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

描述(由申请人提供)：癌症改变了转录程序，导致正常细胞功能的解除调节，并支持癌细胞的特征。癌细胞的先决条件是其核合成活性的增加，以支持增加的蛋白质合成需求。这些过程是由RNA聚合酶I(POL I)控制的，它对外界刺激高度反应，在癌症中通常是过度激活的。然而，它还没有被用作临床相关的靶点。PolI转录和核糖体生物合成是核仁的精细过程。Pol I转录异常导致核仁压力和核仁解体，导致一种独特的、可识别的细胞表型。这 该项目提出了一种适用于高通量筛选(HTS)平台的双细胞高内容成像屏幕，用于核仁压力和Pol I的调节。该屏幕将利用稳定整合到癌细胞中的核仁荧光融合蛋白的易位和Pol I催化亚单位的表达作为可识别标记。这里提供的数据表明，Pol I催化亚单位可以选择性地被降解，其靶向与癌细胞活力下降有关。该项目旨在确定HTS平台读数的可行性，并证明该检测方法用于识别新的调节剂的适宜性和重复性。拟议工作的理由是，它将为调节POL I活性提供所有新的筛查工具。我们的目标是利用筛选来识别核仁活动的抑制物，以及POLI催化亚基的特定调节因子。为了实现这些目标，在目标1中，将建立和测试双重筛查试验，并为命中阈值设定参数。使用当前治疗中使用的已定义的药物化学库，AIM 2已被设计成系统地筛选影响核仁完整性和Pol I转录的药物和化合物，并为高通量平台提供组合设置和重复性的验证。AIM 3旨在通过二级筛查和分析来验证屏幕点击，以确定哪些POL I复杂因子和核仁过程受到影响。这些方法将定义影响POL I的当前药物空间，确定有利于治疗结果的潜在新效应，并支持将屏幕应用于不同的大规模化学图书馆收藏。这项研究将显著增加对抑制rRNA转录如何有助于抑制癌细胞生长的理解，并确定这一过程中的新药和小分子先导分子。

DESCRIPTION (provided by applicant): 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 increaed 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 integrate 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 targetingis associated with decreased cancer cell viability. This project aims to define the feasibility of thee 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 fundamentall novel screening tools for regulation of Pol I activity. The goal is to employ the screen to identif, firstly, inhibitors of nucleolar activities and secondly, specific regulators of the Pol I catalyti 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 assy 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.