LGG-08. TARGETED THERAPY IN BRAFV600E-DRIVEN PEDIATRIC GLIOMAS: UNCOVERING MECHANISMS OF RESISTANCE THROUGH A MULTI-OMIC APPROACH

Abstract

Abstract BRAFV600E is a key oncogenic mutation, detected in both pediatric low-grade gliomas and high-grade gliomas. Targeting the Ras-BRAF-MAPK pathway with BRAFV600E-specific inhibitors, such as Dabrafenib, is a therapeutic strategy increasingly used in clinical practice. However, therapy failure often emerges in patients, and the mechanisms driving drug resistance remain elusive. We apply a multi-omic approach to understand the evolution of BRAFV600E glioma cell lines and ex-vivo patient-derived cultures under BRAF inhibition. To recapitulate the clinical situation and decipher mechanisms of acquired resistance, we chronically expose BRAFV600E-positive glioma cells to a sublethal dose of Dabrafenib (IC20) and generate drug-resistant cell lines. We then profile the kinome (Pamgene microarray) and the transcriptome (bulk RNAseq) of resistant versus wildtype cells: in addition to reactivation of the Ras-BRAF-MAPK pathway, we observe increased activation and expression of PDGFRB and Src family kinases (SFK) in the Dabrafenib-resistant phenotype. To identify and track the longitudinal evolution of drug-resistant states, we further assess the evolution of glioma cell subpopulations under therapeutic pressure and profile consecutively collected samples (day 0; day 2; day 10; day 30 of treatment; and 10 days post-treatment) using single-cell transcriptomics (10x Chromium system, 10x Genomics). By profiling BRAFV600E glioma cells pre-, on- and post-treatment, we find that therapeutic pressure induces changes in cell subpopulations from early stages, and these changes are selected over time. Consistent with our kinome and bulk RNAseq findings, we confirm the role of PDGFRB in driving Dabrafenib resistance in a subset of cells. Furthermore, when off-treatment, the cells recapitulate the untreated status, indicating that resistance is a temporary, drug-dependent, phenomenon relying on the kinome rewiring. By dissecting the pro-survival role of the PDGFR/SFK axis upon Dabrafenib treatment, we will provide valuable mechanistic and therapeutic insights into targeted therapies for children with BRAFV600E gliomas.