ZFTA-RELA Dictates Oncogenic Transcriptional Programs to Drive Aggressive Supratentorial Ependymoma

Abstract

Over 60% of supratentorial (ST) ependymomas harbor a ZFTA-RELA (ZR^fus) gene fusion (formerly C11orf95-RELA). To study the biology of ZR^fus, 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 ZR^fus tumors by CUT&RUN, ChIP, ATAC, and RNA sequencing and compared to human ZR^fus driven ependymoma. In addition to direct canonical NF-κB pathway activation, ZR^fus 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. ZR^fus activates gene expression programs through recruitment of transcriptional co-activators (Brd4, Ep300, Cbp, Pol2) that are amenable to pharmacologic inhibition. Downstream ZR^fus target genes converge on developmental programs marked by Plagl transcription factor proteins, and activate neoplastic programs enriched in Mapk, focal adhesion, and gene imprinting networks.

INTRODUCTION

Ependymoma is an aggressive and chemo-resistant pediatric brain tumor, with treatment limited to surgical resection and radiation (1). Although histologically similar, ependymomas are divided into at least nine molecular subtypes associated with distinct genetic and epigenetic alterations (2–6). Over 60% of supratentorial (ST) ependymomas are characterized by an oncogenic fusion between zinc finger translocation associated (ZFTA, formerly C11orf95) and v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA) (7). Gene fusions involving YAP1 or ZFTA and other gene partners are less frequent (3). ZFTA-RELA fusion (denoted ZR^fus) protein is typically the sole genetic driver detected in ST ependymoma (7). When expressed in the developing mouse brain, or in transplanted neural stem cells (NSCs), ZR^fus is capable of cellular transformation and tumor initiation (7,8). These findings suggest that the ZR^fus protein and its direct target genes may represent candidates for therapy.

RELA (also known as p65 protein) is a transcription factor that mediates NF-κB pathway activation in diverse processes such as inflammation, cellular metabolism, and chemotaxis (9). In a normal cellular context, the majority of RELA is sequestered in the cytosol by IkBα protein. Tumor necrosis factor alpha (TNF-α) or interleukin-1 (IL-1) cytokine induction results in phosphorylation of IkBα, proteasomal degradation, and translocation of RELA to the nucleus. Within the nuclear compartment, RELA undergoes post-translational modifications to recruit transcriptional co-activators, such as BRD4, to activate inflammatory gene expression programs (10). This occurs via binding of RELA to both enhancers and stretch-super enhancers and accumulation of active chromatin modifications, demarcated by H3K27 acetylation (H3K27ac) (10,11). RELA protein interacts as both homo-dimers and hetero-dimers with nuclear factor kappa B subunit 1 (NFKB1/P50) to activate target gene expression (9).

While the normal molecular function of ZFTA protein is currently unknown, its gene fusion with RELA results in constitutive nuclear translocation (7). The fragment of ZFTA, that undergoes gene fusion, contains at least one to four zinc finger domains suggesting that this fragment may contribute to novel DNA binding and transcriptional regulation (7). Additionally, ZFTA fuses to other genes, often transcription factors (TF) or transcriptional co-activators, such as YAP1 and MAML2 in ependymoma, or Myocardin-like 2 (MKL2) in chondroid lipomas (7,12). Our study reveals that ZR^fus modulates the tumor epigenome through de novo binding of DNA at specific motifs. Accompanying ZR^fus binding are transcriptional complexes associated with wide-spread patterns of active chromatin, through recruitment of Brd4, Ep300, and Crebbp(Cbp), for which specific small molecule inhibitors are currently available. Elucidation of transcriptional co-regulators and mapping of ZR^fus downstream genes reveals molecular targets and pathways that may contribute to ependymoma development, and may represent leads for future therapy.

RESULTS

An autochthonous model of mouse ZFTA-Rela ependymoma

To investigate the molecular function of the ZR^fus oncogene in ependymoma, we utilized PiggyBAC (pB) transposon based in utero electroporation (IUE) technology to model the most common Type 1 ZR^fus fusion variant (denoted ZR^fus1, Fig.1A–D). As a control, we introduced green fluorescence protein (GFP) pB vectors by IUE in age-matched mice which did not generate tumors or affect mouse survival. IUE technology provided several advantages: 1) Over-expression of ZR^fus1 in a native context during embryonic brain development, 2) Simultaneous introduction of HA-tagged ZR^fus1 along with DNA constructs: GFP, luciferase, and CRISPR/Cas9 deletion of Trp53, and 3) Study of ZR^fus1 tumor biology all within an immune-competent environment (13). The resulting mouse tumors recapitulated histologic features of ependymoma (Fig.1B) and showed sustained ZR^fus1 nuclear localization (Fig.1C). Consequently, the mice succumbed to brain tumors with a median age of about 60 days (Fig.1D). ZR^fus1 tumors were micro-dissected by GFP positive signal and compared against contralateral normal brain (non-GFP positive) tissue by RNA-seq. Differential gene expression analysis and comparison against an independent RCAS-TVA ZR^fus1 driven ependymoma mouse model demonstrated a significant correlation of neoplastic transcriptional programs compared to matched normal brain (Fig.1E, R=0.79, p < 2.2^e–16, Supplementary Table S1, Supplemental Data Fig.S1) (8). The IUE ZR^fus1 model positively associated with human ZR^fus1 transcriptional programs as opposed to other ependymoma subtypes, including gene expression of Dlk1, Lmx1b, Ephb2, Igf2, Gli2, and Akt1 (Fig.1F–H). To further support the consistency between our mouse model and its reflection of the human disease, we directly compared the transcriptome of our IUE model to different ZR^FUS1 mouse models and human ZR^fus1 datasets described in Kupp et al., (Co-Submitted Cancer Discovery Manuscript)(14) and Zheng et al., (Co-Submitted Cancer Discovery Manuscript)(15). We observed a significant overlap between the datasets and derived a ZR^fus1 93 gene signature consistent across all datasets, including our IUE model (Fig.1H, Supplementary Table S2). These findings demonstrate that the IUE ZR^fus1 driven mouse model of ependymoma recapitulates several key aspects of human ZFTA-RELA ependymoma.

