Coexpression of MYCN and ALK Induces Neuroblastoma-Like Tumors From Human iPS Cell-Derived Cranial Neural Crest Cells [RNA-seq]

Neuroblastoma (NB) is a pediatric solid tumor originating from neural crest cells (NCCs), which are precursors of the sympathetic nervous system. MYCN amplification is a key factor contributing to poor prognosis of NB. Additionally, anaplastic lymphoma kinase (ALK) mutation or amplification drives oncogenic signaling pathways, such as MAPK signaling, which together with MYCN amplification exacerbate NB malignancy. NCCs are mainly classified into cranial NCCs (cNCCs) and trunk NCCs (tNCCs), and recent studies have reported NB development from tNCCs. However, the potential for NB development from cNCCs remains unexplored. In this study, we sought to mimic the tumorigenic process of NB by overexpressing MYCN and ALK in cNCCs derived from human induced pluripotent stem cells. These modified cells when subcutaneously transplanted into immunodeficient mice induced NB -like tumors, and could thus be used an in vitro model to study this tumor. Through extensive gene expression profiling and whole exome sequencing of MYCN/ALK-induced clones, we identified key features of NB, including activation of the MAPK pathway and gain of 17q chromosome, which is critical for malignant tumor development. This model provides a valuable platform for studying the biological mechanisms driving ALK and MYCN amplification in NB derived from cNCCs. Overall design: Total RNA was prepared using ISOGENII (NIPPON GENE). High-throughput sequencing were performed by the Takara Bio INC. Quality-checked mRNA was captured using the SMART-Seq mRNA kit (Takara Bio INC) and sequenced using a NovaSeq 6000 (Illumina, California, USA). RNA sequencing paired-end reads were mapped to the reference genome GRCh38 and the downstream processes were performed by the sequencing service provider.

神经母细胞瘤（Neuroblastoma, NB）是一类起源于神经嵴细胞（neural crest cells, NCCs）的儿童实体瘤，神经嵴细胞是交感神经系统的前体细胞。MYCN扩增是导致NB预后不良的关键因素。此外，间变性淋巴瘤激酶（anaplastic lymphoma kinase, ALK）突变或扩增可驱动致癌信号通路，例如丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）信号通路，该通路与MYCN扩增共同加剧NB的恶性程度。神经嵴细胞主要分为颅神经嵴细胞（cranial NCCs, cNCCs）与躯干神经嵴细胞（trunk NCCs, tNCCs），近期已有研究报道NB可由躯干神经嵴细胞发育而来，但颅神经嵴细胞诱发NB的潜力仍未被探索。本研究旨在通过在人诱导多能干细胞衍生的颅神经嵴细胞中过表达MYCN与ALK，模拟NB的致瘤过程。将这些修饰后的细胞皮下移植至免疫缺陷小鼠体内后，可诱导出NB样肿瘤，因此可作为研究该肿瘤的体外模型。通过对MYCN/ALK诱导的克隆开展全面的基因表达谱分析与全外显子组测序，我们鉴定出NB的关键特征，包括MAPK通路激活以及17号染色体长臂（17q）获得，该特征对恶性肿瘤的发生发展至关重要。该模型为研究颅神经嵴细胞来源的NB中ALK与MYCN扩增的生物学驱动机制提供了宝贵的研究平台。实验整体设计：总RNA采用ISOGENII（NIPPON GENE）提取。高通量测序由Takara Bio INC.完成。经质量质控的mRNA通过SMART-Seq mRNA试剂盒（Takara Bio INC.）捕获，并使用NovaSeq 6000测序仪（Illumina，美国加利福尼亚州）进行测序。RNA测序的双端reads被比对至参考基因组GRCh38，后续分析流程由测序服务供应商完成。