1042 BRAFV600E Mutation Induces TWIST1 Upregulation Associated With Migration and Cerebral Fluid Dissemination in Glioblastoma

INTRODUCTION: BRAFV600E-mutant glioblastoma (GBM) with epithelioid features frequently show cerebrospinal fluid dissemination (CSFD), which results in poor prognoses. BRAF mutation is worth investigating since BRAF or downstream MEK inhibitors show dramatic response for BRAFV600E-mutant GBM including CSFD. This suggests BRAF mutation could play a role for inducing the clinical manifestation of CSFD found in newly diagnosed and recurrent BRAFV600E-mutant GBM. Because, BRAFV600E-mutant GBM often recur with CSFD, elucidating the mechanism by which BRAFV600E-mutant GBM exhibits CSFD is important to improve prognosis. METHODS: We introduced the heterozygous BRAFV600E mutation in human induced pluripotent stem cells (iPSC) in the context of GBM genetic alterations, CDKN2A/2B, and PTEN deletion, TERT promoter mutation and with EGFRvIII overexpression. We implanted these iPSC-derived neural progenitor cells into immunodeficient mouse brains to establish a BRAFV600E-mutant GBM model. At the same time, we created a model with the same genetic background without BRAFV600E. We compared their gene expression, pathology, and migration patterns to identify BRAFV600E specific characteristics. RESULTS: RNA-sequencing revealed BRAFV600E-mutant tumors upregulated migration, and downregulated cell-to-cell adhesion genes. TWIST1 was one of the most upregulated genes. Concomitant with TWIST1 expression we detected downregulation of E-cadherin. The BRAFV600E model histologically showed a cluster-like invasion phenotype, while non-BRAF tumors showed homogenous invasion with typical GBM pathological findings. Immunohistochemical staining showed relatively high TWIST1 expression in cluster-like tumor cells of BRAFV600E tumors. Upon re-engraftment of primary tumor-derived cells, secondary BRAFV600E tumors presented leptomeningeal dissemination. TWIST1 knockdown resulted in a decrease in the migratory ability of our BRAFV600E model in vitro, and is under investigation in vivo. CONCLUSIONS: BRAV600E mutation induces TWIST1 upregulation, and might facilitate migration and CSFD. This work nominates TWIST1 and its regulated genes as targets for BRAFV600E-mutant GBM with CSFD.