Published OnlineFirst September 7, 2016; DOI: 10.1158/0008-5472.CAN-15-2507 Cancer Tumor and Stem Cell Biology Research Integrative Genome-Scale Analysis Identifies Epigenetic Mechanisms of Transcriptional Deregulation in Unfavorable Neuroblastomas Kai-Oliver Henrich1, Sebastian Bender2, Maral Saadati3, Daniel Dreidax1, Moritz Gartlgruber1, Chunxuan Shao4, Carl Herrmann5, Manuel Wiesenfarth3, Martha Parzonka1, Lea Wehrmann1, Matthias Fischer6, David J. Duffy7, Emma Bell8, Alica Torkov1, Peter Schmezer9, Christoph Plass9, Thomas Hofer€ 4, Axel Benner3, Stefan M. Pfister2, and Frank Westermann1 Abstract The broad clinical spectrum of neuroblastoma ranges from MYCN. Transcriptome integration and histone modification– spontaneous regression to rapid progression despite intensive based definition of enhancer elements revealed intragenic multimodal therapy. This diversity is not fully explained by enhancer methylation as a mechanism for high-risk–associated known genetic aberrations, suggesting the possibility of epige- transcriptional deregulation. Furthermore, in high-risk neuro- netic involvement in pathogenesis. In pursuit of this hypothesis, blastomas, we obtained evidence for cooperation between we took an integrative approach to analyze the methylomes, PRC2 activity and DNA methylation in blocking tumor-sup- transcriptomes, and copy number variations in 105 cases of pressive differentiation programs. Notably, these programs neuroblastoma, complemented by primary tumor- and cell could be re-activated by combination treatments, which tar- line–derived global histone modification analyses and epigenetic geted both PRC2 and DNA methylation. Overall, our results drug treatment in vitro. We found that DNA methylation pat- illuminate how epigenetic deregulation contributes to neuro- terns identify divergent patient subgroups with respect to blastoma pathogenesis, with novel implications for its diagnosis survival and clinicobiologic variables, including amplified and therapy. Cancer Res; 76(18); 1–15. Ó2016 AACR. Introduction DNA methylation, an epigenetic modification via methylation of cytosin carbon 5, is a major mechanism in cell differentiation Neuroblastoma originates from precursor cells of the sympa- and neoplastic transformation. Promoter-associated CpG islands thetic nervous system. It is the most frequent solid tumor of early have been identified as frequent targets of transcriptionally relevant childhood with a remarkable variation in clinical and biological methylation events. Accumulating evidence, however, suggests that behavior ranging from spontaneous regression to rapid progres- nonpromoter methylation may be also actively involved in gene sion in spite of intensive multimodal chemotherapy. The molec- regulatory processes and is an abundant phenomenon among ular basis of neuroblastoma pathogenesis is still poorly under- somatically acquired methylation changes in human cancer stood. Genetic alterations seen in high-risk tumors include ampli- (4, 5). In neuroblastoma, candidate-based approaches revealed fication of the proto-oncogene MYCN, activation of the ALK gene, methylation of several genes including CASP8 (6), which plays an heterozygous deletions of 1p or 11q and gain of 17q. However, important role in the TNF-related apoptosis pathway. Global the molecular etiology of a substantial portion of aggressive approaches based on epigenetic drug-induced re-expression anal- neuroblastomas remains largely enigmatic. Recurrent somatic yses and/or affinity-based capture methods in neuroblastoma cell mutations are rare (1–3), suggesting that epigenetic mechanisms lines revealed further potential mechanisms and targets of DNA may drive neuroblastoma development and progression. methylation in neuroblastoma (7, 8). Further reports suggested the 1Neuroblastoma Genomics B087, German Cancer Research Center, Hei- Note: Supplementary data for this article are available at Cancer Research 2 delberg, Germany. Division of Pediatric Neurooncology, German Can- Online (http://cancerres.aacrjournals.org/). cer Consortium (DKTK), German Cancer Research Center, Heidelberg, Germany & Department of Pediatric Oncology, Hematology and Immu- K.-O. Henrich, S. Bender, and M. Saadati contributed equally as co-first authors to nology, Heidelberg University Hospital, Germany. 3Division of Biostatis- this article. tics, German Cancer Research Center, Heidelberg, Germany. 4Division of fi Theoretical Systems Biology, German Cancer Research Center, Heidel- A. Benner, S.M. P ster, and F. Westermann contributed equally as co-senior berg, Germany. 5Division of Theoretical Bioinformatics, German Cancer authors to this article. Research Center, Institute of Pharmacy and Molecular Biotechnology, Corresponding Authors: Kai-Oliver Henrich, German Cancer Research Center Bioquant, University of Heidelberg, Germany. 