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Accelerated discovery and collaborative research continue to highlight the remarkable progress that has been made in the diagnosis and treatment of pediatric cancers. This manuscript highlights important discoveries on how precision oncology is being incorporated into the diagnosis and treatment of childhood cancer at the national level to identify promising new therapies using a tumor-agnostic approach. In addition, we have highlighted three articles that incorporate genomics and cell-free DNA to better classify, monitor and incorporate risk-based therapies for children with medulloblastoma. Finally, we highlighted the important role of monclonal antiobody therapy in the treatment of recurrent B-cell leukemia and newly diagnosed high-risk neuroblastoma.

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.

Glioblastoma (GBM) is a heterogeneous brain tumor entity from infancy through adulthood. ALK gene fusions enriched in congenital and infant GBM have emerged as druggable driver alterations. Understanding the molecular basis and prevalence of ALK gene rearrangements will help define GBM patients that may benefit from ALK targeted therapy.

93 gene signature that characterizes ZFTA-Rela mouse and human tumors.

Figure S2 supporting expression and localization of ZRfus in the mouse IUE model.

Supplemental File 2: Genes common to all resistant U87 cell lines compared to drug naive control

Supplemental File 5

Supplemental Figure 4. Volcano plot representing differentially expressed genes for U87 naive versus resistant cells with a minimum of a 1.5-fold change, P-value < 0.05 and FDR < 0.10 between control and A.

Supplemental Figure 1. Schematic of the approach taken to achieve drug resistance.

Supplemental Figure 3. Drug naive U87 or panobinostat-resistant, bortezomib-resistant, or panobinostat and bortezomib-resistant (P + B) cells were cotreated with panobinostat (25 nmol/L) plus bortezomib (2.5 nmol/L) for 24 h.

Supplemental Figure Legends 1-7

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

Supplemental Figure 6. A. Drug naive and panobinostat and bortezomib-resistant (P + B) U87 cells were treated with gefitinib (2 µmol/L), enzastaurin (2 µmol/L), dasatinib (0.1 µmol/L), dinaciclib (0.18 µmol/L), PI 103 (1 µmol/L), PD 0325901 (2 µmol/L), HSP 990 (0.025 µmol/L), ABT 737 (2 µmol/L), JQ 1 (2 µmol/L), TP 0903 (2 µmol/L), Bay 11-7082 (2 µmol/L), YM 155 (0.025 µmol/L), cucurbitacin-I (0.2 µmol/L), vincristine (0.01 µmol/L), vinblastine (0.01 µmol/L), taxol (0.05 µmol/L), topotecan (0.025 µmol/L), temozolomide (25 µmol/L), gemcitabine (0.25 µmol/L) and 5-fluorouracil (20 µmol/L).

Supplemental Figure 7. Bar graph showing QPRT mRNA expression levels (absolute value) between drug naive and panobinostat + bortezomib resistant DIPG 007, SJG 2 and U87 cells.

Supplementary Figure 5. Assessment of citric acid cycle activity by protein expression analysis of PDHE1a.

Supplementary Table 1 - 7. Supplementary Table 1: Clinical and histopathological data associated with all spinal ependymomas analyzed in this study, along with molecular subgroup assignment; Supplementary Table 2: Table of all antibodies used in this study, their commercial sources, and dilutions used; Supplementary Table 3: Table of differentially expressed genes between spinal grade II and myxopapillary ependymoma; Supplementary Table 4: Summary of focal amplifications detected in spinal ependymoma by GISTIC2; Supplementary Table 5: Summary of focal deletions detected in spinal ependymoma by GISTIC2; Supplementary Table 6: List of significant pathways enriched by Gene Set Enrichment Analysis in spinal grade II ependymoma; Supplementary Table 7: List of significant pathways enriched by Gene Set Enrichment Analysis in spinal myxopapillary ependymoma

Supplementary Figure 1. Unsupervised consensus hierarchical clustering and non-negative matrix factorization of 35 spinal ependymoma gene expression signatures from a rank 2 to 4 classification.

