Abstract 3457: The KIAA1549-BRAF fusion oncogene in pediatric low-grade gliomas undergoes in vitro paradoxical activation in response to RAF specific inhibition

Abstract

Abstract Low-grade gliomas are the most common tumors of the central nervous system in children. We recently described a 7q34 duplication resulting in a novel KIAA1549-BRAF fusion, that includes the C-terminus BRAF kinase domain, exclusively in the majority of pediatric low-grade gliomas. BRAF activating mutations have been well described in human malignancies; however, little is known about KIAA1549. In silico analysis of the fusion gene predicts two putative transmembrane domains in the N-terminus segment of KIAA1549. Clones of the fusion were created, including the long and short forms found in pediatric tumors and a truncated version lacking a transmembrane domain. NIH/3T3 stable cell lines for each clone were then generated using Plat-E retrovirus. Assays were also performed with transient over-expression of the clones in HEK/293T cells. Activity of the fusion clones was compared to BRAF (wild-type, kinase-dead, V600E mutant), KIAA1549 (wild-type), and Rasv12 mutant. The KIAA1549-BRAF fusion gene constructs all demonstrated activation of the MAPK pathway with increased phosphylation of both MEK 1/2 and ERK1/2; malignant transformation in soft agar assay; and increased transcriptional output. In vivo studies are ongoing but preliminary results demonstrate the KIAA1549-BRAF fusion is sufficient to cause tumor formation in a xenograft murine model. Overall, our sets of experiments reveal the truncated fusion has kinase activity comparable to the canonical V600E BRAF mutant. Wild-type KIAA1549 had no effect on MEK1/2 and ERK1/2 phosphorylation nor does over-expression cause cell transformation. Not surprising RAF specific and MEK specific inhibition resulted in decreased MEK1/2 phosphorylation in BRAF (all variants) and KIAA1549-BRAF fusion transient over-expression in HEK/293T cells and in IP/BRAF kinase assays. Interestingly, RAF specific inhibition of the KIAA1549-BRAF fusion NIH/3T3 stable cell lines resulted in increased MEK 1/2 phosphorylation at low drug concentrations (0.01-10 micromolar) consistent with recent reports describing paradoxical activation of the MAPK pathway in wild-type BRAF. Drug concentrations of greater than 20 micromolar demonstrated inhibition of all of the constructs. Our data suggest the KIAA1549-BRAF fusion gene has oncogenic potential and is sufficient for tumor formation; however, BRAF kinase specific inhibition can result in paradoxical activation of the MAPK pathway at lower drug concentrations. Targeting the KIAA1549-BRAF fusion in pediatric low-grade gliomas is likely still a rational approach but further preclinical studies are needed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3457. doi:10.1158/1538-7445.AM2011-3457