Abstract B22: Cell state and lineage specification are controlled by epigenetic landscapes regulating the core transcriptional regulatory circuitry in pediatric neuroblastoma
Authors: Zimmerman Mark W. , Durbin Adam D. , Abraham Brian J. , Berezovskaya Alla , He Shuning , Oppel Felix , Young Richard A. , Look A. Thomas 
Published: August 2020

Abstract
Metazoan cell identity and lineage specification are regulated by a small group of super-enhancer-driven transcription factors unique to each cell type, referred to as the core regulatory circuit (CRC). In pediatric neuroblastoma, the survival of transformed neuroblasts is dependent on the activity of an autoregulatory CRC loop that includes HAND2, ISL1, PHOX2B, GATA3, and TBX2. Treatment of adrenergic neuroblastoma cells with all-trans retinoic (ATRA) induced strong morphologic changes and cell cycle arrest concomitant with differentiation and senescence. Tumor-bearing MYCN-transgenic zebrafish treated with retinoic acid exhibited a 70% reduction in tumor size in vivo. During reprogramming of cellular identity with ATRA, neuroblastoma cells exhibited altered expression of most adrenergic CRC transcription factors. The gene expression and protein levels of MYCN, ISL1, PHOX2B, GATA3, and ASCL1 were substantially reduced in ATRA-treated relative to control cells, while the expression of HAND2 was only modestly suppressed. Conversely, the expression levels of TBX2 and TBX3 were increased by more than 2-fold following ATRA treatment. Altered gene expression was frequently associated with loss or enrichment of H3K27ac-modified chromatin within the topologically associating domains regulating these genes. De novo super-enhancers were created over a subset of elevated transcription factors, indicating the emergence of a novel CRC framework that regulates the identity of differentiated neural crest-derived cells. We evaluated several genes, including RARA, MEIS1, and SOX4, by high-throughput ChIP-seq followed by whole-genome alignment and found that a novel autoregulatory loop of ATRA-regulated transcription factors co-opts the MYCN-driven gene expression program leading to both global transcriptional suppression and upregulation of genes and pathways associated with neuronal differentiation. Activation of MYC or MYCN by enhancer hijacking via translocations involving the HAND2 gene locus, which is not repressed by retinoic acid signaling, circumvents ATRA-induced the loss of MYCN expression and stabilizes the oncogenic transcriptome. These results shed light on the essential role of CRC transcription factors in regulating neuroblastoma cell survival and could guide the development of future transcriptionally directed therapeutics.
Citation Format: Mark W. Zimmerman, Adam D. Durbin, Brian J. Abraham, Alla Berezovskaya, Shuning He, Felix Oppel, Richard A. Young, A. Thomas Look. Cell state and lineage specification are controlled by epigenetic landscapes regulating the core transcriptional regulatory circuitry in pediatric neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B22.