Supplementary Figure 2. Summary of focal copy number alterations detected by GISTIC2 in spinal Grade II and Myxopapillary Ependymoma.

Supplementary Figure 3. Analysis of HIF-1a expression in Spinal Grade II versus Myxopapillary Ependymoma in additional to single sample GSEA analysis of HIF1a transcriptional targets.

Supplementary Figure 4. Metabolic protein expression analysis supporting Figure 4 along with lactate measurements in normal spinal tissue, Grade II spinal ependymoma, and myxopapillary ependymoma.

Purpose: Myxopapillary ependymoma (MPE) is a distinct histologic variant of ependymoma arising commonly in the spinal cord. Despite an overall favorable prognosis, distant metastases, subarachnoid dissemination, and late recurrences have been reported. Currently, the only effective treatment for MPE is gross-total resection. We characterized the genomic and transcriptional landscape of spinal ependymomas in an effort to delineate the genetic basis of this disease and identify new leads for therapy.Experimental Design: Gene expression profiling was performed on 35 spinal ependymomas, and copy number profiling was done on an overlapping cohort of 46 spinal ependymomas. Functional validation experiments were performed on tumor lysates consisting of assays measuring pyruvate kinase M activity (PKM), hexokinase activity (HK), and lactate production.Results: At a gene expression level, we demonstrate that spinal grade II and MPE are molecularly and biologically distinct. These are supported by specific copy number alterations occurring in each histologic variant. Pathway analysis revealed that MPE are characterized by increased cellular metabolism, associated with upregulation of HIF1α. These findings were validated by Western blot analysis demonstrating increased protein expression of HIF1α, HK2, PDK1, and phosphorylation of PDHE1A. Functional assays were performed on MPE lysates, which demonstrated decreased PKM activity, increased HK activity, and elevated lactate production.Conclusions: Our findings suggest that MPE may be driven by a Warburg metabolic phenotype. The key enzymes promoting the Warburg phenotype: HK2, PKM2, and PDK are targetable by small-molecule inhibitors/activators, and should be considered for evaluation in future clinical trials for MPE. Clin Cancer Res; 21(16); 3750–8. ©2015 AACR.

Supplementary Figure 6. A schematic of the glycolytic pathway in myxopapillary ependymoma illustrating deregulation of proteins and metabolites and possible targets for therapeutic intervention.

Supplementary Figure and Table Legends. Legends describing in detail the data shown in supplementary figures and tables pertaining to this manuscript.

Ependymomas comprise approximately 10 percent of childhood CNS malignancies and are associated with dismal outcomes if metastatic, incompletely resected, or recurrent.1 Standard therapy consists of maximal safe surgical resection followed by focal irradiation.2 Recurrent fusion genes involving C11orf95-RELA or YAP1 have been identified in supratentorial ependymomas, but targetable molecular lesions are rare.3,4 Here, we report a sustained response to larotrectinib in a pediatric patient with a histologic diagnosis of recurrent metastatic anaplastic ependymoma, whose molecular testing unexpectedly revealed a KANK1-NTRK2 fusion and methylation profile most consistent with pleomorphic xanthoastrocytoma (PXA). This case highlights the potentially profound clinical implications of integrating genomic profiling into the diagnostic evaluation of pediatric CNS tumors.

Ependymoma is the third most common malignant pediatric brain tumor. Although the biology that drives ependymoma is slowly being unraveled, the ability to translate these findings to clinical care remains an ongoing challenge. Epigenetic alterations appear to play a central role in the development of molecular classification of ependymoma. We reviewed the published literature available describing genetic and epigenetic underpinnings of ependymoma that have been reported to date and have summarized the information regarding genetic drivers of ependymoma that may point us toward therapeutic strategies. Ependymoma is a molecularly heterogeneous disease which has now been divided into at least nine distinct molecular subtypes based on DNA methylation and gene expression profiling. DNA methylation has emerged as an effective tool for classification of brain tumors alongside histopathology and other molecular diagnostics. There have been large retrospective cohorts describing molecular subgroup identity as a powerful independent predictor of outcome. There is limited published data on prospective trials to date however this is forthcoming which will lead to molecular stratification in the next generation of clinical studies. This is a review of recent advancements in our understanding of the epigenetic basis of ependymoma and discussion of how these findings reveal potential therapeutic opportunities.

