Ependymoma Cell Research Articles

THE LINK BETWEEN M6A RNA METHYLATION AND R LOOPS IN PAEDIATRIC HIGH-GRADE GLIOMAS AND EPENDYMOMAS

Abstract AIMS Paediatric high-grade glioma (HGG) and ependymoma (EPN) are two devastating malignant tumours of the central nervous system that unfortunately carry a poor prognosis. As R loops are a known source of genomic instability in eukaryotic cells and have previously been revealed to undergo m6A RNA methylation, we sought to investigate the potential link between m6A methylation and R loops in relation to the pathogenesis of paediatric gliomas. METHOD Paediatric HGG cell lines including KNS42, GCE62 and SF188, and paediatric EPN cell lines including BXD- 1425EPN and EPN9, were utilised for experimental analysis, in addition to normal human astrocytes. Immuno- cytochemistry was performed to assess m6A and R loop levels in tumour cell lines compared to normal human astrocytes. Moreover, DNA damage levels and the effects of anti-cancer drugs on R loop levels were also examined. RESULTS Immunocytochemistry analysis revealed that overall m6A and R loop levels were higher in tumour cell lines compared to normal human astrocytes. Furthermore, differences in R loop levels were observed as a result of pharmacological and biological manipulation, including reduction in R loop abundance with PARP inhibitors. CONCLUSIONS The link between m6A methylation and R loops in relation to the pathogenesis of paediatric HGG and EPN is being investigated. The results of these experiments will be presented.

EPEN-11. CHARACTERIZATION OF THE SPATIAL ARCHITECTURE OF INTRATUMORAL CELL STATES IN PEDIATRIC HIGH-RISK EPENDYMOMA

Abstract By single-cell analyses, we previously uncovered distinct intratumoral transcriptomic ependymoma (EPN) cell states and developmental trajectories correlating with molecular subtype and biological behavior. However, the relation of these cell states to characteristic EPN tissue architecture and tumor microenvironment remains widely unknown. We aimed to characterize the spatial architecture and elucidate regulatory circuits in EPN via spatial transcriptomics (10X Visium) of tumor specimens matching our previously generated single-cell transcriptome data spanning all EPN subtypes (ZFTA, YAP1, PFA, PFB). We investigated whether spatially restricted cell-cell interactions correlate with our previously identified cell states. Analyses revealed distinct spatially resolved cell populations in supratentorial and posterior fossa EPN. All subgroups showed several (5-9) spatially discrete cell clusters. Via non-negative matrix factorization (NMF), cell-specific spatial clusters emerged correlating with previous single-cell programs. ZFTA EPN highly correlates with ZFTA-specific metaprograms whereas all other subgroups did not show ZFTA-specific gene expression. Explicit genes such as DLK1 are exclusively upregulated in ZFTA EPN indicating spatially distinct aberrant Notch pathway signaling. Interestingly, this subgroup revealed spatial zones with upregulation of metabolic genes and endothelial markers anti-correlating with a ZFTA-specific program. Ependymal and neuronal-precursor-like signatures were enriched in our unique YAP1 EPN sample in a spatially restricted pattern, whereas GFAP was ubiquitously expressed. PFA was characterized by a neural stem cell (NSC)-like niche accompanied by metabolic and immune-reactive gene signatures. PFB showed a similar cell niche associated with NSC-like and glial-progenitor-like genes enriched simultaneously, further highlighting the NSC characteristic of posterior fossa tumors. Ependymal signatures were absent in PFA and present in PFB indicating more differentiated cell populations in PFB. Summarizing, we confirm presence of distinct intratumoral cell states within ependymoma tissue and reveal their spatial organization. In depth analysis and functional validation in matched ependymoma models will enable interrogation of underlying tumor promoting signaling events driving intratumoral developmental trajectories.

