EPEN-34. THE CRISPR-Cas9 SYSTEM-MEDIATED ENDOGENOUS GENE REARRANGEMENT INDUCED C11orf95-RELA FUSION IN VITRO AND IN VIVO THAT LED TO THE DEVELOPMENT OF EPENDYMOMA-LIKE TUMOR

Recent large-scale genomic studies of ependymal tumors have identified recurrent RELA and YAP1 fusion genes in supratentorial ependymomas. The formation of the C11orf95-RELA fusion gene has been attributed to massive genomic rearrangement involving chromosome 11q termed Chromothripsis in many cases. However, the causal relationship has not been clarified experimentally. In this study, we developed a system to reproduce the oncogenic gene rearrangement using the CRISPR-Cas9 system and examined whether consequent endogenous ependymoma fusion genes are competent to form brain tumors in mice. Initially, to investigate whether C11orf95-RELA fusion can be formed by inducing the relevant gene rearrangement in vitro, we designed multiple guide RNAs on the human and mouse genomic loci and introduced them into cultured cells. RT-PCR and immunoblot analyses detected endogenous C11orf95-RELA fusion transcript and protein in both human and mouse cultured cells. Subsequently, we lentivirally introduced the gRNAs into a mouse brain. Brain tumor formation was observed from around 2 months after the lentivirus injection, thus indicating successful gene rearrangement followed by C11orf95-RELA fusion expression in vivo. Analysis of the tumor tissue confirmed the expression of the endogenous C11orf95-RELA fusion gene. These results suggested that a gene rearrangement is a primary mechanism to form the C11orf95-RELA fusion which is the direct driver of tumorigenesis. Our system to simulate a genomic event will provide significant insights into the understanding of the tumorigenic mechanism in ependymomas.