Dominant Malignant Clones Leverage Lineage Restricted Epigenomic Programs to Drive Ependymoma Development

ZFTA-RELA is the most recurrent genetic alteration seen in pediatric supratentorial ependymoma (EPN) and is sufficient to initiate tumors in mice. Despite ZFTA-RELAs potent oncogenic potential, ZFTA-RELA gene fusions are observed exclusively in childhood EPN, with tumors located distinctly in the supratentorial region of the central nervous system (CNS). We hypothesized that specific chromatin modules accessible during brain development would render distinct cell lineage programs at direct risk of transformation by ZFTA-RELA. To this end, we performed combined single cell ATAC and RNA-seq analysis (scMultiome) of the developing mouse forebrain as compared to ZR-driven mouse and human EPN. We demonstrate that specific developmental lineage programs present in radial glial cells and regulated by Plagl family transcription factors are at risk of neoplastic transformation. Binding of this chromatin network by ZFTA-RELA or other PLAGL family motif targeting fusion proteins leads to persistent chromatin accessibility at oncogenic loci and oncogene expression. Cross-species analysis of mouse and human EPN reveals significant cell type heterogeneity mirroring incomplete neurogenic and gliogenic differentiation, with a small percentage of cycling intermediate progenitor-like cells that establish a putative tumor cell hierarchy. In vivo lineage tracing studies reveal single neoplastic clones that aggressively dominate tumor growth and establish the entire EPN cellular hierarchy. These findings unravel developmental epigenomic states critical for fusion oncoprotein driven transformation and elucidate how these states continue to shape tumor progression. HIGHLIGHTS1. Specific chromatin modules accessible during brain development render distinct cell lineage programs at risk of transformation by pediatric fusion oncoproteins. 2. Cross-species single cell ATAC and RNA (scMultiome) of mouse and human ependymoma (EPN) reveals diverse patterns of lineage differentiation programs that restrain oncogenic transformation. 3. Early intermediate progenitor-like EPN cells establish a tumor cell hierarchy that mirrors neural differentiation programs. 4. ZFTA-RELA transformation is compatible with distinct developmental epigenetic states requiring precise goldilocks levels of fusion oncoprotein expression. 5. Dominant tumor clones establish the entire EPN cellular hierarchy that reflects normal gliogenic and neurogenic differentiation programs.