Abstract 2341: A study of relationships between signalling output and growth inhibition to personalize combinations of MEK and AKT inhibitors.
Authors: Stewart Adam , Thavasu Parames , de Bono Johann S. , Banerji Udai 
Published: August 2014

Abstract
Purpose
We hypothesized that understanding the relationships between combinatorial inhibition of signalling output and growth in cell line models will help design clinical trials of combinations of MEK and AKT inhibitors which are currently challenged by overlapping toxicity.
Methods
We exposed two BRAF mutant cell lines (WM266.4 and SKMEL-28), two PIK3CA mutant cell lines (MCF-7 and SKOV-3) and a cell line with no BRAF or PIK3CA mutations (Caco2) to increasing concentrations of a MEK inhibitor (MEKi) and an AKT inhibitor (AKTi), PD0325901 and AKT11/2 kinase inhibitor respectively, for 24hrs. ELISAs were used to quantify signalling output downstream of MEK and AKT i.e. p-ERK and p-S6 respectively. Concentrations of MEKi and AKTi that inhibited phosphorylation of 0, 25, 50, 75 and 100% of ERK and S6 respectively were calculated in these models. The cell line panel was then exposed to different functionally inhibitory concentrations of both MEKi and AKTi in combination and effects on growth inhibition was quantified by 96hr sulforhoamine B assays.
Results
In BRAF mutant lines WM266.4 and SKMEL-28, the growth inhibition (GI) by maximally inhibiting p-ERK by MEKi was 83.2 % and 64 % respectively. There was no statistical difference on GI by additionally maximally inhibiting p-S6 in addition to maximal p-ERK inhibition in these cell lines (86.9 vs 83.2 % p=0.34 and 74% vs 64%, p= 0.27 respectively). In PIK3CA mutant cell lines MCF-7 and SKOV3 the GI by maximally inhibiting p-S6 by AKTi was 93 % and 69.4 % respectively. There was no statistical difference on growth inhibition by additionally maximally inhibiting p-ERK in addition to maximal p-S6 inhibition in these cell lines (94.6 vs 93 % p=0.34 and 79.6% vs 69.4%, p= 0.05 respectively). In Caco2 which does not harbour either a BRAF or PIK3CA mutation, the GI by maximally inhibiting p-ERK was 48.9 % and additionally maximally inhibiting p-S6 caused a significantly improved growth inhibition (78.1 vs 48.9%, p= 0.0007). GI by maximally inhibiting p-S6 was 64.1% and additionally maximally inhibiting p-ERK caused a statistically significant improved growth inhibition (78.1 vs 64.1%, p=0.0203) in this cell line model. In Caco2 cells the incremental significant GI caused by the addition of AKTi and MEKi started at concentrations that inhibited 50% of p-S6 and 75% of p-ERK respectively.
Conclusions
In the cell line panel studied, significant additional growth inhibition by maximally inhibiting p-ERK and p-S6 in combination occurred in BRAF and PIK3CA wt cells and not in those that harboured activating mutations in these genes. Titration of the degree of p-ERK and p-S6 inhibition needed to maximally inhibit growth in combination may obviate the necessity to completely inhibit both p-ERK and p-S6. These findings have important implications in the way we design phase I/II trials of combinations of MEK and AKT inhibitors.
Citation Format: Adam Stewart, Parames Thavasu, Johann S. de Bono, Udai Banerji. A study of relationships between signalling output and growth inhibition to personalize combinations of MEK and AKT inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2341. doi:10.1158/1538-7445.AM2013-2341