Abstract 3886: 3D genome conformation analysis in ST-EPN-ZFTA ependymoma identifies RCOR2 as a potential target

Abstract Ependymoma (EPN) is a tumor of the central nervous system that occurs in children and adults. Childhood EPNs are one of the most aggressive pediatric brain tumors, arising both in infratentorial and supratentorial (ST) regions of the brain. Molecular profiling has identified distinct subgroups of ependymomas, including ST-EPN-ZFTA and PF-EPN-A as the most common and most aggressive ones in children. To this date, the only curative and indispensable treatment option is surgical resection followed by radiation. Unfortunately, there are no targeted therapies in clinical use. However, EPN cells have been extremely difficult to culture and expand in vitro, which explains why molecular studies on EPN cells have been hampered. This shortcoming has recently been overcome by establishing EPN models including human cell lines and organoids as well as patient derived xenografts (PDX). 3D genome conformation analysis has emerged as a powerful approach to understand tumor development and to identify novel drug targets for the treatment of different tumors. In this project we have used genome-wide chromosome conformation capture (Hi-C), complemented with H3K27ac (active enhancers) as well as gene expression in primary ependymoma tumors and cell lines to identify regulatory mechanisms underlying aberrant expression of genes that are essential for ependymoma tumorigenesis. Results of this study showed that the fusion between ZFTA and RELA leads to the formation of new regulatory environments that are recurrently associated with aberrant overexpression of RCOR2 only in ST-EPN-ZFTA ependymoma cells. Indeed, compared to all other ependymoma subgroups as well as to normal brain, RCOR2 is significantly upregulated only in ST-EPN-ZFTA patient samples. To test oncogenic relevance of RCOR2 for growth and maintenance, we depleted RCOR2 by RNAi and observed a strongly reduced cell survival in several ST-EPN-ZFTA cell lines but not or to a lesser extent in PF-EPN-A cells. Since RCOR2 is an adaptor protein lacking enzymatic activity, we looked at the expression of the proteins known as an interacting partner of RCOR2. Lysine-histone demethylase 1 (LSD1) is one of them and its expression in ST-EPN-ZFTA significantly correlates with RCOR2 expression. We therefore hypothesized that ST-EPN-ZFTA cells may be sensitive to LSD1 depletion, too. Indeed, depletion of LSD1 by RNAi resulted in a strong growth inhibition in ST-EPN-ZFTA cells, but not in PF-EPN-A cells. However, none of the tested LSD1 inhibitors exerted anti-proliferative activity at clinically reachable doses in the ST-EPN-ZFTA cells indicating a potential LSD1 non-enzymatic function in this particular tumor type. In summary, our study nominates RCOR2 as a oncogenic gene specific for ST-EPN-ZFTA ependymomas. Current studies focus on the identification of other interacting partners of the RCOR2-LSD1 complex and how to target these for therapeutic approaches. Citation Format: Aylin Camgöz, Konstantin Okonechnikov, Owen Chapman, Monika Maurmann, Frank Buchholz, Stefan Pfister, Lukas Chavez, Marcel Kool. 3D genome conformation analysis in ST-EPN-ZFTA ependymoma identifies RCOR2 as a potential target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3886.

EPEN-32. Leveraging cell surface targets as therapeutic vulnerabilities for pediatric ependymoma

Brain tumors are the leading cause of cancer-associated death in children. Ependymoma, an aggressive type of childhood brain tumor, is currently treated with surgery and radiotherapy. Ependymomas are a molecularly heterogeneous group of tumors driven by distinct genetic and epigenetic alterations. In children, 90% of ependymomas arise intracranially, with two thirds occurring in the posterior fossa (PF) and one third in the supratentorial brain (ST). PF ependymomas are divided into at least two groups termed, PFA and PFB, with PFA tumors associated with poor clinical outcomes. Over 70% of ST ependymoma are characterized by an oncogenic fusion between ZFTA and RELA and shown in some cohorts to have poor clinical outcome, particularly in the context of CDKN2A tumor suppressor gene loss. A major challenge in identifying therapies against ependymoma, has been the paucity of genetic abnormalities available for targeting. PFA ependymomas harbor largely balanced genomes with no recurrent CNVs, fusions, or somatic mutations that are amenable to pharmacologic inhibition. ZFTA-RELA ependymoma while representing a clear disease driver, functions as a transcription factor and lacks clear binding surfaces available for direct inhibition using small molecules. Therefore, alternative approaches are needed to identify new targets and effective therapies in ependymoma to be evaluated in pre-clinical models. In both human ependymoma cell culture lines and PDX models, we demonstrate that a multi-omic approach is promising for cell surface target discovery, and further, focused cell surface profiling can identify lead targets that can be rapidly translated for CAR T-cell therapy.

