Epen-12. comparing cellular hierarchies and spatial architecture across molecular subgroups of supratentorial ependymomas

Abstract Ependymomas (EPN) are among the most common and fatal pediatric brain tumors with currently no targeted therapies available. Molecular studies including genome-wide DNA methylation profiling have shown that supratentorial ependymoma is subdivided into subgroups characterized by distinct fusion proteins: ZFTA-RELA fusion-positive (ZFTA-RELA), YAP1 fusion-positive (ST-YAP1), and four alternative ZFTA fusion-positive (ZFTA-Cluster 1, ZFTA-Cluster 2, ZFTA-Cluster 3, and ZFTA-Cluster 4). ZFTA-RELA tumors have been identified as the most common and aggressive molecular group with high intra-tumoral heterogeneity. However, limited sample size and interpatient variability in previous studies highlight a critical need for comprehensive profiling of supratentorial ependymomas across a larger patient cohort. In particular, how ZFTA-RELA compares to other subgroups of supratentorial ependymoma is unknown. Here, we collected and profiled 44 supratentorial patient tumors encompassing ZFTA-RELA (n=21), ZFTA-Cluster 1 (n=6), ZFTA-Cluster 2 (n=4), ZFTA-Cluster 3 (n=7), ZFTA-Cluster 4 (n=2), and ST-YAP1 (n=4). We profiled all tumors by single cell/single nucleus RNA-sequencing, and further characterized ZFTA-RELA by single cell spatial transcriptomics. Within each patient’s tumor we find multiple normal and aberrant differentiation trajectories from neural progenitor-like cells towards glial-like, mesenchymal-like and ependymal-like cells. Using sequencing-based lineage tracing, we experimentally validate these computationally inferred cellular hierarchies in vitro. Notably, compared to ZFTA-RELA ependymomas we observed predominantly higher proportion of early neural progenitor-like cells in ZFTA-Cluster 2 and ZFTA-Cluster 3, while in ST-YAP1 greater representation of mature ependymal-like cells. These findings highlight differences in developmental and aberrant differentiation trajectories driven by combination of oncogenic fusions and cell of origin in supratentorial ependymoma subgroups. In addition, neural progenitor-like cells most highly express ZFTA-fusions and are spatially distinct from other cell states, highlighting oncogenic fusions driving tumorigenesis organized in spatial niches. Together, this study reveals previously unknown intra- and intertumoral heterogeneity across subgroups of supratentorial ependymoma, as well as distinct cellular and spatial architecture.