Abstract 3872: Retinoic acid (RA) relieves EZH2-mediated epigenetic suppression in high-risk neuroblastoma (HR-NB)
Authors: Banerjee Deblina , Liu Zhihui , Oh Doo-Yi , Cam Maggie , Kim Bong-Hyun , Thiele Carol 
Published: July 2017

Abstract
High risk neuroblastoma tumors (HR-NB) are characterized by the presence of undifferentiated histopathology, while tumors from low risk patients have histologic evidence of differentiation and a transcriptome enriched in differentiation genes. Determining the cellular and molecular underpinnings that contribute to this undifferentiated state will lead to better understanding of the disease pathogenesis. Retinoic acid (RA) is known to prime embryonic stem cells for lineage specific differentiation. To elucidate the early events important for reprogramming of NB cells leading to changes in the degree of cellular differentiation, we treated HR-NB cells (KCNR : MYCN amplified) with 5µM RA for 24 hrs. RNA-Seq analysis, to assess early changes in the transcriptome, showed significant changes in gene expression [2fold-up-regulated-446, 2fold-down-regulated-250, FDR≤0.01] which were enriched for biological functions like neuron projection, neuron differentiation, indicating onset of neurogenesis as a marker of differentiation. Gene set enrichment analysis identified significant positive enrichment of NB differentiation (p=0.001) and PRC2 (Polycomb Repressor Complex2) gene sets (p=0.02) along with downregulation of MYCN signature gene set (p=0.001). PRC2 component EZH2 (Histone Methyltransferase) is highly expressed in NB and represses differentiation genes. Though, EZH2 protein levels did not change after treatment, RA caused 40% reduction in H3 bound EZH2. To gain insight into how this reduction affects subsequent gene expression; we evaluated changes in genome wide binding of EZH2 and H3K27me3 after treatment. Indeed, significant correlation was observed between the changes in expression levels of genes after RA treatment and relative binding of EZH2 (p=3.88e-6) and H3K27me3 (p=3.88e-6) at their promoter regions. One-third (n=147) of the upregulated genes (eg. HEY1, RARB) and half (n=117) of the downregulated genes (eg. INSM2, CAMK4) were EZH2 targets. Upregulated genes were related to biological functions like negative regulation of cell growth and transcription, nervous system development. To probe the underlying mechanism, in-silico analysis indicated RA decreased Wnt/ß-catenin, eNOS and PTEN pathways while stimulating cAMP, Integrin linked kinase and G-coupled protein pathways. Evaluation of the cAMP pathway candidates identified 13 genes as EZH2 targets. eg CNR1, DUSP6, PKIA, RSG4. In primary NB tumors, microarray studies indicate that low levels of CNR1 and DUSP6 are associated with poor prognosis. RA stimulates 10-fold and 2-fold increases in CNR1 and DUSP6 levels, respectively. RA treatment inhibits EZH2 binding to its target genes thereby activating pathways involved in reprogramming of HR-NBs to a more differentiated state. Studies are underway to further evaluate the molecular mechanism leading to decreased EZH2 binding upon RA treatment.
Citation Format: Deblina Banerjee, Zhihui Liu, Doo-Yi Oh, Maggie Cam, Bong-Hyun Kim, Carol Thiele. Retinoic acid (RA) relieves EZH2-mediated epigenetic suppression in high-risk neuroblastoma (HR-NB) [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 3872. doi:10.1158/1538-7445.AM2017-3872