EPEN-31. Developmental and Oncogenic Transcription Factor Circuits as Dependencies in Ependymoma

Brain tumors are the most common cause of cancer death in children. ZFTA-RELA gene fusion is one the most potent drivers of cancer and is sufficient to induce tumors when expressed during brain development. ZFTA-RELA (denoted ZRFUS) fusion is the most frequent events that occurs in an aggressive childhood brain tumor called ependymoma (> 70% of cases). ZFTA recruits RELA to novel DNA binding sites and is necessary to activate ependymoma oncogene transcription. There are currently no targetable treatments for ependymoma, thus studying the mechanisms that regulate ZRFUS oncogenic programs may yield opportunities to develop effective therapies. To study proteins that regulate gene expression programs in brain cancer, the Mack lab and others have comprehensively characterized the active chromatin landscapes of several adult and pediatric brain cancers. This genome-wide analysis has identified highly active TFs, termed core regulatory circuit (CRC) TFs that govern gene expression programs such as MYC, GLI2, SOX2, and OLIG1/2, previously described in brain tumors such as glioblastoma and medulloblastoma. Critically, a glial cell fate specification TF, SOX9, showed the highest levels of activity in ependymoma. A functional RNA interference screen of CRC TFs prioritized SOX9 as the top cancer dependency gene required for ZRFUS ependymoma cell proliferation. To study ZRFUS ependymoma, we developed one of the first genetic mouse models of the disease, and show in preliminary data, that SOX9 knockout abolishes tumor initiation. Surprisingly, SOX9 KO has no impact on tumor initiation in an aggressive glioma model, suggesting tumor-specific contributions of SOX9. This concept is supported by our data that shows SOX9 co-recruitment to a vast majority of ZRFUS binding sites in the genome. Our data supports that SOX9 regulates ZFTA-RELA target cistrome; presenting a potential pathway that may be explored for therapeutic benefit.

Neuroaxial Infantile Hemangiomas: Imaging Manifestations and Association with Hemangioma Syndromes

Infantile hemangiomas are common lesions in the pediatric population; in rare cases, an infantile hemangioma can be detected along the neural axis. The purposes of our study included determination of the incidence, location, and imaging appearance of neuroaxial infantile hemangiomas and their syndromic association. We also assessed additional features of cerebral and cardiovascular anomalies that may be associated with neuroaxial lesions. A retrospective cohort study was performed, searching the radiology database for patients with segmental infantile hemangiomas referred for assessment of possible hemangioma syndromes. We retrospectively reviewed brain and spine MR imaging studies, with particular attention paid to neuroaxial vascular lesions, as well as the relevant clinical data. Neuroaxial hemangioma imaging findings were described, and comparison of segmental cutaneous infantile hemangioma location with the imaging findings was performed in patients with confirmed hemangioma syndromes and in patients with isolated skin infantile hemangioma. Ninety-five patients with segmental infantile hemangioma were included in the study, 42 of whom had a hemangioma syndrome; of those, 41 had posterior fossa brain malformations, hemangioma, arterial lesions, cardiac abnormalities, and eye abnormalities (PHACE) syndrome and 1 had diffuse neonatal hemangiomatosis. Neuroaxial involvement was detected in 20/42 patients (48%) with hemangioma syndromes and in no subjects with isolated segmental infantile hemangioma (P < .001). The most common intracranial hemangioma location was within the ipsilateral internal auditory canal (83%). Many pediatric patients with segmental infantile hemangioma in the setting of hemangioma syndromes, especially those with PHACE, had neuroaxial hemangiomas. This finding may potentially lead to requiring additional clinical evaluation and management of these patients.

EPEN-09. SUPER ENHANCER GENES AS MOLECULAR TARGETS IN C11ORF95-RELA FUSION 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.

BLBP Is Both a Marker for Poor Prognosis and a Potential Therapeutic Target in Paediatric Ependymoma.