Figure 1. Establishment of a native ZFTA-Rela driven mouse model by embryonic in utero electroporation (IUE).

(A) Schematic of in utero electroporation technique used for introduction of piggyBAC DNA plasmids at embryonic day 16.5 (E16.5) (B) Luciferase, GFP imaging, and histology of IUE ZR^fus1 murine ependymoma, and (C) confirmation of fusion protein nuclear localization. (D) Survival of mice reaching tumor endpoint as a result of ZR^fus1expression. (E) Pearson correlation of IUE vs RCAS driven ZFTA-RELA mouse ependymoma(8) and (F) association with human ZFTA-RELA human tumor expression (18). (G) Comparison between genes upregulated in mouse IUE- ZR^fus1 tumors vs matched normal brain and upregulated in ZR^fus human ependymoma vs other ependymoma subtypes. Shared genes were restricted to those with a log2 fold change greater than 1. Agreement score represents the product of the fold change in mouse and human comparisons. (H) Waterfall plot of 93 ZFTA-Rela signature genes ordered by agreement score representing the product of Log2 fold change (ZR^fus1/Normal Brain) and Log2 fold change (ZFTA-Rela/Non-Rela ependymoma).

ZR^fus1 engages open and active chromatin at de novo promoter and enhancer loci

Using brain tumors from the IUE ZR^fus1 mouse model, we acutely dissociated neoplastic GFP positive cells to perform two orthogonal methods of chromatin profiling, ChIP followed by sequencing (ChIP-seq) and CUT&RUN (Fig.2A–C, Supplemental Data Fig.S2A). CUT&RUN permitted native cross-linking-free genomic mapping of proteins and histone modifications and reduction of non-specific cross-linking artifacts often present in ChIP-seq experiments (16). ChIP-seq and CUT&RUN directed against the HA tag was performed on HA-tagged ZR^fus1 protein, as well as CUT&RUN against Rela, H3K27ac, and IgG (negative control), and ATAC-seq (to measure accessible chromatin). Combining two independent experiments, we identified 6845 shared ZR^fus1 binding sites. This set of peaks was further filtered to exclude non-specific signal using an IgG control. The resulting 5608 peaks significantly overlapped with regions also identified by ChIP-seq (Fig.2B,D, Supplemental Data Fig.S2). CUT&RUN of HA tagged ZR^fus1 and Rela revealed mostly sub-nucleosomal binding of ZR^fus1 with small fragment patterns (< 120 bp) as expected of transcription factor proteins (Fig.2A,C, Supplemental Data Fig.S2B,C) (16). ATAC-seq and H3K27ac CUT&RUN demonstrated that HA-ZR^fus1 binding was localized to ‘open and accessible’ and active chromatin, respectively (Fig.2D–E) and enriched in both active enhancer and promoter loci (Fig.2F). ZR^fus1 was bound to promoters and proximal enhancers of known ependymoma oncogenes such as Ephb2, Ccnd1, Akt1, and Notch1 (Fig.2G–H) (17,18). Interestingly, expression of the ZR^fus1 protein led to a global elevation of H3K27ac, but also H3K27me3, a mark of poised and repressive chromatin (Supplemental Data Fig.S2D–F). These findings support that ZR^fus1 protein directly engages DNA as a transcription factor oncoprotein, and is associated with open and active chromatin surrounding many genes, including known ependymoma oncogenes.

Figure 2. ZFTA-Rela binds open and active chromatin of promoter and enhancer loci.

(A) Schematic of proteins and epigenetic marks profiled using CUT&RUN, ChIP-seq, or ATAC-seq in IUE ZR^fus1 ependymoma. (B) Venn diagram of the intersection between ZR^fus1 peaks detected using CUT&RUN and ChIP-seq data. (C) CUT&RUN fragment size profiles of HA and Rela (p65). (D-E) Localization of ZR^fus1 by HA-CUT&RUN and Rela-CUT&RUN in open chromatin (ATAC-seq) and actively transcribed chromatin (H3K27ac CUT&RUN). (F) Distribution of ZR^fus1 binding sites at different genomic regions. (G-H) Binding profiles of ZR^fus1 at the promoters and enhancers of ependymoma oncogenes.

