H3 K27M and EZHIP Impede H3K27-Methylation Spreading by Inhibiting Allosterically Stimulated PRC2.

Diffuse midline gliomas and posterior fossa type A ependymomas contain the recurrent histone H3 lysine 27 (H3 K27M) mutation and express the H3 K27M-mimic EZHIP (CXorf67), respectively. H3 K27M and EZHIP are competitive inhibitors of Polycomb Repressive Complex 2 (PRC2) lysine methyltransferase activity. In vivo, these proteins reduce overall H3 lysine 27 trimethylation (H3K27me3) levels; however, residual peaks of H3K27me3 remain at CpG islands (CGIs) through an unknown mechanism. Here, we report that EZHIP and H3 K27M preferentially interact with PRC2 that is allosterically activated by H3K27me3 at CGIs and impede its spreading. Moreover, H3 K27M oncohistones reduce H3K27me3 in trans, independent of their incorporation into the chromatin. Although EZHIP is not found outside placental mammals, expression of human EZHIP reduces H3K27me3 in Drosophila melanogaster through a conserved mechanism. Our results provide mechanistic insights for the retention of residual H3K27me3 in tumors driven by H3 K27M and EZHIP.

Molecular heterogeneity and CXorf67 alterations in posterior fossa group A (PFA) ependymomas.

Of nine ependymoma molecular groups detected by DNA methylation profiling, the posterior fossa type A (PFA) is most prevalent. We used DNA methylation profiling to look for further molecular heterogeneity among 675 PFA ependymomas. Two major subgroups, PFA-1 and PFA-2, and nine minor subtypes were discovered. Transcriptome profiling suggested a distinct histogenesis for PFA-1 and PFA-2, but their clinical parameters were similar. In contrast, PFA subtypes differed with respect to age at diagnosis, gender ratio, outcome, and frequencies of genetic alterations. One subtype, PFA-1c, was enriched for 1q gain and had a relatively poor outcome, while patients with PFA-2c ependymomas showed an overall survival at 5 years of > 90%. Unlike other ependymomas, PFA-2c tumors express high levels of OTX2, a potential biomarker for this ependymoma subtype with a good prognosis. We also discovered recurrent mutations among PFA ependymomas. H3 K27M mutations were present in 4.2%, occurring only in PFA-1 tumors, and missense mutations in an uncharacterized gene, CXorf67, were found in 9.4% of PFA ependymomas, but not in other groups. We detected high levels of wildtype or mutant CXorf67 expression in all PFA subtypes except PFA-1f, which is enriched for H3 K27M mutations. PFA ependymomas are characterized by lack of H3 K27 trimethylation (H3 K27-me3), and we tested the hypothesis that CXorf67 binds to PRC2 and can modulate levels of H3 K27-me3. Immunoprecipitation/mass spectrometry detected EZH2, SUZ12, and EED, core components of the PRC2 complex, bound to CXorf67 in the Daoy cell line, which shows high levels of CXorf67 and no expression of H3 K27-me3. Enforced reduction of CXorf67 in Daoy cells restored H3 K27-me3 levels, while enforced expression of CXorf67 in HEK293T and neural stem cells reduced H3 K27-me3 levels. Our data suggest that heterogeneity among PFA ependymomas could have clinicopathologic utility and that CXorf67 may have a functional role in these tumors.

Identification of a novel, recurrent MBTD1-CXorf67 fusion in low-grade endometrial stromal sarcoma.

Endometrial stromal sarcomas (ESSs) are a genetically heterogeneous group of rare uterine neoplasms that are commonly driven by recurrent gene rearrangements. In conventional low-grade ESS, JAZF1-SUZ12, PHF1-JAZF1, EPC1-PHF1 and MEAF6-PHF1, and recently described ZC3H7-BCOR chimeric fusions have been reported in > 50% of cases. Conversely, oncogenic t(10;17)(q22;p13) translocation yields YWHAE-FAM22A/B chimeric proteins that are associated with histologically high-grade and clinically more aggressive ESS. Integrating whole-transcriptome paired-end RNA sequencing with fluorescence in situ hybridization (FISH) and banding cytogenetics, we identified MBTD1 (malignant brain tumor domain-containing 1) and CXorf67 (chromosome X open reading frame 67) as the genes involved in the novel reciprocal t(X;17)(p11.2;q21.33) translocation in two independent low-grade ESS of classical histology. The presence of the MBTD1-CXorf67 fusion transcript was validated in both cases using reverse-transcription polymerase chain reaction followed by Sanger sequencing. A specific FISH assay was developed to detect the novel t(X;17) translocation in formalin-fixed paraffin-embedded material, and resulted in identification of an additional low-grade ESS case positive for the MBTD1-CXorf67 fusion among 25 uterine stromal tumors [14 ESS and 11 undifferentiated endometrial sarcomas (UESs)] that were negative for JAZF1 and YWHAE rearrangements. Gene expression profiles of seven ESS (including three with YWHAE and two with JAZF1 rearrangements) and four UES without specific chromosomal aberrations indicated clustering of tumors with MBTD1-CXorf67 fusion together with low-grade JAZF1-associated ESS. The chimeric MBTD1-CXorf67 fusion identifies yet another cytogenetically distinct subgroup of low-grade ESS and offers the opportunity to shed light on the functions of two poorly characterized genes.

