Abstract 2425: Genome-wide mapping of MYCN, MYC, and MAX binding across neuroblastoma cell lines identifies novel transcriptional targets

Abstract

Abstract Arising from the sympathetic nervous system, neuroblastoma (NBL) is the most common extracranial solid tumor in children. Although the 5-year survival rate for children in the low and intermediate risk groups ranges between 90 to over 95%, children with high-risk disease have only a 40-50% likelihood of survival and relapse of high-risk disease is currently largely incurable. The best-characterized genetic determinant of high-risk disease is amplification of MYCN. Members of the MYC protein family are highly homologous and share redundant biological functions, each able to form heterodimers with MAX at consensus E-box motifs (CANNTG) to regulate transcription. While MYCN is known to transcriptionally activate genes involved in malignancy and repress expression of genes that antagonize metastases, amplification of MYCN alone is likely not sufficient for transformation. To identify MYCN and MYC DNA binding partners and ultimately, therapeutic targets in the high-risk NBL subset, we performed chromatin immunoprecipitation and next-generation sequencing (ChIP-Seq) for MYCN, MYC, and MAX across three MYCN amplified and three non-MYCN amplified cell lines (n = 2 ChIPs per protein). Peak calling was performed using MACS2 and annotated using bedtools and/or ChIPseeker. MYCN displays genome-wide binding in all three amplified cell lines, suggesting global transcriptional regulation in NBL. ChIPenrich and TOPPgene were used to determine enrichment of peaks based on gene ontology. As expected, we show MYCN ChIP peaks were significantly enriched for MYC and MAX interactions, as well as DNA-binding, in the MYCN amplified subset of cell lines. Genes containing peaks within 1 kb of gene promoters were intersected with genes differentially expressed based on MYCN amplification status in high-risk NBL cases based on RNA-Seq analysis. This set of over 4000 genes was significantly enriched (p < 1E-10) for multiple categories of RNA binding and ribosomal proteins, as well as interactions with known oncogenes and/or cooperating partners of MYCN or MYC: CUL7, VCAM1, SUMO2, and CDK2 (p < 1E-20). Finally, we performed de novo motif discovery on MYCN ChIP peaks using Homer2 and found significant enrichment (p < 1E-50) of 15-20 motifs, including the canonical MYCN basic helix loop helix E-box motif (CCACGTGGCN, ∼23% enriched), an ELK1 ETS motif (∼44%), an AHR motif (∼29%), and a SMAD4 (MAD) motif (∼21%). To our knowledge, this combination of RNA- and ChIP-Seq represents the first global epigenomic analysis of MYCN, MYC, and MAX binding across multiple NBL cell lines, revealing MYCN-targeted transcriptional upregulation and/or putative repression. This and further epigenomic characterization of known and novel MYC and MYCN transcriptional targets will enable prioritization of potential therapeutic targets for treatment of the high-risk NBL subset. Citation Format: Jo Lynne Harenza, Robyn Sussman, Derek Oldridge, John Maris. Genome-wide mapping of MYCN, MYC, and MAX binding across neuroblastoma cell lines identifies novel transcriptional targets. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2425.