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The aggressive nature of glioblastoma multiforme (GBM) may be attributed to the dysregulation of pathways driving both proliferation and invasion. EphrinB2, a membrane-bound ligand for some of the Eph receptors, has emerged as a critical target regulating these pathways. In this study, we investigated the role of ephrinB2 in regulating proliferation and invasion in GBM using intracranial and subcutaneous xenograft models. The Cancer Genome Atlas analysis suggested high transcript and low methylation levels of ephrinB2 as poor prognostic indicators in GBM, consistent with its role as an oncogene. EphrinB2 knockdown, however, increased tumor growth, an effect that was reversed by ephrinB2 Fc protein. This was associated with EphB4 receptor activation, consistent with the data showing a significant decrease in tumor growth with ephrinB2 overexpression. Mechanistic analyses showed that ephrinB2 knockdown has anti-invasive but pro-proliferative effects in GBM. EphB4 stimulation following ephrinB2 Fc treatment in ephrinB2 knockdown tumors was shown to impart strong anti-proliferative and anti-invasive effects, which correlated with decrease in PCNA, p-ERK, vimentin, Snail, Fak, and increase in the E-cadherin levels. Overall, our study suggests that ephrinB2 cannot be used as a sole therapeutic target. Concomitant inhibition of ephrinB2 signaling with EphB4 activation is required to achieve maximal therapeutic benefit in GBM.

Loss of SMARCB1 is the hallmark genetic event that characterizes rhabdoid tumors in children. Rhabdoid tumors of the brain (ATRT) occur in young children and are particularly challenging with poor long-term survival. SMARCB1 is a member of the SWI/SNF chromatin remodeling complex that is responsible for determining cellular pluripotency and lineage commitment. The mechanisms by which SMARCB1 deletion results in tumorigenesis remain unclear. Recent studies demonstrate that ATRT consists of 3 genomic subgroups with a subset of poor outcome tumors expressing high BMP and MYC pathway activation. Here we show that MYC occupies distinct promoter loci in ATRT compared to embryonic stem (ES) cells. Furthermore, using human ATRT cell lines, patient-derived cell culture, ex vivo patient-derived tumor, and orthotopic xenograft models, we show that MYC inhibition is a molecular vulnerability in SMARCB1-deleted tumors and that such inhibition effectively suppresses BMP and pluripotency-associated genomic programs, attenuates tumor cell self-renewal, promotes senescence, and inhibits ATRT tumor growth in vivo. Transgenic expression of Omomyc (a bona-fide MYC dominant negative) or chemical inhibition of MYC transcriptomic programs with the BET inhibitor JQ1 phenocopy genetic depletion of MYC, effectively restricting ATRT tumor growth and opening a promising therapeutic avenue for rhabdoid tumors in children.

Background Atypical teratoid rhabdoid tumors (AT-RT) are pediatric tumors of the central nervous system with limited treatment options and poor survival rate. We investigated whether enhancing chemotherapy toxicity by depleting intracellular glutathione (GSH; a key molecule in cisplatin resistance) with high dose acetaminophen (AAP), may improve therapeutic efficacy in AT-RT in vitro. Procedure BT16 (cisplatin-resistant) and BT12 (cisplatin-sensitive) AT-RT cell lines were treated with combinations of AAP, cisplatin, and the anti-oxidant N-acetylcysteine (NAC). Cell viability, GSH and peroxide concentrations, mitochondrial damage, and apoptosis were evaluated in vitro. Results AAP enhanced cisplatin cytotoxicity in cisplatin-resistant BT16 cells but not cisplatin-sensitive BT12 cells. Baseline GSH levels were elevated in BT16 cells compared to BT12 cells, and AAP decreased GSH to a greater magnitude in BT16 cells than BT12 cells. Unlike BT12 cells, BT16 cells did not have elevated peroxide levels upon treatment with cisplatin alone, but did have elevated levels when treated with AAP + cisplatin. Both cell lines had markedly increased mitochondrial injury when treated with AAP + cisplatin relative to either drug treatment alone. The enhanced toxic effects were partially reversed with concurrent administration of NAC. Conclusions Our results suggest that AAP could be used as a chemo-enhancement agent to potentiate cisplatin chemotherapeutic efficacy particularly in cisplatin-resistant AT-RT tumors with high GSH levels in clinical settings. Pediatr Blood Cancer 2014;61:120–127. © 2013 Wiley Periodicals, Inc.

