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Background Ependymoma (EPN) posterior fossa group A (PFA) has the highest rate of recurrence and the worst prognosis of all EPN molecular groups. At relapse, it is typically incurable even with re-resection and re-irradiation. The biology of recurrent PFA remains largely unknown, however, the increasing use of surgery at first recurrence has now provided access to clinical samples to facilitate a better understanding of this. Methods In this large longitudinal international multicenter study, we examined matched samples of primary and recurrent disease from PFA patients to investigate the biology of recurrence. Results DNA methylome derived copy number variants (CNVs) revealed large scale chromosome gains and losses at recurrence. CNV changes were dominated by chromosome 1q gain and/or 6q loss, both previously identified as high-risk factors in PFA, which were present in 23% at presentation but increased to 61% at 1 st recurrence. Multivariate survival analyses of this cohort showed that cases with 1q gain or 6q loss at 1 st recurrence were significantly more likely to recur again. Predisposition to 1q+/6q- CNV changes at recurrence correlated with hypomethylation of heterochromatin-associated DNA at presentation. Cellular and molecular analyses revealed that 1q+/6q- PFA had significantly higher proportions of proliferative neuroepithelial undifferentiated progenitors and decreased differentiated neoplastic subpopulations. Conclusions This study provides clinically and preclinically-actionable insights into the biology of PFA recurrence. The hypomethylation predisposition signature in PFA is a potential risk-classifier for trial stratification. We show that the cellular heterogeneity of PFAs evolves largely because of genetic evolution of neoplastic cells.

Over 60% of supratentorial (ST) ependymomas harbor a gene fusion between C11orf95, an uncharacterized gene, and RELA (also known as p65), a main component of the NF-ĸB family of transcription factors. While its sufficiency to drive tumor has been established, the mechanism of tumorigenesis remains elusive. To tackle this question, we developed a natively forming mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain and performed integrative epigenomic and transcriptomic mapping. Our findings indicate that in addition to direct canonical NF-ĸB pathway activation, C11orf95-RELA (CRfus) dictates a neoplastic transcriptional program and binds to unique sites across the genome enriched with Plagl family transcription factor motifs. CRfus modulates the transcriptional landscape by recruiting transcription co-activators (Brd4, EP300, Cbp, Pol2) which are amenable to pharmacologic inhibition. Downstream CRfus target genes converge on developmental programs marked by Plagl family of transcription factors and activate neoplastic programs enriched in Mapk, focal adhesion, and gene imprinting networks, many of which contain previously unreported therapeutic leads in C11orf95-RELA ependymoma.

Genomic sequencing has driven precision-based oncology therapy; however, genetic drivers remain unknown or non-targetable for many malignancies, demanding alternative approaches to identify therapeutic leads. Ependymomas comprise histologically similar tumor entities driven by distinct molecular mechanisms, such as fusion oncoproteins, genome-wide chromosomal instability, or disruption of DNA methylation patterns. Despite these differences, ependymomas resist chemotherapy and lack available targeted agents for clinical trial development. Based on our previous findings, we hypothesized that mapping chromatin landscapes and super enhancers (SE) could uncover transcriptional dependencies as targets for therapy (Mack, Pajtler, Chavez et al., Nature, 2018). To functionally test the requirement of these SE genes for ependymoma cellular growth, we designed a pooled RNA interference screen against 267 SE associated genes. Our screen was performed in one C11ORF95-RELA-fusion model and two PF-EPN-A models as controls in biological triplicates. As an indication that our screen was successful, positive controls scored among lead hits including KIF11, BUB1B, PHF5A and MYC. Importantly, we identified many subtype specific dependencies in both C11ORF95-RELA-fusion and PF-EPN-A models, thus revealing novel pathways that potentially govern subgroup-specific ependymoma cell growth. Further, several candidates detected across all ependymoma lines were also identified as pan-cancer dependencies or glioma/glioblastoma specific essential genes from the DepMap Cancer Dependency Gene Resource. Our findings reveal novel targets and pathways that are essential for ependymoma cell growth, which may provide new insight into therapeutic strategies against these aggressive brain tumors.