PIK3CA/PIK3R1 mutational status is associated with increased ARQ 092 and ARQ 751 sensitivity.

<p>Somatic mutation analysis was performed for ARQ 092 and ARQ 751 sensitive (GI₅₀<1 μM) and resistant (GI₅₀ ≥1 μM) groups for 212 cancer cell lines. Twenty-eight of 240 cells were excluded due to unavailability of mutation status. (A) Scatter plot shows the correlation between ARQ 092 and ARQ 751sensitivity and PIK3CA/R1 mutations in comparison to WT-PIK3CA/PIK3R1. (B) Scatter plot shows the correlation between ARQ 092 and ARQ 751 sensitivity and PTEN mutations in comparison to WT-PTEN.</p

Determination of <i>K</i>_<i>d</i> values for the allosteric inhibitors ARQ 092, ARQ 751, and MK-2206 and pathway inhibition by ARQ 092 and ARQ 751 in transfected 293T cells.

<p>(A) The <i>K</i>_<i>d</i> values of ARQ 092, ARQ 751, and MK-2206 were determined against AKT-WT and AKT1-E17K. (B) 293T cells were transiently transfected with pcDNA-E17K-GFP and then treated with various concentrations of ARQ 092 or ARQ 751 for 2 hours. pAKT(S473) and total AKT were assessed. Densitometry analysis was performed and relative pAKT(S473) level was shown as percentage (%) of pAKT/total AKT.</p

Comparison of AKT pathway inhibition by ARQ 092, ARQ 751, MK-2206, and GDC-0068 in MDA-MB-453, NCI-H1650, and KU-19-19 cells.

<p>(A) MDA-MB 453 breast cancer cell line (PIK3CAH1047R; Her2 amp), (B) NCI-H1650 NSCLC cell line (PTEN null), and (C) KU-19-19 bladder cancer cell line (AKT1-E17K&E49K; NRas Q61R) were treated with various concentrations (1 = 0, 2 = 0.012, 3 = 0.037, 4 = 0.11, 5 = 0.33, and 6 = 1 μM) of ARQ 092, ARQ 751, MK-2206 or GDC-0068 for 2 hours. pAKT(S473), pAKT(T308), pPRAS40(T246), pFOXO1(T24) /3a(T36), pGSK3β(S9), pAS160(S318), pBAD(S136), pS6(S235/236) and p4E-BP1(S65) and phospho ERK were assessed by western blot analysis.</p

Plasma membrane translocation experiments conducted in AKT1-WT and E17K mutant forms of AKT1.

<p>NIH 3T3 cells were transiently transfected with either pcDNAAKT-WT-GFP or pcDNA-E17K-GFP, starved for 24 hours and then were treated with (A) DMSO (B) ARQ 092 or ARQ 751 or (C) MK-2206 or GDC-0068 at 1 μM for 2 hours. After cells were stimulated with PDGF-BB at 50 ng/ml for 10 minutes, membrane translocation of AKT1-WT and AKT1-E17K was detected by a fluorescent microscope.</p

Antitumor activity in endometrial PDX mouse xenograft models after treatment with ARQ 092 or ARQ 751.

<p>An endometrial PDX mouse xenograft model, ARQ 092 dosed at 50, 75 or 100 mg/kg or ARQ 751 dosed at 25, 50, 75 mg/kg in a schedule of 5 days on and 2 days off for 20 days. (A) Plot of tumor growth inhibition. (B) Plot of tumor regrowth after removal of drug. (Day 20 to Day 40).</p

ARQ 087 biochemical activity.

<p>ARQ 087 biochemical activity.</p

ARQ 092 and ARQ 751 kinase selectivity against AKT-WT.

<p>The biochemical IC₅₀ values of ARQ 092 against 303 kinases were determined (Carna Biosciences). The percent kinase inhibition of ARQ 751 was determined at a concentration of 5μM against a panel of 245 kinases.</p><p>ARQ 092 and ARQ 751 kinase selectivity against AKT-WT.</p

ARQ 087 inhibits FGFR phosphorylation.

<p>COS-1 cells ectopically expressing FGFR1, FGFR2, FGFR3 or FGFR4 were treated with the indicated concentrations of ARQ 087 for 2 hours followed by stimulation with 100 pM of FGF1/2/7 for 15 minutes. Total and phospho-FGFR was assessed by Western blot analyses. β-Actin was used as a loading control. The EC₅₀ values of individual FGFR family members are shown.</p

Determination of the sensitivity of ARQ 092 and ARQ 751 with different cancer cell types.

<p>Plots of cancer type versus drug sensitivity and number of cell lines per cancer type were prepared for ARQ 092 (left panel) and ARQ 751 (right panel).</p

ARQ 092 inhibits AKT signaling in AN3CA mouse xenografts, <i>In Vivo</i>.

<p>(A) Tumor samples were assessed by IHC for p-AKT(S473) and p-PRAS40(T246). Veh: Vehicle. (B) p-AKT(S473) and (T308), and p-PRAS40(T246) were assessed by western blot analysis from tumor tissues from and AN3CA mouse xenograft after treatment with 100 or 200 mg/kg. The percentage remaining of the phosphorylated proteins is shown and the vehicle group was designated as 100%.</p