Mechanisms and Vulnerabilities of Aberrant Transcriptional Enhancers in Cancer

项目总结/摘要 人类肿瘤中许多重要的癌基因具有正常的编码序列和氨基酸序列， 结构，但由于产生强转录增强子的基因组异常而成为致癌的。 尽管这些异常的增强子在恶性转化中起着中心作用，但对它们的深入了解仍然是一个难题。 作用机制主要基于相关的染色体异常，如染色体异常， 易位最近，在T细胞急性淋巴细胞白血病（T-ALL）中癌基因TAL 1的研究中， 我们发现了一种新的机制，有望改变人们对异常细胞作用的传统看法。 癌症中的转录增强子。我们发现小的，体细胞获得的插入突变， 在精确的非编码位点引入MYB转录因子的结合基序， 转录增强子驱动高水平的TAL 1表达。这一发现提供了机会， 研究突变和小插入是如何在人类癌细胞中形成的，以及它们是如何创造新的 增强子来驱动对细胞生长和存活至关重要的癌基因的表达。此类信息将 在两个方面至关重要。(1)它将为正常增强子如何调节细胞特性提供一个概念框架， 不同组织的发展和（2）它将有助于澄清区分的分子机制 正常的致癌增强子，从而指导新的靶向癌症治疗的发展。我 工作假设是选择癌细胞中由体细胞突变形成的异常增强子， 提供了在给定的肿瘤细胞内促进恶性转化所需的癌基因表达的精确水平， 细胞谱系我的长期目标是实验性地剖析特定的DNA序列，转录因子， 转录衔接子和介体，以及形成所需的表观遗传阅读器，写入器和擦除器 并维持能够驱动高水平关键癌基因表达的异常转录增强子， 人类癌细胞为了实现这一目标，我将首先关注人类T-ALL和AML的分析， 建议（i）鉴定与关键基因的过表达相关的增强子突变的各种实例， 这些白血病中的癌基因;（ii）使用CRISPR-cas9诱变来评估肿瘤的依赖性 对癌基因表达和恶性细胞生长和存活的新增强子突变的细胞;和（iii） 通过RNAi定义表观遗传机制中的哪些蛋白质（例如，BRD 4、CDK 7和CDK 9）， 靶癌基因在肿瘤细胞中过表达和下调的表型后果 表情最后，我将构建斑马鱼动物模型和小鼠原发性患者来源的异种移植物模型 研究异常转录增强子在体内的分子发病机制。有了这些动物模型， 测试策略，以消除这些肿瘤细胞的治疗，旨在专门针对机制， 潜在的异常转录增强子。这项研究计划的成功将催化一种范式转变 在理解致癌增强子及其作为靶向治疗候选者的潜力方面。

Project Summary/Abstract Many important oncogenes in human cancers have normal coding sequences and amino acid structures, but become oncogenic due to genomic abnormalities that create strong transcriptional enhancers. Despite the central role of these aberrant enhancers in malignant transformation, insights into their mechanisms of action are based largely on associated chromosomal abnormalities, such as chromosomal translocations. Very recently, in studies of the oncogene TAL1 in T cell acute lymphoblastic leukemia (T-ALL), we discovered a new mechanism that promises to revise conventional perceptions of the role of aberrant transcriptional enhancers in cancer. We found that small, somatically acquired insertion mutations introduce binding motifs for the MYB transcription factor in a precise noncoding site, creating an aberrant transcriptional enhancer that drives high levels of TAL1 expression. This finding opens the opportunity to investigate how mutations and small insertions are formed in human cancer cells and how they create new enhancers to drive the expression of oncogenes critical for cell growth and survival. Such information will be vital on two fronts. (1) It will provide a conceptual framework for how normal enhancers regulate cell identity in the development of diverse tissues and (2) it will help to clarify the molecular mechanisms that distinguish normal from oncogenic enhancers, thus guiding the development of new targeted cancer treatments. My working hypothesis is that aberrant enhancers formed by somatic mutation in cancer cells are selected to provide the precise levels of oncogene expression needed to promote malignant transformation within a given cell lineage. My long-term goal is to experimentally dissect the specific DNA sequences, transcription factors, transcriptional adaptors and mediators, and epigenetic readers, writers and erasers that are required to form and sustain aberrant transcriptional enhancers capable of driving high levels of key oncogene expression in human cancer cells. To achieve this goal, I will focus initially on the analysis of human T-ALL and AML, and I propose to (i) identify diverse examples of enhancer mutations associated with the overexpression of key oncogenes in these leukemias; (ii) use CRISPR-cas9 mutagenesis to assess the dependence of the tumor cells on novel enhancer mutations for oncogene expression and malignant cell growth and survival; and (iii) define by RNAi which proteins in the epigenetic machinery (e.g., BRD4, CDK7, and CDK9) are required for the target oncogene to be overexpressed in tumor cells and the phenotypic consequences of downregulated expression. Finally, I will construct animal models in zebrafish and primary patient derived xenografts in mice to study molecular pathogenesis of aberrant transcriptional enhancers in vivo. With these animal models I will test strategies to eliminate these tumor cells with therapies designed to specifically target the mechanisms underlying aberrant transcriptional enhancers. Success in this research program will catalyze a paradigm shift in the understanding of oncogenic enhancers and their potential as candidates for targeted therapy.