DNA Methylation & Chromatin Modifications: Mechanisms & Applications in Cancer*

描述(申请人提供)：本计划项目资助申请的长期目标是促进我们对癌细胞中基因表达的表观遗传调节的基本理解，并迅速将分子机制的基本发现转化为慢性淋巴细胞白血病(CLL)患者的临床试验。该方案由五个高度互动和相互依存的项目组成，由三个核心支持。它的基础假设是，特定基因的异常甲基化在肿瘤的发展中起着关键作用，这些基因的重新激活可以作为临床上阻止肿瘤生长的有效手段。项目1重点是使用针对表观遗传修饰的试剂进行早期治疗试验，并结合详细的药代动力学和药效学研究，最终目标是重新激活CLL中抑制的基因。该项目的发展在很大程度上依赖于靶基因的确定(项目2和3)、组蛋白修饰的鉴定(项目4)以及染色质重塑复合体在调节拟议的表观遗传靶向治疗的生物效应中的重要性。(项目5)。项目2旨在识别甲基化沉默的慢性淋巴细胞性白血病基因亚型中的新基因，选定基因的甲基化图谱与临床参数的相关性，基于对慢性淋巴细胞性白血病发病和进展的潜在重要性详细研究选定基因的功能，以及这些基因在慢性淋巴细胞性白血病中的重新激活(与项目1相互作用)。项目3将探索CLL中受体类型蛋白酪氨酸磷酸酶(PTPRO)的甲基化和抑制，PTPRO启动子的甲基化和在CLL遗传亚型中表达的减少，PTPRO的新的肿瘤抑制功能，阐明该基因的DNA甲基化导致其抑制的分子机制，以及它在CLL中的重新激活(与项目1的相互作用)。项目4将集中于通过灵敏质谱学确定核心组蛋白翻译后修饰的新位置，与癌症相关的特定组蛋白修饰，以及用靶向染色质结构的药物治疗慢性淋巴细胞性白血病患者所导致的组蛋白修饰变化的特征(与项目1的相互作用)。项目5将集中于BRG1和hBrm相关PRMT5的活性和底物特异性的生化特征，鉴定在临床试验中使用的药物治疗前后CLL中由含有PRMTS的BRG1和hBrm染色质重塑复合体调控的共同靶基因(与项目1相互作用)。除了所有项目之间的相互依存关系外，这些项目的协同和互动因素也反映在但不限于a)试剂、b)特征良好的初级CLL样本和c)技术和技术(限制性地标基因组测序、亚硫酸盐基因组测序、芯片分析、实时聚合酶链式反应)的共同使用，以实现实时翻译研究的共同目标。

DESCRIPTION (provided by applicant): The long-term objective of this Program Project Grant application is to advance our basic understanding of the epigenetic regulation of gene expression in cancer cells and to rapidly translate the basic discovery of the molecular mechanisms into clinical trials in patients with chronic lymphocytic leukemia (CLL). The Program consists of five highly interactive and interdependent Projects supported by three Cores. It is based on the hypothesis that aberrant methylation of specific genes plays a key role in tumor development and that reactivation of these genes can be used as an effective means to clinically arrest tumor growth. Project 1 focuses on early therapeutic trials using agents that target epigenetic modifications, and incorporate detailed pharmacokinetic and pharmacodynamic studies with the ultimate goal of re-activating genes suppressed in CLL. The development of this project is heavily dependent on the determination of target genes (Project 2 and 3), identification of histone modifications (Project 4) and importance of chromatin remodeling complexes in mediating the biologic effect of the proposed epigenetically targeted therapies. (Project 5). Project 2 is aimed at identification of novel genes in genetic subtypes of CLL silenced by methylation, correlation of the methylation profile of selected genes with clinical parameters, detailed study of the function of selected genes based upon potential importance to the pathogenesis and progression of CLL, and re-activation of these genes in CLL (interaction with Project 1). Project 3 will explore the methylation and suppression of the receptor-type protein tyrosine phosphatase (PTPRO) in CLL, PTPRO promoter methylation and reduced expression in genetic subtypes of CLL, the novel tumor suppressor function of PTPRO, elucidation of the molecular mechanisms by which DNA methylation of this gene leads to its suppression, and its re-activation in CLL (interaction with Project 1). Project 4 will focus on identification of novel sites of post-translational modifications on core histones by sensitive mass spectrometry, specific histone modifications that are associated with cancer, and characterization of the changes in histone modifications that result from the treatment of CLL patients with agents that target chromatin structure (interaction with Project 1). Project 5 will focus on biochemical characterization of the activity and substrate specificity of the Brg1 and hBrm-associated PRMT5, identification of common target genes regulated by PRMTS-containing Brg1 and hBrm chromatin remodeling complexes in CLL before and after treatment with the agents used in clinical trials (interaction with Project 1). In addition to the interdependence of all projects, the synergistic and interactive elements of the projects are also reflected in but not limited to the common use of a) reagents, b) well characterized primary CLL samples, and c) techniques & technologies (Restriction Landmark Genomic Sequencing, Bisulfite genomic sequencing, chIP assays, Real-time PCR) in order to accomplish the common goal of real-time translational research.