EZHIP links neuro-epithelial and mesenchymal tumor programs during progression of PFA ependymoma

Abstract High-risk ependymomas (EPN) arise throughout the neuroaxis, with aggressive posterior fossa group A (PFA EPN) and supratentorial tumors harboring zinc finger translocation-associated protein fusions (ST-ZFTA) predominating in pediatric patients. Among these, PFA EPN are characterized by distinctly poor overall survival based on high tumor recurrence rates and limited response to therapies. This underscores an urgent need to identify molecular drivers and mechanisms underlying the aggressive behavior of this high-risk EPN subtype. Besides a high abundance of stem- and progenitor-like cell populations, EZH2 inhibitory protein (EZHIP) is suggested to act as a driver of PFA EPN-associated epigenetic reprogramming. However, the impact of EZHIP on cellular differentiation during PFA EPN progression remains to be elucidated. In this study, we explored the interaction between EZHIP and cilia-associated as well as mesenchymal-like marker genes through integrative analysis of RNA expression ( n = 58), DNA methylation ( n = 37), ChIP sequencing profiling of H3K27me3, H3K27ac, and EZHIP ( n = 4), and multiplex immunohistochemistry ( n = 15) in a cohort of PFA EPN tissue specimens complemented by publicly available datasets. Here, we demonstrate increased expression of cilia-related genes, particularly CAPS and DNAAF1 , in PFA EPN compared to ST-ZFTA. Notably, EZHIP binds to CAPS gene loci, resulting in significantly reduced CAPS levels upon EZHIP knockdown in PFA EPN cells. This suggests an association of EZHIP with neuro-epithelial differentiation programs. Even further, a strong correlation between CAPS and EZHIP and the chromosome 1q gain was identified, and this association was particularly enriched in progressive diseases. During PFA EPN progression, tissue reorganization led to layered pseudorosette structures in which CA9-positive mesenchymal compartments encircled CAPS-positive ciliated cells, with both cell types being positive for EZHIP. Summarizing, our study advances the understanding of PFA EPN tumor biology by uncovering epigenetic and spatial mechanisms associated with EZHIP and neuro-epithelial- as well as mesenchymal-like differentiation during PFA EPN progression.