Determining the significance of the N-MYC-WDR5 interaction in neuroblastoma.

项目总结/摘要 神经母细胞瘤（NB）是最常见的颅外实体瘤发现婴儿，几乎所有的情况下， 在五岁的时候被诊断出来。最严重的神经母细胞瘤病例与 N-MYC癌基因，在所有NB中约占20%。N-MYC扩增与~50% 尽管有积极的多模式治疗，但总生存率仍然很高，这突出了对新靶向治疗的需求， 对抗这种儿科癌症。与任何MYC蛋白一样，N-MYC必须能够识别并结合染色质 以激活其致癌潜力。这个项目是建立在一个前提下，一个重要的辅助因素， 被称为WDR 5的MYC蛋白可以将N-MYC募集到染色质中的基因上， 神经母细胞瘤功能，靶向N-MYC-WDR 5相互作用可以作为抗神经母细胞瘤的焦点。 基于N-MYC的治疗。支持这一假设的是已发表的数据，显示WDR 5是一种 在WDR 5结合的多种细胞环境中的蛋白质合成基因的保守调节因子， 通过其所谓的“WIN”位点调节这些位点。此外，MYC家族成员c-MYC需要 WDR 5结合与生物量积累和翻译相关的基因，以及c-MYC-WDR 5结合与生物量积累和翻译相关的基因。 相互作用对于淋巴瘤小鼠模型中的肿瘤维持至关重要。在N-MYC扩增的NB细胞中， 线，初步数据显示，靶向WDR 5的WIN-site使用小分子抑制剂，导致 WDR 5结合的全基因组减少，随后在特异性N-MYC- WDR 5共结合基因。而且，重要的是，使用遗传突变体破坏N-MYC-WDR 5相互作用， 削弱N-MYC驱动锚定独立生长的能力，提供证据表明， 与N-MYC相关的重要肿瘤功能需要与WDR 5相互作用。这些数据提供了一个 N-MYC需要WDR 5来结合和调节N-MYC-WDR 5共结合的概念的坚实基础 这些基因对于驱动N-MYC特异性活性很重要。这个项目的目标是询问N- 在N-MYC扩增的神经母细胞瘤细胞系中MYC-WDR 5相互作用，并确定 WDR 5具有N-MYC驱动的转录过程和神经母细胞瘤功能， 化学扰动具体目标1将采用遗传学和基因组学方法来确定基因 与N-MYC和WDR 5结合，并确定N-MYC的募集是在哪些基因上进行的。 依赖于N-MYC-WDR 5相互作用。Specific Aim 2将联合收割机高分辨率转录组学 用细胞和体内功能测定进行分析，以挑战N-MYC-WDR 5的意义 互动在这些研究完成后，我们将确定N-MYC在其中的精确基因。 结合和转录受N-MYC-WDR 5相互作用的调节，并直接挑战了 N-MYC-WDR 5相互作用在NB肿瘤发生的多个方面的意义。

PROJECT SUMMARY/ABSTRACT Neuroblastoma (NB) is the most common extracranial solid tumor found in infants, with almost all cases being diagnosed by the age of five. The most severe cases of neuroblastoma are linked to amplification of the N-MYC oncogene, which occurs in ~20% of all NB. N-MYC amplification is associated with ~50% overall survival despite aggressive multimodal therapies, highlighting the need for new targeted therapies to combat this pediatric cancer. N-MYC, like any MYC protein, must be able to recognize and bind chromatin in order to activate its oncogenic potential. This project is built on the premise that an essential co-factor for MYC proteins called WDR5 acts to recruit N-MYC to chromatin at genes that work to maintain neuroblastoma function, and that targeting the N-MYC-WDR5 interaction can serve as a focal point for anti- N-MYC based therapies. Support for this hypothesis comes from published data showing that WDR5 is a conserved regulator of protein synthesis genes across multiple cellular contexts where WDR5 binds and regulates these loci through its so-called “WIN”-site. In addition, the MYC family member, c-MYC, requires WDR5 to bind genes associated with biomass accumulation and translation, and the c-MYC-WDR5 interaction is essential for tumor maintenance in lymphoma mouse models. In an N-MYC amplified NB cell line, preliminary data reveal that targeting the WIN-site of WDR5 using small molecule inhibitors results in a genome-wide decrease in WDR5 binding, with a subsequent loss of N-MYC binding at specific N-MYC- WDR5 co-bound genes. And, importantly, disrupting the N-MYC-WDR5 interaction using genetic mutants impairs the ability of N-MYC to drive anchorage-independent growth, providing evidence that there are essential tumor functions tied to N-MYC that require interaction with WDR5. Together these data provide a solid foundation for the notion that N-MYC requires WDR5 to bind and regulate N-MYC-WDR5 co-bound genes that are important for driving N-MYC specific activities. The goal of this project is to interrogate the N- MYC-WDR5 interaction in N-MYC amplified neuroblastoma cell lines and determine the influence that WDR5 has on N-MYC driven transcriptional processes and neuroblastoma function using genetic and chemical perturbations. Specific Aim 1 will employ genetic and genomic approaches to identify the genes that are bound by N-MYC and WDR5, and determine at which genes the recruitment of N-MYC is dependent on the N-MYC-WDR5 interaction. Specific Aim 2 will combine high-resolution transcriptomic analyses with cellular and in vivo functional assays to challenge the significance of the N-MYC-WDR5 interaction. At the completion of these studies we will have identified the precise genes in which N-MYC binding and transcription is regulated by the N-MYC-WDR5 interaction, and have directly challenged the significance of the N-MYC-WDR5 interactions on multiple facets of NB tumorigenesis.