PDCT-02. COMBINED INHIBITION OF MTORC1/C2 AND MEK PATHWAY IS SYNERGISTIC IN PRECLINICAL TESTING OF PEDIATRIC LOW-GRADE GLIOMA INCLUDING A NOVEL PATIENT-DERIVED NF1 PILOCYTIC ASTROCYTOMA CELL LINE

摘要

Pediatric low-grade glioma (PLGG) is the most common brain tumor of childhood. We and others have identified mTOR and MEK-pathway activation in PLGG. The dual mTORC1/2-inhibitor, TAK228, and the FDA approved MEK-inhibitor, trametinib, are promising candidates for targeted PLGG therapy. We treated five different patient-derived PLGG cell models with TAK228 and/or trametinib: JHH_NF1_PA1 (NF1 mutation), BT66_SV40/hTERT (BRAF:KIAA1549 fusion), BT40 (BRAFV600E) Res186 (PTEN deletion) and Res259 (PDGFRα amplification and CDKN2A deletion). Treatment with TAK228 or trametinib reduces cell proliferation in a dose and time depended manner investigated via MTS-assay. Both drugs exert a synergistic effect at 5-20nM in JHH_NF1_PA1, Res186, and Res259 cells as calculated by the method of Chou-Talay. BT66_SV40/hTERT cells have a 70% reduction in cell growth with 10nM TAK228 (p < 0.001 by ANOVA) but not in combination with trametinib. In all cell lines trametinib treatment leads to a pERK inactivation at low nM levels. TAK228 treatment leads to inactivation of mTORC1 and mTORC2. Apoptosis induction was examined through cleaved PARP via western blot and CC-3 via immunocytochemistry. The combination of TAK228 and trametinib increased apoptosis by up to 127% (p < 0.001) in Res186, Res259, and JHH_NF1_PA1 cells. We tested trametinib and TAK228 against the BRAFV600E mutant BT40 patient derived xenograft in immunodeficient mice. In combination TAK228 and trametinib decreased significantly BT40 tumor growth compared to vehicle or either agent alone, (p < 0.01 by ANOVA). Striking was the reduced vascularization of the tumor tissue after combination treatment compare the vehicle control and single agent treatment. Vascularization differences will be evaluated with immunohistochemistry staining for PDGFα and PECAM-1. Our results show that PLGG-derived cell lines are sensitive to TORC1/2 kinase inhibition and MEK inhibition. Further, our in vivo experimentation provides the first strong rationale for combination therapy of these agents in aggressive PLGG.