Abstract 703: Combinatory treatment for pediatric low grade glioma with the dual mTORC1/2 inhibitor TAK228 and MEK inhibitor Trametinib
Authors: Arnold Antje , Rodriguez Fausto , Eberhart Charles George , Raabe Eric Hutton 
Published: July 2017

Abstract
Pediatric low grade glioma (PLGG) is one of the most common childhood tumors. If the tumor is located in a region of the brain that is not accessible for surgical resection or if the tumor recurs after surgery, additional therapies are needed. Recent studies highlighted the important role of mTORC and MEK-activation in PLGG. The dual mTORC1/2-inhibitor, TAK-228, and the FDA approved MEK-inhibitor, Trametinib, have good brain penetration and are promising candidates for targeted therapy PLGG. We hypothesized that TAK-228 and Trametinib would show synergistic effects both in vitro and in vivo in PLGG models. We treated the PLGG derived cell lines Res186 and Res259 with TAK-228, Trametinib, and vehicle. Cell growth was investigated using MTT-assay over different days and compared to the treatment with the vehicle. DNA replication was measured through bromodeoxyuridine (BrdU) incorporation assay and apoptosis was evaluated through cleaved caspase 3 (CC-3) staining. Cells were analyzed and counted with ImageJ. Activation of MAPK pathway was detected via Western Blot by phosphorylated pERK compared to total pERK, and β-actin. Treatment of Res186 and Res259 with TAK-288 or Trametinib reduces cell growth and proliferation in a dose and time depended manner. Res186 have a significant reduction in cell growth after 4 days treatment (TAK-288: 20nM, **p&lt;0.01; Trametinib: 20nM, **p&lt;0.01). Res259 showed a significant cell reduction at the same time point, but with a higher drug dose (TAK-288: 50nM, *p&lt;0.05; Trametinib: 50nM, **p&lt;0.01). IC50 values for TAK-288 was 15nM for Res186 and 20nM for Res259 cells on day 4. IC50 values for Trametinib was 50nM for Res186 and 100nM for Res259 cells. Staining for CC-3 showed a significant increase for apoptosis in Res259 cells after treatment with TAK-288 or Trametinib (**p &lt; 0.01). No positive CC-3 staining was detected in Res186 cells after drug treatment. MAPK pathway was activated in a dose-dependent manner as determined by phosphorylated pERK Western Blot after TAK-288 and inactivated after Trametinib treatment. No change in total ERK concentration was detected, suggesting that cells attempt to compensate for loss of mTOR signaling by upregulating MAP kinase signaling. Our preliminary results show that the PLGG-derived cell lines are sensitive to TAK-228 and Trametinib treatment. All cell lines showed decreased proliferation at various doses of either inhibitor. The increased MAP kinase activity we identified after TAK-228 treatment suggests a compensatory mechanism that may render these cells especially sensitive to treatment with both TORC1/2 and MEK inhibitors. We will now investigate both drugs in vivo. Evidence of activity in murine models will be necessary to provide a pre-clinical rationale for combination therapy of these agents in aggressive PLGG.
Citation Format: Antje Arnold, Fausto Rodriguez, Charles George Eberhart, Eric Hutton Raabe. Combinatory treatment for pediatric low grade glioma with the dual mTORC1/2 inhibitor TAK228 and MEK inhibitor Trametinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 703. doi:10.1158/1538-7445.AM2017-703