Dual High-Throughput Imaging Screen for Nucleolar Integrity and RNA Transcription

癌症改变了导致正常细胞放松调控的转录程序 功能和支持癌细胞的特性。癌细胞的先决条件是 增加其核合成活性，以支持增加的蛋白质合成需求。这些 过程由RNA聚合酶I(Pol I)控制，它对外部环境高度响应 刺激，通常在癌症中过度激活。然而，它还没有被用作临床上的 相关目标。PolI转录和核糖体的生物合成是 核仁。Pol I转录异常导致核仁应激和细胞解体 核仁导致一种独特的、可识别的细胞表型。这个项目阐述了一种双重的 基于细胞的高内容成像屏幕，适用于高通量筛选(HTS)平台， 对于核仁应激和Pol I的调节剂，筛查将利用 稳定整合在癌细胞中的核仁荧光融合蛋白的易位和 POL I催化亚基的表达数据表明，POL I催化 亚基可以选择性地被靶向降解，并且它的靶向与 降低了癌细胞的活力。该项目旨在确定这些读数的可行性 HTS平台，并证明该方法的适用性和重复性，以鉴定新的 监管者。拟议工作的基本原理是它将从根本上提供新奇的 用于调节POL I活性的筛选工具。我们的目标是利用屏幕识别，首先， 核仁活性的抑制物，其次是Pol I催化亚基的特定调节剂。 为实现这些目标，在目标1中，将建立、测试和 将设置命中阈值的参数。使用已定义的药物化学库 目前的治疗用途，Aim 2已经被设计成系统地筛选药物和化合物 影响核仁完整性和Pol I转录，并提供检测的验证 高吞吐量平台的设置和重现性。AIM 3的设计目的是验证 通过二级筛查和化验来确定哪些POL I复杂因素 核仁突起受到影响。这些方法将定义当前的毒品领域 影响Pol I，确定对治疗结果有利的潜在新影响，并提供支持 屏幕在各类大型化工图书馆藏书中的应用。这项研究将 显著提高对抑制rRNA转录如何有助于抑制 癌细胞的生长，并在这一过程中识别新药和小分子铅分子。

Cancers have altered transcriptional programs that lead to deregulation of normal cellular functions and support the cancer cell characteristics. A prerequisite for the cancer cell is an increase in its ribosynthetic activity to support the increased protein synthetic needs. These processes are governed by RNA polymerase I (Pol I), which is highly responsive to external stimuli and commonly hyperactivated in cancer. Yet, it has not been exploited as a clinically relevant target. Pol I transcription and ribosome biosynthesis are exquisite processes of the nucleolus. Aberration of Pol I transcription leads to nucleolar stress and disintegration of the nucleolus leading to a distinctive, identifiable cellular phenotype. This project sets forth a dual cell-based high-content imaging screen, adaptable to high-throughput screening (HTS) platforms, for regulators of nucleolar stress and of Pol I. The screen will utilize, as identifiable marks, the translocation of a nucleolar fluorescent fusion protein stably integrated in cancer cells and the expression of the catalytic subunit of Pol I. Data presented here show that the Pol I catalytic subunit can be selectively targeted for degradation and that its targeting is associated with decreased cancer cell viability. This project aims to define the feasibility of these readouts for HTS platforms, and to demonstrate the suitability and reproducibility of the assay to identify novel regulators. The rationale for the proposed work is that it will provide fundamentally novel screening tools for regulation of Pol I activity. The goal is to employ the screen to identify, firstly, inhibitors of nucleolar activities and secondly, specific regulators of the Pol I catalytic subunit. Towards these goals, in Aim 1, the dual-screening assay will be established, tested and parameters will be set for hit thresholds. Using a defined chemical library of pharmaceuticals in current therapy use, Aim 2 has been devised to systematically screen for drugs and compounds that affect the nucleolar integrity and Pol I transcription, and to provide verification of the assay set-up and reproducibility for high-throughput platforms. Aim 3 has been designed to validate the screen hits through secondary screens and assays that determine which Pol I complex factors and nucleolar processes are affected. These approaches will define the current drug space affecting Pol I, identify potential new effects beneficial for therapeutic outcome, and support application of the screen to diverse large-scale chemical library collections. The study will significantly increase the understanding how inhibition of rRNA transcription contributes to curb growth of cancer cells, and identify new drugs and small-molecule lead molecules in this process.