Abstract
Pleomorphic xanthoastrocytoma (PXA) is a type of astrocytic glial neoplasm classified as WHO grade II and anaplastic PXA as WHO grade III. These tumors are known to commonly harbor BRAF (p.V600E, c.1799T>A) mutation leading to dysregulation of mitogen-activated protein kinase (MAPK) pathway. Using targeted next-generation sequencing analysis, two gene fusions ATG7-RAF1/CRAF and NRF1-BRAF were identified in BRAF p.V600 wild-type anaplastic PXA patients. We sought to functionally characterize above two fusions using the heterologous cell model systems, which stably expressed ATG7-RAF1 and NRF1-BRAF. Immunoblot analysis showed increased phosphorylation of PI3K/mTOR and MAPK pathways in both the cell lines expressing ATG7-RAF1 and NRF1-BRAF fusions. In protein-protein interaction analysis of ATG7-RAF1 and NRF1-BRAF by co-immunoprecipitation we found both fusions to dimerize suggesting activation of PI3K/mTOR and MAPK pathways due to dimerization. ATG7-RAF1 and NRF1-BRAF preferred homo dimerization compared to hetero dimerization to the full form CRAF and BRAF partners. Soft agar proliferation assay showed significant increase in colony formation in both the fusion expressing cell lines compared to the control (p<0.0005) which was significantly inhibited by PLX8394 RAF inhibitor in the NRF1-BRAF fusion but not in the ATG7-RAF1. By combinatorial inhibition of both MAPK and PI3K/mTOR using trametinib (MEK inhibitor) and everolimus (mTOR inhibitor) we found suppressed phospho-ERK and phospho-S6 expression in both fusions. In summary, our preliminary data suggests differential responsiveness to targeted therapies in the BRAF and CRAF fusions. Our study suggests that further functional characterization of BRAF and CRAF fusions may provide a better therapeutic intervention for the PXA patients.