Transcriptional co-activators and RNA polymerase are recruited to ZR^fus1 bound genomic loci

To assess whether ZR^fus1 activates gene expression programs, we performed RNA-seq. The ZR^fus1 bound genes showed higher expression levels as compared to mouse contralateral normal brain (non-GFP⁺) tissue (Fig.3A). 920 ZR^fus1 bound genes that were at least 2-fold upregulated were identified including common ZR^fus1 signature genes such as: Ccnd1, Ephb2, and Gli2 (Fig.3B, p < 0.05, Supplementary Table S3). We reasoned that localization of ZR^fus1 to enhancers and promoters might indicate aberrant recruitment of transcriptional activation complexes, particularly proteins involved in transcriptional initiation and elongation such as Ep300, Cbp, Brd4, and phosphorylated forms of RNA polymerase II (Serine 2 and 5 phosphorylation of the carboxy terminal domain). Supporting this, in the ZR^fus1 IUE model, CUT&RUN signals demonstrated co-recruitment of Brd4, Ep300, Cbp, and Ser2/5 phosphorylated RNA polymerase II at most ZR^fus1 binding sites. (Fig.3C, Supplemental Data Fig.S3). These findings are consistent with a manuscript co-submitted to Cancer Discovery by Kupp et al.,(14) in which ZR^fus1 was shown to physically bind both Brd4 and Ep300 to promote gene transcription, and ZFTA was reported to facilitate Ep300 mediated gene activation.

Figure 3. Recruitment of transcriptional activation proteins to ZFTA-Rela binding sites.

(A-B) Gene expression of ZR^fus1 targets in mouse tumors versus contralateral normal brain (C) Recruitment of transcriptional co-regulators Brd4, Ep300, Crebbp, Ser2-Pol2, and Ser5-Pol2 to ZR^fus1 binding sites as compared to IgG control. (D) CRISPR-CAS9 KO of ZR^fus1 and Rela (E) Pie chart depicting the number of genes that were down-regulated, up-regulated, or exhibited no change in expression between non-targeting and Rela KO (F) Heatmap of selected genes in the NF-kB, ZR^fus1 targets, and neuronal differentiation pathway that were down-regulated or up-regulated (G) Differential gene expression between non-targeting and Rela KO experiments across three cell passages. Represented in the waterfall plot is the Log2 fold-change between KO versus non targeting across n = 24,428 genes/transcripts. (H) Pathway analysis of the top down-regulated genes following Rela-KO as compared to non-targeting controls.

To determine whether the effects on gene transcription were direct, we generated knockout (KO) models of cell lines derived from IUE:ZR^fus1 using CRISPR/Cas9 deletion of Rela and ZR^fus1 with varying efficiencies (Fig.3D). Using sgRNA:Rela targeting constructs, ZR^fus1 KO resulted in decreased expression of 872 (23%) of ZR^fus1 target genes while 173 (4%) were up-regulated, and 2780 (73%) unchanged (Fig.3E). Loss of ZR^fus1 and Rela did not result in a global change in transcriptional output as measured by RNA ERCC spike-in controls (Supplemental Data Fig.S3A,B). Down-regulated genes included several ZR^fus1 specific genes such as Dlk1, Lmx1b, and Akt1, and also members of the NF-kB pathway such as Cxcl2, Icam1, and Stat1 (Fig.3F–G, Supplementary Table S4, Supplemental Data Fig.S3C). Most notably, the predominant pathway down-regulated upon ZR^fus1 and Rela KO included genes involved in nervous system development and neural cell differentiation (Fig.3H). The top genes down-regulated were markers of oligodendrocyte (Olig1/2) and radial glial cell identity (Fabp7), while the genes marking neuronal cells (Dlx5/6 and Gchfr) were up-regulated (19,20) (Fig 3F). Similarly, chemical inhibition of Ep300/Cbp using inhibitors A485 versus A486 (inactive isomeric control) significantly impaired expression of ZR^fus1 target genes and was associated with decreased cellular viability at 72 hours of treatment (Supplemental Data Fig.S3 D–E) (21). Our data demonstrates that ZR^fus1 recruits transcriptional activation complexes, including Brd4, Ep300, Cbp, and RNA Pol2 to drive neoplastic gene transcription. Genetic ablation of ZR^fus1 or chemical inhibition of transcriptional co-regulators impairs ZR^fus1 target gene expression and cell identity programs.

ZR^fus1 binds Plagl TF family DNA motifs to promote neoplastic gene expression

We hypothesized that ZR^fus1, which harbors a C2H2 zinc finger domain, would promote aberrant DNA binding in addition to its function to drive nuclear localization (7). To mark and remove canonical NF-κB binding sites from tumor-specific sites, we compared ZR^fus1 binding against mouse embryonic fibroblasts treated with TNF-α, a method of stimulating Rela nuclear localization and studying inflammatory gene expression (22). The majority of Rela-bound sites in the context of inflammation were absent or weakly bound by ZR^fus1 (Fig.4A). Tumor-specific DNA binding of ZR^fus1 was enriched for the Plagl1 or Plagl2 transcription factor family motifs consisting of a core ‘GGGCC’ DNA sequence (Fig.4B, Enrichment: p < 1e⁻¹⁴⁸⁸, Supplementary Table S5). Also, shared programs between tumor and inflammatory programs were enriched for both Rela and Plagl2 motifs (Fig.4B, Rela: 1e⁻³⁸⁷ and Plagl2: 1e⁻¹¹⁸, Supplementary Table S6). An enrichment of Plagl TF family motifs was also observed in the ZR^fus1 HA ChIP-seq data thus further validating our findings using an orthogonal technique (Supplemental Data Fig. S4A–C). Interestingly, in a small number of cases, we identified Rela binding sites directly flanked by adjacent Plagl family motifs suggesting possible co-recruitment and utilization of endogenous Rela proteins (Supplemental Data Fig.S4D–G). Further, Tead family motifs were found to be enriched at a subset of tumor-specific and shared inflammatory loci, and also bound by H3K27ac, Brd4, Ep300 and Pol2 (Supplemental Data Fig.S4C). This is potentially relevant as other subtypes of supratentorial ependymoma are driven by YAP1 gene fusions, suggesting potentially shared oncogenic mechanisms(3,23).

