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

项目摘要 肾母细胞瘤是儿童最常见的肾癌，也是儿童第三常见的实体肿瘤。 肾母细胞瘤的治疗采用手术、化疗和放射治疗相结合的方法，而大多数儿童 虽然治愈了，但晚期患者的存活率仍然很低。化疗的不良后遗症 这些疾病很常见，并影响幸存者的生活质量。需要对肾母细胞瘤有更好的分子知识 开发更有效、毒性更小的新疗法。已知的驱动因素突变(WT1、WTX和 CTNNB1)仅在三分之一的肾母细胞瘤中发现。最近，我们对一大群Wilms进行了测序 肿瘤标本，并发现DROSHA酶的反复出现的体细胞杂合性错义突变， 这与WT1和CTNNB1中已知的驱动突变是互斥的。DROSHA是一种核糖核酸酶 这对于microRNAs(MiRNAs)的生物发生的第一步是必不可少的，miRNAs是发挥关键作用的小RNA 在癌症的生物学方面。Wilms瘤是第一个发生DROSHA突变的人类癌症 已确认身份。然而，目前还不清楚DROSHA突变是如何影响miRNA的生物发生和贡献的 肾母细胞瘤的发病机制。Wilms肿瘤中的DROSHA突变发生在保守的金属结合点或附近 核糖核酸酶(RNase)III结构域中的残基，并损害miRNA的加工。重要的是，我们发现 具有DROSHA突变的Wilms肿瘤表现出显著降低的特定亚类 MiRNAs，包括let-7肿瘤抑制因子miRNA家族的多个成员。让-7 miRNAs调控 肾母细胞瘤中已知的几个癌基因，包括MCYN和LIN28，因此失去了 在这种情况下，这些miRNAs可能有助于肿瘤的发生。以确定其临床意义。 肾母细胞瘤的分子亚型，我们将把已知和新突变的存在与临床联系起来 大量临床注释的肾母细胞瘤标本的结果。我们的初步数据表明 杂合子DROSHA突变通过显性-负性机制起作用。我们假设 杂合子DROSHA RNase III错义突变通过损害肿瘤的生物发生而促进肿瘤的发生 一组特定的肿瘤抑制因子miRNA，重新编程miRNA表达程序正在开发中 肾脏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。我们将识别并验证 特定的miRNAs，其表达失调导致肿瘤的发生。我们将测试一部有针对性的小说 使用基因工程细胞系和Wilms肿瘤小鼠模型的治疗策略。澄清： 这些机制将为Wilms瘤提供关键的分子洞察力，并可能揭示新的 基于miRNA传递或下游通路的药物调节的治疗方法。

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.