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 Three replicates were generated for each condition - control, ATRA treated, and withdrawn.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总数的15%以上。高危患者的生存率不足50%。维甲酸（Retinoic acid）治疗是神经母细胞瘤患者维持治疗的组成部分，但并非所有肿瘤都能对维甲酸介导的细胞分化产生应答。在神经母细胞瘤中，研究人员已根据其超级增强子（super-enhancer）图谱与转录核心调控环路（transcriptional core regulatory circuitries），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）。我们提出假说：这些不同肿瘤细胞中存在的特异性超级增强子，可能介导了对维甲酸的差异性应答。为此，我们对四种不同的神经母细胞瘤细胞系（涵盖ADRN型[含MYCN扩增与非扩增亚型]与MES型）施加维甲酸处理，并分别在处理后及移除维甲酸后，对其超级增强子图谱进行分析。我们采用H3K27ac ChIP-seq与RNA-seq联合测序技术，对比了对维甲酸介导的分化产生应答的细胞与未发生分化的细胞之间的超级增强子差异。我们鉴定出与细胞分化相关的特异性超级增强子；然而，即便在对处理产生应答的细胞中，移除维甲酸后仍存在异质性：MYCN扩增的细胞维持分化状态，而MYCN非扩增的细胞则恢复至增殖状态。本研究证实，调控性超级增强子是维甲酸介导的细胞分化产生差异性应答的潜在机制。整体实验设计：我们针对三种神经母细胞瘤细胞系的H3K27ac图谱进行检测，以探究维甲酸处理及撤除的影响。每种条件——对照组、ATRA处理组、撤除组——均设置三次生物学重复。输入文库取自对照样本。