6Department of Pediatric (DKFZ), Im Neuenheimer Feld 280, Heidelberg 69120, Germany. Phone: 4962- Oncology, University Children's Hospital, and Center for Molecular Med- icine Cologne (CMMC), University of Cologne, Cologne, Germany. 7Sys- 2142-3279; Fax: 4962-2142-3277; E-mail: k.henrich@dkfz; and Frank Wester- tems Biology Ireland, University College Dublin, Belfield, Dublin, Ireland. mann, [email protected] 8 Department of Pathology, University of Cambridge, Cambridge, United doi: 10.1158/0008-5472.CAN-15-2507 Kingdom. 9Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center, Heidelberg, Germany. Ó2016 American Association for Cancer Research. www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst September 7, 2016; DOI: 10.1158/0008-5472.CAN-15-2507 Henrich et al. presence of a CpG island methylator phenotype (CIMP), defined MYCN levels, stable neuroblastoma cell models were used as widespread simultaneous CpG island methylation, which is where MYCN can be either up- or downregulated upon addition significantly associated with poor outcome (9, 10). Genome-wide of tetracycline [SH-SY5Y-MYCN and IMR5-75-shMYCN (small DNA methylation analyses in primary neuroblastomas using hairpin RNA targeting MYCN), respectively; refs. 16, 17]. IMR5- methylated DNA immunoprecipitation (MeDIP) or DNA meth- 75-shMYCN with and without MYCN knockdown (ÆTet) were ylation arrays identified methylation events representing new synchronized via thymidine (2 mmol/L) block for 18 hours candidate epigenetic biomarkers (11–14). An integrated approach, before release by wash out. combining methylome, transcriptome, and genome data from a large cohort of primary neuroblastomas with chromatin modifi- Array-based gene expression and DNA methylation profiling cation analyses has, however, not yet been applied. Expression profiles were generated using customized 4 Â 44 Here, we analyzed DNA methylation in 105 neuroblastomas K oligonucleotide microarrays (Agilent Technologies) and using the Illumina 450k methylation array, which covers pro- genome-wide DNA methylation was assessed using Infinium moter and gene body sites of 99% of Refseq-annotated genes. HumanMethylation450 (450k) BeadChips (Illumina). See These data were complemented by expression profiles and copy Supplementary Materials and Methods for details. number information from the same tumors and combined with Genomic annotation of CpGs was done using the assign- primary neuroblastoma- and cell line–derived global histone GenomeAnnotation program of the HOMER tool suite (http:// modification data. We identified functional programs targeted homer.salk.edu/homer). GpGs with gene context annotations by epigenetic mechanisms in high-risk neuroblastomas and pro- "EXON", "INTRON", "3UTR" and "TTS" (transcription termina- vide evidence for methylation of intragenic enhancers being a tion site) were defined as intragenic (gene body), whereas CpGs mechanism of high-risk–associated transcriptional dysregulation. with annotations "PROMOTER-TSS" and "5UTR" were defined as Furthermore, our data suggest an active contribution of PRC2 50-associated. For association of DNA methylation and gene components to the downregulation of tumor-suppressive genes expression data, only CpGs with gene context annotation were that are hypermethylated in high-risk patients. In line with this, a considered. MassARRAY analysis (Sequenom) at two selected combination of drugs targeting the repressive effect of both DNA CpGs within CDKN2D revealed strong correlation with methyl- methylation and PRC2 efficiently reinduced programs epigenet- ation levels assessed by 450k arrays [Pearson correlation coeffi- ically impaired in high-risk disease. Our results provide funda- cients: 0.85 (95% confidence interval (CI), 0.79–0.90) and 0.89 mental new insights into epigenetic deregulation of aggressive (95% CI, 0.84–0.92; ref. 18]. neuroblastoma and may open new diagnostic and therapeutic avenues for children with high-risk disease. Detection of copy number alterations Copy number alterations were assessed in the 450k Infinium Patients and Methods array data using a previously described custom approach inte- Patients grating both methylated and unmethylated signals (19). Result- fi All patients were enrolled in the German Neuroblastoma Trial ing pro les were manually curated and overall genomic patterns fi (NB97þNB2004) and diagnosed between 1998 and 2011. Treat- used for patient risk strati cation according to Janoueix-Lerosey ment was according to the trial guidelines. Informed consent was and colleagues (20). obtained from the patients' parents and