Abstract 3108: Inhibition of the mTOR and MAPK pathways suppresses growth, induces apoptosis, and decreases vascularity in pediatric low grade glioma
Authors: Arnold Antje , Harris Lauren , Yuan Ming , Rodriguez Fausto , Eberhart Charles , Raabe Eric 
Published: August 2019

Abstract
Pediatric low-grade glioma (pLGG) is the most common brain tumor in children. We and others have recently identified constitutive activation of the mTOR and MEK-pathway in pLGG. This led us to investigate the antitumor activity of TAK228, a second generation mTORC1/2-inhibitor, and the FDA approved MEK-inhibitor, trametinib, in mono- and combination therapy in pLGG. We examined antitumor activity in five different patient-derived pLGG cell models treated with TAK228, trametinib, and combination: JHH_NF1_PA1NF1mut, BT66_SV40BRAF:KIAA1549, BT40BRAFV600E, Res186PTEN-/- and Res259CDKN2A-/-. In all cell lines, as measured by western blot (WB), trametinib treatment led to suppression of phospho-ERK at low levels (1-5nM). Similarly, TAK228 treatment led to inhibition of mTORC1 and mTORC2 in the low nanomolar range. Treatment with TAK228 or trametinib reduced cell proliferation in a dose and time dependent manner (MTS-assay). The combination treatment exerted a synergistic effect at 5-20nM in JHH_NF1_PA1, Res186, and Res259 cells (by Chou-Talay method). The BT66 cell line had a 70% reduction in cell growth with 10nM TAK228 (p&lt;0.001 by 1-way ANOVA), but not in combination, or mono-treatment with trametinib in clinically relevant dose range. The combination of TAK228 and trametinib increased apoptosis by up to 127% (p&lt;0.001) in Res186, Res259, and JHH_NF1_PA1 cells as measured by cleaved caspase 3 immunocytochemistry. We tested trametinib and TAK228 against the mutant BT40BRAFV600E patient-derived xenograft cell line in immunodeficient mice. The combination of TAK228 with trametinib showed greater antitumor activity than that of either mono-treatment in vivo. BT40 tumor growth was significantly decreased in combination compared to vehicle or either agent alone, (p&lt;0.01). Immunohistochemistry (IHC) imaging showed a significant decrease in proliferation marker Ki67 with combination treatment (p&lt;0.0001), and trametinib mono-treatment (p=0.0466), compared to TAK228 and vehicle. TAK228 and combination treatment decreased the mTORC1 phosphorylation of S6 ribosomal protein (WB & IHC). Combination treatment strikingly reduced the vascularization of the tumor after combination treatment analyzed by VEGF protein expression (WB, p=0.0076), compared to the vehicle control, and mono-treatment. IHC confirmed a reduction in vascularization by CD31 staining. Our study provides evidence that pLGG-derived cell lines in vitro and in vivo are sensitive to mTORC1/2 kinase inhibition and MEK inhibition. Combination treatment with TAK228 and trametinib had a significant anti-tumor activity in vivo shown in survival rate, decreased tumor size, and reduced vascularity. These results provide the first strong rationale for combination therapy with TAK228 and trametinib for clinical consideration in pLGG in the near future.
Citation Format: Antje Arnold, Lauren Harris, Ming Yuan, Fausto Rodriguez, Charles Eberhart, Eric Raabe. Inhibition of the mTOR and MAPK pathways suppresses growth, induces apoptosis, and decreases vascularity in pediatric low grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3108.