Figure 4. ZFTA-Rela engages NF-κB and Plagl (‘GGGCC’) motifs.

(A) Comparison of HA- and Rela- CUT&RUN binding in ZR^fus1 ependymoma against mouse embryonic fibroblasts cells treated with TNF-α. Motif enrichment analysis (B) and Distribution of peaks at different genomic regions (C) for tumor-specific, shared, and inflammation-specific binding sites. (D) Example of ZR^fus1 binding at the Lmx1b locus, recruitment of Brd4 and Ep300, and presence of multiple Plagl motifs. (E) Lmx1b gene expression in IUE ZR^fus1 tumor versus normal brain. (F) Comparison of DNA motifs identified in human ZFTA-RELA and non-ZFTA-RELA ependymoma using HOMER (see methods).

ZR^fus1 binding profiles in a tumor-specific or shared context was comparable between enhancers and promoters, unlike enhancer and super enhancer centric profiles observed in canonical TNF-α driven Rela activation (Fig.4C) (10). As an example, we observed ZR^fus1 binding at the proximal enhancer of Lmx1b, along with Brd4 and Ep300 recruitment and presence of three central ‘GGGCC’ motifs (Fig.4D). Lmx1b expression was elevated in ZR^fus1-driven mouse tumors and human ependymoma, both associated with the presence of a proximal ZR^fus1-specific super enhancer (Fig.4E). To confirm our findings in human tumor tissue, we mapped nucleosome-free regions of H3K27ac ChIP data in ZFTA-RELA vs non-ZFTA-RELA primary ependymoma samples (18). We found that PLAGL family TF motifs were the top-ranking DNA motif observed in a ZFTA-RELA specific context (Fig.4F, Supplemental Data Fig. S4H, S5A–E). Therefore, our findings demonstrate that ZR^fus1 directly binds a tumor-specific program distinct from the canonical NF-κB pathway, and converges on activation of PLAGL TF target genes in both mouse and human ependymoma.

ZR^fus1 regulates transcriptional regulatory circuits to drive super enhancer gene expression

We have previously shown that enhancers and super enhancers (SE) regulate subtype-specific gene expression programs in human ependymoma, including ZFTA-RELA tumors (18). To elucidate the mechanisms of oncogene activation in ZR^fus1 ependymoma, we mapped super enhancers by Ranking of Super Enhancer (ROSE) analysis and identified several genes such as Dlk1, Lmx1b, and Ephb2 which also harbor SEs in human ZFTA-RELA ependymoma (Fig.5A, Supplementary Table S7) (18). Of note, 73% (511 of 703) of SEs identified harbored at least one overlapping ZR^fus1 binding site, suggesting a significant contribution of ZR^fus1 binding to SE driven gene expression (Fig.5B–C). As an example, we observed a 5’ SE predicted to regulate Dlk1, which contained ZR^fus1 binding as well as Brd4 recruitment to sites of open chromatin (Fig.5D). Dlk1 was over-expressed in IUE:ZR^fus1 tumors compared to normal brain, and had significantly higher expression in human ZFTA-RELA fusion driven ependymoma in comparison to other ependymoma subtypes. (Fig.5E). These findings are in line with single-cell RNA sequencing characterization of human ependymoma that pinpoints DLK1 as a top signature gene in a unique and tumor cell specific transcriptional program in ZR^fus driven human tumors (24).

Figure 5. ZFTA-RELA disrupts core regulatory circuitry programs to drive SE gene expression.

(A) Hockey-stick plot depicting super enhancers detected in IUE: ZR^fus1 murine ependymoma (B) Venn diagram showing overlap of HA: ZR^fus1 peaks and SEs (C) Box-violin plot depicting up-regulation of SE associated genes in tumor vs. normal brain (D) HA-ZR^fus1 CUT&RUN, ATAC-seq, Brd4 CUT&RUN, and H3K27ac CUT&RUN shown at the Dlk1 locus (E) Dlk1 expression in mouse and human ZR^fus ependymoma compared to normal brain and other ependymoma subtypes, respectively (F) Schematic of identification of core regulatory circuit (CRC) transcription factors (G) Top CRC module enriched in IUE-ZR^fus1 ependymoma including predicted binding sites and engagement of ZR^fus1 at Plagl1/2 and Rela motifs (H) CUT&RUN localization of Plagl2, HA-ZR^fus1 and Rela at super enhancers that harbor the Plagl1/2 or Rela motif.

To dissect the transcription factors that regulate ZR^fus1 SEs, we conducted core regulatory circuitry analysis (CRC, Fig.5F). This method searches regions of open chromatin (demarcated by ATAC-seq) within H3K27ac-defined SEs (i.e. nucleosome-free regions). Core transcriptional circuits are defined by TFs that are regulated by SEs, which are transcribed and translated, and subsequently bind other SEs (25,26). CRC analysis identified the top-ranking set of eleven TFs that form a putative regulatory circuit including: Plagl2, Rela, Sox7, Sp2, Sox3, Foxo1, Srf, Smad1, Stat3, Zfp3, and Hic1 (Fig.5G, Rank = 1, Score = 284.091, Supplementary Table S8). To distinguish between ZR^fus1 and Plagl2 binding at SEs and regulation of CRC transcriptional circuits, we performed CUT&RUN of Plagl2 (Fig.5H, Supplemental Data Fig. S5F). While ZR^fus1 shared some binding sites with Plagl2, the occupancy at SEs, containing a Plagl2 motif or Rela motif, was largely a result of ZR^fus1 binding. These findings support that ZR^fus1 may act as an oncogenic TF by invading a CRC network, binding directly to Plagl1/2 and Rela motifs, and engaging SEs that regulate downstream ependymoma gene expression programs.