Background Rhabdoid tumors (RTs) are aggressive tumors of early childhood that occur most often in brain (AT/RTs) or kidney (KRTs). Regardless of location, they are characterized by loss of functional SMARCB1 protein, a component of the SWI/SNF chromatin remodeling complex. The aim of this study was to determine genes and biological process dysregulated in common to both AT/RTs and KRTs. Procedure Gene expression for AT/RTs was compared to that of other brain tumors and normal brain using microarray data from our lab. Similar analysis was performed for KRTs and other kidney tumors and normal kidney using data from GEO. Dysregulated genes common to both analyses were analyzed for functional significance. Results Unsupervised hierarchical clustering of RTs identified three major subsets: two comprised of AT/RTs, and one of KRTs. Compared to other tumors, 1,187, 663, and 539 genes were dysregulated in each subset, respectively. Only 14 dysregulated genes were common to all three subsets. Compared to normal tissue, 5,209, 4,275, and 2,841 genes were dysregulated in each subset, with an overlap of 610 dysregulated genes. Among these genes, processes associated with cell proliferation, MYC activation, and epigenetic dysregulation were common to all three RT subsets. Conclusions The low overlap of dysregulated genes in AT/RTs and KRTs suggests that factors in addition to SMARCB1 loss play a role in determining subsequent gene expression. Drugs which target cell cycle or epigenetic genes may be useful in all RTs. Additionally, targeted therapies tailored to specific RT subset molecular profiles should be considered. Pediatr Blood Cancer 2013; 60: 10951102. (c) 2013 Wiley Periodicals, Inc.

Enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 that catalyzes the trimethylation of histone H3 on Lys 27, and represses gene transcription. EZH2 enhances cancer-cell proliferation and regulates stem cell maintenance and differentiation. Here, we demonstrate that EZH2 is highly expressed in medulloblastoma, a highly malignant brain tumor of childhood, and this altered expression is correlated with genomic gain of chromosome 7 in a subset of medulloblastoma. Inhibition of EZH2 by RNAi suppresses medulloblastoma tumor cell growth. We show that 3-deazaneplanocin A, a chemical inhibitor of EZH2, can suppress medulloblastoma cell growth partially by inducing apoptosis. Suppression of EZH2 expression diminishes the ability of tumor cells to form spheres in culture and strongly represses the ability of known oncogenes to transform neural stem cells. These findings establish a role of EZH2 in medulloblastoma and identify EZH2 as a potential therapeutic target especially in high-risk tumors.

Background A better understanding of pediatric brain tumor biology is needed to assist in the development of less toxic therapies and to provide better markers for disease stratification. MicroRNAs (miRNA) may play a significant role in brain tumor biology. The present study provides an initial survey of miRNA expression in pediatric central nervous system (CNS) malignancies including atypical teratoid/rhabdoid tumor, ependymoma, glioblastoma, medulloblastoma, and pilocytic astrocytoma. Procedure MicroRNA expression in pediatric brain tumors and normal tissue controls was examined by microarray. Three aberrantly expressed miRNAs were further studied in a larger cohort by quantitative real-time PCR (qRT-PCR). Results MicroRNA-129, miR-142-5p, and miR-25 were differentially expressed in every pediatric brain tumor type compared to normal tissue controls as measured by microarray. When further examined by qRT-PCR, these miRNAs demonstrated differential expression that significantly correlated with the microarray findings. Distinctive miRNA expression profiles were also observed in the different pediatric brain tumor types. Conclusions MicroRNAs are differentially expressed between pediatric CNS neoplasms and normal tissue suggesting that they may play a significant role in oncogenesis. A greater understanding of aberrant miRNA expression in pediatric brain tumors may aid in the development of novel therapies. The characterization of tumor-specific miRNA signatures may aid in the discovery of biomarkers with diagnostic or prognostic utility.

