Discovery of a Highly Potent and Selective Indenoindolone Type 1 Pan-FLT3 Inhibitor

For a subpopulation of acute myeloid leukemia (AML) patients, the mutationally activated tyrosine kinase FLT3, has emerged as a promising target for therapy. The development of drug resistance due to mutation is a growing concern for mutant FLT3 inhibitors, such as PKC412, Quizartinib, PLX3397, and Crenolanib. Thus, there is a need to develop novel FLT3 inhibitors that overcome these mutations. Here we report the development of a novel type I ATP competitive inhibitor, JH-IX-179, that is extremely potent and selective for FLT3. JH-IX-179 also has the highest affinity for three constitutively active isoforms of FLT3 (FLT3-ITD, FLT3-N841I, and FLT3-D835V) compared to a panel 456 other kinases. The unique and specific kinase inhibition profile suggests that this chemotype may represent an attractive starting point for the development of further improved FLT3 inhibitors with therapeutic potential in tumors harboring deregulated FLT3 activity

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

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

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

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