Mechanisms and Vulnerabilities of Aberrant Transcriptional Enhancers in Cancer

项目摘要/摘要 人类癌症中的许多重要癌基因都有正常的编码序列和氨基酸 结构，但由于产生强烈转录增强子的基因组异常而变得致癌。 尽管这些异常的增强子在恶性转化中起着核心作用，但对其 作用机制主要基于相关的染色体异常，如染色体。 易位。最近，在对T细胞急性淋巴细胞白血病(T-ALL)癌基因TAL1的研究中， 我们发现了一种新的机制，有望改变人们对反常角色的传统看法 癌症中的转录增强剂。我们发现小的，体细胞获得性插入突变 在精确的非编码区引入MYB转录因子的结合基序，造成异常 转录增强子，驱动高水平的TAL1表达。这一发现为我们提供了一个机会 研究人类癌细胞中的突变和小插入是如何形成的，以及它们是如何产生新的 驱动对细胞生长和存活至关重要的癌基因表达的增强剂。这样的信息将是 在两条战线上至关重要。(1)它将为正常增强剂如何调节细胞身份提供一个概念性框架。 不同组织的发育和(2)它将有助于阐明区分 从致癌增强剂中获得正常，从而指导新的靶向癌症治疗方法的开发。我的 工作假说是，癌细胞中由体细胞突变形成的异常增强剂被选择来 提供在给定范围内促进恶性转化所需的癌基因表达的精确水平 细胞谱系。我的长期目标是通过实验剖析特定的DNA序列，转录因子， 转录适配子和介体，以及表观遗传读取器、写入器和擦除器，它们是形成 并维持能够驱动关键癌基因高水平表达的异常转录增强子 人类癌细胞。为了实现这个目标，我首先将重点放在人类T-ALL和AML的分析上，我 建议(I)确定与KEY过表达相关的增强子突变的不同例子 这些白血病的癌基因；(Ii)使用CRISPR-Cas9突变来评估肿瘤的依赖性 细胞对癌基因表达和恶性细胞生长和存活的新的增强子突变；以及(Iii) 由RNAi定义表观遗传机制中的哪些蛋白质(例如，BRD4、CDK7和CDK9)是 靶基因在肿瘤细胞中的过表达及其下调的表型后果 表情。最后，我将在斑马鱼和小鼠体内建立原始患者来源的异种移植动物模型。 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，我会 通过专门针对这些机制的治疗来消除这些肿瘤细胞的测试策略 潜在的异常转录增强子。这项研究计划的成功将促进范式的转变 了解致癌增强剂及其作为靶向治疗候选者的潜力。

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.