Background Medulloblastoma comprises approximately 20% of all primary pediatric brain tumors. Despite recent advances, the survival rate for high-risk patients and the morbidity associated with these treatments remains suboptimal. To improve outcomes and decrease morbidity, more targeted therapy is required. One possible target is the Aurora Kinase family. The objective of this study was to evaluate the impact of Aurora Kinase A inhibition in medulloblastoma cell lines. Procedure Cell proliferation was measured using an MTS assay after adding an Aurora Kinase inhibitor (C1368) at different concentrations. Cell cycle analysis was carried out by Flow Cytometry using propidium iodide (PI). RNAi experiments were performed using siRNA oligonucleotides. Luciferase experiments were carried out using the Cignal Finder 10 Pathway Reporter Arrays. Results Inhibition of Aurora Kinase A induces cell death in medulloblastoma cells and lowers the IC50 of other chemotherapeutic agents (etoposide and cisplatin) used in medulloblastoma treatment. Cell arrest at G2/M phase was significantly increased in medulloblastoma cell lines treated with C1368 Sigma at IC30 or transfected siRNA. Inhibition of Aurora Kinase A resulted in decreased activity of pro-proliferative signaling pathways including Wnt, Myc, and RB as measured by luciferase reporter assays. Conclusions These data indicate that inhibition of Aurora Kinase A inhibits cell growth in medulloblastoma through inhibition of pro-proliferative signaling pathways Wnt, Myc, and RB. Additionally, combining Aurora Kinase A inhibition with other chemotherapeutic agents significantly lowers their IC50, which make it a promising small molecule target for medulloblastoma therapy. Pediatr Blood Cancer 2010;55:35–41. © 2010 Wiley-Liss, Inc.

Background Posterior fossa (PF) ependymomas (EPNs) in infants less than 1 year of age (iEPN-PF) have a poorer clinical outcome than EPNs in older children. While radiation therapy is the standard of care for the latter, it is withheld in infants to avoid neurotoxicity to immature brain. It is unknown whether the adverse outcome in iEPN-PFs is due to treatment differences or aggressive biology. We examined this question using molecular profiling. Methods Six anaplastic iEPN-PFs were subjected to transcriptomic analysis and FISH for p16 loss and gains of 1q, and compared with anaplastic PF EPNs from older children. Results were validated by immunohistochemistry (IHC). Results All six iEPN-PFs were grouped within EPN PF subgroup A (PFA). E2F targets and G2M checkpoint were identified as the most enriched gene sets in iEPN-PF, which was validated in a larger independent cohort. Accordingly, MIB-1 IHC demonstrated a higher mitotic rate in iEPN-PFs than noninfant anaplastic EPN PFA. Genetic and protein analyses demonstrated that p16 loss and low p16 protein expression is a hallmark of iEPN-PF, and that none harbored 1q gains. Kaplan–Meier analysis confirmed the poorer clinical outcome of the iEPN-PF cohort. Conclusions Biological differences, characterized by loss of p16 expression without gains of 1q in iEPN-PFs, as well as deregulated E2F target gene transcription, are indicative of deregulated p16-CDK4/6-pRB-E2F pathway activity. This may underlie the poor clinical outcome seen in this group of iEPN-PFs, rather than the withholding of radiation therapy. Results suggest a potential actionable therapy for iEPN-PF, namely cyclin-dependent kinase 4/6 (CDK4/6) inhibitors.