Simple SummaryEpendymomas are the second most common paediatric brain tumour, and the 5-year survival rate remains no higher than 50%. Identifying new prognostic markers and targets for therapy in ependymoma is an important area of research. In this study, we demonstrate that brain lipid binding protein (BLBP, FABP7) is expressed in a sub-population of cells in ependymoma patient samples, consistent with it being a cancer stem cell marker. BLBP expression was associated with both significantly reduced event free survival and overall survival. BLBP can be functionally inhibited by PPAR antagonists, and we demonstrated that these antagonists can reduce ependymoma cell migration, invasion and chemo-resistance. The BLBP ligand docosohexanoic acid also attenuated these three hallmark characteristics of ependymomas, leading us to conclude that BLBP is not just a prognostic marker for poor ependymoma survival, but that it represents a druggable target in ependymoma therapy.Paediatric ependymomas are aggressive, treatment-resistant tumours with a tendency towards relapse, consistent with a sub-population of therapy-resistant cancer stem cells. These cells are believed to derive from brain lipid binding protein (BLBP)-expressing radial glia, hence we proposed that BLBP may be a marker for ependymoma therapy resistance. BLBP protein expression correlated with reduced overall survival (OS) in patients from two trials (CNS9204, a chemotherapy-led infant trial—5 y OS 45% vs. 80%, p = 0.011—and CNS9904, a radiotherapy-led trial—OS 38% vs. 85%, p = 0.002). All ependymoma cell lines examined by qRT-PCR expressed BLBP, with expression elevated in stem cell-enriched neurospheres. Modulation of BLBP function in 2D and 3D assays, using either peroxisome proliferator activated receptor (PPAR) antagonists or BLBP’s fatty acid substrate docosahexaneoic acid (DHA), potentiated chemotherapy response and reduced cell migration and invasion in ependymoma cell lines. BLBP is therefore an independent predictor of poor survival in paediatric ependymoma, and treatment with PPAR antagonists or DHA may represent effective novel therapies, preventing chemotherapy resistance and invasion in paediatric ependymoma patients.

IMMU-20. EVALUATION OF CAR T CELLS IN EPENDYMOMA

BACKGROUNDEpendymoma is the third most common pediatric brain tumor and current treatment still results in a 10-year relapse rate of over 70% in the highest risk groups. The treatment refractory nature of ependymoma to standard therapies strongly supports the development of novel interventions. Ependymoma tumor cells express HER2 and there are active clinical trials treating children with ependymoma using local delivery of second-generation HER2 CAR T cells.METHODSTwo high-risk patient-derived ependymoma cell lines, MAF811 and MAF928, that display HER2 surface expression are used for testing. We tested second-generation HER2-BBz CAR T cells in vitro and in vivo.RESULTSHER2 CAR T cells effectively kill ependymoma tumor cells in culture, but this strategy cannot eradicate the same tumor cells in mice when implanted in the fourth ventricle of the brain. HER2 CAR T cells proliferate and traffic into the tumor, but this causes a dramatic influx of immune cells, tumor swelling and lethal toxicity in a subset of mice. Mice that survive this initial tumor swelling, display significant tumor shrinkage but all tumors eventually start growing again. Ependymoma tumor cells release high amounts of inflammatory chemokines that strongly attract neutrophils and monocytes to the tumor, compared to other brain tumors, and can downregulate HER2 expression to escape recognition by CAR T cells.CONCLUSIONThe immunosuppressive microenvironment as well as tumor heterogeneity make HER2 CAR T cells ineffective in ependymoma. Studying these two hurdles in CAR T cell therapy is critical to effectively treat brain tumors with CAR T cells.

EPEN-21. IMPAIRED NEURONAL-GLIAL FATE SPECIFICATION IN PEDIATRIC EPENDYMOMA REVEALED BY SINGLE-CELL RNA-SEQ

Ependymoma represents a heterogeneous disease affecting the entire neuraxis. Extensive molecular profiling efforts have identified molecular ependymoma subgroups based on DNA methylation. However, the intratumoral heterogeneity and developmental origins of these groups are only partially understood, and effective treatments are still lacking for about 50% of patients with high-risk tumors. We interrogated the cellular architecture of ependymoma using single cell/nucleus RNA-sequencing to analyze 24 tumor specimens across major molecular subgroups and anatomic locations. We additionally analyzed ten patient-derived ependymoma cell models and two patient-derived xenografts (PDXs). Interestingly, we identified an analogous cellular hierarchy across all ependymoma groups, originating from undifferentiated neural stem cell-like populations towards different degrees of impaired differentiation states comprising neuronal precursor-like, astro-glial-like, and ependymal-like tumor cells. While prognostically favorable ependymoma groups predominantly harbored differentiated cell populations, aggressive groups were enriched for undifferentiated subpopulations. Projection of transcriptomic signatures onto an independent bulk RNA-seq cohort stratified patient survival even within known molecular groups, thus refining the prognostic power of DNA methylation-based profiling. Furthermore, we identified novel potentially druggable targets including IGF- and FGF-signaling within poorly prognostic transcriptional programs. Ependymoma-derived cell models/PDXs widely recapitulated the transcriptional programs identified within fresh tumors and are leveraged to validate identified target genes in functional follow-up analyses. Taken together, our analyses reveal a developmental hierarchy and transcriptomic context underlying the biologically and clinically distinct behavior of ependymoma groups. The newly characterized cellular states and underlying regulatory networks could serve as basis for future therapeutic target identification and reveal biomarkers for clinical trials.