To improve therapeutic responses in patients with glioma, new combination therapies that exploit a mechanistic understanding of the inevitable emergence of drug resistance are needed. Intratumoral heterogeneity enables a low barrier to resistance in individual patients with glioma. We reasoned that targeting two or more fundamental processes that gliomas are particularly dependent upon could result in pleiotropic effects that would reduce the diversity of resistant subpopulations allowing convergence to a more robust therapeutic strategy. In contrast to the cytostatic responses observed with each drug alone, the combination of the histone deacetylase inhibitor panobinostat and the proteasome inhibitor bortezomib synergistically induced apoptosis of adult and pediatric glioma cell lines at clinically achievable doses. Resistance that developed was examined using RNA-sequencing and pharmacologic screening of resistant versus drug-naïve cells. Quinolinic acid phosphoribosyltransferase (QPRT), the rate-determining enzyme for de novo synthesis of NAD+ from tryptophan, exhibited particularly high differential gene expression in resistant U87 cells and protein expression in all resistant lines tested. Reducing QPRT expression reversed resistance, suggesting that QPRT is a selective and targetable dependency for the panobinostat–bortezomib resistance phenotype. Pharmacologic inhibition of either NAD+ biosynthesis or processes such as DNA repair that consume NAD+ or their simultaneous inhibition with drug combinations, specifically enhanced apoptosis in treatment-resistant cells. Concomitantly, de novo vulnerabilities to known drugs were observed. Implications: These data provide new insights into mechanisms of treatment resistance in gliomas, hold promise for targeting recurrent disease, and provide a potential strategy for further exploration of next-generation inhibitors.

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.

Advances in genomic, transcriptomic and epigenomic profiling now identifies pediatric ependymoma as a defined biological entity. Molecular interrogation has segregated these tumors into distinct biological subtypes based on anatomical location, age and clinical outcome, which now defines the need to tailor therapy even for histologically similar tumors. These findings now provide reasons for a paradigm shift in therapy, which should profile future clinical trials focused on targeted therapeutic strategies and risk-based treatment. The need to diagnose and differentiate the aggressive variants, which include the posterior fossa group A and the supratentorial RELA fusion subtypes, is imperative to escalate therapy and improve survival.

Diffuse midline gliomas (DMGs) bearing driver mutations of histone 3 lysine 27 (H3K27M) are incurable brain tumors with unique epigenomes. Here, we generated a syngeneic H3K27M mouse model to study the amino acid metabolic dependencies of these tumors. H3K27M mutant cells were highly dependent on methionine. Interrogating the methionine cycle dependency through a short-interfering RNA screen identified the enzyme methionine adenosyltransferase 2A (MAT2A) as a critical vulnerability in these tumors. This vulnerability was not mediated through the canonical mechanism of MTAP deletion; instead, DMG cells have lower levels of MAT2A protein, which is mediated by negative feedback induced by the metabolite decarboxylated S-adenosyl methionine. Depletion of residual MAT2A induces global depletion of H3K36me3, a chromatin mark of transcriptional elongation perturbing oncogenic and developmental transcriptional programs. Moreover, methionine-restricted diets extended survival in multiple models of DMG in vivo. Collectively, our results suggest that MAT2A presents an exploitable therapeutic vulnerability in H3K27M gliomas.

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

Ependymomas are aggressive central nervous system tumors that resist chemotherapy. In this issue of Cancer Cell, Gojo et al. dissect the single cell transcriptional landscapes of ependymoma to define cellular programs that mediate therapeutic resistance, tumor aggressiveness, and potential targets for therapy.

