The role of dynamics in defining the limits of normal developmental signaling.

 描述(申请人提供)：RAS/ERK信号通路控制细胞的增殖、迁移和分化。这一途径的正确功能对人类发育和动态平衡至关重要。该通路的零星突变在许多癌症中起着核心作用，而遗传性突变会导致一系列先天性综合征，称为Rasopathies，从而导致认知发育障碍、心脏畸形和癌症风险增加。一个主要悬而未决的问题是，如何区分正常和病理性的RAS/ERK信号。众所周知，ERK活动的动态模式--包括其活动的强度、频率和持续时间--对于正确的信号传递是必不可少的。然而， 测量ERK活性的标准方法缺乏解决这些关键细节所需的单细胞精确度。在这个项目中，我们将使用活细胞成像，它允许几乎连续地同时监测数千个细胞，以收集关于突变驱动的ERK信号的数据，这些数据比以前更准确和详细。我们将把重点放在Rasopathies中发现的突变上，在Rasopathies中，它们作为单个基因在发育中驱动病理效应的作用比在癌症中更明确，而在癌症中，许多其他基因的突变是一种并发症。我们将首次使用我们的成像平台在单细胞水平上比较致病突变导致的ERK信号变化。使用多个体外系统复制参与发育的细胞过程，我们将确定这些变化如何改变细胞的增殖、迁移和分化。我们会 然后，使用一类直接整合到人类细胞基因组中的新型记者，在基因表达水平上剖析这些表型变化背后的机制。在激酶动力学、基因表达和细胞行为的水平上，我们将量化 突变细胞如何对多种RAS途径抑制剂做出反应，这些抑制剂现在被认为是Rasopathies的治疗方法。这项工作将有几个重要的结果。首先，它将揭示与正常功能兼容的信号行为的量化边界，使我们能够理解RAS途径突变是如何导致疾病的，以及为什么某些突变比其他突变更严重。其次，它将使我们能够理性地选择给不同突变患者服用哪些药物，这样治疗就可以个性化，最好地使每个人的特定信号模式正常化。最后，这将导致一个数学模型之间的联系激酶活性和下游的基因表达程序，将使我们能够更好地了解发育程序和设计所需的细胞反应使用现有的药物，针对激酶活性。

 DESCRIPTION (provided by applicant): Signaling by the Ras/ERK pathway controls cell proliferation, migration, and differentiation. Proper function of this pathway is essential for human development and homeostasis. Sporadic mutations in the pathway play a central role in many cancers, while hereditary mutations cause a series of congenital syndromes, termed RASopathies, which result in impaired cognitive development, cardiac malformations, and increased risk of cancer. A major unanswered question is what differentiates normal from pathological Ras/ERK signaling. It is known that the dynamic pattern of ERK activity - including the strength, frequency, and duration of its activity - are essential to proper signaling. However, the standard methods for measuring ERK activity lack the single-cell precision needed to resolve these essential details. In this project, we will use live-cell imaging, which allows nearl continuous monitoring of thousands of cells simultaneously, to collect data on mutant-driven ERK signaling that is far more accurate and detailed than was previously available. We will focus on the mutations found in the RASopathies, where their role as a single gene driving pathological effects in development is more clearly defined than in cancer, where mutations in many other genes are a complication. We will use our imaging platform to compare for the first time the changes in ERK signaling resulting from disease-causing mutations at the single cell level. Using multiple in vitro systems to replicate cellular processes involved in development, we will determine how these changes modify cell proliferation, migration, and differentiation. We will then dissect the mechanisms underlying these phenotypic changes at the level of gene expression, using a new class of reporters that are integrated directly into the genomes of human cells. At the levels of kinase kinetics, gene expression, and cell behavior, we will quantify how mutant cells respond to multiple Ras pathway inhibitors, which are now being considered as treatments for the RASopathies. This work will have several important outcomes. First, it will reveal the quantitative boundaries of signal behavior that are compatible with normal function, allowing us to understand how Ras pathway mutations lead to disease, and why some mutations are more severe than others. Secondly, it will allow us to make rational choices about which drugs to give to patients with different mutations, so that treatment can be personalized to best normalize each individual's specific signaling patterns. Finally, it will result in a mathematical model of the link between kinase activity and downstream gene expression programs that will allow us to better understand developmental programs and engineer desired cellular responses using existing drugs that target kinase activity.