EPEN-16. TRANSCRIPTIONAL REGULATORY CIRCUITRIES AS MOLECULAR TARGETS IN 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.

EPCO-35. SINGLE-CELL RNA-SEQ OF PEDIATRIC EPENDYMOMA REVEALS PROGNOSTIC IMPACT OF IMPAIRED NEURONAL-GLIAL FATE SPECIFICATION

Abstract Ependymoma represents a heterogeneous disease affecting the entire neuraxis. Extensive molecular profiling efforts have identified molecular ependymoma subgroups based on DNA methylation. However, the intratumoral heterogeneity and developmental origins of these groups are only partially understood, and effective treatments are still lacking for about 50% of patients with high-risk tumors. We interrogated the cellular architecture of ependymoma using single cell/single nucleus RNA-sequencing to analyze 24 tumor specimens across major molecular subgroups and anatomic locations. We additionally analyzed ten patient-derived ependymoma cell models and two patient-derived xenografts (PDXs). Interestingly, we identified an analogous cellular hierarchy across all ependymoma groups, originating from undifferentiated neural stem cell-like populations towards different degrees of impaired differentiation states comprising neuronal precursor-like, astro-glial-like, and ependymal-like tumor cells. While prognostically favorable ependymoma groups predominantly harbored differentiated cell populations, aggressive groups were enriched for undifferentiated subpopulations. Projection of transcriptomic signatures onto an independent bulk RNA-seq cohort stratified patient survival even within known molecular groups, thus refining the prognostic power of DNA methylation-based profiling. Furthermore, we identified novel potentially druggable targets such as IGF- and FGF-signaling within poorly prognostic transcriptional programs. Ependymoma-derived cell models/PDXs widely recapitulated the transcriptional programs identified within fresh tumors and are leveraged to validate identified target genes in functional follow-up analyses. Taken together, our analyses reveal a developmental hierarchy and transcriptomic context underlying the biologically and clinically distinct behavior of ependymoma groups. The newly characterized cellular states and underlying regulatory networks could serve as basis for future therapeutic target identification and reveal biomarkers for clinical trials.

Abstract A30: Alisertib acts synergistically with sonidegib by modulating primary cilia assembly in a pediatric RELA ependymoma cell line