Clear cell sarcoma of the kidney (CCSK) is the second most common malignant pediatric renal tumor. Two of the recurrent somatic alterations reported in CCSK are BCL-6 corepressor (BCOR) internal tandem duplication (ITD) and YWHAE-NUTM2B/E gene fusion. A minority of patients with CCSKs have other rare somatic alterations. We report two patients with CCSK showing BCOR-CCNB3 (where CCNB3 is cyclin B3) fusion, who had similar clinical presentation of a large renal mass with tumor thrombus extending through the inferior vena cava into the right atrium and a favorable response to chemotherapy. We recommend BCOR-CCNB3 fusion testing for all patients with CCSK who lack BCOR-ITD or YWHAE-NUTM2B/E gene fusions.

Genomic sequencing has driven precision-based oncology therapy; however, the genetic drivers of many malignancies remain unknown or non-targetable, so alternative approaches to the identification of therapeutic leads are necessary. Ependymomas are chemotherapy-resistant brain tumours, which, despite genomic sequencing, lack effective molecular targets. Intracranial ependymomas are segregated on the basis of anatomical location (supratentorial region or posterior fossa) and further divided into distinct molecular subgroups that reflect differences in the age of onset, gender predominance and response to therapy1,2,3. The most common and aggressive subgroup, posterior fossa ependymoma group A (PF-EPN-A), occurs in young children and appears to lack recurrent somatic mutations2. Conversely, posterior fossa ependymoma group B (PF-EPN-B) tumours display frequent large-scale copy number gains and losses but have favourable clinical outcomes1,3. More than 70% of supratentorial ependymomas are defined by highly recurrent gene fusions in the NF-κB subunit gene RELA (ST-EPN-RELA), and a smaller number involve fusion of the gene encoding the transcriptional activator YAP1 (ST-EPN-YAP1)1,3,4. Subependymomas, a distinct histologic variant, can also be found within the supratetorial and posterior fossa compartments, and account for the majority of tumours in the molecular subgroups ST-EPN-SE and PF-EPN-SE. Here we describe mapping of active chromatin landscapes in 42 primary ependymomas in two non-overlapping primary ependymoma cohorts, with the goal of identifying essential super-enhancer-associated genes on which tumour cells depend. Enhancer regions revealed putative oncogenes, molecular targets and pathways; inhibition of these targets with small molecule inhibitors or short hairpin RNA diminished the proliferation of patient-derived neurospheres and increased survival in mouse models of ependymomas. Through profiling of transcriptional enhancers, our study provides a framework for target and drug discovery in other cancers that lack known genetic drivers and are therefore difficult to treat., This work was supported by an Alex's Lemonade Stand Young Investigator Award (S.C.M.), The CIHR Banting Fellowship (S.C.M.), The Cancer Prevention Research Institute of Texas (S.C.M., RR170023), Sibylle Assmus Award for Neurooncology (K.W.P.), the DKFZ-MOST (Ministry of Science, Technology & Space, Israel) program in cancer research (H.W.), James S. McDonnell Foundation (J.N.R.) and NIH grants: CA154130 (J.N.R.), R01 CA169117 (J.N.R.), R01 CA171652 (J.N.R.), R01 NS087913 (J.N.R.) and R01 NS089272 (J.N.R.). R.C.G. is supported by NIH grants T32GM00725 and F30CA217065. M.D.T. is supported by The Garron Family Chair in Childhood Cancer Research, and grants from the Pediatric Brain Tumour Foundation, Grand Challenge Award from CureSearch for Children’s Cancer, the National Institutes of Health (R01CA148699, R01CA159859), The Terry Fox Research Institute and Brainchild. M.D.T. is also supported by a Stand Up To Cancer St. Baldrick’s Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT1113).