H3K27me3 deficiency in dedifferentiated carcinoma and carcinosarcoma of the endometrium.

The present study immunohistochemically investigated trimethylation of lysine 27 of histone 3 (H3K27me3) expression in 769 endometrial carcinomas and 196 uterine mesenchymal tumors. One dedifferentiated endometrial carcinoma (DEC) and one carcinosarcoma showed H3K27me3 deficiency that was limited to undifferentiated and sarcomatous components, respectively. Switch/sucrose nonfermenting (SWI/SNF) complex subunits (SMARCA4, SMARCB1, and ARID1A/1B) and mismatch repair proteins were proficient in both tumors. The dimethylation of H3K27 (H3K27me2) was deficient in the undifferentiated component, whereas the sarcomatous component had scattered H3K27me2-positive cells. CXorf67, which inhibits PRC2 function, was diffusely expressed in the sarcomatous component. CXorf67 was negative in the undifferentiated component, which was submitted to a genetic analysis and showed no alterations in PRC2 core subunits or H3K27. The present results suggest H3K27 methylation dysregulation as a cause of SWI/SNF-proficient DEC and carcinosarcoma and imply differences in their level of and the mechanisms underlying H3K27 methylation dysregulation.

EZH2 inhibitory protein (EZHIP/Cxorf67) expression correlates strongly with H3K27me3 loss in posterior fossa ependymomas and is mutually exclusive with H3K27M mutations.

The PFA molecular subgroup of posterior fossa ependymomas (PF-EPNs) shows poor outcome. H3K27me3 (me3) loss by immunohistochemistry (IHC) is a surrogate marker for PFA wherein its loss is attributed to overexpression of Cxorf67/EZH2 inhibitory protein (EZHIP), C17orf96, and ATRX loss. We aimed to subgroup PF-EPNs using me3 IHC and study correlations of the molecular subgroups with other histone related proteins, 1q gain, Tenascin C and outcome. IHC for me3, acetyl-H3K27, H3K27M, ATRX, EZH2, EZHIP, C17orf96, Tenascin-C, and fluorescence in-situ hybridisation for chromosome 1q25 locus were performed on an ambispective PF-EPN cohort (2003-2019). H3K27M-mutant gliomas were included for comparison. Among 69 patients, PFA (me3 loss) constituted 64%. EZHIP overexpression and 1q gain were exclusive to PFA seen in 72% and 19%, respectively. Tenascin C was more frequently positive in PFA (p = 0.02). H3K27M expression and ATRX loss were noted in one case of PFA-EPN each. All H3K27M-mutant gliomas (n = 8) and PFA-EPN (n = 1) were EZHIP negative. C17orf96 and acetyl-H3K27 expression did not correlate with me3 loss. H3K27me3 is a robust surrogate for PF-EPN molecular subgrouping. EZHIP overexpression was exclusive to PFA EPNs and was characteristically absent in midline gliomas and the rare PFA harbouring H3K27M mutations representing mutually exclusive pathways leading to me3 loss.

Elevated CXorf67 Expression in PFA Ependymomas Suppresses DNA Repair and Sensitizes to PARP Inhibitors.

Ependymoma is the third most common pediatric tumor with posterior fossa group A (PFA) being its most aggressive subtype. Ependymomas are generally refractory to chemotherapies and thus lack any effective treatment. Here, we report that elevated expression of CXorf67 (chromosome X open reading frame 67), which frequently occurs in PFA ependymomas, suppresses homologous recombination (HR)-mediated DNA repair. Mechanistically, CXorf67 interacts with PALB2 and inhibits PALB2-BRCA2 interaction, thereby inhibiting HR repair. Concordantly, tumor cells with high CXorf67 expression levels show increased sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors, especially when combined with radiotherapy. Thus, our findings have revealed a role of CXorf67 in HR repair and suggest that combination of PARP inhibitors with radiotherapy could be an effective treatment option for PFA ependymomas.