Usta, D;
Sigaud, R;
Buhl, JL;
Selt, F;
Marquardt, V;
Pauck, D;
Jansen, J;
... Milde, T; + view all
(2020)
A Cell-Based MAPK Reporter Assay Reveals Synergistic MAPK Pathway Activity Suppression by MAPK Inhibitor Combination in BRAF-Driven Pediatric Low-Grade Glioma Cells.
Molecular Cancer Research
, 19
(8)
pp. 1736-1750.
10.1158/1535-7163.MCT-19-1021.
Preview |
Text
A cell-based MAPK reporter assay reveals synergistic MAPK pathway activity suppression by MAPK inhibitor combination in BRAF-driven pediatric low-grade glioma cells.pdf - Accepted Version Download (12MB) | Preview |
Abstract
Pilocytic astrocytomas (PAs) as well as other pediatric low-grade gliomas (pLGGs) exhibit genetic events leading to aberrant activation of the MAPK pathway. The most common alterations are KIAA1549:BRAF fusions, BRAFV600E and NF1 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 pre-clinical 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 BRAFV600E 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 screen 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 IC50s, followed by ERK and next-generation RAF inhibitors. Combination treatments with different MAPKi classes showed synergistic effects in BRAF fusion as well as BRAFV600E mutation backgrounds. We here report a novel reporter assay for medium- to high-throughput pre-clinical drug testing in pLGG cell lines. The assay confirmed MEK, ERK and next-generation RAF inhibitors as potential treatment approaches for KIAA1549:BRAF and BRAFV600E mutated pLGGs. In addition, the assay revealed that combination treatments synergistically suppressed MAPK pathway activity.
| Type: | Article |
|---|---|
| Title: | A Cell-Based MAPK Reporter Assay Reveals Synergistic MAPK Pathway Activity Suppression by MAPK Inhibitor Combination in BRAF-Driven Pediatric Low-Grade Glioma Cells |
| Location: | United States |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1158/1535-7163.MCT-19-1021 |
| Publisher version: | https://doi.org/10.1158/1535-7163.MCT-19-1021 |
| Language: | English |
| Additional information: | This version is the author accepted manuscript. For information on re-use, please refer to the publisher's terms and conditions. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health > Developmental Biology and Cancer Dept |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10098855 |
Archive Staff Only
 |
View Item |
