Abstract 4629: Synergistic growth inhibitor effect on a patient derived NF1 pilocytic astrocytoma cell line with the dual mTORC1/2 inhibitor TAK228 and MEK inhibitor trametinib
Authors: Arnold Antje , Yuan Ming , Rodriguez Fausto J. , Eberhart Charles G. , Raabe Eric H. 
Published: August 2018

Abstract
Pediatric low grade glioma (PLGG) is the most common brain tumor of childhood. If the tumor is located in a region of the brain that is not accessible for surgical resection, additional therapies are needed. We and others have identified mTORC and MEK-activation in PLGG. The dual mTORC1/2-inhibitor, TAK228, and the FDA approved MEK-inhibitor, trametinib, are promising candidates for targeted PLGG therapy. We hypothesized that TAK228 and trametinib would show synergistic effects in vitro and in vivo PLGG models. We treated in vitro four different patient-derived PLGG cell lines with TAK228, trametinib, or vehicle control: JHH_NF1_PA1 (NF1 mutation), BT66_SV40/hTERT (BRAFfusion), Res186 and Res259 (both cell lines with mTOR and MAPK activation). In vivo, BT40 (BRAFV600E) tumor cells were investigated with both agents in immunodeficient mice. Cell growth was investigated using MTS-assay compared to vehicle control. Activation of MAPK pathway was detected via Western Blot by phosphorylated ERK compared to total ERK, and β-actin. mTOR pathway was investigated with pAKT, pS6, and p4E-BP1 compared to the total protein amount and β-actin. In all of the cell lines, treatment with TAK228 or trametinib reduces cell growth and proliferation in a dose and time depended manner. We have found a robust synergistic (via Chou-Talalay method) effect for JHH_NF1_PA1, Res186, and Res259 cells in clinically relevant doses of both drugs (5nM, 10nM, 20nM). BT66_SV40/hTERT cells have a significant reduction in cell growth under TAK228 treatment after 4 days by up to 70% (p&lt;0.001; via one-way ANOVA), but not under trametinib treatment. Interestingly for this cell line, the MAPK pathway was inactivated in all tested trametinib doses (≥1nM) and in combination treatment. In all cell lines trametinib treatment leads to a pERK inactivation at low nM levels. TAK228 leads to an inactivation of mTORC1 and mTORC2 in all four tested cells lines. In TAK228 treated cells, there was compensatory activation of pERK, which was reduced when trametinib was added. Apoptosis induction was verified through cleaved PARP via western blot and CC-3 via immunocytochemistry. The combination of TAK228 and trametinib increased apoptosis by up to 127% (p&lt;0.001). After determination the optimal dosing schedule for TAK228 (1mg/kg/every other day), trametinib (3mg/kg/daily) and combination, BT40 transplanted nude mice are investigated for tumor size and survival. Our results show that PLGG-derived cell lines are sensitive to TAK228 and trametinib treatment. The increased MAP kinase activity we identified after TAK228 treatment, suggests a compensatory mechanism that may render these cells especially sensitive to treatment with both TORC1/2 and MEK inhibitors. The ongoing in vivo experimentation will provide a pre-clinical rationale for combination therapy of these agents in PLGG.
Citation Format: Antje Arnold, Ming Yuan, Fausto J. Rodriguez, Charles G. Eberhart, Eric H. Raabe. Synergistic growth inhibitor effect on a patient derived NF1 pilocytic astrocytoma cell line with the dual mTORC1/2 inhibitor TAK228 and MEK inhibitor trametinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4629.