The transcriptional co-repressor Runx1t1 is essential for N-Myc-driven neuroblastoma tumorigenesis [RNA-Seq 1]

Changes in epigenetic regulation are believed to be a major contributing factor to neuroblastoma development. Using a large-scale in vivo mutagenesis screen in Th-MYCN transgenic mice, we identified a single point mutation in the transcriptional corepressor Runx1t1, that can block N-Myc-driven neuroblastoma tumorigenesis. The loss of function mutation disrupts a highly conserved zinc finger domain (NHR4) within Runx1t1. Crossing an independent Runx1t1 knockout model with Th-MYCN mice, demonstrated that Runx1t1 haploinsufficiency is enough to prevent neuroblastoma development and reverse ganglia hyperplasia. Silencing RUNX1T1 in human neuroblastoma cells resulted in decreased colony formation in vitro, and significant inhibition of tumor growth in vivo. Our results show that RUNX1T1 forms part of a transcriptional LSD1-CoREST3-HDAC repressive complex that regulates the epigenomic landscape and chromatin accessibility, to control neuron-specific pathway genes and maintain an undifferentiated state. Runx1t1 thus represents an entirely novel and highly promising target not previously described in neuroblastoma. Overall design: KELLY cells containing doxycycline-inducible shRNA to Runx1t1 were cultured with and without treatment. After 72 hours, Total RNA was isolated using the RNeasy Micro kit (50), # 74004 kit and RNA quality was assessed prior to being used (Agilent 2100 Bioanalyzer, Agilent Technologies). Only RNA with an RNA Integrity Number (RIN) of >9.0 was used. Whole transcriptome sequencing was performed at the Ramaciotti Centre for Genomics with SMARTer Stranded Total RNA-seq v2 preparation kit. Prepared libraries were pooled and sequenced on NovaSeq 6000 S1 2x100bp lane generating an average of 60 million reads per sample.

表观遗传调控异常被认为是神经母细胞瘤发生的主要致病因素之一。本研究通过在Th-MYCN转基因小鼠中开展大规模体内诱变筛选，在转录共抑制因子Runx1t1中鉴定出一处单核苷酸点突变，该突变可阻断N-Myc介导的神经母细胞瘤发生。该功能丧失型突变破坏了Runx1t1内高度保守的锌指结构域（NHR4）。将独立构建的Runx1t1敲除模型与Th-MYCN小鼠杂交后证实，Runx1t1单倍体剂量不足足以抑制神经母细胞瘤发生，并逆转神经节增生。在人神经母细胞瘤细胞中沉默RUNX1T1，可在体外降低细胞集落形成能力，并在体内显著抑制肿瘤生长。本研究结果显示，RUNX1T1是转录抑制复合物LSD1-CoREST3-HDAC的组成部分，该复合物可调控表观基因组景观与染色质可及性，从而调控神经元特异性通路基因的表达，并维持细胞未分化状态。因此，Runx1t1是一种此前未在神经母细胞瘤中被报道过的全新且极具应用前景的治疗靶点。实验整体设计：将携带多西环素诱导型Runx1t1靶向shRNA的KELLY细胞分为给药组与对照组进行培养。培养72小时后，使用RNeasy Micro试剂盒（50，#74004）提取总RNA，并在使用前通过Agilent 2100生物分析仪（安捷伦科技）评估RNA质量。仅选用RNA完整性数（RIN）大于9.0的RNA样本。随后在拉马乔蒂基因组学中心（Ramaciotti Centre for Genomics）使用SMARTer Stranded Total RNA-seq v2制备试剂盒开展全转录组测序。将构建好的测序文库混合后，在NovaSeq 6000 S1 2×100bp测序通道上进行测序，每个样本平均生成6000万条reads。