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Molecular Cancer Therapeutics
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Models and Technologies

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

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
Diren Usta
1Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany.
2Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
3KiTZ Clinical Trial Unit (ZIPO), Department of Pediatric Hematology and Oncology, Heidelberg University Hospital, Heidelberg, Germany.
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  • ORCID record for Diren Usta
Romain Sigaud
1Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany.
2Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
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Juliane L. Buhl
1Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany.
2Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
4Faculty of Biosciences, Heidelberg University, Heidelberg, Germany.
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Florian Selt
1Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany.
2Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
3KiTZ Clinical Trial Unit (ZIPO), Department of Pediatric Hematology and Oncology, Heidelberg University Hospital, Heidelberg, Germany.
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Viktoria Marquardt
5Department 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.
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David Pauck
5Department 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.
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Jennifer Jansen
6Institute 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.
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Stefan Pusch
7Department of Neuropathology, Heidelberg University Hospital, Heidelberg, Germany.
8Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
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Jonas Ecker
1Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany.
2Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
3KiTZ Clinical Trial Unit (ZIPO), Department of Pediatric Hematology and Oncology, Heidelberg University Hospital, Heidelberg, Germany.
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Thomas Hielscher
9Division of Biostatistics, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
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Johanna Vollmer
1Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany.
2Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
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Alexander C. Sommerkamp
1Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany.
4Faculty of Biosciences, Heidelberg University, Heidelberg, Germany.
10Pediatric Glioma Research Group, German Cancer Research Center (DKFZ), Heidelberg, Germany.
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Tobias Rubner
11Flow Cytometry Unit, German Cancer Research Center (DKFZ), Heidelberg, Germany.
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Darren Hargrave
12Neurooncology and Experimental Therapeutics, Great Ormond Street Hospital for Children, London, United Kingdom.
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Cornelis M. van Tilburg
1Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany.
2Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
3KiTZ Clinical Trial Unit (ZIPO), Department of Pediatric Hematology and Oncology, Heidelberg University Hospital, Heidelberg, Germany.
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Stefan M. Pfister
1Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany.
3KiTZ Clinical Trial Unit (ZIPO), Department of Pediatric Hematology and Oncology, Heidelberg University Hospital, Heidelberg, Germany.
13Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
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David T.W. Jones
1Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany.
10Pediatric Glioma Research Group, German Cancer Research Center (DKFZ), Heidelberg, Germany.
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Marc Remke
5Department 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.
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Tilman Brummer
6Institute 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.
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Olaf Witt
1Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany.
2Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
3KiTZ Clinical Trial Unit (ZIPO), Department of Pediatric Hematology and Oncology, Heidelberg University Hospital, Heidelberg, Germany.
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Till Milde
1Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany.
2Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
3KiTZ Clinical Trial Unit (ZIPO), Department of Pediatric Hematology and Oncology, Heidelberg University Hospital, Heidelberg, Germany.
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  • For correspondence: t.milde@kitz-heidelberg.de
DOI: 10.1158/1535-7163.MCT-19-1021 Published August 2020
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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.

This article is featured in Highlights of This Issue, p. 1573

Footnotes

  • Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

  • Mol Cancer Ther 2020;19:1736–50

  • Received November 6, 2019.
  • Revision received April 11, 2020.
  • Accepted May 15, 2020.
  • Published first May 25, 2020.
  • ©2020 American Association for Cancer Research.
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Molecular Cancer Therapeutics: 19 (8)
August 2020
Volume 19, Issue 8
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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
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
Mol Cancer Ther August 1 2020 (19) (8) 1736-1750; DOI: 10.1158/1535-7163.MCT-19-1021

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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
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
Mol Cancer Ther August 1 2020 (19) (8) 1736-1750; DOI: 10.1158/1535-7163.MCT-19-1021
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