TY  - JOUR
AU  - Usta, Diren
AU  - Sigaud, Romain
AU  - Buhl, Juliane L
AU  - Selt, Florian
AU  - Marquardt, Viktoria
AU  - Pauck, David
AU  - Jansen, Jennifer
AU  - Pusch, Stefan
AU  - Ecker, Jonas
AU  - Hielscher, Thomas
AU  - Vollmer, Johanna
AU  - Sommerkamp, Alexander C
AU  - Rubner, Tobias
AU  - Hargrave, Darren
AU  - van Tilburg, Cornelis Martinus
AU  - Pfister, Stefan M
AU  - Jones, David T W
AU  - Remke, Marc
AU  - Brummer, Tilman
AU  - Witt, Olaf
AU  - Milde, Till
TI  - A cell-based MAPK reporter assay reveals synergistic MAPK pathway activity suppression by MAPK inhibitor combination in BRAF-driven pediatric low-grade glioma cells.
JO  - Molecular cancer therapeutics
VL  - 19
IS  - 8
SN  - 1538-8514
CY  - Philadelphia, Pa.
PB  - AACR
M1  - DKFZ-2020-01284
SP  - 1736-1750
PY  - 2020
N1  - 2020 Aug;19(8):1736-1750#EA:B310#LA:B310#
AB  - 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.
LB  - PUB:(DE-HGF)16
C6  - pmid:32451331
DO  - DOI:10.1158/1535-7163.MCT-19-1021
UR  - https://inrepo02.dkfz.de/record/156979
ER  -