American Association for Cancer Research
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15357163mct191021-sup-231430_2_supp_6267050_q9l19q.xlsx (13.26 kB)

Suppl. Table S3 from A Cell-Based MAPK Reporter Assay Reveals Synergistic MAPK Pathway Activity Suppression by MAPK Inhibitor Combination in BRAF-Driven Pediatric Low-Grade Glioma Cells

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posted on 2023-04-03, 18:26 authored by 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, Till Milde

Suppl. Table S3 shows "Screening for false positive results using the reporter assay without ELK-1 binding site in DKFZ-BT66 and BT-40 cells".

Funding

The Brain Tumour Charity

The German National Academic Foundation

PLGA Foundation

History

ARTICLE 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.