A Cell-Based MAPK Reporter Assay Reveals Synergistic MAPK Pathway Activity Suppression by MAPK Inhibitor Combination in BRAF-Driven Pediatric Low-Grade Glioma Cells
- 1 August 2020
- journal article
- research article
- Published by American Association for Cancer Research (AACR) in Molecular Cancer Therapeutics
- Vol. 19 (8), 1736-1750
- https://doi.org/10.1158/1535-7163.MCT-19-1021
Abstract
Pilocytic astrocytomas as well as other pediatric low-grade gliomas (pLGG) exhibit genetic events leading to aberrant activation of the MAPK pathway. The most common alterations are KIAA1549:BRAF fusions and BRAF(V600E) and Nig mutations. Novel drugs targeting the MAPK pathway (MAPKi) are prime candidates for the treatment of these single-pathway diseases. We aimed to develop an assay suitable for preclinical testing of MAPKi in pLGGs with the goal to identify novel MAPK pathway-suppressing synergistic drug combinations. A reporter plasmid (pDIPZ) with a MAPK-responsive ELK-1-binding element driving the expression of destabilized firefly luciferase was generated and packaged using a lentiviral vector system. Pediatric glioma cell lines with a BRAF fusion (DKFZ-BT66) and a BRAF(V600E) mutation (BT-40) background, respectively, were stably transfected. Modulation of the MAPK pathway activity by MAPKi was measured using the luciferase reporter and validated by detection of phosphorylated protein levels. A screening of a MAPKi library was performed, and synergy of selected combinations was calculated. Screening of a MAPKi library revealed MEK inhibitors as the class inhibiting the pathway with the lowest IC(50)s, followed by ERK and next-generation RAF inhibitors. Combination treatments with different MAPKi classes showed synergistic effects in BRAF fusion as well as BRAF(V600E) mutation backgrounds. Here, we report a novel reporter assay for mediumto high-throughput preclinical drug testing in pLGG cell lines. The assay confirmed MEK, ERK, and next-generation RAF inhibitors as potential treatment approaches for KIAA1549:BRAF and BRAF(V600E)-mutated pLGGs. In addition, the assay revealed that combination treatments synergistically suppressed MAPK pathway activity.Other Versions
Funding Information
- The Brain Tumour Charity (GN-000382)
- the German National Academic Foundation
- PLGA Foundation
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