ZR^fus1 tumor transcriptomes match radial glial cells that may give rise to ependymoma

Ependymomas are thought to arise from a heterogeneous population of radial glial cells during embryonic brain development (17,27). To investigate the developmental origins in our IUE:ZR^fus1 model, we compared tumor transcriptomes against a single-cell RNA sequencing atlas of the mouse developing cortex (Supplemental Data Fig.S5G–I) (28). As compared to normal brain, we found that cells with the closest and most specific enrichment were embryonic day E15 dorsal and ventral radial glial cells (Supplemental Data Fig.S5G). Astrocytes of early post-natal stages showed varied degrees of enrichment. Plagl1 and Plagl2 gene expression was also found to be present and modestly elevated during this embryonic stage (Supplemental Data Fig.S5G). We observed more restricted patterns in the human developing brain cortex by querying the CoDEx database, and found Plagl1 expression to be enriched in outer radial glial cells (Supplemental Data Fig.S5J) (29). These findings support a possible hypothesis that ZR^fus1 may bind, maintain, and/or activate Plagl target gene expression in developing radial glial cells that subsequently give rise to ependymoma.

ZR^fus1 cistrome uncovers oncogenic pathways and molecular targets

The Plagl1 and Plagl2 proteins have diverse roles in both development (30) and tumorigenesis and have been shown across several cell types and tissues to regulate and express an imprinted gene network (including Axl, Dlk1, Igf2), Wnt pathway (Wnt and Fzd family genes) and ECM target genes (Integrin family) (30–33). Within ZR^fus1 tumors, we detected direct binding enrichment of genes associated with NF-κB pathway activation such as TNF-α signaling, Epstein-Barr Virus Infection, Toxoplasmosis, and Osteoclast differentiation (Supplemental Data Fig. S6A–E). In the tumor-specific program, we identified genes elevated and associated with Wnt signaling pathway activity, Cushing syndrome, Hepatocellular carcinoma, focal adhesion, and members of the MAPK signaling pathway (Fig.6A). Many of the identified target genes identified contained previously unreported therapeutic leads in ZFTA-RELA ependymoma such as Cdk4/6, Gsk3B, Akt1, Mapkapk2/3, Fgfr4, Ngfr, Brd7, and Ep300. Further integration of ZR^fus1 tumor-specific programs with the Washington University Drug Gene Interaction Database (34) revealed downstream candidate genes that are possibly clinically actionable (Akt1, Cdk4/6, Fgfr1/4), mapped in the ‘druggable’ genome space (Axl, Csf1r, Dlk1, Ephb2/4, Notch1), protein kinases (Cdk1/4/6, Igf2r, Ccnd1/3), proteases (Adamts enzymes, Stat3, Smad3), methyltransferases (Prdm1/16), and cell surface proteins (Acvrl1, Cd40/44/63, Pdgfrb) (Fig.6B). Our findings demonstrate that genes regulated by ZR^fus1 represent a novel set of candidates relevant to ependymoma development which may represent possible therapeutic leads for future characterization.

Figure 6. Pathway enrichment of ZFTA-RELA specific and inflammatory-shared target genes.

(A) Tumor-specific programs enriched with Plagl TF motifs. Color of nodes highlight differential gene expression between mouse ZR^fus1 and contralateral brain. (B) Integration of ZR^fus1 bound and over-expressed genes with the Washington University Drug-Gene Interaction database to identify candidate drugs and small molecule inhibitors as potential therapeutic candidates against ependymoma.

DISCUSSION

Supratentorial ependymomas are often initiated by a single gene fusion (7,8,23,35). ZFTA-RELA is the most common alteration, however YAP1 and other gene fusions have been reported (See Zheng et al., co-submitted Cancer Discovery manuscript)(15). Together, ependymoma gene fusions have been shown to promote nuclear localization, and are thought to lead to promiscuous transcription factor activity, aberrant chromatin regulation, and upregulation of oncogenes. This activity may be tied to developmental programs that are essential for ependymoma cell growth (18). Our study demonstrates, in a natively forming brain tumor mouse model, that ZR^fus1 directly engages accessible DNA and active chromatin. ZR^fus1 binding is associated with expression of known ependymoma oncogenes, such as Ephb2, Notch1 and Ccnd1 and expression patterns are highly consistent with different mouse and human datasets of ZR^fus driven tumors (See Kupp et al., and Zheng et al., Cancer Discovery Manuscript)(14,15). Our collective analysis across these different studies led to synthesis of a core set of 93 signature genes elevated in ZR^fus driven mouse and human tumors.