BACKGROUND Patients with metastatic pediatric ependymoma have limited therapeutic options and poor outcomes. Approximately ¾ of supratentorial ependymomas are driven by C11ORF95-RELA fusions, and the remaining by a heterogeneous group of fusion events. We present a six year-old male diagnosed with supratentorial ependymoma with leptomeningeal carcinomatosis harboring an NTRK2-fusion. Local and distant multifocal, intracranial and intraspinal tumor recurrence occurred seven months following gross total resection of the primary lesion and proton beam craniospinal irradiation. METHODS DNA and RNA from FFPE tumor were used for targeted sequencing using an 81-gene fusion panel and 124-gene mutation panel. Separately, capture transcriptome sequencing, exome sequencing, and copy number array were performed as part of the Texas KidsCanSeq study, an NHGRI/NCI-funded Clinical Sequencing Evidence-Generating Research (CSER) consortium project. All sequencing was carried out in CLIA-certified laboratories. RESULTS An in-frame fusion between 5’ exons 1–3 of KANK1 and 3’ exons 16–21 of NTRK2, predicted to retain the kinase domain, was identified. At tumor recurrence, therapy was initiated with Larotrectinib, an FDA-approved pan-TRK inhibitor. Clinical improvement in cognitive speed, motor strength, and coordination was observed at two weeks with significant tumor response on MRI at two and four months. CONCLUSION TRK gene fusions have not previously been reported in ependymoma. Further tumor characterization by methylation profiling is underway and will be of diagnostic interest given the apparent discordance between tumor histology and molecular findings. This case highlights the potential impact of clinical genomic analysis for children with CNS tumors.

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.

To improve therapeutic responses in glioma patients, new combination therapies that exploit a mechanistic understanding of the inevitable emergence of drug resistance are needed. Intra-tumoral heterogeneity enables a low barrier to resistance in individual glioma patients. We reasoned that targeting two or more fundamental processes that gliomas are particularly dependent upon could result in pleiotropic effects that would reduce the diversity of resistant subpopulations allowing convergence to a more robust therapeutic strategy. In contrast to the cytostatic responses observed with each drug alone, the combination of the HDAC inhibitor panobinostat and the proteasome inhibitor bortezomib synergistically induced apoptosis of adult and pediatric glioma cell lines at clinically achievable doses. Resistance that developed was examined using RNA sequencing and pharmacological screening of resistant versus drug naive cells. Quinolinic acid phosphoribosyltransferase (QPRT), the rate-determining enzyme for de novo synthesis of nicotinamide adenine dinucleotide (NAD+) from tryptophan, exhibited particularly high differential gene expression in resistant U87 cells and protein expression in all resistant lines tested. Reducing QPRT expression reversed resistance, suggesting that QPRT is a selective and targetable dependency for the panobinostat-bortezomib resistance phenotype. Pharmacological inhibition of either NAD+ biosynthesis or processes such as DNA repair that consume NAD+ or their simultaneous inhibition with drug combinations, specifically enhanced apoptosis in treatment-resistant cells. Concomitantly, de novo vulnerabilities to known drugs were observed.

Mack et al. defined active chromatin landscapes of glioblastoma stem cells (GSCs) and primary tumor specimens, revealing novel transcriptional regulatory circuits and therapeutic targets. Super-enhancers identified essential transcription factors that underlie GSC identity and intertumoral diversity, potentially informing precision medicine., Glioblastoma is an incurable brain cancer characterized by high genetic and pathological heterogeneity. Here, we mapped active chromatin landscapes with gene expression, whole exomes, copy number profiles, and DNA methylomes across 44 patient-derived glioblastoma stem cells (GSCs), 50 primary tumors, and 10 neural stem cells (NSCs) to identify essential super-enhancer (SE)–associated genes and the core transcription factors that establish SEs and maintain GSC identity. GSCs segregate into two groups dominated by distinct enhancer profiles and unique developmental core transcription factor regulatory programs. Group-specific transcription factors enforce GSC identity; they exhibit higher activity in glioblastomas versus NSCs, are associated with poor clinical outcomes, and are required for glioblastoma growth in vivo. Although transcription factors are commonly considered undruggable, group-specific enhancer regulation of the MAPK/ERK pathway predicts sensitivity to MEK inhibition. These data demonstrate that transcriptional identity can be leveraged to identify novel dependencies and therapeutic approaches., Graphical Abstract

