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Ependymomas are classified into nine molecular groups by DNA methylation profiling. PFA tumors are most prevalent, present mainly in infants and have poor outcomes. Genomic studies have not identified a recurrent driver mutation in PFA ependymomas. However, PFA ependymomas are characterized by lack of H3 K27 trimethylation (H3K27-me3) and overexpression of CXorf67, a novel gene of unknown function. A review of PF ependymoma sequencing data revealed recurrent mutations in CXorf67. Subsequent analysis of a large series of ependymomas (n=263) showed that CXorf67 mutations occur in PFA ependymomas at a frequency of 9.4%, but not in non-PFA ependymomas. Targeted sequencing also revealed H3 K27M mutations in PFA ependymomas at a frequency of 3.9%. H3 and CXorf67 mutations were mutually exclusive. We used immunoprecipitation (IP) / mass spectrometry (MS) to study proteins bound to CXorf67 in the Daoy cell line, which overexpresses CXorf67. EZH2, SUZ12, and EED, core components of the PRC2 complex were identified among enriched peptides and validated through complementary SUZ12 IP/MS. We also showed that modulation of CXorf67 influences H3K27-me3 levels downstream of PRC2, suggesting a link between overexpression of CXorf67 and the altered global epigenetic state in PFA ependymomas through its interaction with PRC2. Enforced reduction of CXorf67 in Daoy cells restored H3K27-me3 levels, while enforced expression of CXorf67 in HEK293T and neural stem cells reduced H3K27-me3 levels. Our results suggest that CXorf67 overexpression may have an oncogenic role in PFA ependymomas, but the selective advantage of CXorf67 mutations is yet to be explained.

BACKGROUND: The nuclear factor-kB (NF-kB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-kB signaling is present in most human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-kB activity in cancer. METHODS: To identify additional genetic alterations that drive ependymoma, we sequenced the whole genomes (WGS) of 41 tumours and matched normal blood, and the transcriptomes (RNAseq) of 77 tumours. The transforming significance of alterations were tested in mouse NSCs that we showed previously to be cells of origin of ependymoma. RESULTS: Here, we show that more than two thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-kB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95-RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95-RELA fusion proteins translocated spontaneously to the nucleus to activate NF-kB target genes, and rapidly transformed neural stem cells—the cell of origin of ependymoma—to form these tumours in mice. CONCLUSIONS: Our data identify the first highly recurrent genetic alteration of RELA in human cancer, and the C11orf95-RELA fusion protein as a potential therapeutic target in supratentorial ependymoma. SECONDARY CATEGORY: Neuropathology & Tumor Biomarkers.