Convergent Drivers of Tumor Evolution at the Mitotic Spindle

有丝分裂纺锤体肿瘤进化的趋同驱动因素

• 批准号: 10654843
• 负责人: Ruhee Dere
• 金额: $ 56.03万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10654843
• 关键词: Address; Alleles; Automobile Driving; Biochemical; Biological Assay; Biology; Cell division; Cells; Centrosome; Chromatin; Chromosome 3; Chromosome abnormality; Chromosomes; Clear cell renal cell carcinoma; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cytoskeleton; Data; Defect; Development; Disease; Disease Progression; Dropout; Equilibrium; Etiology; Evolution; Failure; Genes; Genome Stability; Genomic Instability; Genomics; Heterozygote; Histones; Human; Intervention; Kidney; Link; Loss of Heterozygosity; Lysine; Malignant Neoplasms; Mediating; Methylation; Methyltransferase; Microtubules; Mitosis; Mitotic; Mitotic spindle; Modeling; Molecular; Mutation; Oncogenic; Organoids; Pathway interactions; Patients; Phenotype; Phosphorylation; Phosphotransferases; Proteins; Publishing; Reader; Regulation; Regulatory Element; Reporting; Role; Route; Specimen; Testing; Therapeutic Intervention; Time; Tubulin; Tumor Suppression; Tumor Suppressor Proteins; Work; aurora kinase; aurora kinase A; carcinogenesis; chromatin remodeling; chromosome 5q loss; combinatorial; genome integrity; histone methyltransferase; innovation; insight; loss of function; member; mutant; novel; novel therapeutic intervention; pharmacologic; preservation; prevent; recruit; screening; targeted treatment; therapeutic evaluation; tool; tumor; ubiquitin isopeptidase; ubiquitin-protein ligase

Clear cell renal cell carcinoma (ccRCC) is characterized by two major chromosome abnormalities, loss of 3p and gain of 5q, which occur nearly universally in this disease. Here we focus on the unique insights these defects provide into activities at the nexus of chromatin and cytoskeletal biology: coordinated activity of chromatin remodelers on spindle microtubules needed for the integrity of mitosis. Colocalized on chromosome 3p are the chromatin remodelers SETD2, PBRM1, and BAP1 and the E3 ligase VHL. Our groups together have pioneered the concept that SETD2 and PBRM1 have active roles on the cytoskeleton that regulate mitotic fidelity, with impacts on genomic integrity that are only now being revealed. SETD2 is a methyltransferase for both histones and spindle microtubules. Using mutant alleles, we isolated loss of microtubule methylation as underlying the genomic instability tied to SETD2 loss. We further discovered that PBRM1, a substrate recognition member of the PBAF chromatin remodeler, specifically recognizes the SETD2 methyl mark on microtubules, and like the mark it “reads”, is required for genomic stability. Finally, we identified the mitotic kinase AURKA as a new target for VHL-mediated degradation, linking this canonical protein (VHL) to complex regulation of mitotic spindle assembly in ccRCC. In Preliminary Data, we find in addition to VHL loss stabilizing AURKA, AURKA regulates SETD2 via phosphorylation on S2080, connecting VHL and SETD2 for the first time in a common oncogenic pathway. We have also discovered another chromatin remodeler, the chromosome 5q histone methyltransferase NSD1, is also acting at the spindle, and excitingly scores in a CRISPR synthetic lethality screen with SETD2 loss. Our Overarching Hypothesis for this application is that VHL and RCC-associated 3p and 5q chromatin remodelers coordinately regulate methylation of spindle microtubules to maintain genomic stability, which when defective, offers unique opportunities for therapeutic intervention. To address our Overarching Hypothesis, we offer three Specific Aims: We will 1) dissect the convergence on the mitotic spindle of 3p and 5q ccRCC chromatin remodelers, 2) define the features of their interactions at the molecular and biochemical levels that promote mitotic integrity or failure, and 3) mechanistically evaluate points of intervention that lend insight into the controls governing mitotic spindle integrity. To accomplish these aims, we will use innovative tools and strategies that precisely evaluate combinatorial mono- and bi-allelic loss of 3p and 5q genes that occur during progression of ccRCC, as well as a rich pipeline of primary ccRCC organoids. The classic genomic features of ccRCC provide new insights on how cytoskeletal activities of chromatin remodelers converge to maintain genomic stability, the principles of study are broadly applicable to many other cancers and have important ramifications for the development of targeted therapies for this disease.

透明细胞肾细胞癌（ccRCC）的特征是两个主要的染色体异常，3p缺失， 和5q的增加，这几乎普遍发生在这种疾病中。在这里我们着重对这些缺陷进行独到的见解 在染色质和细胞骨架生物学的联系中提供活动：染色质的协调活动 有丝分裂的完整性所需的纺锤体微管的重塑。共定位于染色体3p 是染色质重塑物SETD2、PBRM 1和BAP 1以及E3连接酶VHL。我们的团队一起 开创了SETD 2和PBRM 1在调节有丝分裂保真度的细胞骨架上具有积极作用的概念， 对基因组完整性的影响现在才被揭示出来。SETD 2是甲基转移酶， 组蛋白和纺锤体微管。使用突变等位基因，我们分离出微管甲基化缺失， 这是与SETD 2丢失相关的基因组不稳定性的基础。我们进一步发现，PBRM 1，一种底物， PBAF染色质重塑剂的识别成员，特异性识别SETD 2甲基标记， 微管，就像它“读取”的标记一样，是基因组稳定性所必需的。最后，我们确定了有丝分裂 激酶AURKA作为VHL介导的降解的新靶点，将这种典型蛋白质（VHL）与复合物 调控ccRCC中的有丝分裂纺锤体组装。在初步数据中，我们发现，除了VHL损失稳定 AURKA，AURKA通过S2080上的磷酸化调节SETD2，首次将VHL和SETD2连接起来 共同的致癌途径。我们还发现了另一个染色质重塑者，染色体5q 组蛋白甲基转移酶NSD1也作用于纺锤体，并在CRISPR合成中令人兴奋地得分。 具有SETD2损失的致死率筛选。 我们对此应用的总体假设是，VHL和RCC相关的3p和5q染色质 重塑者协调调节纺锤体微管的甲基化以维持基因组稳定性， 当其有缺陷时，为治疗干预提供了独特的机会。解决我们 概括这一假说，我们提出了三个具体的目的：1）解剖有丝分裂纺锤体上的会聚 的3p和5q ccRCC染色质重塑，2）定义其在分子和 促进有丝分裂完整性或失败的生化水平，以及3）机械地评估干预点 这有助于我们深入了解有丝分裂纺锤体完整性的控制机制。为了实现这些目标，我们将使用 精确评估3p和5q基因的组合单和双等位基因缺失的创新工具和策略 这发生在ccRCC的进展，以及丰富的管道主要ccRCC类器官。 ccRCC的经典基因组特征为染色质的细胞骨架活动提供了新的见解。 重塑者会聚以维持基因组稳定性，研究的原则广泛适用于许多其他 癌症，并对这种疾病的靶向治疗的发展具有重要的影响。