Super-enhancer-associated core regulatory circuits mediate susceptibility to retinoic acid in neuroblastoma cells [ChIP-Seq]. Super-enhancer-associated core regulatory circuits mediate susceptibility to retinoic acid in neuroblastoma cells [ChIP-Seq]

Neuroblastoma is a pediatric tumor that accounts for more than 15% of cancer-related deaths in children. Survival chances for high-risk patients are less than 50%. Retinoic acid treatment is part of the maintenance therapy given to neuroblastoma patients; however, not all tumors respond to retinoic acid-mediated differentiation. Among neuroblastoma tumors, two phenotypically distinct cell types-adrenergic (ADRN) and mesenchymal (MES), have been identified based on their super-enhancer landscape and transcriptional core regulatory circuitries. We hypothesized that distinct super-enhancers in these different tumor cells could mediate differential response to retinoic acid. To this end, we treated four different neuroblastoma cell lines, comprising both ADRN (MYCN amplified and non-amplified) and MES subtypes, with retinoic acid and studied the super-enhancer landscape upon treatment and after removal of retinoic acid. Using H3K27ac ChIP-seq paired with RNA-seq, we compared the super-enhancers in cells that respond to retinoic acid-mediated differentiation versus those that fail to differentiate. We identified unique super-enhancers associated with cells differentiation; however, even among cells that respond to treatment, there was heterogeneity upon removal of retinoic acid, with MYCN amplified cells remaining differentiated whereas MYCN non-amplified cells reverted to a proliferative state. This study identifies regulatory super-enhancers as a plausible mechanism behind the differential response to retinoic acid-mediated differentiation. Overall design: The H3K27ac landscape was determined in three neuroblastoma cell lines to investigate the effect of retinoic acid treatment and withdrawal. Three replicates were generated for each condition - control, ATRA treated, and withdrawn. Input libraries were derived from control samples.

神经母细胞瘤（Neuroblastoma）是一种儿童肿瘤，占儿童癌症相关死亡病例的15%以上。高危患者的生存率不足50%。维A酸（retinoic acid）治疗是神经母细胞瘤患者维持治疗的组成部分，但并非所有肿瘤均能对维A酸介导的分化产生应答。研究人员已基于超级增强子（super-enhancer）图谱与转录核心调控环路（transcriptional core regulatory circuitries），在神经母细胞瘤中鉴定出两种表型迥异的细胞类型——肾上腺素能型（adrenergic, ADRN）与间质型（mesenchymal, MES）。
我们假设，上述不同肿瘤细胞中特有的超级增强子，可介导对维A酸的差异应答。为此，我们对四种不同的神经母细胞瘤细胞系施加维A酸处理，这些细胞系涵盖MYCN扩增型与非扩增型的ADRN亚型，以及MES亚型，并分别探究了处理后及撤除维A酸后的超级增强子图谱。我们联合使用H3K27ac染色质免疫共沉淀测序（H3K27ac ChIP-seq）与RNA测序（RNA-seq），对比了对维A酸介导的分化产生应答的细胞与未能发生分化的细胞的超级增强子差异。我们鉴定出了与细胞分化相关的独特超级增强子；然而，即便在对治疗产生应答的细胞中，撤除维A酸后仍存在异质性：MYCN扩增型细胞维持分化状态，而MYCN非扩增型细胞则恢复至增殖状态。本研究证实，调控性超级增强子或是维A酸介导的分化产生差异应答的潜在合理机制。
实验整体设计：我们在三种神经母细胞瘤细胞系中检测了H3K27ac图谱，以探究维A酸处理与撤除的影响。每种实验条件均设置三次生物学重复，分别为对照组、全反式维A酸（ATRA）处理组与维A酸撤除组。输入文库（Input libraries）源自对照样本。