Phospho-Akt mediates synergy observed between allosteric Akt inhibitor, KIN001-102, and PKC412.

<p>Immunoblots of protein lysates prepared from MOLM14-luc+ cells treated for 2 hours with PKC412 (40 nM), KIN001-102 (165, 330, 660 nM), or a combination of the two agents in the presence of 50% SCM. Data shown are representative of two independent experiments in which similar results were achieved.</p

Selective inhibitors of AKT positively combine with AC220 in RPMI+10% FBS against MOLM14-luc+ cells.

<p>(A–D) Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with AC220 in RPMI+10% FBS. (E) Calcusyn combination indices. The cut-off for nearly additive effects (C.I.: 1.1) is marked by a dashed line.</p

Effects of PKC412 and KIN001-102, alone and combined, on MOLM14-luc+ cell cycle progression (following 24 hours of treatment) and apoptosis (following 48 hours of treatment) when cells are cultured in the presence of 50% HS-5 SCM.

<p>Details of the assays used for these studies are provided in the Materials and Methods section.</p

Ability of Akt inhibitors to positively combine with PKC412 or AC220 against AML patient samples in the presence of cytoprotective SCM.

<p>(A) Approximately two-day proliferation study performed with a selective Akt inhibitor in combination with PKC412 in the presence of HS-5 SCM against mutant FLT3-positive AML#2. (B) Approximately two-day combination studies: AC220 (0.4 nM) +/− selective AKT inhibitors (660 nM) against MOLM14-luc+ cells in the presence of 50% HS-5 SCM. (C) Approximately two-day combination studies: AC220 (0.4 nM) +/− selective AKT inhibitors (660 nM) against MOLM14-luc+ cells in the presence of RPMI+10% FBS. (D) Approximately two-day combination studies: PKC412 (40 nM)+/− selective AKT inhibitors (660 nM) against primary AML patient cells in the presence of 50% HS-5 SCM. (E) Approximately two-day combination studies: AC220 (0.4 nM) +/− selective AKT inhibitors (660 nM) against primary AML patient cells in the presence of 50% SCM. (F) Ability of Akt inhibitors to positively combine with PKC412 or AC220 against primary AML cells in the presence of cytoprotective SCM. Patient information is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056473#pone.0056473.s010" target="_blank">Table S1</a>.</p

Selective inhibitors of AKT positively combine with PKC412 in the presence of adherent HS-5 stroma against MOLM14-luc+ cells.

<p>(A) Approximately two-day proliferation study performed with MOLM14-luc+ cells cultured in the presence of adherent HS-5 stroma testing the combination of PKC412 and KIN001-102 versus each agent alone. (B) MOLM14-luc+ cells cultured in the presence of adherent HS-5 stroma for approximately two days: PKC412 (40 nM)+/− Akt inhibitors (660 nM). (C) Approximately two-day PKC412 treatment of MOLM14-luc+ cells cultured in the absence and presence of human stroma. (D) Approximately two-day treatment of adherent HS-5 stroma: PKC412 (40 nM) +/− Akt inhibitors (660 nM). (E) Calcusyn combination indices derived from 4-point concentration proliferation experiments. The cut-off for nearly additive effects (C.I.: 1.1) is marked by a dashed line.</p

Effects of PKC412 and KIN001-102, alone and combined, on MOLM14-luc+ cell cycle progression (following 24 hours of treatment) and apoptosis (following 48 hours of treatment) when cells are cultured in the presence of RPMI+10% FBS.

<p>Details of the assays used for these studies are provided in the Materials and Methods section.</p

Calcusyn software-derived combination indices.

<p>Data shown here correspond to dose-response curves shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056473#pone-0056473-g005" target="_blank">Figures 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056473#pone-0056473-g006" target="_blank">6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056473#pone.0056473.s008" target="_blank">Figure S8</a>. Interpretation of combination indices is provided in the Materials and Methods section.</p

Selective inhibitors of AKT positively combine with PKC412 in the presence of SCM against MOLM14-luc+ cells.

<p>(A–D) Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in the presence of HS-5 SCM. (E) Approximately two-day PKC412 treatment of MOLM14-luc+ cells cultured in the absence or presence of HS-5 SCM (n = 2). (F) Calcusyn combination indices derived from the 4-point concentration proliferation experiments shown in A-D. The cut-off for nearly additive effects (C.I.: 1.1) is marked by a dashed line.</p

Selective inhibitors of AKT positively combine with PKC412 in RPMI+10% FBS against MV4,11 and Ba/F3-FLT3-ITD cells.

<p>(A–C) Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in RPMI+10% FBS against MV4,11 cells. (D–F) Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in RPMI+10% FBS against Ba/F3-FLT3-ITD cells.</p

Selective inhibitors of AKT positively combine with PKC412 in the absence and presence of HS-5 SCM against MOLM13-luc+ cells.

<p>(A–B) Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in the presence of RPMI+10% FBS. (C–D) Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in 80% HS-5 SCM. (E) Treatment of MOLM13-luc+ cells with PKC412 in either RPMI+10% FBS or 80% HS-5 SCM.</p