Molecular groups of supratentorial ependymomas comprise tumors with ZFTA-RELA or YAP1-involving fusions and fusion-negative subependymoma. However, occasionally supratentorial ependymomas cannot be readily assigned to any of these groups due to lack of detection of a typical fusion and/or ambiguous DNA methylationbased classification. An unbiased approach with a cohort of unprecedented size revealed distinct methylation clusters composed of tumors with ependymal but also various other histological features containing alternative translocations that shared ZFTA as a partner gene. Somatic overexpression of ZFTA-associated fusion genes in the developing cerebral cortex is capable of inducing tumor formation in vivo, and cross-species comparative analyses identified GLI2 as a key downstream regulator of tumorigenesis in all tumors. Targeting GLI2 with arsenic trioxide caused extended survival of tumor-bearing animals, indicating a potential therapeutic vulnerability in ZFTA fusion-positive tumors. (Word count: 131) SignificanceZFTA-RELA fusions are a hallmark feature of supratentorial ependymoma. We find that ZFTA acts as a partner for alternative transcriptional activators in oncogenic fusions of supratentorial tumors with various histological characteristics. Establishing representative mouse models, we identify potential therapeutic targets shared by ZFTA fusion-positive tumors, such as GLI2. (Word count: 48)Research.

More than 60% of supratentorial ependymomas harbor a ZFTA–RELA (ZRfus) gene fusion (formerly C11orf95–RELA). To study the biology of ZRfus, we developed an autochthonous mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain. Integrative epigenomic and transcriptomic mapping was performed on IUE-driven ZRfus tumors by CUT&RUN, chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin sequencing, and RNA sequencing and compared with human ZRfus-driven ependymoma. In addition to direct canonical NFκB pathway activation, ZRfus dictates a neoplastic transcriptional program and binds to thousands of unique sites across the genome that are enriched with PLAGL family transcription factor (TF) motifs. ZRfus activates gene expression programs through recruitment of transcriptional coactivators (Brd4, Ep300, Cbp, Pol2) that are amenable to pharmacologic inhibition. Downstream ZRfus target genes converge on developmental programs marked by PLAGL TF proteins, and activate neoplastic programs enriched in Mapk, focal adhesion, and gene imprinting networks. Significance: Ependymomas are aggressive brain tumors. Although drivers of supratentorial ependymoma (ZFTA- and YAP1-associated gene fusions) have been discovered, their functions remain unclear. Our study investigates the biology of ZFTA–RELA-driven ependymoma, specifically mechanisms of transcriptional deregulation and direct downstream gene networks that may be leveraged for potential therapeutic testing. This article is highlighted in the In This Issue feature, p. 2113

ZFTA (C11orf95)—a gene of unknown function—partners with a variety of transcriptional coactivators in translocations that drive supratentorial ependymoma, a frequently lethal brain tumor. Understanding the function of ZFTA is key to developing therapies that inhibit these fusion proteins. Here, using a combination of transcriptomics, chromatin immunoprecipitation sequencing, and proteomics, we interrogated a series of deletion-mutant genes to identify a tripartite transformation mechanism of ZFTA-containing fusions, including: spontaneous nuclear translocation, extensive chromatin binding, and SWI/SNF, SAGA, and NuA4/Tip60 HAT chromatin modifier complex recruitment. Thereby, ZFTA tethers fusion proteins across the genome, modifying chromatin to an active state and enabling its partner transcriptional coactivators to promote promiscuous expression of a transforming transcriptome. Using mouse models, we validate further those elements of ZFTA-fusion proteins that are critical for transformation—including ZFTA zinc fingers and partner gene transactivation domains—thereby unmasking vulnerabilities for therapeutic targeting. Significance: Ependymomas are hard-to-treat brain tumors driven by translocations between ZFTA and a variety of transcriptional coactivators. We dissect the transforming mechanism of these fusion proteins and identify protein domains indispensable for tumorigenesis, thereby providing insights into the molecular basis of ependymoma tumorigenesis and vulnerabilities for therapeutic targeting. This article is highlighted in the In This Issue feature, p. 2113

2020) Neuropathology and Applied Neurobiology 46, 48-56 The role and contribution of transcriptional enhancers in brain cancer Genetic alterations identified across several paediatric and adult brain tumours reveal recurrent disruption of active chromatin landscapes and dysregulation of transcriptional programmes. Noncoding elements, specifically enhancers, are central to these mechanisms, and are influenced by developmental and neural gene regulatory signatures. Epigenomic and transcriptomic methods and techniques have facilitated detection of active enhancers, and characterization of brain tumours integrated with genomic structural information. These datasets have provided new insights into the mechanisms of transcriptional control that are profoundly altered in childhood and adult brain cancer; offering new ideas and molecular targets for therapeutic intervention. This review summarizes recent advances in our understanding of active transcriptional programmes of brain cancer, their impact on tumour development, and research areas for further exploration.

Introduction Ependymoma is an aggressive type of pediatric brain tumor resistant to chemotherapy, with treatment to date limited to surgical resection and radiation. Thus, identification and validation of molecular targets that can translate into clinical trials in ependymoma is desperately needed to improve patient outcomes. Over 70% of supratentorial ependymoma are driven by an oncogenic fusion between C11orf95 and Rela (denoted CRFUS). CRFUS expression initiates ependymoma development in mice by potentially acting as an oncogenic transcription factor and disrupting gene expression programs. We hypothesized that specific CRFUS interacting proteins are required for tumor formation and could represent lead therapeutic targets. Methods To study CRFUS ependymoma, a natively-forming tumor model of CRFUS generated by in utero electroporation of the developing mouse brain was utilized. Tumor cells were isolated and then subjected to nuclear Rapid Immunoprecipitation and Mass Spectrometry Analysis of Endogenous Proteins (RIME) of HA-tagged CRFUS protein. Immunoprecipitation and Western Blot (IP-WB) were utilized to probe for leading protein interactions. Results We identified NF-kB proteins consistent with canonical Rela mediated transcription (NFKB1 and NFKB2) as well as novel protein interactomes that converged on RNA splicing and translational regulation. In addition, we identified a large series of novel chromatin-binding proteins as candidates potentially required for CRFUS mediated tumorigenesis. Conclusions Further study is ongoing to validate key CRFUS protein interaction dependency on tumor development. ChIP-Seq (chromatin immunoprecipitation with massively parallel DNA sequencing) and CUT&RUN (cleavage under target and release using nuclease) assays have been employed to further analyze the functional role of canonical Rela pathway members. By interrogating these mechanisms, novel therapeutic targets and pathways may be identified in parallel with dissecting the molecular basis of CRFUS driven ependymoma.

<p>Domain mapping of ZFTA-RELAFUS1 and ZFTA-YAP1FUS driven nuclear translocation in mNSCs</p>

<p>Additional Methods for online</p>

<p>ZFTA-YAP1 interactome</p>