WT1 as an oncogene and therapeutic target in anaplastic Wilms tumor

WT1 作为间变性肾母细胞瘤的癌基因和治疗靶点

• 批准号: 10320453
• 负责人: Andrew J Murphy
• 金额: $ 26.51万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10320453
• 关键词: 17p13.1; Anaplasia; Apoptosis; Biological Models; Biology; Biometry; CRISPR/Cas technology; Cancer Patient; Cell Culture Techniques; Cell Line; Cell Survival; Cells; Cessation of life; Chemicals; Childhood Renal Neoplasm; Chromosomes; Code; Complex; DNA; DNA Binding Domain; DNA Damage; Data; Detection; Diffuse; Disease; Dominant-Negative Mutation; Dose; Enzymes; Epigenetic Process; Evaluation; Event; Exhibits; Genetic Transcription; Genomics; Germ-Line Mutation; Goals; Histology; Histones; Holoenzymes; Human; Impairment; In Vitro; Knock-out; Lead; Libraries; Malignant Childhood Neoplasm; Malignant childhood renal neoplasm; Mediating; Mentors; Microscopic; Modeling; Mus; Mutation; Nephroblastoma; Oncogenes; Outcome; Outcome Study; Pathway interactions; Patients; Preclinical Testing; Prognosis; Proteins; Research; Resistance; Role; Sampling; TERT gene; TP53 gene; Tamoxifen; Telomerase; Telomere Maintenance; Telomere Shortening; Testing; Training; Transgenic Mice; Transgenic Organisms; Tumor Biology; Tumor Suppressor Proteins; WAGR Syndrome; WT1 gene; Xenograft Model; behavior in vitro; cancer cell; career development; cell behavior; chemical genetics; chemotherapy; combat; demethylation; epigenetic regulation; experience; experimental study; genetic manipulation; high risk; high risk population; histone demethylase; improved; inhibitor; insight; knock-down; loss of function; loss of function mutation; mouse model; mutant; neoplastic cell; nephron progenitor; novel; novel therapeutics; overexpression; pre-clinical; pre-clinical therapy; promoter; small hairpin RNA; stem cells; targeted agent; targeted treatment; telomere; therapeutic target; therapy resistant; transcription factor; treatment strategy; tumor; tumor xenograft

PROJECT SUMMARY/ABSTRACT Wilms tumor is the most common kidney cancer in children. High-risk patient groups continue to have dismal outcomes. The greatest predictor of a poor outcome is microscopic detection of diffuse anaplasia (unfavorable histology). This finding accounts for 50% of deaths from this disease and is associated with treatment resistance. Diffuse anaplasia (unfavorable histology) is caused by mutation in the TP53 gene that occurs late in a tumor with previously favorable histology. While TP53 mutation is associated with treatment resistance and poor outcomes, it could also result in new tumor vulnerabilities. The long-term goal of our research is to identify and exploit new therapeutic vulnerabilities in anaplastic Wilms tumor using targeted approaches. Wilms tumors with WT1 mutation do not go on to develop TP53 mutation and do not develop anaplasia. Previous evidence shows that the gene WT1 serves as a tumor suppressor in Wilms tumor. However, our central hypothesis is that WT1 functions as an oncogene in anaplastic Wilms tumor. Tumors with WT1 mutation do not develop anaplasia and WT1 and TP53 mutations do not occur in the same pediatric cancer patient tumors of any histology. Therefore, we hypothesize that WT1 mutations and TP53 mutations in the same Wilms tumor cell could be lethal events to the cancer cell. Aim 1 will test the hypothesis that loss of function WT1 and TP53 mutations are synthetic lethal events in Wilms tumor cancer cells. To test this hypothesis, we will model knockout of WT1 in TP53-mutant anaplastic Wilms tumor cells in vitro. We will also introduce a TP53 mutation into a mouse Wilms tumor model that has loss of WT1 function. Our preliminary data show that WT1 is necessary for activation of the telomerase pathway in Wilms tumor. Telomerase (critical portion coded by TERT gene) adds DNA repeats to the ends of chromosomes to counteract chromosomal shortening caused by rapidly dividing cancer cells. Aim 2 will test the hypothesis that functional WT1 is necessary for increased TERT expression and telomerase activity in anaplastic Wilms tumor. By knocking out WT1 in vitro and in a mouse model system, we aim to determine if WT1 is necessary for gain of telomerase function. We also will test the telomere targeted therapy 6-Thio-dG in mouse xenograft models using human patient tumor samples. We have identified that the histone demethylase KDM6B (an enzyme that modifies how DNA is bound to histones) regulates WT1 levels in Wilms tumor cells. We have shown that inhibition of KDM6B lowers WT1 levels. Aim 3 will test the hypothesis that KDM6B upregulates WT1 in Wilms tumor and can therefore be exploited as a therapeutic target. We plan to perform in vitro experiments which impair KDM6B function on the chemical and genetic levels in order to determine its effect on WT1 and anaplastic Wilms tumor cell behavior. We also aim to test the KDM6B inhibitor GSK-J4 in mouse xenograft models using human patient tumor samples. The expected outcome of these studies will be a greatly improved understanding of how WT1 functions in anaplastic Wilms tumor and the evaluation of WT1, telomerase, and KDM6B as preclinical therapeutic targets in anaplastic Wilms tumor.

