Convergent Drivers of Tumor Evolution at the Mitotic Spindle

肾透明细胞癌(Ccrcc)以两种主要的染色体异常为特征，即3p丢失。 和5q的增加，几乎在本病中普遍存在。在这里，我们重点关注这些缺陷的独特见解 在染色质和细胞骨架生物学的结合点提供INTO活动：染色质的协调活动 纺锤体微管上的重构器是有丝分裂的完整性所必需的。共定位于染色体3p 是染色质重构体SETD2、PBRM1和BAP1以及E3连接酶VHL。我们的团体在一起 开创了SETD2和PBRM1在调节有丝分裂保真度的细胞骨架上发挥积极作用的概念， 对基因组完整性的影响，这些影响直到现在才被揭示。SETD2是两者的甲基转移酶 组蛋白和纺锤体微管。利用突变的等位基因，我们分离出微管甲基化的丢失 SETD2缺失是基因组不稳定的根本原因。我们进一步发现，PBRM1，一种底物 PBAF染色质重构体的识别成员，特异性识别SETD2甲基标记 微管和它“读取”的标记一样，是基因组稳定所必需的。最后，我们鉴定了有丝分裂 激酶AURKA作为VHL介导的降解的新靶点，将该规范蛋白(VHL)连接到复合体 慢性肾细胞癌中有丝分裂纺锤体组装的调节。在初步数据中，我们发现除了VHL损失外， AURKA，AURKA通过S2080上的磷酸化调控SETD2，首次连接VHL和SETD2 在共同的致癌途径中。我们还发现了另一种染色质重构体，即染色体5q 组蛋白甲基转移酶NSD1也作用于纺锤体，并且令人兴奋地在CRISPR合成中得分 带有SETD2丢失的致命性筛选。 我们对这一应用的主要假设是VHL和RCC相关的3p和5q染色质 重构体协调调节纺锤体微管的甲基化以维持基因组的稳定， 当有缺陷时，它提供了独特的治疗干预机会。向我们的 在总体假设中，我们提供了三个具体目标：1)解剖有丝分裂纺锤体上的会聚 在3p和5q的ccRCC染色质重构体中，2)定义了它们在分子和 促进有丝分裂完整或失败的生化水平，以及3)机械地评估干预点 这有助于深入了解控制有丝分裂纺锤体完整性的因素。为了实现这些目标，我们将利用 精确评估3p和5q基因组合单等位基因和双等位基因丢失的创新工具和策略 发生在ccRCC进展过程中，以及丰富的初级ccRCC有机体管道。 CcRCC的经典基因组特征为了解染色质的细胞骨架活性提供了新的见解 重构体融合以维持基因组稳定性，研究原理广泛适用于许多其他 并对这种疾病的靶向治疗的发展具有重要的影响。

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.