Abstract Background Neuroblastoma (NB) is a pediatric tumor derived from precursor cells of the sympathetic nervous system. NB accounts for 12% of all childhood cancer deaths with ~50% high-risk cases which frequently harbor amplified proto-oncogene MYCN. Evidence accumulates that epigenetic deregulation, including aberrant DNA methylation in high-risk disease or oncogene activation by enhancer hijacking, plays a prominent role in NB. The present study applies a comprehensive approach integrating chromatin modification data with genomic and expression data to elucidate NB subtype specific super-enhancer (SE) landscapes and core regulatory circuitries (CRCs) consisting of lineage-specific interconnected loops of SE-driven, auto-regulatory master transcription factors. Methods Chromatin immunoprecipitation sequencing (ChIP-seq) of histone 3 lysine 27 acetylation (H3K27ac) was used to identify active enhancer elements in 23 primary NBs. A validation cohort consisting of 16 NB cell lines and two human neural crest cell lines was used. ChIPmentation was applied to validate predicted transcription factor (TF) binding events. Circular chromatin conformation capture sequencing (4C-seq) was used to assay physical promoter-enhancer interactions. Results Unsupervised clustering of 23 primary NBs according to H3K27ac signal intensity at the most variable SEs (genome-wide) revealed two main subgroups, MYCN-amplified (n = 8) and MYCN single copy tumors (n = 15), with distinctive activity patterns. Calling of CRCs in the 23 primary NBs yielded a core set of NB master TFs (CRC TFs). Amongst the top ten of them are HAND2, PHOX2B and MYCN, all of which are implicated in NB biology and playing essential roles in the development of the sympathetic nervous system. In line with this, gene ontology analyses of the top 50 CRC TFs converge on biological processes like development of neural crest cells, sympathetic nervous system and peripheral nervous system neurons. ChIPmentation analyses of selected CRC TFs confirmed auto-binding to their assigned SEs and those of other CRC TFs in their respective network. Interactions between promoters and SEs of selected CRC TFs were verified via 4C-seq. Intriguingly, expression analysis of the top 50 CRC TFs in a cohort of 498 primary NBs revealed that less than 20% of the CRC TFs are up-regulated in MYCN-amplified tumors while the remaining 80% are down-regulated in that subgroup. This suggests a superordinate role of MYCN in differentially orchestrating NB master TFs. Conclusion The study identifies the core set of NB master transcription factors and assigns established NB regulators like HAND2, PHOX2B and MYCN to well-defined CRCs. It reveals an association of MYCN amplification with the global SE landscape of primary NBs and suggests a role for MYCN in differentially controlling subsets of CRC TFs and their networks. Specific targeting of the SE-dependent CRC networks may open a therapeutic window for epigenetic drugs, including BET inhibitors, CDK7 or EZH2 inhibition, in NB. Citation Format: Moritz Gartlgruber, Daniel Dreidax, Daria Doncevic, Sebastian Steinhauser, Stefan Gröschel, Kai Oliver Henrich, Young-Gyu Park, Carl Herrmann, Frank Westermann. Core transcriptional regulatory circuitries in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-083. doi:10.1158/1538-7445.AM2017-LB-083