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.
Highlights
Pediatric low-grade gliomas are the most common brain tumors in children [1] and comprise various World Health Organization grade I–II entities, including pilocytic astrocytomas [2]
Metabolic activity was measured using an ATP-based assay in the KIAA1549:BRAF fusion– positive pilocytic astrocytoma cell line DKFZ-BT66 and the BRAFV600E mutation–positive pediatric glioma cell line BT-40 after treatment with various MAPKis for 72 hours (Supplementary Fig. S1)
Discussion Pediatric low-grade gliomas (pLGG) are chronic condition often associated with multiple recurrences and therapeutic interventions in the course of a patient's lifetime, and new effective drug treatments are urgently needed
Summary
Pediatric low-grade gliomas (pLGG) are the most common brain tumors in children [1] and comprise various World Health Organization grade I–II entities, including pilocytic astrocytomas [2]. Complete surgical resection is the therapy of choice, but in case of unresectable tumors, chemo- or radiotherapy is applied (e.g., SIOP LGG 2004 trial, NCT00276640). The overall survival is good, with a 10year survival rate of more than 90% [3, 4]. Recurrences occur frequently, leading to a poor 10-year event-free survival rate of only around 45% in this population [5]. The clinical course can be variable, requiring repeated periods of treatment. This often leads to chronic morbidity of the affected patients with significant neurologic sequelae [6, 7]. In spite of a good overall survival, the management of pLGGs requires novel therapeutic approaches to tackle disease- and therapy-related morbidity
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