RATIONALE Over 70% of supratentorial (ST) ependymoma are characterized by an oncogenic fusion between C11ORF95 and RELA. C11ORF95-RELA fusion is frequently the sole genetic driver detected in ST ependymoma, thus ranking this genomic event as a lead target for therapeutic investigation. RELA is a transcription factor (TF) central to mediating NF-kB pathway activation in processes such as inflammation, cellular metabolism, and chemotaxis. HYPOTHESIS: We posited that C11ORF95-RELA acts as an oncogenic TF that aberrantly shapes the tumor epigenome to drive aberrant transcription. Approach: To this end we developed an in utero electroporation (IUE) mouse model of ependymoma to express C11ORF95-RELA during embryonic development. Our IUE approach allowed us to develop C11ORF95-RELA driven tumor models and cell lines. We comprehensively characterized the epigenome and transcriptome of C11ORF95-RELA fusion driven mouse cells by H3K27ac ChIP-seq, ATAC-seq, and RNA-seq. RESULTS This data revealed that: 1) C11ORF95-RELA directly engages ‘open’ chromatin and is enriched at regions with known RELA TF binding sites as well as novel genomic loci/motifs, 2) C11ORF95-RELA preferentially binds to both H3K27ac (active) enhancers and promoters, and 3) Bound C11ORF95-RELA promoter loci are associated with increased transcription of genes shared with human ependymoma. CONCLUSION Our findings shed light on the transcriptional mechanisms of C11ORF95-RELA, and reveal downstream targets that may represent cancer dependency genes and molecular targets.

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.

High-grade gliomas (HGG) defined by histone 3 K27M driver mutations exhibit global loss of H3K27 trimethylation and reciprocal gain of H3K27 acetylation, respectively shaping repressive and active chromatin landscapes. We generated tumor-derived isogenic models bearing this mutation and show that it leads to pervasive H3K27ac deposition across the genome. In turn, active enhancers and promoters are not created de novo and instead reflect the epigenomic landscape of the cell of origin. H3K27ac is enriched at repeat elements, resulting in their increased expression, which in turn can be further amplified by DNA demethylation and histone deacetylase inhibitors providing an exquisite therapeutic vulnerability. These agents may therefore modulate anti-tumor immune responses as a therapeutic modality for this untreatable disease.

Purpose: Myxopapillary ependymoma (MPE) is a distinct histologic variant of ependymoma arising commonly in the spinal cord. Despite an overall favorable prognosis, distant metastases, subarachnoid dissemination, and late recurrences have been reported. Currently, the only effective treatment for MPE is gross-total resection. We characterized the genomic and transcriptional landscape of spinal ependymomas in an effort to delineate the genetic basis of this disease and identify new leads for therapy. Experimental Design: Gene expression profiling was performed on 35 spinal ependymomas, and copy number profiling was done on an overlapping cohort of 46 spinal ependymomas. Functional validation experiments were performed on tumor lysates consisting of assays measuring pyruvate kinase M activity (PKM), hexokinase activity (HK), and lactate production. Results: At a gene expression level, we demonstrate that spinal grade II and MPE are molecularly and biologically distinct. These are supported by specific copy number alterations occurring in each histologic variant. Pathway analysis revealed that MPE are characterized by increased cellular metabolism, associated with upregulation of HIF1α. These findings were validated by Western blot analysis demonstrating increased protein expression of HIF1α, HK2, PDK1, and phosphorylation of PDHE1A. Functional assays were performed on MPE lysates, which demonstrated decreased PKM activity, increased HK activity, and elevated lactate production. Conclusions: Our findings suggest that MPE may be driven by a Warburg metabolic phenotype. The key enzymes promoting the Warburg phenotype: HK2, PKM2, and PDK are targetable by small-molecule inhibitors/activators, and should be considered for evaluation in future clinical trials for MPE. Clin Cancer Res; 21(16); 3750–8. ©2015 AACR.

