Abstract 1431: Therapeutic exploitation of mutant BRAF in childhood glioma

Abstract

Abstract Low grade gliomas (LGG) are the most common tumors of the central nervous system in children, accounting for about 50% of all brain tumors. They represent a heterogeneous group of grade I and II tumors according to the WHO classification. Pediatric LGG, is associated with activation of BRAF through a tandem duplication that results in the KIAA1549-BRAF fusion or through an activating point mutation of BRAF (predominantly V600E). More recent findings suggest that the KIAA1549-BRAF fusion is restricted to Grade 1 tumors (70-90%) whereas BRAF(V600E) occurs more frequently in Grade 2-4 tumors (~23%). Findings for BRAF mutation, similar to other tumors with activated BRAF (e.g. melanoma), and the phase I activity of MEK inhibitor in the PBTC-029 protocol, suggest that activated BRAF may provide a validated drug target. Previous studies from our lab indicate that, in the context of mutant BRAF, inhibition of MEK inhibits TORC1 signaling and may induce a ‘BRCA-like’ phenotype, through depletion of FANCD2. Potentially suppression of TORC1 could have effects on other DNA damage response pathways that could compensate for loss of FANCD2. To understand the consequences of MEK inhibition in the context of BRAF abreation in LGG we surveyed DNA repair genes that may be regulated via the MEK/TORC1 pathway in BRAF mutant cells. The objective of this particular study was to examine the mechanism and significance of MEK inhibition to the repair of DNA damage by the homologous recombination (HR) pathway. For this study, we have used glioma cell lines having BRAF(V600E) mutation; the BT40 cell line was developed from patient-derived astrocytoma xenograft (PDX) model in mice in our lab and AM38c1 and DBTRG-05MG cells were generously provided by T. Nicolades. Annexin binding assay demonstrated that MEKi treatment significantly increased the percentage of apoptosis and necrosis of glioma cells. Human DNA repair PCR Array analysis identified MEKi induced down regulation of genes involved in Base Excision Repair (BER), Nucleotide Excision Repair (NER), Mismatch Repair (MMR) and Double-Strand Break (DSB) repair pathways. DSB repair genes were further validated by real time qPCR analysis. Immunoblot analysis of glioma cell lines indicate that MEKi treatment enhanced the gamma-H2AX levels. To elucidate the mechanism of DSB repair pathway in presence of MEKi, clonogenic assay, nuclear foci formation assay, and GFP reporter assay for homologous recombination (HR) and non-homologous end-joining (NHEJ) are ongoing. Collectively, these findings demonstrate for the first time a previously unknown role for MEKi in treatment of glioma cells that involves inhibition of DNA double-strand break repair pathways. Further, we will examine the mechanism and significance of MEK/ERK and TORC1 signaling axis in regulation of DNA repair genes and its effect on LGG. Understanding how MEK plays a role in DNA repair pathways will be useful in maximizing treatment opportunities for childhood glioma. Citation Format: Sudipa Saha Roy, Terry Shackleford, Peter Houghton. Therapeutic exploitation of mutant BRAF in childhood glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1431. doi:10.1158/1538-7445.AM2017-1431