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

癌症改变了转录程序，导致正常细胞的失调， 功能和支持癌细胞的特性。癌细胞的先决条件是 增加其核糖合成活性以支持增加的蛋白质合成需求。这些 这些过程由RNA聚合酶I（Pol I）控制，该聚合酶I对外界刺激高度敏感。 在癌症中通常过度激活。然而，它还没有被利用作为临床 相关目标Pol I的转录和核糖体的生物合成是细胞的精细过程， 核仁Pol I转录的异常导致核仁应激和细胞分裂。 核仁导致独特的、可识别的细胞表型。该项目提出了一个双重 基于细胞的高含量成像屏幕，适用于高通量筛选（HTS）平台， 核仁应激和Pol I的调节剂。屏幕将使用，作为可识别的标记， 稳定整合在癌细胞中的核仁荧光融合蛋白的易位， Pol I催化亚基的表达。这里提供的数据表明，Pol I催化 亚基可以选择性地靶向降解，并且其靶向与 降低癌细胞活力。该项目旨在确定这些读数的可行性， HTS平台，并证明该测定法用于鉴定新的 监管部门拟议工作的理由是，它将提供从根本上新颖的 用于调节Pol I活性的筛选工具。我们的目标是利用屏幕来识别，首先， 核仁活性的抑制剂，其次是Pol I催化亚基的特异性调节剂。 为了实现这些目标，在目标1中，将建立、测试和 将为命中阈值设置参数。使用定义的药物化学库， 目前的治疗用途，目标2已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，并提供测定的验证 高通量平台的设置和再现性。目标3旨在验证 通过二级筛选和测定筛选命中，确定哪些Pol I复合因子 和核仁过程受到影响。这些方法将定义当前的药物空间 影响Pol I，确定对治疗结果有益的潜在新效应，并支持 筛选应用于不同的大型化学图书馆馆藏。这项研究将 显著增加了对抑制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先导分子。

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.