PIK-75 disrupts the interaction of Bak with the prosurvival proteins, Mcl-1 and Bcl-x_L, to induce apoptosis via a Bak-dependent mechanism. (A) The ability of PIK-75 to promote cell death was assessed in hemopoietic FDM cell lines derived from wt mice or mice lacking proapoptotic members of the Bcl-2 family: Bid^−/−, Puma^−/−, Bad^−/−Bim^−/−, Puma^−/−Bim^−/−, Noxa^−/−Puma^−/−, Noxa^−/−Puma^−/−Bim^−/−, Bak^−/−, Bax^−/−, Bax^−/−Bak^−/−, and p53^−/−. FDM lines were plated in 0.25 ng/mL mIL-3 and cultured for 24 hours in the presence of PIK-75 and cell survival measured by flow cytometric enumeration of PI-negative cells. (B) The IC50 for PIK-75 for 8 primary human AML samples was determined as in Figure 1D and plotted against Bak protein expression levels as determined by western blotting and laser densitometry quantification. Regression analysis was performed (R² = 0.74) and a Pearson linear correlation coefficient was calculated (r = −0.86), indicating a strong inverse linear relationship between Bak expression and PIK-75 IC50 (P = .006 for a non-zero slope). (C) Primary AML blasts were transfected with 100 nM of the indicated siRNAs and/or treated with 1 μM of either GDC-0941 (GDC) or A66 and cell survival was measured at 48 hours. (D) MV4;11 cells were transfected with constructs for the expression of eGFP and either an empty vector or Mcl-1. After 24 hours, cells were treated with 50 nM PIK-75 and live GFP+ cells were counted using Flow-Count Fluorospheres by flow cytometry at 48 hours. (E-F) MV4;11 cells were treated with either (E) 100 nM SNS-032 or (F) 1 μM GDC-0941 for the indicated times following which cell lysates were subjected to immunoprecipitation (IP) using anti-Bak antibodies followed by immunoblotting with the indicated antibodies. Whole cell lysates (WCLs) were also immunoblotted as indicated. The asterisks indicate cross-reactivity of the IP IgG light chain with detection antibodies used in the western blots. (G) Primary human AML blasts were treated for 6 hours with either 100 nM SNS-032 or 1 μM GDC-0941 following which cell lysates were immunoprecipitated using anti-Bak antibodies and immunoblotted with the indicated antibodies. (H) Primary AML blasts were transfected with either a single targeting siRNA (50 nM targeting siRNA plus 50 nM control siRNA) or with 2 targeting siRNAs (50 nM of each targeting siRNA) and cell survival was measured at 72 hours. (I) Primary AML blasts were transfected with siRNAs as in (H) and Bak activation was measured by intracellular staining with anti-Bak antibodies (Ab-1) and flow cytometry (see supplemental Methods) at 48 hours. (J) MV4;11 cells were transfected with constructs for the expression of eGFP and either empty vector (Ctl) or human myr-Akt. After 24 hours, cells were either nontreated (-) or treated (+) with 50 nM of PIK-75 for 6 hours following which cell lysates were subjected to IP (bottom 2 panels) and immunoblotting with the indicated antibodies. WCLs (top 2 panels) were also immunoblotted as indicated. (K) MV4;11 cells transfected as in panel (J) were incubated in the absence (-) or presence (+) of PIK-75 and cell survival was examined after 48 hours. Results are typical of at least 2 experiments. Error bars represent standard deviations with asterisks indicating P < .05.