Previous reports have suggested aberrant NF-κB activity as a central driver and mechanism of transformation in ZFTA-RELA ependymoma (7). This hypothesis has prompted clinical trial concepts to inhibit NF-κB signaling using known small molecules against the canonical NF-κB pathway. While our data reports a shared inflammatory program directly activated by ZR^fus1, a major component of ZR^fus1 binding is seen in a tumor-specific context and not observed in canonical TNF-α driven gene expression. This suggests that the ZFTA fragment of the ZR^fus1 fusion protein confers oncogenic properties and is supported by several points: 1) ZFTA is fused with other genes in ependymoma (Reference to Zheng et al., co-submitted Cancer Discovery manuscript)(15), and 2) Hyperactive Rela is unable to drive tumorigenesis (8,12). On the other hand, the ZFTA fragment alone is unable to initiate tumors, therefore tying its function to its fusion partner, often a transcription factor and/or co-activator (7). Indeed, Kupp et al.,(14) have demonstrated a high similarity between ZFTA-Rela and ZFTA-Ep300 transcriptional programs, consistent with our data that reveals co-recruitment of transcriptional machinery including Brd4, Ep300, Cbp, and Pol2. These findings suggest that therapeutic strategies that disrupt Rela transcriptional activity may be more effective than drugs that target cytosolic regulation of Rela (i.e. IKK inhibitors). An alternate approach could be the disruption of transcriptional co-factors such as BRD4 and EP300/CBP that are recruited and associated with transcription of ZR^fus1 bound genes. Indeed, ZFTA-RELA driven patient-derived xenograft (PDX) models show sensitivity to BET bromodomain inhibitors, such as JQ1, and IUE:ZR^fus1 tumor cells to Ep300/Cbp inhibitors (18). It is not yet clear whether ZFTA-RELA tumors, in vivo, are more sensitive to perturbation of oncogene transcription (i.e. Transcriptional Addiction) to an extent where a therapeutic window can be achieved that spares effects on normal cells.

Our data points to tumor-specific DNA binding of ZR^fus1 and convergence on the core binding sequence of Plagl family TFs. This group of TFs has been shown to be expressed in a variety of cancer types with diverse tumor suppressive and oncogenic roles and regulate an imprinted gene network along with Wnt pathway activation (30,36–41). Plagl2, specifically, has been reported to be amplified and over-expressed in glioblastoma and positively contributes to glioma-genesis through inhibition of cell differentiation (33). While our data does not exclude the functional role of Plagl1/2, it does suggest that ZR^fus1 may be utilizing shared DNA binding sites for target gene activation. ZR^fus1 directed transcriptional programs converge on upregulation of Plagl-associated Mapk, focal adhesion, imprinted and Wnt pathway genes. These Plagl genesets are enriched for putative molecular targets that are potentially clinically actionable, and hold promise for future lead development, such as druggable genome candidates, kinases, and cell surface proteins.

We hypothesize that Plagl1/2 programs may be active during embryonic brain development. Supporting this, we show that ZR^fus1 tumor expression matches Plagl1/2 positive radial glial cells (RGCs) of the developing cortex, and consistent with data that RGCs contain cells of origin of ependymoma (27,42). In line with other pediatric brain tumors, we hypothesize that our data supports a model of stalled development where stem- or progenitor- cell gene expression programs are aberrantly sustained (28). Related to this, unclear is the role and expression patterns of ZFTA in normal development and whether its cis-regulatory elements facilitate cell and temporal specific expression of ZR^fus1 expression during tumorigenesis. Indeed, several examples in other pediatric brain tumors exemplify how structural alterations not only activate oncogenes but also ‘hijack’ developmental enhancers to promote gene expression(43–45).

Our findings shed light on the transcriptional programs de-regulated by ZFTA-RELA and reveal potential downstream molecular targets that may hold important cellular vulnerabilities. As a single genetic driver, ‘drugging’ the ZFTA-RELA fusion specifically, as opposed to co-regulators and downstream targets would likely be the most effective. Solving the structure of ZFTA-RELA fusion with emerging protein crystallography approaches and identification of binding surfaces of the ZR^fus protein could facilitate the design of proteolysis targeting chimeras for inhibition and/or protein degradation. However, a crucial question as to whether ZR^fus is still required for tumor maintenance in vivo following transformation and establishment of epigenetic marks, remains unanswered. Lessons learned from studying ZFTA-RELA are likely to apply to many other fusion-driven pediatric and adult tumors in terms of developmental modeling, epigenetic characterization, and pre-clinical therapeutic testing.

METHODS

Western blot and Immunohistochemistry

Western blot analysis was performed using standard techniques. Antibodies used included Anti-HA tag antibody (Abcam catalogue no. ab9110, RRID:AB_307019), Lamin B1 (Abcam catalogue no. ab16048), β-Tubulin (CST 86298), β-Actin (Millipore catalogue no. A1978), BRD4 (Bethyl, Cat# A301–985100A), Crebbp (Cbp) (Cell Signaling Technologies, Cat#7389s) and EP300(Abcam, Cat# ab10485). Immunohistochemistry was performed on deparaffinized FFPE sections with antigen retrieval in sodium citrate (10mM pH6) at 95C for 15 min. Sections were blocked with 5% normal horse serum in PBS-T for 1h and primary antibodies added for an overnight incubation at 4C. IHC for the antibodies H3K27me3 (CST9733 1:250–1:500 dilution), H3K27ac (Abcam 1:250–1:500 dilution), and HA (Sigma – 11867431001 at 1:1000 dilution).

In Utero Electroporation (IUE) and tumor cell dissociation

All animal procedures in this study were performed with IACUC approval. In utero electroporation was performed as described previously, and plasmids provided as a gift from Dr. Joseph LoTurco PhD (13). After anesthesia with 5% isoflurane, pregnant mice, at E16.5, were subjected to abdominal incision to expose the uterus. DNA plasmid cocktail (pBCAG-HA-ZR^FUS1, pbCAG-eGFP, pX330-sgTp53, GLAST-PBase, pBCAG-Luc) was injected into the lateral ventricles with a glass pipette. Electric pulses were then delivered to the embryos by gently clasping their heads with forceps-shaped electrodes. Six 33V pulses of 55 ms were applied at 100 ms intervals. The uterus was then repositioned into the abdominal cavity and the abdominal wall and the skin were then sutured. Following birth, pups were traced on a weekly basis by bioluminescent imaging to monitor brain tumor formation. Mouse tumors were collected based on isolation of GFP+ tumors and dissociation into single-cells using a Brain Tumor Dissociation kit (MACS, #130-095-942) following manufacturer’s instructions.

