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

项目摘要/摘要 神经母细胞瘤(NB)是婴儿最常见的颅外实体瘤，几乎所有病例都是如此 在五岁的时候被诊断出来。最严重的神经母细胞瘤病例与放大的 N-MYC癌基因，约占所有NB的20%。N-MYC扩增与~50%相关 尽管积极的多模式治疗，总体存活率，突显了新的靶向治疗的必要性 与这种儿科癌症作斗争。像任何MYC蛋白一样，N-MYC必须能够识别和结合染色质 以激活其致癌潜能。这个项目是建立在这样一个前提下的，即 名为WDR5的MYC蛋白作用于将N-MYC招募到染色质中，这些基因的作用是维持 神经母细胞瘤的功能，靶向N-MYC-WDR5相互作用可作为抗肿瘤治疗的焦点 基于N-MYC的疗法。对这一假设的支持来自已公布的数据，数据表明WDR5是一种 WDR5与WDR5结合的多种细胞环境中蛋白质合成基因的保守调节因子 通过其所谓的“Win”-Site调节这些基因座。此外，MYC家族成员c-MYC需要 WDR5结合与生物量积累和翻译相关的基因，以及c-myc-WDR5 在淋巴瘤小鼠模型中，相互作用对于维持肿瘤是必不可少的。在N-MYC扩增的NB细胞中 LINE，初步数据显示，使用小分子抑制剂靶向WDR5的WIN位点会导致 WDR5结合在全基因组范围内减少，随后在特定的N-MYC-上失去N-MYC结合。 WDR5共同结合的基因。而且，重要的是，使用遗传突变来破坏N-MYC-WDR5的相互作用 削弱N-MYC驱动非锚定生长的能力，提供证据表明存在 与N-MYC相关的基本肿瘤功能需要与WDR5相互作用。这些数据一起提供了一个 N-MYC需要WDR5来结合和调节N-MYC-WDR5共同结合这一概念的坚实基础 对驱动N-MYC特异性活动非常重要的基因。这个项目的目标是审问N- MYC-WDR5在N-MYC扩增的神经母细胞瘤细胞系中的相互作用及其影响 WDR5对N-MYC驱动的转录过程和神经母细胞瘤功能具有影响 化学扰动。特殊目标1将使用遗传和基因组方法来识别基因 它们由N-MYC和WDR5结合，并决定N-MYC的募集是在哪些基因 取决于N-MYC-WDR5相互作用。《特定目标2》将结合高分辨率转录 用细胞和体内功能分析挑战N-MYC-WDR5的意义 互动。在这些研究完成后，我们将确定N-MYC 结合和转录受N-MYC-WDR5相互作用的调节，并直接挑战 N-MYC-WDR5相互作用在神经母细胞瘤发生的多方面意义

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.