Development of a novel assay suitable for pre-clinical testing of MAPK inhibitors in low-grade glioma

Introduction:

Pilocytic astrocytoma (PA), the most frequent pediatric brain tumor, exhibits an activation of MAPK-signaling by genetic aberration in nearly 100% of the cases, with activating KIAA1549:BRAF-fusions being the most frequent alteration of this pathway (60 – 80%). Surgery, chemotherapy and irradiation can cause significant sequelae without being able to control the disease with certainty. As a result, new therapeutic options with better anti-tumor and fewer side effects are urgently required for the treatment of patients with PA. The aim of this project is to develop an assay suitable for pre-clinical testing of MAPK inhibitors in PA with the ultimate goal to generate pre-clinical data for translational purposes.

Material and methods:

A reporter construct (pDIPZ) was cloned for the use in medium- to high-throughput screening of MAPKi libraries, with a MAPK-responsive ELK1-binding element driving the expression of destabilized GFP (desGFP) and destabilized luciferase (desLuc). Experiments are ongoing to transduce pDIPZ into LGG models (e.g. DKFZ-BT66 with a KIAA1549:BRAF fusion activating the MAPK signaling) using a lentiviral vector. GFP and luciferase activity will be determined by flow cytometry and a luminescence plate reader. MAPKi are used to modulate MAPK activity.

Results:

The pDIPZ reporter construct was successfully packaged in the lentiviral vector. Experiments are ongoing evaluating the dynamic range of the reporter construct's signal and modulation thereof by MAPKi.

Discussion:

After successful generation of a MAPK reporter construct for the use in PA model cells, a full characterization of functional properties will be performed, followed by application of the assay in a MAPKi compound library screening.

 

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