Chemical Genetic Analysis of the Human Cell Cycle

人类细胞周期的化学遗传分析

基本信息

批准号：
8479753
负责人：
ROBERT P FISHER
金额：
$ 32.07万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8479753
关键词：
Ablation Active Sites Alleles Antineoplastic Agents Apoptosis Benefits and Risks Binding Binding Sites Biological Process Cell Cycle Cell Cycle Regulation Cell Fraction Cell Proliferation Cell Proliferation Regulation Cell division Cells Cessation of life Checkpoint kinase 1 Colon Carcinoma Complement Complex Coupled Cyclin-Dependent Kinase Inhibitor Cyclin-Dependent Kinases Cyclins DNA Damage DNA Double Strand Break DNA biosynthesis Development Discrimination Dissection Ensure Enzymes Event Family Feedback Financial compensation Gene Expression Genes Genetic Techniques Genomics Goals Growth and Development function Human Human Engineering Individual Ionizing radiation Logic Malignant Neoplasms Mammalian Cell Mitogens Mus Mutate Mutation Nijmegen Breakage Syndrome Normal tissue morphology Organism Pathway interactions Phosphotransferases Physiological Population Proteins Regulation Role S Phase Specificity Stress Testing Time Toxic effect Work adenine analog cancer cell cell killing cell type chemical genetics chemotherapy cyclin-dependent kinase-activating kinase falls flexibility gain of function genetic analysis inhibitor/antagonist insight knockout gene novel strategies premature prevent public health relevance repaired response scaffold small molecule tumor

项目摘要

DESCRIPTION (provided by applicant): Proper regulation of cell division is required for normal growth, development and genomic integrity; aspects of this control are lost or disrupted in human cancers. The cyclin-dependent kinases (CDKs) drive all major transitions in the cell division cycle. Mammalian cells have multiple downstream effector CDKs but, as we show, a single upstream CDK-activating kinase (CAK), Cdk7. Gene knockout and silencing studies suggested redundancy in the CDK network, by showing that Cdk2-long thought to be the principal driver of DNA synthesis (S) phase-was dispensable for viability. However, our work-using a chemical-genetic approach that selectively inactivates catalytic function of the targeted CDK while sparing non-catalytic, "scaffold" functions-uncovered strict requirements for Cdk2 activity in proliferation of both transformed and non- transformed human cells. An important function of Cdk2 is to prevent premature activation of Cdk1, and thereby ensure coordinated progression through S phase. That function depends on a kinetically more favorable pathway for activation of Cdk2, compared to Cdk1, due in part to different mechanisms of recognition by Cdk7. I hypothesize that specialized functions of Cdk2-in regulating gene expression, coordinating S phase and responding to DNA damage or replication stress-emerge from its unique mode of activation. I propose, moreover, that another distinct pathway by which Cdk7 activates Cdk4 and Cdk6-which function prior to cell-cycle commitment-is directly or indirectly coupled to mitogen-sensing pathways. The specific aims are: 1. To dissect the G1/S regulatory network comprising Cdk7, Cdk2, Cdk4 and Cdk6 2. To probe specific functions of Cdk7 and Cdk2 in the DNA damage response 3. To target the Cdk2 activation pathway with small molecule inhibitors Our preliminary studies reveal a unitary CAK-CDK network that achieves regulatory flexibility through kinetically distinct modes of kinase activation and inactivation. I ow propose to probe specific functions of CAK and the downstream CDKs in coordinating constitutive cell-cycle events and ensuring effective responses to genotoxic insults; and to test a new paradigm for inhibiting a specific CDK by selective targeting of its activation pathway. These studies promise to advance basic understanding of cell-cycle control, and to reveal novel strategies for anti-CDK therapy of human cancer.

描述（由申请人提供）：正常生长、发育和基因组完整性需要细胞分裂的适当调节；这种控制的某些方面在人类癌症中丢失或被破坏。细胞周期蛋白依赖性激酶 (CDK) 驱动细胞分裂周期中的所有主要转变。哺乳动物细胞具有多个下游效应 CDK，但正如我们所展示的，只有一个上游 CDK 激活激酶 (CAK)，即 Cdk7。基因敲除和沉默研究表明，CDK 网络存在冗余，表明 Cdk2（长期以来被认为是 DNA 合成 (S) 阶段的主要驱动因素）对于生存能力来说是可有可无的。然而，我们的工作——使用化学遗传方法选择性地灭活目标CDK的催化功能，同时保留非催化“支架”功能——发现了转化和非转化人类细胞增殖中对Cdk2活性的严格要求。 Cdk2 的一个重要功能是防止 Cdk1 过早激活，从而确保 S 期的协调进展。与 Cdk1 相比，该功能依赖于动力学上更有利的 Cdk2 激活途径，部分原因是 Cdk7 的识别机制不同。我推测 Cdk2 的特殊功能——调节基因表达、协调 S 期以及响应 DNA 损伤或复制应激——源于其独特的激活模式。此外，我还提出，Cdk7 激活 Cdk4 和 Cdk6 的另一种独特途径（其在细胞周期定型之前发挥作用）直接或间接与有丝分裂原感应途径偶联。具体目标是： 1. 剖析由 Cdk7、Cdk2、Cdk4 和 Cdk6 组成的 G1/S 调控网络 2. 探讨 Cdk7 和 Cdk2 在 DNA 损伤反应中的特定功能 3. 用小分子抑制剂靶向 Cdk2 激活途径我们的初步研究揭示了一个单一的 CAK-CDK 网络，该网络通过动力学上不同的激酶激活和失活模式实现了调节灵活性。我现在建议探索 CAK 和下游 CDK 在协调组成性细胞周期事件和确保对基因毒性损伤的有效反应中的特定功能；并测试一个通过选择性靶向其激活途径来抑制特定 CDK 的新范例。这些研究有望增进对细胞周期控制的基本了解，并揭示人类癌症的抗 CDK 治疗的新策略。