Cyclin-dependent kinase control of cell-division and transcription cycles

细胞分裂和转录周期的细胞周期蛋白依赖性激酶控制

• 批准号: 10378005
• 负责人: ROBERT P FISHER
• 金额: $ 46.47万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10378005
• 关键词: Antineoplastic Agents; Cell Cycle; Cell Cycle Arrest; Cell Cycle Regulation; Cell division; Cells; Chemicals; Colon Carcinoma; Complex; Coupling; Cyclin-Dependent Kinases; Dependence; Disease; Drug Combinations; Drug Targeting; Elongation Factor; Ensure; Enzymes; Event; Failure; Fission Yeast; Gene Expression; Gene Expression Regulation; Genetic; Genetic Transcription; Goals; Human; Human Development; Kinetics; Lead; Link; Malignant Neoplasms; Modeling; Pathway interactions; Pharmaceutical Preparations; Phase; Phosphorylation; Polyadenylation; Polymerase; Positive Transcriptional Elongation Factor B; Protein phosphatase; Proteins; RNA Polymerase II; RNA Processing; Regulation; Signal Transduction; Specificity; Speed; TP53 gene; Testing; Therapeutic Agents; Time; Transcription Elongation; Transcriptional Regulation; Yeasts; analog; cancer cell; chemical genetics; cyclin T1; cyclin-dependent kinase-activating kinase; drug discovery; functional genomics; genetic approach; inhibitor; mutant; novel; promoter

Project Summary Cyclin-dependent kinases (CDKs) regulate cell division and transcription. To understand the mechanisms of this regulation, or to target the CDK network in cancer cells, we need to identify the functions and substrates of specific CDKs. To achieve this goal, we pioneered a chemical-genetic approach combining the specificity of genetics with the speed and reversibility of chemical inhibition, by replacement of wild-type with analog- sensitive (AS) mutant CDKs in living cells. In the next five years, we will combine chemical genetics with functional genomics in novel ways, to establish new paradigms of cell-cycle and transcriptional control, and to discover new pathways that interact with the CDK network, which might be targeted in cancer. The CAK-CDK network in cell-cycle control: Our studies revealed distinct activation pathways for CDKs that act in different phases of the cell cycle; a goal for the next five years is to understand how those pathways are regulated by upstream signaling and linked to cell cycle-regulated transcription. We will investigate how a cascade comprising the CDK-activating kinase (CAK) Cdk7 and its target Cdk4 is switched on in G1 when quiescent cells re-enter the division cycle, and how Cdk7 is specifically down-regulated as cells exit the cycle. CDK regulation of the transcription cycle: Inhibiting transcriptional CDKs perturbs RNA polymerase (Pol) II dynamics in ways reminiscent of cell-cycle arrests and checkpoint failures, but the precise mechanisms still need to be defined. Cdk7 is required to establish a promoter-proximal pause in the transition from initiation to elongation, and to promote pause release by activating positive transcription elongation factor b (P-TEFb, a Cdk9/cyclin T1 complex). We showed that normal Pol II elongation rates depend on Cdk9 activity in fission yeast, and defined sets of human and yeast Cdk9 substrates, which are enriched for proteins implicated in RNA processing. Over the next five years, we will test the idea that Cdk9 acts on different substrates to stimulate both transcription elongation and RNA processing, to ensure their kinetic coupling. Defining a transcription exit network: CDK regulation persists to the end of the transcription cycle; we validated the termination enzyme Xrn2 and protein phosphatase 1 (PP1, implicated in cleavage and polyadenylation) as bona fide Cdk9 substrates. Phosphorylation by Cdk9 activates Xrn2 but inhibits PP1, which is required to dephosphorylate the elongation factor (and Cdk9 target) Spt5. In the next five years we will test the emergent model of a bistable Cdk9-PP1 switch that controls the elongation-termination transition. Chemical-genetic discovery of synthetic-lethal interactions CDKs have emerged as targets of drugs that exploit transcriptional dependencies unique to certain cancer cells. We induced such a dependency in colon cancer cells by combining activators of the tumor suppressor p53 with inhibitors of Cdk7 to achieve synthetic lethality. In the next five years, we will uncover novel pathways that interact with the CDK network—and might lead to anti-cancer drug combinations—by synthetic-lethal screens in human cells dependent on AS CDKs.

项目摘要 细胞周期蛋白依赖性激酶（CDKS）调节细胞分裂和转录。了解机制 该调节，或针对癌细胞中的CDK网络，我们需要确定 特定的CDK。为了实现这一目标，我们开创了一种化学基因方法，结合了 遗传学具有化学抑制的速度和可逆性，通过用类似物代替野生型 活细胞中的敏感（AS）突变CDK。在接下来的五年中，我们将将化学遗传学与 以新颖方式进行功能基因组学，建立细胞周期和转录控制的新范式，并 发现与CDK网络相互作用的新途径，该途径可能针对癌症。 细胞周期控制中的CAK-CDK网络：我们的研究揭示了CDK的不同激活途径 在细胞周期的不同阶段作用； a goal for the next five years is to understand how those pathways are 由上游信号传导调节，并与细胞周期调节的转录相关。我们将研究如何 完成CDK激活激酶（CAK）CDK7及其目标CDK4的级联反应在G1中打开 静止的细胞重新进入分裂周期，以及当细胞退出周期时，CDK7如何被特别下调。 转录周期的CDK调节：抑制转录CDKS Perturbs RNA聚合酶（POL）II 动力学以使人联想到细胞周期逮捕和检查点故障的动态，但仍然存在精确的机制 需要定义。 CDK7需要在从启动到 伸长，并通过激活正转录伸长因子B促进暂停释放（p-tefb，a CDK9/Cyclin T1复合物）。我们表明，正常的pol II伸长率取决于裂变中的CDK9活性 酵母以及定义的人和酵母CDK9底物，这些蛋白丰富了实施的蛋白质 RNA处理。在接下来的五年中，我们将测试CDK9对不同基板作用的想法 刺激转录伸长和RNA处理，以确保其动力学耦合。 定义转录出口网络：CDK调节持续到转录周期的结束； 验证了终止酶XRN2和蛋白质磷酸酶1（pp1，在裂解和 多腺苷酸化）作为真正的CDK9底物。 CDK9磷酸化激活XRN2，但抑制PP1， 这是脱磷酸化的伸长因子（和CDK9靶）SPT5所需的。在接下来的五年中，我们将 测试控制伸长终止过渡的双轴CDK9-PP1开关的新兴模型。 合成致死相互作用CDK的化学遗传发现CDK已成为药物的靶标 利用某些癌细胞独有的转录依赖性。我们引起了这种颜色的依赖性 癌细胞通过将抑制p53的激活剂与CDK7的抑制剂相结合以实现合成 致死性。在接下来的五年中，我们将发现与CDK网络相互作用的新颖途径，并且可能 通过依赖于CDK的人类细胞中的合成致命筛选，导致抗癌药物组合。