项目概要/摘要 肾母细胞瘤是儿童中最常见的肾癌。高危患者群体持续惨淡 结果。不良结果的最大预测因素是显微镜下检测弥漫性退行性变（不利 组织学）。这一发现占该疾病死亡人数的 50%，并且与治疗耐药性相关。 弥漫性退行性变（组织学不良）是由 TP53 基因突变引起的，该突变发生在肿瘤晚期 先前有利的组织学。虽然 TP53 突变与治疗抵抗和不良结果相关， 它还可能导致新的肿瘤脆弱性。我们研究的长期目标是识别和开发新的 使用靶向方法治疗间变性肾母细胞瘤的脆弱性。 WT1 肾母细胞瘤 突变不会继续发展为 TP53 突变，也不会发展为退行性变。先前的证据表明 WT1 基因在肾母细胞瘤中充当肿瘤抑制因子。然而，我们的中心假设是 WT1 在间变性肾母细胞瘤中充当癌基因。具有 WT1 突变的肿瘤不会发生退行性变 WT1和TP53突变不会发生在任何组织学的同一儿童癌症患者肿瘤中。 因此，我们假设同一肾母细胞瘤细胞中的 WT1 突变和 TP53 突变可能是致命的 事件对癌细胞的影响。目标 1 将检验以下假设：WT1 和 TP53 突变导致功能丧失 肾母细胞瘤癌细胞中的合成致死事件。为了检验这一假设，我们将在以下模型中对 WT1 的敲除进行建模： TP53 突变型间变性肾母细胞瘤细胞在体外。我们还将向小鼠 Wilms 中引入 TP53 突变 WT1 功能丧失的肿瘤模型。我们的初步数据表明 WT1 对于激活 肾母细胞瘤中的端粒酶途径。端粒酶（由 TERT 基因编码的关键部分）添加 DNA 重复序列 到染色体末端以抵消癌细胞快速分裂引起的染色体缩短。目的 2 将检验以下假设：功能性 WT1 对于增加 TERT 表达和端粒酶活性是必需的 在间变性肾母细胞瘤中。通过在体外和小鼠模型系统中敲除 WT1，我们的目的是确定是否 WT1 对于获得端粒酶功能是必需的。我们还将测试端粒靶向治疗 6-Thio-dG 使用人类患者肿瘤样本的小鼠异种移植模型。我们已经确定组蛋白去甲基化酶 KDM6B（一种改变 DNA 与组蛋白结合方式的酶）调节肾母细胞瘤细胞中的 WT1 水平。 我们已经证明抑制 KDM6B 会降低 WT1 水平。目标 3 将检验 KDM6B 的假设 在肾母细胞瘤中上调 WT1，因此可用作治疗靶点。我们计划演出于 在化学和基因水平上损害 KDM6B 功能的体外实验以确定其 对 WT1 和间变性肾母细胞瘤细胞行为的影响。我们还旨在测试 KDM6B 抑制剂 GSK-J4 使用人类患者肿瘤样本的小鼠异种移植模型。这些研究的预期结果将是 极大地提高了对 WT1 如何在间变性肾母细胞瘤中发挥作用以及 WT1 评估的理解， 端粒酶和 KDM6B 作为间变性肾母细胞瘤的临床前治疗靶点。