Project 4: Prognostic Significance and Therapeutic Potential of DROSHA Mutations in Wilms Tumor

项目摘要 肾母细胞瘤是最常见的儿童肾癌，也是儿童第三常见的实体瘤。 肾母细胞瘤的治疗方法是手术、化疗和放疗，而大多数儿童 虽然晚期癌症患者的存活率仍然很低。化疗的晚期不良反应 这是常见的，影响幸存者的生活质量。对肾母细胞瘤的分子生物学认识是必要的 开发出更有效、毒性更小的新疗法。已知的驱动突变（WT 1、WTX和 CTNNB 1）仅在三分之一的Wilms肿瘤中被鉴定。最近，我们对一大批威尔姆斯 肿瘤标本和确定的复发，体细胞杂合错义突变的酶DROSHA， 这些突变与WT 1和CTNNB 1中已知的驱动突变相互排斥。DROSHA是核糖核酸酶 这是微小RNA（miRNAs）生物发生的第一步，小RNA在其中起着关键作用， 在癌症生物学中。Wilms肿瘤是第一个DROSHA突变的人类癌症， 鉴定然而，目前尚不清楚DROSHA突变如何影响miRNA的生物发生， 威尔姆斯瘤发病机制。肾母细胞瘤中的DROSHA突变发生在保守的金属结合位点或附近 核糖核酸酶（RNase）III结构域中的残基，并损害miRNA加工。重要的是，我们发现 具有DROSHA突变的Wilms肿瘤表现出一个特定的亚类的表达大大降低， miRNA，包括let-7肿瘤抑制miRNA家族的多个成员。let-7 miRNAs调控 Wilms肿瘤中的几种已知癌基因，包括MCYN和LIN 28，因此表达缺失， 这些miRNAs可能在这种情况下促进肿瘤发生。确定以下临床意义 在Wilms肿瘤的分子亚型中，我们将把已知的和新的突变的存在与临床 在大量临床注释的Wilms肿瘤标本中获得结果。我们的初步数据显示， 杂合DROSHA突变通过显性负性机制起作用。我们假设 杂合DROSHA RNA酶III错义突变通过损害肿瘤细胞的生物发生来驱动肿瘤发生。 一组特定的肿瘤抑制miRNA，在发育过程中重新编程miRNA表达程序， 肾促进肾母细胞瘤的发展。我们将使用最先进的基因组编辑来验证这一假设 在Wilms肿瘤细胞中重建肿瘤特异性DROSHA突变的技术。我们会确认 特异性miRNAs，其表达失调驱动肿瘤发生。我们将测试一本小说， 使用基因工程细胞系和Wilms肿瘤小鼠模型的治疗策略。阐明 这些机制将为肾母细胞瘤提供关键的分子见解，并可能揭示新的 基于miRNA递送或下游途径的药理学调节的治疗方法。

Project Summary Wilms tumor is the most common pediatric kidney cancer and the 3rd most common solid tumor of childhood. Wilms tumor is treated with a combination of surgery, chemotherapy, and radiation, and while most children are cured, survival remains poor in those with advanced-stage disease. Adverse late effects of chemotherapy are common and affect the quality of life of survivors. Better molecular knowledge of Wilms tumor is necessary to develop novel therapies that are more effective and less toxic. Known driver mutations (WT1, WTX, and CTNNB1) are identified in only one-third of Wilms tumors. Recently, we sequenced a large cohort of Wilms tumor specimens and identified recurrent, somatic heterozygous missense mutations in the enzyme DROSHA, which were mutually exclusive with known driver mutations in WT1 and CTNNB1. DROSHA is a ribonuclease that is essential for the first step in the biogenesis of microRNAs (miRNAs), small RNAs that play critical roles in the biology of cancer. Wilms tumor is the first human cancer in which DROSHA mutations have been identified. However, it is currently unknown how DROSHA mutations impact miRNA biogenesis and contribute to Wilms tumor pathogenesis. DROSHA mutations in Wilms tumors occur at or near conserved metal-binding residues in the ribonuclease (RNase) III domains, and impair miRNA processing. Importantly, we discovered that Wilms tumors with DROSHA mutations exhibit greatly reduced expression of a specific sub-class of miRNAs, including multiple members of the let-7 tumor suppressor miRNA family. let-7 miRNAs regulate several known oncogenes in Wilms tumors, including MCYN and LIN28, and therefore loss of expression of these miRNAs is likely to contribute to tumorigenesis in this setting. To establish the clinical significance of molecular subtype in Wilms tumors, we will correlate the presence of known and novel mutations with clinical outcome in a large collection of clinically-annotated Wilms tumor specimens. Our preliminary data suggest that heterozygous DROSHA mutations operate through a dominant-negative mechanism. We hypothesize that heterozygous DROSHA RNase III missense mutations drive tumorigenesis by impairing the biogenesis of a specific set of tumor suppressor miRNAs, reprogramming the miRNA expression program in the developing kidney to facilitate Wilms tumor development. We will test this hypothesis using state-of-the-art genomic editing techniques to recreate tumor-specific DROSHA mutations in Wilms tumor cells. We will identify and validate specific miRNAs whose dysregulated expression drives tumorigenesis. We will test a novel targeted therapeutic strategy using genetically-engineered cell lines and mouse models of Wilms tumor. Elucidation of these mechanisms will provide critical molecular insights into Wilms tumor and potentially reveal new therapeutic approaches based on miRNA delivery or pharmacologic modulation of downstream pathways.