Targeting Multiple Colorectal Cancer-Specific Enhancers

项目摘要/摘要 许多癌症形成所需的基因在正常细胞中也发挥着必要的功能。消除这种情况 因此，基因可能会对正常细胞产生不幸的副作用。该领域的最新发展 基因调控的研究为这个问题提供了一个可能的解决方案。转录通常通过以下方式激活 增强子，携带一组结合的转录激活因子的调节DNA元件。增强剂可以 延伸到数百个碱基对上，通常位于数百甚至数千个碱基对上 从它的目标基因。一个给定的基因通常是由一种细胞类型的增强子驱动的，而另一种细胞类型的基因是由不同的 其他细胞类型中的增强子(包含不同的转录因子，结合在不同的DNA位置)。我们 其他人描述了在正常细胞S中没有发现的肿瘤细胞中的癌基因增强剂。 灭活或消除这种肿瘤特异性增强子(同时不损害其非肿瘤细胞中增强子的形成 我们可以在不影响正常细胞生长或功能的情况下抑制肿瘤细胞的生长。 最近的三个进展表明了一条识别和灭活特定增强子的途径。首先，我们 其他人已经开发出在肿瘤和正常细胞中识别全基因组增强剂的分析方法。 第二，CRISPR技术的最新发展表明，我们可以通过一种手段 同时消除(摧毁)多个独立的增强剂。第三，已经开发出方法来 生长器官，比以前更接近体内细胞(正常细胞和肿瘤细胞)的细胞群 描述的程序。本申请的主要人员，斯隆·凯特琳癌症纪念医院的斯科特·洛博士 中心(MSKCC)，已经在使用有机物建立癌症模型方面取得了开创性的进展。我们的合作私家侦探迈克尔博士 Roehrl在MSKCC是公认的内科科学家和病理学家，在结直肠方面具有特殊的专业知识 癌症(CRC)。其他关键人员，作为贡献者，如Christina Leslie博士也在 MSKCC是计算生物学方面的专家，将协助我们进行数据分析。 我们将在结直肠癌肿瘤及其衍生的有机化合物中鉴定结直肠癌特异的增强子。我们会申请 CRISPR技术，以确定这些增强子成对激活的哪些基因需要 CRC有机化合物的生长。然后，我们将使用CRISPR平铺突变来鉴定增强子序列 表达这些基因所需的基因。根据这些知识，我们将设计成对的引导RNA，通过 同时攻击关键的增强剂，将特异性地抑制CRC有机物的形成。

PROJECT SUMMARY/ ABSTRACT Many genes required for cancer formation also perform essential functions in normal cells. Elimination of such genes would, therefore, likely evoke unfortunate side effects on normal cells. Recent developments in the field of gene regulation suggest a possible solution to this problem. Transcription is typically activated by enhancers, regulatory DNA elements bearing a group of bound transcriptional activators. An enhancer can extend over many hundreds of base pairs, and is often positioned hundreds or even thousands of base pairs from its target gene. A given gene is typically driven by an enhancer in one cell type, and by a different enhancer (comprising different transcription factors, bound at a different DNA location) in other cell types. We and others have described enhancers of oncogenes in tumor cells that are not found in normal cell s. Were we to inactivate or eliminate such tumor-specific enhancers (while not damaging enhancer formation in their non- tumor counterparts), we would inhibit tumor cell growth without affecting growth or function of normal cells. Three recent developments have suggested a path to identifying and inactivating specific enhancers. First, we and others have developed assays that identify enhancers genome-wide in tumors and in normal cells. Second, recent developments in CRISPR technology have suggested a means by which we can simultaneously eliminate (destroy) multiple independent enhancers. Third, methods have been developed to grow organoids, groups of cells that more closely mimic cells (normal and tumor) in vivo than previously described procedures. Key personnel on this application, Dr. Scott Lowe at Memorial Sloan Kettering Cancer Center (MSKCC), has made seminal advances in modeling cancer, using organoids. Our co -PI, Dr. Michael Roehrl at MSKCC, is a recognized physician-scientist and pathologist with special expertise in colorectal cancers (CRCs). Additional key personnel, serving as contributors, such as Dr. Christina Leslie also at MSKCC, is an expert in computational biology and will assist us in data analysis. We will identify CRC-specific enhancers in CRC tumors and organoids derived therefrom. We will apply CRISPR technology to identify which of the genes activated by these enhancers, in pairs, are required for growth of CRC organoids. We will then use CRISPR tiling mutagenesis to identify enhancer sequences required for expression of these genes. From this knowledge, we will design pairs of guide RNAs that, by simultaneously attacking crucial enhancers, will specifically inhibit CRC organoid formation.