Background: Cellular heterogeneity within tumors is increasingly recognized as a source of therapeutic failure. However, the cis-regulatory landscapes driving transcriptional states of intra-tumor heterogeneity, drug-resistance and relapse remain elusive.

Results: Here, using H3K27Ac chromatin immunoprecipitation followed by sequencing (ChIP-seq) we characterized the active super-enhancer (SE) landscape in neuroblastoma, a pediatric cancer of the sympathetic nervous system. Analysis of differentially active SEs identified cis-regulatory modules associated with distinct transcriptional states in material derived from individual patients. These transcriptional states associated with two phenotypically divergent cellular subtypes.

One subtype is referred to as adrenergic (ADN) and expresses classic neuroblastoma markers from the peripheral sympathetic nervous system. In contrast, the other subtype referred to as mesenchymal (MES) has similarity to neural crest cells, expresses mesenchymal genes, is motile and lacks adrenergic markers. In contrast to ADN cells, MES-type cells are resistant to a wide variety of chemotherapeutics used in clinical management of neuroblastoma.

Computational reconstruction identified core transcription factor modules associated with ADN- and MES-type cells. DNA binding profiles of adrenergic TFs MAML3 and GATA3 suggest feed-forward activation of the adrenergic SE-associated TFs.

Interconversion of FACS-sorted MES- and ADN-type cells is observed in vitro. Induction experiments with the mesenchymal TF PRRX1 efficiently converted ADN-type cells to an induced-MES (iMES) state. These iMES cells acquired many features of MES-cells including motility, mesenchymal gene expression and -histone modifications as well as chemo-resistance.

Primary neuroblastoma biopsies included a small fraction of PRRX1-positive MES-type cells, as determined by immunohistochemistry. Importantly, the proportion of both cell types appears dynamic upon therapy and in relapse development, suggesting selective pressure of treatment.

Conclusions: Here we establish that intra-tumor heterogeneity in neuroblastoma follows a bi-phasic structure characterized by two different SE-associated TF programs that reflects stages of the normal developmental programs. The detailed understanding of core regulatory modules and pathways may redesign strategies for therapeutic intervention.

Citation Format: Johan van Nes, Tim van Groningen, Linda J. Valentijn, Danny Zwijnenburg, Jan J. Molenaar, Bart A. Westerman, Ellen M. Westerhout, Mohamed Hamdi, Godelieve A. Tytgat, Jan Koster, Rogier Versteeg. Active enhancers delineate intra-tumor heterogeneity of developmental states 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 3876. doi:10.1158/1538-7445.AM2017-3876