Abstract 2618: PTEN loss as a putative biomarker of synergistic growth inhibitory activity of combined MEK/ERK and PI3K/mTOR pathway blockade
Authors: Ciuffreda Ludovica , Falcone Italia , Matteoni Silvia , Sacconi Andrea , Malusa Federico , Luca Teresa D. , Incani Ursula Cesta , Curatolo Anais Del , Konopleva Marina , Andreeff Michael , Eramo Adriana , Maria Ruggero D. , Bufalo Donatella Del , Cognetti Francesco , Milella Michele 
Published: May 2015

Abstract
Background. Complex feedback loops and crosstalk between the MEK/ERK and PI3K/mTOR pathways, thus molecular predictors of sensitivity/resistance and synergistic/antagonistic interactions are urgently needed for the effective clinical development of combination strategies. Here we hypothesize that PTEN status critically modulates the growth inhibitory activity of single and combined MEK and PI3K/mTOR inhibition. Methods. Molecular and functional effects of single and combined MEK (trametinib, T) and mTOR (everolimus, E) blockade were assessed in a panel of 29 cancer cell lines with different molecular ‘drivers’. Pharmacologic interactions were analyzed by conservative isobologram analysis. PTEN role was mechanistically assessed by either silencing PTEN expression by shRNA or overexpressing a functional PTEN protein by stable transfection. Specific proteins and their phosphorylation states were analyzed using Kinexus Antibody Microarrays. Results. In the cell line panel analyzed, the presence of a wt-PTEN was the only significant predictor of sensitivity to T (p=0.001), while BRAF mutations were significantly related to E resistance (p=0.015). PTEN silencing slightly increased resistance to T in the wt-PTEN melanoma cell line M14; conversely, wt-PTEN overexpression in the PTEN-del WM115 cell line, dramatically increased sensitivity to T. E-induced growth inhibition was not significantly affected. Combined MEK and mTOR inhibition resulted in a striking growth inhibitory synergism in cells lacking PTEN expression (CI: 0.4-0.0005), but not in those with an intact PTEN (CI: 1.2-107; p=0.001). However, PTEN silencing restored growth inhibitory synergism of combined T and E in M14 cells (CI:0.36); similarly, the slope of the CI/fraction affected curve was dramatically altered in PTEN-overexpressing WM115 cells, as compared to their control-transfected counterpart, again indicating that PTEN expression/function causally influences functional response to combined MEK and mTOR inhibition. Similar results in terms of synergistic/antagonistic pharmacologic interactions were obtained when either double PI3K/mTOR kinase inhibitors (PF-5212384) or AKT allosteric inhibitors (MK-2206) were used to block the PI3K/mTOR pathway in combination with T. Proteomic analysis indicated that a greater modification of protein expression/phosphorylation profiles in response to MEK or combined MEK/mTOR inhibition occurs in cells lacking a functional PTEN, as compared to the wt-PTEN-expressing ones; preliminary analysis suggests that AKT phosphorylation and NF-κB activation maybe be crucial mediators of synergistic growth-inhibitory interactions occurring with combined treatment. Conclusions. PTEN loss may constitute a suitable genetic/molecular marker of synergistic activity interactions between MEK/ERK and PI3K/mTOR pathway inhibitors.
Citation Format: Ludovica Ciuffreda, Italia Falcone, Silvia Matteoni, Andrea Sacconi, Federico Malusa, Teresa De Luca, Ursula Cesta Incani, Anais Del Curatolo, Marina Konopleva, Michael Andreeff, Adriana Eramo, Ruggero De Maria, Donatella Del Bufalo, Francesco Cognetti, Michele Milella. PTEN loss as a putative biomarker of synergistic growth inhibitory activity of combined MEK/ERK and PI3K/mTOR pathway blockade. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2618. doi:10.1158/1538-7445.AM2014-2618