WT1 as an oncogene and therapeutic target in anaplastic Wilms tumor

项目总结/摘要 肾母细胞瘤是儿童最常见的肾癌。高风险患者群体继续令人沮丧 结果。预后不良的最大预测因素是显微镜下检测到弥漫性间变性（不利 组织学）。这一发现占该疾病死亡人数的50%，并与治疗耐药性有关。 弥漫性间变性（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， 以前有利的组织学。虽然TP53突变与治疗抗性和不良结局相关， 它也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 利用靶向方法研究间变性肾母细胞瘤的治疗弱点。Wilms瘤伴WT1 突变不继续发展TP 53突变，也不发展间变性。此前的证据显示， WT1基因在肾母细胞瘤中作为肿瘤抑制因子。然而，我们的中心假设是WT1 在间变性肾母细胞瘤中作为癌基因发挥作用。WT1突变的肿瘤不发生间变性 WT1和TP53突变不发生在任何组织学的同一儿科癌症患者肿瘤中。 因此，我们假设在同一Wilms肿瘤细胞中WT1突变和TP53突变可能是致命的 对癌细胞的影响。目的1将检验WT1和TP53突变的功能丧失是 在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： 体外TP53突变体间变性Wilms肿瘤细胞。我们还将TP53突变引入小鼠Wilms WT1功能丧失的肿瘤模型。我们的初步数据表明，WT1是激活 肾母细胞瘤端粒酶通路的研究进展端粒酶（由TERT基因编码的关键部分）添加DNA重复序列 在染色体的末端，以抵消癌细胞快速分裂引起的染色体缩短。目的 2将检验功能性WT1对于增加的TERT表达和端粒酶活性是必需的这一假设 间变性肾母细胞瘤通过在体外和小鼠模型系统中敲除WT1，我们旨在确定 WT1是获得端粒酶功能所必需的。我们还将测试端粒靶向治疗6-Thio-dG， 使用人患者肿瘤样品的小鼠异种移植物模型。我们已经发现组蛋白去甲基化酶 KDM6B（一种改变DNA与组蛋白结合方式的酶）调节Wilms肿瘤细胞中的WT1水平。 我们已经表明，抑制KDM6B会降低WT1水平。目标3将检验KDM6B 在肾母细胞瘤中上调WT1，因此可以作为治疗靶点。我们计划在 在化学和遗传水平上损害KDM6B功能的体外实验，以确定其 对WT1和间变性Wilms肿瘤细胞行为影响。我们还旨在测试KDM6B抑制剂GSK-J4， 使用人患者肿瘤样品的小鼠异种移植物模型。这些研究的预期结果将是 极大地提高了对WT1在间变性肾母细胞瘤中的功能的理解和对WT1的评价， 端粒酶和KDM6B作为间变性肾母细胞瘤的临床前治疗靶点。

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.