Cell Culture

Cells were grown in neural basal medium (Invitrogen) supplemented with sodium pyruvate, glutamine, B27, N2, bFGF (10 ng/mL) and rhEGF (20ng/mL). Cells were grown on treated cell culture dishes coated with Matrigel (Corning). All lines tested negative for mycoplasma contamination, as assessed using a PCR based approach. Tumor-derived cell lines were confirmed to be authentic and unique by STR fingerprinting.

Fractionation of nuclear and cytoplasmic extracts.

Mouse tumor cells were grown to full confluency and washed with ice-cold PBS and then collected with a cell scraper in ice-cold E1 buffer (50mM HEPES-KOH, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1 mM DTT supplemented with 1X protease inhibitor cocktail) for each volume of cell pellet. Resuspended cells were spun down at 1,100g at 4°C for 2 minutes and supernatant (cytoplasm fraction) was collected in a fresh tube. Cell pellet was resuspended in the same volume of the E1 buffer and spun down at 1,100 xg for 2 minutes. Supernatant was discarded and pellet was resuspended in the same volume of the E1 buffer and incubated on ice for 10 minutes before spinning down at 1,100 xg for 2 minutes at 4°C. Supernatant was discarded and pellet was gently resuspended in 2 volumes of ice-cold E2 buffer (10 mM Tris-HCl, 200 mM NaCl, 1mM EDTA, 0.5 mM EGTA, supplemented with 1x protease inhibitor cocktail) and centrifuged at 1,100 xg for 2 min at 4°C. The supernatant (nuclear fraction) was collected in a fresh tube.

CRISPR Knock-out (KO) line generation

sgRNA sequences targeting Rela and non-targeting control (D98) were designed using CRISPOR tool and the constructs were generated by plasmid cloning following GECKO protocol (RRID:SCR_015935). sgRNA sequences are provided in Supplementary Table S9. To produce lentiviral particles, HEK293T cells were seeded 24 hours prior to transfection and were transfected with 5 ug pMD2.G, 5 ug psPAX2 and 5 ug Lenti-CRISPR-v2 using calcium phosphate transfection method (RRID:Addgene_12260). Medium was refreshed 24 hours post-transfection. The media containing the viral particles were collected at 48 and 72 hours time-points and filtered with 0.45-μm filters. Mouse tumor cells were seeded into 10-cm plates 24hr prior to infection and were infected with freshly collected lenti-viruses with 8 μg/mL Polybrene for 24 hours. Infected cells were selected with 2 μg/mL puromycin for 3 days. Knock-out efficiencies were tested with western blot analysis.

RNA preparation for qRT-PCR and RNA-seq

RNA was extracted using TRIzol or Qiagen miRNA easy kit according to the manufacturer’s instructions and cDNA was prepared from ~1ug total RNA using iScript^™ gDNA clear cDNA Synthesis Kit (Bio-Rad, #1725034). Quantitative PCR was performed using iTaq Universal SYBR^® Green Supermix (Bio-Rad, #172–5120) using the manufacturer’s recommended protocol. Primers are listed in Supplementary Table S7. In the case of RNA-seq, ERCC spike-ins (Thermofisher) were added according to manufactures recommendations and protocols before proceeding with library preparation. RNA-seq library preps were performed using Kapa RNA Hyperprep kit with RiboErase (HMR) according to manufacturer’s recommendations.

CUT&RUN

CUT&RUN assay was performed as described in Skene and Henikoff. (2018)(16). Briefly, 0.5–1 million cells were captured with BioMagPlus Concanavalin A beads and incubated with primary antibody for 10–20 mins at room temperature. After washing away the EDTA in the buffer and unbound antibody with dig-wash buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.5mM Spermidine, 1x Complete Protease Inhibitor EDTA-Free and 0.05% Digitonin), protein A-MNase was added and incubated for 10–20mins. The cells were washed again and placed in an ice-water pre-chilled metal block at least 5mins. CaCl₂ was added to the final concentration of 2 mM to activate protein A-MNase for 30mins on the ice-water chilled metal block. The reaction was stopped by addition of equal volume of 2XSTOP buffer (340 mM NaCl, 20mM EDTA, 4mM EGTA, 0.02% Digitonin, 5μL/ml RNase A, 50μg/ml glycogen and 2 pg/ml heterologous spike-in DNA). The protein-DNA complex was released and DNA was extracted with Gel and PCR Clean-up kit (Macherey-Nagel NucleoSpin®, cat. no.740609.250) or Phenol-chloroform-isoamyl alcohol precipitation (for small fragment DNA), followed by Qubit fluorometer and Agilent 4200 Tapestation quality and size distribution control.

