Abstract B57: Effects of MEK inhibition alone or in combination with PI3K-mTOR pathway inhibitors in pancreatic ductal adenocarcinoma in vitro and on an innovative ex vivo fresh tumor tissue culture model
Authors: Neuzillet Cindy , Tijeras-Raballand Annemilaï , Sadanandam Anguraj , Bourgoin Pierre , Bourget Philippe , Poudel Pawan , Serova Maria , Gramont Armand De , Ruszniewski Philippe , Paradis Valérie , Raymond Eric , Cros Jérôme , Hammel Pascal 
Published: December 2016

Purpose: Activating KRAS mutations are frequent (&gt;90%) in pancreatic ductal adenocarcinoma (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. While RAS and BRAF mutations, EMT, PI3K-mTOR activation, and pERK inhibition have been suggested as predictive markers for MEKi efficacy, they are not validated in PDAC. We aimed to explore the cellular and molecular effects of MEKi GSK1120212 (GSK212) alone or in combination with PI3K-mTOR inhibitors, in PDAC cell lines and on an innovative ex vivo system.

Methods: GSK212 is an allosteric non-ATP competitive MEKi, everolimus (Ev) 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 assays were performed at different drug concentrations on cultures of fresh tumor tissue slices prepared from patient surgical specimens. Apoptosis and proliferation were assessed by cleaved caspase 3 (Cas-3) and MIB-1 (Ki67) immunostainings, respectively; MEKi sensitivity was defined as cleaved Cas-3 expression in 30% or more of cancer cells. Correlations between protein expressions were explored using linear regression and Pearson’s R2 calculation. Samples will be classified into PDAC subtypes according to Collisson’s, Moffit’s and Bailey’s transcriptomic signatures using non-negative matrix factorization and correlation methods.

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 (CI&lt;1) in MIAPaCa-2 (MEKi sensitive) but not in PANC-1. GSK212 (0.1µM) treatment resulted in pERK extinction in both cell lines but in decreased pS6 expression only in MIAPaCa-2. Combination therapy led to extinction of pERK and pS6 in both cell lines. Apoptosis induction in MIAPaCa-2 was confirmed by PARP cleavage. Eleven tumor specimens were cultured ex vivo. We observed that: (a) GSK212 (0.1µM) treatment for 48h induced significant (≥30%) apoptosis concomitantly with a decrease in pS6 expression in MEKi sensitive tumors; (b) Ev (1µM) or BKM120 (0.1µM) exerted antiproliferative effects but did not induce apoptosis; (c) combinations resulted in higher apoptosis induction associated with a higher decrease in pS6 expression compared to MEKi alone in MEKi sensitive tumors. In sensitive tumors, cleaved Cas-3 induction was inversely correlated with pS6 expression under treatment by MEKi +/- Ev or BKM120. A R2&gt;0.50 discriminated between sensitive and resistant tumors. There was no correlation with RAS/RAF mutation status. Predictive value of transcriptomic signatures for MEKi response will be presented.

Conclusion: Response to combined MEK/mTOR pathway inhibition was not correlated with known biomarkers of response to MEKi. Besides the therapeutic potential of MEK/mTOR pathway inhibition in PDAC, this work provides the first evidence of feasibility of pharmacodynamic biomarker monitoring on fresh PDAC tissue slices.

Citation Format: Cindy Neuzillet, Annemilaï Tijeras-Raballand, Anguraj Sadanandam, Pierre Bourgoin, Philippe Bourget, Pawan Poudel, Maria Serova, Armand De Gramont, Philippe Ruszniewski, Valérie Paradis, Eric Raymond, Jérôme Cros, Pascal Hammel.{Authors}. Effects of MEK inhibition alone or in combination with PI3K-mTOR pathway inhibitors in pancreatic ductal adenocarcinoma in vitro and on an innovative ex vivo fresh tumor tissue culture model. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B57.