Abstract PR08: Overexpression and mutations of CXorf67 in “infant-type” posterior fossa type-A ependymomas
2018
Our study aims to establish whether CXorf67 has a role in the pathogenesis of posterior fossa (PF) ependymoma. Among molecular groups of ependymoma defined by DNA methylation profiling, PF type-A (PFA) is the commonest. PFA ependymomas present mainly in infants and are difficult to treat, having a 10-year overall survival of approximately 40%. Published genomic studies have not identified a recurrent driver mutation in PFA ependymomas, but they do show widespread epigenetic alterations, including global loss of histone H3 K27-trimethlyation (H3K27-me3). Another childhood PF tumor, the diffuse pontine glioma (DPG), also shows loss of H3K27-me3. In DPGs, loss of H3K27-me3 is associated with H3 K27M mutation, but the mechanism in PFA ependymomas is not yet established. We reexamined published PF ependymoma sequencing data and discovered recurrent mutations in a novel gene, CXorf67 . Targeted sequencing in a series of PFA ependymomas revealed CXorf67 mutations in 22/234 (9.4%). CXorf67 is a single-exon gene of unknown function. Its protein product is predicted to be “disordered,” apart from one region towards the N terminus. Mutations in PFA ependymomas are missense, and there is a mutation hotspot in the “ordered” region. CXorf67 mutations are not present in other molecular groups of ependymoma and are rare in other cancers. Analyzing the DNA methylation profiles of 675 PFA ependymomas, we have discovered two subgroups, PFA-1 and PFA-2, and nine subtypes among these two subgroups. All PFA subtypes harbor CXorf67 mutations with the exception of PFA-1f and PFA-2c. Targeted sequencing in our tumor series also revealed H3 K27M mutations in PFA ependymomas at a frequency of 3.9%. Two thirds of mutations in HIST1H3B , HIST1H3C , and H3F3A were found in PFA-1f ependymomas, among which H3 mutations were present at a frequency of 35%. Mutations in H3 genes and CXorf67 were mutually exclusive across our series of PFA ependymomas. We used Affymetrix u133v2 arrays to establish that CXorf67 is expressed at high levels in PFA ependymomas, in contrast to low levels in other ependymomas from the PF and supratentorial compartments. A mechanism for CXorf67 overexpression was revealed in a similar comparative analysis of CpG island methylation profiles, which showed that the promoter region of CXorf67 is hypomethylated in PFA tumors, but not in other ependymomas. Using immunohistochemical preparations, we detected expression of CXorf67 at the protein level in the nuclei of PFA ependymomas; PFB and supratentorial tumors were immunonegative. CXorf67 overexpression is found in all PFA molecular subtypes, except PFA-1f, which shows similar levels to those in supratentorial and PFB ependymomas. CXorf67 expression is unrelated to mutation status. Elevated CXorf67 expression is found in the Daoy and U2-OS cancer cell lines. We used immunoprecipitation (IP)/mass spectrometry (MS) to study proteins bound to CXorf67 in Daoy and U2-OS. Analysis of enriched peptides following immunoprecipitation of CXorf67 indicated that it binds EZH2, SUZ12, and EED, three core components of the PRC2 complex. Complementary immunoprecipitation of SUZ12 detected CXorf67. Detecting CXorf67 mutations in almost 10% of PFA ependymomas led to the discovery of overexpression related to promoter region hypomethylation in these tumors. Overexpression of CXorf67 was detected in all PFA ependymoma subtypes except PFA-1f, 35% of which harbor an H3 K27M mutation. The protein product of CXorf67 is found in tumor cell nuclei, where our IP/MS data suggest it is bound to PRC2. Our findings suggest that global loss of H3K27-me3 in PFA ependymomas could be related to overexpression of CXorf67 and its interaction with PRC2, except in tumors where alterations in histone H3 genes are responsible. However, the selective advantage of CXorf67 mutation is yet to be explained. Citation Format: Ji Wen, Jens-Martin Hubner, Wilda Orisme, Gang Wu, Bo Tang, Sujuan Jia, John Easton, Kelly Haupfear, Brian D. Freibaum, Hong Joo Kim, Anthony High, Junmin Peng, Ruth G. Tatevossian, J. Paul Taylor, Stefan M. Pfister, Jinghui Zhang, Kristian W. Pajtler, Marcel Kool, David W. Ellison. Overexpression and mutations of CXorf67 in “infant-type” posterior fossa type-A ependymomas [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR08.
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