Screening Assays for Small Molecules Targeting Oncogenic eIF4E Expression

描述（由申请人提供）：真核翻译起始因子4E （eIF4E）在人类癌症中经常过表达，并通过选择性地驱动对癌细胞生长和生存至关重要的基因的翻译（例如，c-myc， VEGF, BCL-2）来促进癌症的发生和进展。在过去的几十年里，越来越多的证据表明，下调癌细胞中eIF4E的表达可以成为癌症治疗干预的有效策略。事实上，eIF4E反义寡核苷酸已被证明能抑制肿瘤生长而对宿主动物没有毒性，并已进入临床试验。然而，由于缺乏可用于高通量药物筛选的可靠检测方法，目前还没有能够抑制癌细胞中eIF4E表达的小分子可用于临床试验。该申请是为了响应美国国立卫生研究院项目公告PA-10-213，“开发用于探针和治疗前发现的高通量筛选检测方法”，并将解决高通量筛选（HTS）检测的迫切需求，以发现靶向癌细胞中eIF4E表达的药物先导。具体来说，我们的目标是利用新兴的细胞工程技术开发一种创新的检测方法，其中整合在天然eIF4E基因位点的生物发光报告基因在内源性eIF4E启动子的控制下表达。这种检测有望克服当前报告基因检测的局限性，这些检测大多基于克隆的转基因启动子，这些启动子通常缺乏必要的远端/内含子顺式调控元件，而这些启动子存在于外部染色质环境中。因此，创新的检测方法将忠实地再现内源性eIF4E基因对化学处理的反应，从而在高通量搜索eIF4E小分子抑制表达方面更加可靠。因此，该项目的具体目标是：(1)开发在天然eIF4E基因位点中含有报告基因的细胞系，用于高通量药物筛选；(2)开发用于鉴定假阳性和筛选命中优先级的二次分析，以进一步开发药物。这项研究的完成将产生一种创新的HTS检测方法，可用于开发通过抑制eIF4E表达来治愈癌症的治疗药物。因此，拟议的研究将影响数百万癌症患者的健康。这一应用也将为一种创新的药物发现策略提供概念证明，该策略可用于搜索针对癌症中任何特定基因异常表达的药物，从而显著推进靶向癌症治疗。

DESCRIPTION (provided by applicant): The eukaryotic translation initiation factor 4E (eIF4E) is frequently overexpressed in human cancers and contributes to cancer development and progression by selectively driving translation of genes that are essential for cancer cell growth and survival (e.g., c-myc, VEGF, BCL-2). In the past decades accumulating evidence has demonstrated that down-regulation of eIF4E expression in cancer cells can be an effective strategy for therapeutic intervention of cancer. Indeed, eIF4E antisense oligonucleotides have been shown to inhibit tumor growth without toxicity to host animals, and have entered clinical trials. However, because of the lack of a reliable assay that can be used in high-throughput drug screening, no small molecule that can inhibit eIF4E expression in cancer cells is currently available for clinical testing. This application is in response to the NIH Program Announcement PA-10-213, "Development of Assays for High-Throughput Screening for Use in Probe and Pre-therapeutic Discovery", and will address the imperative need of a high-throughput screening (HTS) assay for discovery of drug leads that target eIF4E expression in cancer cells. Specifically, our objective is to utilize emerging cell-engineering technologies to develop an innovative assay in which a bioluminescent reporter integrated in the native eIF4E gene locus is expressed under control of the endogenous eIF4E promoter. Such an assay is expected to overcome limitations of current reporter assays that are mostly based on cloned, transgenic promoters, which often lack essential distal/intronic cis-regulatory elements while residing in foreign chromatin environments. Therefore, the innovative assay would faithfully reproduce responses of the endogenous eIF4E gene to chemical treatments and thus be more reliable in high-throughput search for small molecules inhibitory for eIF4E expression. Accordingly, the specific aims of this project are: (1) to develop cell lines harboring a reporter gene in the native eIF4E gene locus for high-throughput drug screening, (2) to develop secondary assays for identification of false positives and prioritization of screening hits for further drug development. Completion of the proposed research will result in an innovative HTS assay useful for developing therapeutic agents that can cure cancer through inhibiting eIF4E expression. Therefore, the proposed studies will impact health of millions of people afflicted with cancer. This application will also provide a proof of concept for an innovative drug-discovery strategy that can be applied to search for agents targeting any given gene aberrantly expressed in cancer thereby significantly advancing targeted cancer therapy.