Abstract
Posterior fossa A (PFA) ependymomas comprise one out of nine molecular groups of ependymoma. PFA tumors are mainly diagnosed in infants and young children, show a poor prognosis and are characterized by a lack of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark. Recently, we reported overexpression of CXorf67 as hallmark of PFA ependymoma and that CXorf67 can interact with polycomb repressive complex 2 (PRC2) thereby inhibiting EZH2. However, a detailed description of the functional domains of CXorf67 and the exact mechanism of EZH2 inhibition is still lacking. We now have performed mass spectrometry (MS) analyses of different CXorf67 protein truncates to identify functional domains in the CXorf67 protein involved in the binding of PRC2 core components. Our results show that PRC2 components like EZH2 and SUZ12 specifically bind to the C-terminal part of the CXorf67 protein. Overexpression of this C-terminal part of CXorf67 was sufficient to induce a strong downregulation of H3K27me3 in HEK293 cell lines. Upon inspection of the amino acid sequence in the C-terminus we discovered that a small, highly conserved amino acid sequence is responsible for binding and inhibition of EZH2 resulting in the perturbation of PRC2 function. A synthetic 21 amino acids long peptide containing these conserved amino acids completely abolished PRC2 activity in a methyltransferase assay. Strikingly, in silico analyses showed that the CXorf67 peptide mimics the H3K27M peptide and both can bind to the SET domain of EZH2, suggesting that the mechanism of EZH2 inhibition by CXorf67 was highly similar to the mechanism observed in pediatric high-grade gliomas harboring H3K27M mutations. Taken together, our findings shed light on the functional domains of CXorf67 and reveal a crucial oncogenic mechanism that may be exploited for targeted therapy in PFA ependymoma.