Identifying Mechanisms of Aurora Kinase A in Centrosome Clustering Using Chemical Genetics

利用化学遗传学鉴定中心体聚类中 Aurora 激酶 A 的机制

• 批准号: 10538615
• 负责人: Gina M Tomarchio
• 金额: $ 4.77万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-11 至 2023-12-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10538615
• 关键词: Address; Alleles; Aneuploidy; Binding; Biological Assay; CRISPR/Cas technology; Cells; Centrosome; Chromosome Segregation; Chromosomes; Complex; Data; Defect; Development; Dimerization; Diploid Cells; Dynein ATPase; Ensure; Genomic Instability; Hematologic Neoplasms; Human; Impairment; In Vitro; Influentials; Kinesin; Literature; Malignant Neoplasms; Masks; Mediating; Microscopy; Microtubule-Organizing Center; Microtubules; Mitosis; Mitotic; Mitotic Spindle Apparatus; Mitotic spindle; Motor; Movement; Mutation; Pathway interactions; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Positioning Attribute; Prognosis; Proteins; Publishing; Role; Sirolimus; Sister Chromatid; Solid; Somatic Cell; System; Testing; Total Internal Reflection Fluorescent; Transgenic Organisms; Work; analog; attenuation; aurora kinase A; cancer cell; cancer survival; cancer therapy; cell cortex; cell motility; chemical genetics; chromosome missegregation; daughter cell; design; dynactin; experimental study; inhibitor; insight; interest; mutant; overexpression; patient prognosis; phosphoproteomics; prevent; scaffold; small molecule inhibitor; spatiotemporal; targeted cancer therapy; tool; tumorigenesis

SUMMARY ABSTRACT Mitotic kinases and phosphatases are essential for preventing errors in chromosome segregation that can lead to aneuploidy and cancer. Aurora Kinase A (AurA) is an essential mitotic kinase that is often upregulated in human cancers. AurA has a well-studied role in assembly of the mitotic spindle apparatus, a complex microtubule array responsible for separating sister chromatids into two identical daughter cells. It is challenging to study AurA function outside of spindle assembly because inhibition causes disruption of the mitotic spindle which can mask other relevant phenotypes. Further, most AurA small molecule inhibitors can be promiscuous. We have developed a system that circumvents these issues by allowing for specific and conditional inhibition of AurA activity. Using cultured human cells, we have inactivated endogenous AurA with CRISPR-Cas9 and rescued its activity with a genetically modified, transgenic AurA allele (AurA-AS). The genetic alteration makes this allele specifically sensitive to modified ATP competitive inhibitors that cannot inhibit any other cellular kinases. Our objective is to use this tool to study the connection between AurA and cancer. Specifically, we are interested in the connection between AurA and centrosome number amplifications, which are common in both solid and hematological malignancies. Extra centrosomes can create mitotic spindles with more than two poles thus causing devastating chromosome mis-segregation. AurA inhibition has been associated with formation of multipolar spindles in the literature. Our preliminary data suggests that AurA activity prevents this multipolar spindle phenotype by promoting the “clustering” of extra centrosomes into two spindle poles. This led us to our central hypothesis that AurA phosphorylation is essential for bi-polar spindle formation in the presence of extra centrosomes. Based on evidence in the literature, we hypothesize that AurA mediates centrosome clustering via phosphorylation of spindle-pole focusing kinesin HSET (Aim 1) and via promoting localization of mitotic spindle positioning factor NuMA to the cell cortex (Aim 2). To determine if AurA-phosphorylation of HSET is important for efficiency of centrosome clustering, we will use in vitro kinase assays to show direct phosphorylation (1.1). We will use HSET phospho-site mutants to determine if this phosphorylation is relevant to centrosome clustering (1.2) and to perform in vitro microtubule motility assays to determine how AurA phosphorylation effects HSET’s activity (1.3). Further, we will use microscopy to determine if loss of AurA impairs mitotic spindle positioning or NuMA localization to the cell cortex (2.1) and if altered NuMA localization effects efficiency of centrosome clustering (2.2). All proposed experiments use our established system for studying AurA. This work is of particular interest as both solid and hematological malignancies often have increased number of centrosomes; by clustering them into two spindle poles, they can prevent division of their chromosomes into multiple, inviable daughter cells. This work will describe a connection between AurA and a unique cancer survival mechanism and will therefore be informative for design and implementation of AurA-specific cancer therapies.

摘要摘要 有丝分裂酶和磷酸酶对于防止染色体分离中的错误是必不可少的，这种错误可能导致 非整倍体和癌症。Aurora Kinase A(AURA)是一种重要的有丝分裂酶，通常在 人类癌症。AURA在有丝分裂纺锤体(一种复杂的微管)的组装中具有广泛研究的作用。 负责将姐妹染色单体分离成两个相同的子细胞的阵列。研究气场是具有挑战性的。 在纺锤体组装之外发挥作用，因为抑制会导致有丝分裂纺锤体的破坏，而有丝分裂纺锤体可以掩盖 其他相关表型。此外，大多数AURA小分子抑制剂可能是混杂的。我们有 开发了一个系统，通过允许特定的和有条件的抑制Aura来绕过这些问题 活动。使用培养的人类细胞，我们用CRISPR-Cas9灭活了内源性AURA并挽救了它 具有转基因AURA等位基因(AURA-AS)的活性。基因的改变使这个等位基因 对不能抑制任何其他细胞激酶的修饰的ATP竞争性抑制剂特别敏感。我们的 目的是利用这一工具研究先兆与癌症之间的联系。具体来说，我们感兴趣的是 灵气和中心体数字放大之间的联系，这在固体和 恶性血液病。额外的中心体可以产生具有两个以上极的有丝分裂纺锤体，因此 造成毁灭性的染色体错误分离。气场抑制与形成 文献中的多极纺锤形。我们的初步数据表明，光环活动阻止了这种多极 纺锤体表型通过促进额外的中心体“聚集”成两个纺锤体极来实现。这让我们找到了我们的 中枢假说：AURA的磷酸化是在额外存在的情况下形成双极纺锤体的关键 中心体。基于文献中的证据，我们假设AURA通过 HSET中纺锤极聚焦运动的磷酸化及促进有丝分裂纺锤体的定位 定位因子NUMA到细胞皮质(目标2)。确定HSET的AURA-磷酸化是否重要 为了提高中心体聚集的效率，我们将使用体外激酶检测来显示直接磷酸化(1.1)。 我们将使用HSET磷酸化位点突变来确定这种磷酸化是否与中心体聚集有关 (1.2)，并进行体外微管运动分析，以确定AURA磷酸化如何影响HSET 活动(1.3)。此外，我们将使用显微镜来确定先兆的丧失是否会损害有丝分裂纺锤体的定位或 NUMA定位于细胞皮质(2.1)，如果改变NUMA定位会影响中心体的效率 集群(2.2)。所有提议的实验都使用我们建立的光环研究系统。这项工作具有特殊的意义 感兴趣，因为实体和血液系统的恶性肿瘤通常都会增加中心体的数量； 将它们聚集成两个纺锤体极，可以防止它们的染色体分裂成多个不能存活的染色体 子代细胞。这项工作将描述先兆和独特的癌症生存机制之间的联系，以及 因此，将为AURA特定癌症疗法的设计和实施提供信息。