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

Abstract

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, BRAF V600E and NF1 mutations. Novel drugs targeting the MAPK pathway (MAPKi) are prime candidates for the treatment of these singlepathway 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 BRAF V600E 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 BRAF V600E 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 BRAF V600E mutated pLGGs. In addition, the assay revealed that combination treatments synergistically suppressed MAPK pathway activity.

Cerebrospinal Fluid Penetration and Combination Therapy of Entrectinib for Disseminated ROS1/NTRK-Fusion Positive Pediatric High-Grade Glioma

“…In contrast, the effect of trametinib alone in the short-term exposure experiment showed IC 50 values exceeding 10 µM. Interestingly, in the MAPK pathway-activated pLGG IC 50 values for trametinib are in the low nm range suggesting activation of alternative pathways such as mTOR in fusion-positive pHGG [ 44 ]. Moreover, in the long-term exposure, already 1 µM of trametinib led to a distinct decrease in cell viability and markedly exceeded the effect of entrectinib alone.…”

Response to trametinib treatment in progressive pediatric low-grade glioma patients

“…The universal activation of the MAPK pathway makes pLGGs uniquely suited for targeted treatment approaches. Pre-clinical data points to robust inhibition of the MAPK pathway by MEK inhibitors (MEKi) such as selumetinib and trametinib in pLGG cells with KIAA1549:BRAF-fusion or BRAF V600E mutation [15,16]. Selumetinib has been studied in a series of clinical trials: 20% of patients with recurrent or progressive pLGG showed sustained partial response in a phase I clinical trial [17] and 36-40% of progressive pLGG patients experienced a sustained partial response in a phase II trial [18].…”

High-Throughput profiling of SH2 domains using Affimer reagents: unravelling protein interaction networks

“…This intracellular functionality was utilised to screen the SH2 A mer toolbox in a pERK translocation assay. Popular methods for investigating MAPK signalling include western blotting and SRE luciferase assays, which can be slow and labour intensive [39] so high-content imaging was used together with GFP-tagged SH2-binding A mer constructs yielding a simple, time e cient and sensitive assay. This approach is easily modi ed to screen different endpoints, for example modulation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway by using AKT phosphorylation as the measurable endpoint [40], or any other phenotypic change that can be imaged or measured.…”