Fig 1.
Determination of Kd 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.
(A) The Kd 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.
Fig 2.
Plasma membrane translocation experiments conducted in AKT1-WT and E17K mutant forms of AKT1.
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.
Table 1.
ARQ 092 and ARQ 751 potency.
Table 2.
ARQ 092 and ARQ 751 kinase selectivity against AKT-WT.
Fig 3.
ARQ 092 inhibits AKT signaling in AN3CA mouse xenografts, In Vivo.
(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%.
Fig 4.
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.
(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.
Fig 5.
Determination of the sensitivity of ARQ 092 and ARQ 751 with different cancer cell types.
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).
Fig 6.
PIK3CA/PIK3R1 mutational status is associated with increased ARQ 092 and ARQ 751 sensitivity.
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.
Fig 7.
Antitumor activity in endometrial PDX mouse xenograft models after treatment with ARQ 092 or ARQ 751.
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).
Fig 8.
Antitumor activity in various mouse xenograft models after treatment with ARQ 092 or ARQ 751.
(A) AN3CA xenograft model treated with ARQ 092, (B) AN3CA xenograft model treated with ARQ 751, (C) KPL-4 xenograft model treated with ARQ 092, (D) ZR-75-1 xenograft model treated with ARQ 092, (E) KPL-4 xenograft model treated with ARQ 092 as a single agent or combined with trastuzumab or paclitaxel, (F) A PDX melanoma model (ST052C) that contained Braf V600E and PIK3CA (H1047R) mutations treated with ARQ 092 as a single agent or combined with trametinib.
Fig 9.
Proposed AKT pathway inhibition by ARQ 092 (and ARQ 751).