Abstract 2677: Decrease in phospho-S6 expression under MEK inhibitor (MEKi) treatment as a potential predictive biomarker of response to MEKi alone or in combination with PI3K-mTOR pathway inhibitor in pancreatic adenocarcinoma in vitro and ex vivo models
Authors: Neuzillet Cindy , Tijeras-Raballand Annemilaï , Cros Jérôme , Bourgoin Pierre , Bourget Philippe , Serova Maria , De Gramont Armand , Faivre Sandrine , Raymond Eric , Ruszniewski Philippe , Hammel Pascal 
Published: August 2015

Context: Activating KRAS mutations are frequent (&gt;90%) in PDAC and drive downstream deregulation of both MAPK and PI3K-mTOR pathways. MEK inhibitors (MEKi) are under clinical evaluation in PDAC, in combination with other agents including PI3K-mTOR inhibitors. RAS and BRAF mutations, EMT, PI3K-mTOR activation, and pERK inhibition under treatment have been suggested as predictive markers for MEKi, but remain unvalidated in PDAC. We explored the cellular and molecular effects of MEKi GSK1120212 (GSK212) alone or in combination with PI3K-mTOR inhibitors, in PDAC cell lines and ex vivo culture system.

Material and methods: GSK212 is an allosteric non-ATP competitive MEKi. Everolimus (Ev) is a mTORC1 inhibitor and BKM120 a pan-class PI3K inhibitor. Effects on proliferation were evaluated by MTT assay. Combinations were analyzed by the Chou-Talalay method. Protein expression was assessed by Western blot. Ex vivo drug evaluation used selected concentrations of drugs on cultures of fresh tissue slices from patient surgical specimens. Apoptosis and proliferation were evaluated by caspase 3 and MIB-1 (Ki67) immunostaining, respectively. Correlations between protein expressions were explored using linear regression.

Results: MIAPaCa-2 and PANC-1 are two mesenchymal KRAS mutated/BRAF wild-type PDAC cell lines with very different response to GSK212, MIAPaCa-2 being sensitive (72h-IC50 = 0.009μM) and PANC-1 resistant (72h-IC50 = 33μM). MIAPaCa-2 was more sensitive than PANC-1 to Ev (IC50 = 23.3μM vs 47.0μM) and BKM120 (IC50 = 4.19μM vs 31.6μM). Combination of GSK212 and Ev or BKM120 for 72h resulted in synergistic effects in MIAPaCa-2 sensitive cell line but not in PANC-1. GSK212 (0.1μM) treatment resulted in pERK extinction in both cell lines but in decreased pS6 expression in MIAPaCa-2 sensitive cell line only. Combination therapy resulted in extinction of pERK and pS6 expression in both cell lines. Apoptosis induction in MIAPaCa-2 was confirmed by PARP cleavage from 48h. Two tumor specimens were cultured ex vivo: (a) GSK212 (0.1 μM) treatment for 48h induced apoptosis (caspase 3 expression) concomitantly with a decrease in pS6 expression; (b) Ev (1μM) or BKM120 (0.1μM) exerted antiproliferative effects but did not induce apoptosis; (c) combinations resulted in higher caspase 3 expression associated with a higher decrease in pS6 expression compared to MEKi alone. Linear regression showed significant correlation between caspase 3 and pS6 (R2 = 0.5798, p = 0.0282, and R2 = 0.9423, p = 0.013, for tumor 1 and 2, respectively).

Conclusion: Response to combined MEK/mTOR pathway inhibition was not correlated with “classical” biomarkers of response to MEKi. Decrease in pS6 expression under MEKi treatment may be an early monitoring biomarker of response to MEKi and MEK/mTOR pathway inhibitor combination.

Citation Format: Cindy Neuzillet, Annemilaï Tijeras-Raballand, Jérôme Cros, Pierre Bourgoin, Philippe Bourget, Maria Serova, Armand De Gramont, Sandrine Faivre, Eric Raymond, Philippe Ruszniewski, Pascal Hammel. Decrease in phospho-S6 expression under MEK inhibitor (MEKi) treatment as a potential predictive biomarker of response to MEKi alone or in combination with PI3K-mTOR pathway inhibitor in pancreatic adenocarcinoma in vitro and ex vivo models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2677. doi:10.1158/1538-7445.AM2015-2677