Abstract Introduction: Recently we found that RELA ependymomas (EPN) demonstrate inappropriate activation of the Hedgehog (Hh) pathway. However, how this activation is modulated remains to be investigated. Primary cilia are essential to positive and negative modulation of Hh signaling and changes in ciliation seem to be linked to Hh drug resistance in pediatric cancers. In contrast, targeting cilia integrity by molecules that prevents ciliary disassembly, such as Aurora-A (AURKA), has been shown to overcome this resistance. However, the role of the Hh pathway and the prevalence and function of cilia in pediatric RELA ependymoma (EPN) subgroup have not been examined. Thus, we explored the effect of Hh inhibitor, sonidegib, alone or in combination with Alisertib, AURKA inhibitor, in EPN RELA cell line. Methods: Hh pathway activation of RELA cell line (BXD-1425) was evaluated by immunostaining using ARL13B, a marker specific for ciliary membranes, proto-oncogene Smoothened (SMO) and Hh effector, GLI, antibodies in untreated cells or after stimulation with SMO agonist (SAG). Western blot was performed for protein expression of GLI1, GLI2, and full-length (FL) and repressor form (R) of Hh transcriptional program GLI3. Cell proliferation was assessed by CCK8 assay. Cell apoptosis was detected with Annexin V-FITC by flow cytometry. Results: We showed that EPN RELA cell line is frequently ciliated. Fluorescence intensity showed SMO and GLI recruitment to cilia in both BXD untreated cells and after stimulation with SAG, implicating in activation of Hh signals. Surprisingly, sonidegib treatment reduced ciliary formation in BXD cell line. Cilia loss was followed by increase of GLI recruitment to cilia and GLI2 expression, potentially by reduction of GLI3R. Our results indicate that impaired GLI processing probably by cilia disruption could explain persistent activation of downstream Hh pathway. To test this hypothesis, we targeted the cilia integrity through the combination of sonidegib with alisertib, to see if the cilia maintenance might affect Hh function and overcome sonidegib resistance. The combination prevented the primary cilia disruption followed by upregulation of GLI3R and downregulation of GLI recruitment to cilia, GLI2, and GLI3FL expression. Our data point towards to a synergistic combination that enhances sonidegib efficiency by primary cilia preservation, reducing cell proliferation and increasing apoptosis BXD cells compared to drugs alone treatment. Conclusion: Our findings suggest a synergistic combination of sonidegib with alisertib that enhances the inhibition of Hh major effectors such as GLIs (GLI2 and GLI3FL) and induces GLI3R expression. Our data suggest that combination of these agents may represent a novel approach for treatment of ST-EPN-RELA tumors to overcome chemoresistance and these mechanisms are potentially mediated by primary cilia, a key regulator for Hh activity. Citation Format: Taciani de Almeida Magalhães, Gustavo Alencastro Veiga Cruzeiro, Kleiton Silva Borges, Cherry Liu Yulu, Graziella Ribeiro de Souza, Keteryne Rodrigues da Silva, Carlos Alberto Scridelli, Adrian Salic, Luiz Gonzaga Tone. Alisertib acts synergistically with sonidegib by modulating primary cilia assembly in a pediatric RELA ependymoma cell line [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A30.

In vitro benchmarking of NF-κB inhibitors

Dysregulated activity of the transcription factors of the nuclear factor κb (NF-κB) family has been implicated in numerous cancer types, inflammatory diseases, autoimmune disease, and other disorders. As such, selective NF-κB pathway inhibition is an attractive target to researchers for preclinical and clinical drug development. A plethora of commercially and clinically available inhibitors claim to be NF-κB specific; however, such claims of specificity are rarely quantitative or benchmarked, making the biomedical literature difficult to contextualize. This imprecision is worsened because some NF-κB reporter systems have low signal-to-noise ratios. Herein, we use a robust, defined, commercially available reporter system to benchmark NF-κB agonists and antagonists for the field. We also functionally characterize a RELA fusion-positive ependymoma cell culture with validated NF-κB inhibitor compounds.

Notch pathway in ependymoma RELA-fused subgroup: upregulation and association with cancer stem cells markers expression.

RELA-fused supratentorial (ST) ependymoma (EPN) is an aggressive subgroup with poor prognosis. Considering the putative role of Notch signaling in the maintenance of the cancer stem cells (CSC) phenotype in RELA-fused EPN, we investigated the expression of Notch pathway and its target genes in this subgroup. We also evaluated the effects of two Notch inhibitors (DAPT and RO4929097) on cell proliferation, apoptosis, colony formation, and CSCs markers gene expression on EPN cell line of the RELA-fused subgroup (BXD-1425). In addition, in silico signatures of the Notch genes and CSCs markers were analyzed on a large clinical dataset from GSE64415 study. We found that among the ST-EPN subgroups the Notch signaling (NOTCH1, JAG1, JAG2, and HES4) is specifically activated in the ST-EPN-RELA. Furthermore, treatment of the RELA-fused EPN cell line with the Notch inhibitors impaired the Notch signaling expression and revealed that Notch axis is not essential for cell proliferation and survival in this setting. NOTCH1 expression in ST-EPN was correlated with the CSCs markers VEGFA and L1CAM overexpression and JAG1 expression was correlated with the CCND1 and CDK6 overexpression. In addition, in vitro treatment with Notch inhibitors induced downregulation of CSCs markers. These findings indicate that Notch signaling can be involved in the ST-EPN-RELA CSCs maintenance by modulating the expression of genes responsible for cell phenotype and cell fate.

