Abstract
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.