Ependymoma is the third most common pediatric brain tumor, yet because of the paucity of effective therapeutic interventions, 45% of patients remain incurable. Recent transcriptional and copy number profiling of the disease has identified few driver genes and in fact points to a balanced genomic profile. Candidate gene approaches looking at hypermethylated promoters and genome-wide epigenetic arrays suggest that DNA methylation may be critical to ependymoma pathogenesis. This review attempts to highlight existing and emerging evidence implicating the ependymoma epigenome as a key player and that epigenetic modifiers may offer new targeted therapeutic avenues for patients.

SummaryDespite the histological similarity of ependymomas from throughout the neuroaxis, the disease likely comprises multiple independent entities, each with a distinct molecular pathogenesis. Transcriptional profiling of two large independent cohorts of ependymoma reveals the existence of two demographically, transcriptionally, genetically, and clinically distinct groups of posterior fossa (PF) ependymomas. Group A patients are younger, have laterally located tumors with a balanced genome, and are much more likely to exhibit recurrence, metastasis at recurrence, and death compared with Group B patients. Identification and optimization of immunohistochemical (IHC) markers for PF ependymoma subgroups allowed validation of our findings on a third independent cohort, using a human ependymoma tissue microarray, and provides a tool for prospective prognostication and stratification of PF ependymoma patients.

A newborn female with Down syndrome initially presented to the neonatal intensive care unit with difficulty feeding, failure to thrive, and respiratory difficulty that required oxygen supplementation. She also had atrioventricular canal defect that was diagnosed prenatally. On physical examination, she had hepatosplenomegaly, as well as dysmorphic features typical of Down syndrome phenotype. Hemoglobin was 16.9, platelet count was 276 000, and white blood cells (WBC) count was 62 400 with the smear showing myelocytes, promyelocytes, and myeloblasts. WBC went as high as 80 000 with blast count as high as 44% and platelets as high as 771 000. Subsequent peripheral smear showed blasts, promyelocytes, metamyelocytes, and macrocytosis of red blood cells with normal platelets. The patient’s liver enzymes, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase were mildly elevated. Abdominal ultrasound revealed mild hepatosplenomegaly, minimal ascites, and gallbladder wall thickening. Further investigation with genetic and molecular biologic workup confirmed trisomy 21 as a somatic mutation and revealed GATA-1 mutation. The patient was negative for JAK2 V617 mutation. On flow cytometry, blasts were positive for CD34, CD117, HLADR, CD4, CD71, and CD9. Markers for megakaryocytic differentiation, CD41 and CD61, were partially expressed. Fluorescence in situ hybridization studies were inconclusive and attempts at obtaining additional genetic markers, specifically RUNX1, were unsuccessful.

© Impact Journals, LLC. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., Advances in the molecular biology of medulloblastoma revealed four genetically and clinically distinct subgroups. Group 3 medulloblastomas are characterized by frequent amplifications of the oncogene MYC, a high incidence of metastasis, and poor prognosis despite aggressive therapy. We investigated several potential small molecule inhibitors to target Group 3 medulloblastomas based on gene expression data using an in silico drug screen. The Connectivity Map (C-MAP) analysis identified piperlongumine as the top candidate drug for non-WNT medulloblastomas and the cyclin-dependent kinase (CDK) inhibitor alsterpaullone as the compound predicted to have specific antitumor activity against Group 3 medulloblastomas. To validate our findings we used these inhibitors against established Group 3 medulloblastoma cell lines. The C-MAP predicted drugs reduced cell proliferation in vitro and increased survival in Group 3 medulloblastoma xenografts. Alsterpaullone had the highest efficacy in Group 3 medulloblastoma cells. Genomic profiling of Group 3 medulloblastoma cells treated with alsterpaullone confirmed inhibition of cell cycle-related genes, and down-regulation of MYC. Our results demonstrate the preclinical efficacy of using a targeted therapy approach for Group 3 medulloblastomas. Specifically, we provide rationale for advancing alsterpaullone as a targeted therapy in Group 3 medulloblastoma., This study was supported by the Canadian Cancer Society (Grant #2011-70051), the Pediatric Brain Tumor Foundation of the United States, the Brain Tumour Foundation of Canada, Meagan’s Walk, b.r.a.i.n.child and the Wiley Fund at the Hospital for Sick Children.