Library Preparation and Sequencing for CUT&RUN

KAPA Hyper prep kit (Cat# KK8504, KAPA Biosystems)and KAPA Dual-indexed adapter kit (Cat# KK8722 KAPA Biosystems) were used to construct the CUT&RUN DNA library for sequencing on an Illumina platform following the manufacturer’s instructions. After adaptor ligation, 2x volume of AMPure XP beads was used to recovery the small fragments. After 12–14 cycles of PCR amplification, the product was cleaned up with AMPure XP beads. 3% gel purification was performed if the PCR dimers were too much. Perform paired-end Illumina sequencing on the barcoded libraries following the manufacturer’s instructions. Briefly, the mixed libraries were denatured according to the standard protocol from Illumina. 1.3 mL of 1.8 pM diluted library pool was loaded to a NextSeq 500/550 Mid Output Kit v2 (150 cycles), and sequenced in the NextSeq 500 platform. Paired-end sequencing was then performed (2×75 bp, 8 bp index).

Cut & Run and ChIP data processing

Paired-end reads were adapter and quality trimmed using Trimgalore (v0.6.5, default parameters, http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) (RRID:SCR_011847) and aligned to mouse genome mm10 using Bowtie2 (v2.3.5.1, parameters: --local -D 20 -R 3 -N 0 -L 20 -i S,1,0.50 --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700) (RRID:SCR_016368). Duplicated reads were then marked and removed using picard MarkDuplicates (v2.21.1) (http://broadinstitute.github.io/picard/), and Samtools (v1.9) (RRID:SCR_002105), respectively with default parameters. For Rela and HA samples, only fragments of size less than 120bp were retained. Deeptools (v3.4.3) (RRID:SCR_016366) was used to convert all the resulting BAM files to Bigwig format for visualization. MACS2 (v2.2.7.1) was used to call peaks, on the resulting BAM files, with a p-value threshold of 1e-3. A set of 6845 peaks were inferred in the HA CUT&RUN by overlapping the called peaks from the two independent mice using bedtools2. This set was further filtered to remove any overlaps with non-specific IgG peak signals (from both mice), resulting in 5608 peaks. The peaks were then annotated to nearest genomic features using annotatePeaks.pl from Homer (v4.11.1)(46).

RNA sequencing and ATAC sequencing analysis

RNA-seq and ATAC-seq analysis was performed using Genialis Expression software (https://www.genialis.com) deployed locally on BCM computational infrastructure. Briefly, the RNA-seq pipeline run on the Genialis platform comprised the following steps. The raw reads were filtered to remove adapters and poor-quality reads using BBDuk (v37.9, https://sourceforge.net/projects/bbmap/). The resulting reads were mapped to the reference genomes (ENSEMBL 92) using STAR (v2.7.0) (RRID:SCR_015899). FeatureCounts (v1.6.3) (RRID:SCR_012919) was used for gene expression level quantification followed by DEseq2 (RRID:SCR_000154) for differential gene expression analysis (47). Lowly expression genes with expression count summed over all samples below 10 were filtered out from the input matrix to DESeq2. The paired-end reads from ATAC-seq were trimmed using BBDuk (v37.9) and mapped to the reference genome mm10 using Bowtie2 (v2.3.4.1). MACS2 (v2.1.1.20160309) was then used to call peaks on the aligned reads using p-value cutoff of 0.01 (parameters --shift -75 --extsize 150 --nomodel --call-summits --nolambda --keep-dup all -p 0.01)

DNA Motif analysis

To find motifs enriched around the binding sites, Homer findMotifsGenome.pl and MEME suite (v5.1.0) (48) were used. Further, we utilized the Rela ChIP -seq data in the GEO accession GSM3895240 (48) to differentiate the tumor-specific binding profile of ZR^fus1 from the canonical inflammatory context. For this, the overlaps between the peaks in GSM3895240 and the 5608 HA CUT&RUN peaks were inferred using bedtools2. Homer motif analysis was performed on each of the peaks sets; unique to HA:ZR^fus1, shared with and unique to the GSM3895240 Rela peaks. Enrichment of specific pathways in each of these groups was then computed using the R package Clusterprofiler (RRID:SCR_016884). To identify DNA motifs in human H3K27ac datasets, first peaks were identified in H3K27ac peaks then valleys (nucleosome-free regions) mapped which contain TF binding sites. Human ZFTA-RELA specific H3K27ac valley regions were compared against non-RELA ependymoma (as background) using HOMER (RRID:SCR_010881).

Super Enhancers and Core Regulatory Circuitry Analysis

Super enhancers were identified based on H3K27ac and IgG control CUT&RUN data using ROSE (49) with a stitching distance of 12.5 kb and exclusion of peaks within 2.5 kb of a promoter. Regions of H3K27ac overlapping ATAC peaks, along with the identified super enhancers, were provided as an input to determine core regulatory circuitry (https://github.com/linlabcode/CRC) (25).

Developmental Mapping Analysis

To project ZR^fus1 tumor and normal brain transcriptomes to the scRNAseq atlas of the developing mouse forebrain(28), we evaluated enrichment of the gene signature of each atlas cell type in each sample, using single-sample sample gene set enrichment analysis (ssGSEA)(50). The most enriched cell type signatures in ZR^fus1 tumors were selected for visualization by computing the median rank of each cell type signature across ZR^fus1 tumor samples based on ssGSEA scores, and taking the top 15.

STATEMENT OF SIGNIFICANCE.

Ependymomas are aggressive brain tumors. While drivers of supratentorial ependymoma (ZFTA- and YAP1- associated gene fusions) have been discovered, their functions remains unclear. Our study investigates the biology of ZFTA-RELA driven ependymoma, specifically, mechanisms of transcriptional de-regulation and direct downstream gene networks that may be leveraged for potential therapeutic testing.