LGG-07. Novel CRISPR/Cas9 induced KIAA1549:BRAF fusion model for preclinical studies of pediatric gliomas

Abstract

BACKGROUND: Pediatric Low Grade Gliomas (pLGG) are the most common group of central nervous system (CNS) tumors in children and cause significant morbidities. Pilocytic astrocytoma (PA) is the most frequent pLGG. KIAA1549:BRAF fusion is a well-established oncogenic driver in PA. Oncogene induced senescence (OIS) has prevented establishing PA cultures for in vitro and in vivo studies. Here we look at a novel NIH-3T3 fibroblast model harboring KIAA1549:BRAF fusion gene via CRISPR/Cas9 somatic genome engineering technology. OBJECTIVES: Establishing that the CRISPR/Cas9 edited NIH-3T3 fibroblast model with the KIAA1549:BRAF fusion is valuable for in vitro and in vivo studies without early OIS. DESIGN/METHOD: CRISPR/Cas9 editing technology was used to establish a KIAA1549:BRAF fusion positive cell model. This cell model was studied in vitro with MEK inhibitor cobimetinib (GDC-0973) and using WST-1 viability assay, clonogenic assay, senescence β-galactosidase staining, and western blot. In vivo murine models with subcutaneous fusion positive NIH-3T3 fibroblast tumors were treated with GDC-0973. Survival studies and tissue studies were subsequently done. RESULTS: A fusion positive NIH-3T3 fibroblast model was successfully established. Increased BRAF cDNA expression and higher levels of p-ERK were observed. In vitro studies showed decreased viability with GDC-0973. Clonogenic assay showed qualitative and quantitative decreases in viable cells. P-ERK target inhibition was established without induction of senescence. In vivo studies demonstrated successful subcutaneous tumor implantation, therapy efficacy, and target inhibition. CONCLUSION: CNS tumors, most commonly pLGG, in children cause significant morbidities. KIAA1549:BRAF fusion is an oncogenic driver in PA. In vitro and in vivo studies are important for pre-clinical models. OIS has prevented establishing adequate fusion positive animal cell models. Here we have demonstrated a successful CRISPR/Cas9 edited fusion positive NIH-3T3 fibroblast model.