Abstract 3565: Abrogation of the paradoxical activation of the MAPK pathway following RAF inhibition by ARQ 680, a potent inhibitor of the RAF family of serine/threonine kinases
Authors: Chen Chang-Rung , Szwaya Jeffrey , Lapierre Jean-Marc , Ashwell Mark A. , France Dennis S. 
Published: June 2012

Abstract
Recent reports have documented the paradoxical activation of the MAPK pathway in response to pharmacological RAF inhibition in cells bearing either wild-type (wt) or mutant RAS, as evidenced by hyperphosphorylation of ERK in human tumor cells exposed to BRAF inhibitors in vitro. ARQ 680 is a pan-RAF kinase inhibitor (IC50 values of 2.6, 2.7, & 7.3 nM respectively for BRAF, mutant BRAF(V600E), and CRAF) which is the active moiety of the highly soluble pro-drug ARQ 736, whose biological properties and anti-tumor activities have been described previously. Here we compared the activity of ARQ 680 and PLX 4032, the latter a structurally distinct pan-RAF inhibitor currently in advanced clinical trials, in various cell lines with various RAS/RAF genotypes. ARQ 680 and PLX 4032 show comparable degrees of selectivity across the human kinome, with ARQ 680 inhibiting 11 other kinases within 100-fold of its activity against BRAF. Interestingly, there is no discernable overlap between the off-target kinases of ARQ 680 and PLX 4032. In human melanoma cell lines (A375, SK-MEL-28) bearing mutant BRAF(V600E), both compounds exhibited comparable concentration-dependent inhibition of phospho-ERK as shown by quantitative western blotting (EC50 ∼ 10 – 30 nM). In 3 colon cancer cell lines harboring a mutant kras gene (HCT-116, SW480, & DLD-1), both ARQ 680 and PLX 4032 induced robust phosphorylation of ERK (1.8 to 5-fold increases) at varying concentrations, with ARQ 680 exhibiting the phenomenon at lower concentrations than PLX 4032. Importantly, ARQ 680 induced a concentration-dependent decrease in, and ultimately a nearly complete inhibition of, ERK phosphorylation at concentrations of 3 μM and above in all KRAS-mutant cell lines. In contrast, PLX 4032 exerted no inhibition of ERK phosphorylation below untreated controls at concentrations up to 100 μM, with the exception of the SW480 cell line, where the elevated p-ERK levels decreased to control levels at 10 to 100 μM. In an NRAS-mutant melanoma cell line (SK-MEL-2), ARQ 680 exerted a concentration-dependent inhibition of ERK phosphorylation (EC50 ∼ 400 nM) with no paradoxical activation of p-ERK at any concentration, while PLX 4032 induced an increase in p-ERK at concentrations up to 10 μM. Finally, in a wt RAS/RAF colon epithelial cell line (NCM-460), both ARQ 680 and PLX 4032 doubled p-ERK levels at sub-micromolar concentrations, with ARQ 680 exhibiting a concentration-dependent abrogation of this effect at concentrations between 1 and 10 μM while PLX 4032 sustained a 2-fold increase in p-ERK at concentrations as high as 100 μM. In summary, we have delineated the pharmacodynamic profile for a novel RAF inhibitor, ARQ 680. The molecular mechanisms by which ARQ 680 inhibits pERK in KRAS and NRAS mutant cell lines are under investigation and may be due in part to the suite of kinases inhibited by ARQ 680 in cells.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3565. doi:10.1158/1538-7445.AM2011-3565