%0 Journal Article
%A Usta, Diren
%A Sigaud, Romain
%A Buhl, Juliane L
%A Selt, Florian
%A Marquardt, Viktoria
%A Pauck, David
%A Jansen, Jennifer
%A Pusch, Stefan
%A Ecker, Jonas
%A Hielscher, Thomas
%A Vollmer, Johanna
%A Sommerkamp, Alexander C
%A Rubner, Tobias
%A Hargrave, Darren
%A van Tilburg, Cornelis Martinus
%A Pfister, Stefan M
%A Jones, David T W
%A Remke, Marc
%A Brummer, Tilman
%A Witt, Olaf
%A Milde, Till
%T A cell-based MAPK reporter assay reveals synergistic MAPK pathway activity suppression by MAPK inhibitor combination in BRAF-driven pediatric low-grade glioma cells.
%J Molecular cancer therapeutics
%V 19
%N 8
%@ 1538-8514
%C Philadelphia, Pa.
%I AACR
%M DKFZ-2020-01284
%P 1736-1750
%D 2020
%Z 2020 Aug;19(8):1736-1750#EA:B310#LA:B310#
%X 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.
%F PUB:(DE-HGF)16
%9 Journal Article
%$ pmid:32451331
%R 10.1158/1535-7163.MCT-19-1021
%U https://inrepo02.dkfz.de/record/156979