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  3. A Cell-Based MAPK Reporter Assay Reveals Synergistic MAPK Pathway Activity Suppression by MAPK Inhibitor Combination in BRAF-Driven Pediatric Low-Grade Glioma Cells

A Cell-Based MAPK Reporter Assay Reveals Synergistic MAPK Pathway Activity Suppression by MAPK Inhibitor Combination in BRAF-Driven Pediatric Low-Grade Glioma Cells

  • Mol Cancer Ther. 2020 Aug;19(8):1736-1750. doi: 10.1158/1535-7163.MCT-19-1021.
Diren Usta 1 2 3,  Romain Sigaud 1 2,  Juliane L Buhl 1 2 4,  Florian Selt 1 2 3,  Viktoria Marquardt 5,  David Pauck 5,  Jennifer Jansen 6,  Stefan Pusch 7 8,  Jonas Ecker 1 2 3,  Thomas Hielscher 9,  Johanna Vollmer 1 2,  Alexander C Sommerkamp 1 4 10,  Tobias Rubner 11,  Darren Hargrave 12,  Cornelis M van Tilburg 1 2 3,  Stefan M Pfister 1 3 13,  David T W Jones 1 10,  Marc Remke 5,  Tilman Brummer 6,  Olaf Witt 1 2 3,  Till Milde 14 2 3
Affiliations

Affiliations

  • 1 Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany.
  • 2 Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
  • 3 KiTZ Clinical Trial Unit (ZIPO), Department of Pediatric Hematology and Oncology, Heidelberg University Hospital, Heidelberg, Germany.
  • 4 Faculty of Biosciences, Heidelberg University, Heidelberg, Germany.
  • 5 Department of Pediatric Oncology, Hematology, and Clinical Immunology, Medical Faculty, University Hospital Düsseldorf, Germany, and Department of Pediatric Neuro-Oncogenomics, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
  • 6 Institute of Molecular Medicine and Cell Research (IMMZ), Faculty of Medicine, University of Freiburg, Freiburg, Germany, Centre for Biological Signalling Studies BIOSS, University of Freiburg, Comprehensive Cancer Center Freiburg (CCCF) and German Consortium for Translational Cancer Research (DKTK), Freiburg, and German Cancer Research Center (DKFZ), Heidelberg, Germany.
  • 7 Department of Neuropathology, Heidelberg University Hospital, Heidelberg, Germany.
  • 8 Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
  • 9 Division of Biostatistics, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
  • 10 Pediatric Glioma Research Group, German Cancer Research Center (DKFZ), Heidelberg, Germany.
  • 11 Flow Cytometry Unit, German Cancer Research Center (DKFZ), Heidelberg, Germany.
  • 12 Neurooncology and Experimental Therapeutics, Great Ormond Street Hospital for Children, London, United Kingdom.
  • 13 Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
  • 14 Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany. [email protected].
PMID: 32451331 DOI: 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 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 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 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 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 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. Here, we report a novel reporter assay for medium- to 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 BRafV600E-mutated pLGGs. In addition, the assay revealed that combination treatments synergistically suppressed MAPK pathway activity.

Figures
Products
  • Cat. No.
    Product Name
    Description
    Target
    Research Area
  • HY-18972
    99.94%, BRAF Inhibitor