A role for ABCB1 in prognosis, invasion and drug resistance in ependymoma

Three of the hallmarks of poor prognosis in paediatric ependymoma are drug resistance, local invasion and recurrence. We hypothesised that these hallmarks were due to the presence of a sub-population of cancer stem cells expressing the multi-drug efflux transporter ABCB1. ABCB1 gene expression was observed in 4 out of 5 paediatric ependymoma cell lines and increased in stem cell enriched neurospheres. Functional inhibition of ABCB1 using vardenafil or verapamil significantly (p ≤ 0.05–0.001) potentiated the response to three chemotherapeutic drugs (vincristine, etoposide and methotrexate). Both inhibitors were also able to significantly reduce migration (p ≤ 0.001) and invasion (p ≤ 0.001). We demonstrate that ABCB1 positive patients from an infant chemotherapy-led trial (CNS9204) had a shorter mean event free survival (EFS) (2.7 versus 8.6 years; p = 0.007 log-rank analysis) and overall survival (OS) (5.4 versus 12 years; p = 0.009 log-rank analysis). ABCB1 positivity also correlated with reduced event free survival in patients with incompletely resected tumours who received chemotherapy across CNS9204 and CNS9904 (a radiotherapy-led SIOP 1999-04 trial cohort; p = 0.03). ABCB1 is a predictive marker of chemotherapy response in ependymoma patients and vardenafil, currently used to treat paediatric pulmonary hypertension in children, could be repurposed to reduce chemoresistance, migration and invasion in paediatric ependymoma patients at non-toxic concentrations.

Abstract 2869: Zika virus is a potent oncolytic agent against aggressive human ependymoma

Abstract Ependymoma is a metastatic CNS tumor responsible for 10% of all pediatric brain tumors. Displaying a poor survival rate of only 32.5%, the conventional ependymoma treatment is surgery and radiation therapy associated to significant morbidity. Previous studies found significantly increased expression of NESTIN, a neural progenitor cell (NPC) marker, in aggressive ependymoma. Zika virus (ZIKV) has recently emerged as an oncolytic therapy against adult and pediatric CNS tumors due to its ability to infect NPCs and neural stem-like tumor cells. We showed for the first time that Brazilian Zika virus strain efficiently destroyed pediatric CNS tumor cells in vitro and in vivo, resulting in significantly mice longer survival and fewer metastasis. Once tumor cells overexpressing NPCs markers are more susceptible to ZIKV infection, here we evaluated the oncolytic properties of Brazilian ZIKV against human ependymoma. A patient-derived ependymoma cell line (USP21-EPE) has been established and found to abnormally express the pluripotency markers CD133, SOX2 and NESTIN. USP21-EPE monolayer culture showed massive cell death three days after ZIKV infection in four tested MOIs: 0.01, 0.1, 1 and 2. At MOI 0,1 50% of cell population decreased viability while at higher MOIs was observed 100% of cell death 72 hours post ZIKV infection. Moreover, ZIKV significantly disrupted CNS ependymoma tumorspheres reinforcing its oncolytic effect against the ependymoma tumor cells. Considering the poor effectiveness and severe side effects of available treatments for ependymoma and that most ZIKV infections are associated to very mild or no symptoms, our findings open new avenues for novel therapies against this aggressive pediatric tumor. Citation Format: Carolini Kaid, Patrícia Semedo-Kuriki, Ernesto Goulart, Luiz Caires-Júnior, Renato Astray, Oswaldo Keith Okamoto, Mayana Zatz. Zika virus is a potent oncolytic agent against aggressive human ependymoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2869.

Bryostatin inhibits proliferation of ependymoma cells by suppressing expressions of cyclooxygenase-2 and interleukin-8

