either 2.5 μg/mL of temsirolimus (TEM), 5μM PTL, either alone or in combination. Primary AML specimens are shown in the left panel and primary normal marrow specimens are shown in the right panel (n = 3; AML4, AML6, AML8). (F) Percentage of engraftment of AML1 achieved in NOD/SCID mice receiving AML (left panel) or normal BM (right panel) cells after 18 hours of culture with or without 2.5 μg/mL TEM and 5μM PTL. Each symbol represents a single animal analyzed at 6 to 8 weeks after transplantation. Mean engraftment is indicated by the horizontal bars. Normal CD34⁺ (untreated vs PTL + TEM), P = .280. Normal CD33⁺ (untreated vs PTL + TEM), P = .084; normal CD19⁺ (untreated vs PTL + TEM), P = .009. “% human cells” indicates the percentage of total bone marrow cells positive for labeling with hCD45 antibody. “%CD34⁺” is gated on hCD45⁺ cells.