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A cell-based MAPK reporter assay reveals synergistic MAPK pathway activity suppression by MAPK inhibitor combination in BRAF-driven pediatric low-grade glioma cells

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 ca...

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Hauptverfasser: Usta, Diren (VerfasserIn) , Sigaud, Romain (VerfasserIn) , Buhl, Juliane (VerfasserIn) , Selt, Florian (VerfasserIn) , Marquardt, Viktoria (VerfasserIn) , Pauck, David (VerfasserIn) , Jansen, Jennifer (VerfasserIn) , Pusch, Stefan (VerfasserIn) , Ecker, Jonas (VerfasserIn) , Hielscher, Thomas (VerfasserIn) , Vollmer, Johanna (VerfasserIn) , Sommerkamp, Alexander C. (VerfasserIn) , Rubner, Tobias (VerfasserIn) , Hargrave, Darren (VerfasserIn) , Tilburg, Cornelis M. van (VerfasserIn) , Pfister, Stefan (VerfasserIn) , Jones, David T. W. (VerfasserIn) , Remke, Marc (VerfasserIn) , Brummer, Tilman (VerfasserIn) , Witt, Olaf (VerfasserIn) , Milde, Till (VerfasserIn)
Dokumenttyp: Article (Journal)
Sprache:Englisch
Veröffentlicht: May 25, 2020
In: Molecular cancer therapeutics
Year: 2020, Jahrgang: 19, Heft: 8, Pages: 1737-1750
ISSN:1538-8514
DOI:10.1158/1535-7163.MCT-19-1021
Online-Zugang:Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1158/1535-7163.MCT-19-1021
Verlag, lizenzpflichtig, Volltext: https://mct.aacrjournals.org/content/early/2020/05/20/1535-7163.MCT-19-1021
Volltext
Verfasserangaben:Diren Usta, Romain Sigaud, Juliane L. Buhl, Florian Selt, Viktoria Marquardt, David Pauck, Jennifer Jansen, Stefan Pusch, Jonas Ecker, Thomas Hielscher, Johanna Vollmer, Alexander C. Sommerkamp, Tobias Rubner, Darren Hargrave, Cornelis M. van Tilburg, Stefan M. Pfister, David T.W. Jones, Marc Remke, Tilman Brummer, Olaf Witt, and Till Milde
Beschreibung
Zusammenfassung: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.</p>
Beschreibung:Gesehen am 12.10.2020
Beschreibung:Online Resource
ISSN:1538-8514
DOI:10.1158/1535-7163.MCT-19-1021

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