Purpose: To investigate the effect of bryostatin on the proliferation of ependymoma cells, and the underlying mechanism(s).Methods: Ependymoma cell lines (SC-EPN1 and SC-EPN2) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and streptomycin (10 mg/ml) in a humidified incubator at 37 °C and 5 % CO2 atmosphere. Rhe cells were randomly assigned to six groups: control group and five bryostatin groups treated with increasing concentrations of bryostatin (10 - 50 μM). Cell proliferation was determined by MTT assay, while real-time quantitative polymerase chain reaction (qRT-PCR) was used to determine the levels of expressions of apoptosisrelated genes. Expressions of cyclooxygenase-2 (COX-2), interleukin-8 (IL-8), Bcl-2, Bax and Pglycoprotein were determined using Western blotting.Results: Treatment with bryostatin significantly and concentration-dependently down-regulated COX-2 and IL-8 mRNAs expressions (p &lt; 0.05). On the other hand, proliferation of SC-EPN1 and SC-EPN2 cells were significantly and concentration-dependently inhibited by bryostatin, relative to control group (p &lt; 0.05). After 72 h of treatment with bryostatin (50 μM), the extent of apoptosis was significantly higher in SC-EPN1 (57.43 %) and SC-EPN2 cells (52.29 %) than in control group (2.37 %, p &lt; 0.05). The results of Western blotting showed that treatment with bryostatin significantly reduced the expressions of Bcl-2 in ependymoma cells, relative to the control group (p &lt; 0.05). However, there were no significant differences in the expression of Bax among the groups (p &gt; 0.05). P-glycoprotein expression was significantly higher in bryostatin groups than in control group (p &lt; 0.05). The results of flow cytometric analysis of rhodamine-123 (rh123) fluorescence showed that after 72 h of treatment with bryostatin (50 μM), rhl23 fluorescence significantly decreased in SC-EPN1 (8.10 %) and SC-EPN2 cells (10.11 %), relative to control group (20.83 %, p &lt; 0.05).Conclusion: Bryostatin exerts anti-proliferative and apoptotic effects on ependymoma cells by suppressing COX-2 and IL-8 expressions. Thus, the inhibition of COX-2 expression may constitute an effective chemotherapeutic strategy for ependymoma treatment.Keywords: Bryostatin, Ependymoma cells, Proliferation, Apoptosis, Expression

Limitations of current in vitro models for testing the clinical potential of epigenetic inhibitors for treatment of pediatric ependymoma.

BackgroundEpigenetic modifications have been shown to play an important role in the classification and pathogenesis of the pediatric brain tumor ependymoma, suggesting they are a potential therapeutic target.ResultsAgents targeting epigenetic modifications inhibited the growth and induced the death of ependymoma cells with variable efficiency. However, this was often not at clinically achievable doses. Additionally, DNA methylation profiling revealed a lack of similarity to primary ependymomas suggesting alterations were induced during culture. Toxicity to fetal neural stem cells was also seen at similar drug concentrationsConclusionsAgents targeting epigenetic modifications were able to inhibit the growth and induced the death of ependymoma cells grown in vitro. However, many agents were only active at high doses, outside clinical ranges, and also resulted in toxicity to normal brain cells. The lack of similarity in DNA methylation profiles between cultured cells and primary ependymomas questions the validity of using in vitro cultured cells for pre-clinical analysis of agents targeting epigenetic mechanisms and suggests further investigation using models that are more appropriate should be undertaken before agents are taken forward for clinical testing.Materials and MethodsThe effects of agents targeting epigenetic modifications on the growth and death of a panel of ependymoma cell lines was investigated, as well as toxicity to normal fetal neural stem cells. The ependymoma cell lines were characterized using DNA methylation profiling.

Identification of FDA-Approved Oncology Drugs with Selective Potency in High-Risk Childhood Ependymoma.

Children with ependymoma (EPN) are cured in less than 50% of cases, with little improvement in outcome over the last several decades. Chemotherapy has not affected survival in EPN, due in part to a lack of preclinical models that has precluded comprehensive drug testing. We recently developed two human EPN cell lines harboring high-risk phenotypes which provided us with an opportunity to execute translational studies. EPN and other pediatric brain tumor cell lines were subject to a large-scale comparative drug screen of FDA-approved oncology drugs for rapid clinical application. The results of this in vitro study were combined with in silico prediction of drug sensitivity to identify EPN-selective compounds, which were validated by dose curve and time course modeling. Mechanisms of EPN-selective antitumor effect were further investigated using transcriptome and proteome analyses. We identified three classes of oncology drugs that showed EPN-selective antitumor effect, namely, (i) fluorinated pyrimidines (5-fluorouracil, carmofur, and floxuridine), (ii) retinoids (bexarotene, tretinoin and isotretinoin), and (iii) a subset of small-molecule multireceptor tyrosine kinase inhibitors (axitinib, imatinib, and pazopanib). Axitinib's antitumor mechanism in EPN cell lines involved inhibition of PDGFRα and PDGFRβ and was associated with reduced mitosis-related gene expression and cellular senescence. The clinically available, EPN-selective oncology drugs identified by our study have the potential to critically inform design of upcoming clinical studies in EPN, in particular for those children with recurrent EPN who are in the greatest need of novel therapeutic approaches. Mol Cancer Ther; 17(9); 1984-94. ©2018 AACR.