Epigenetic Targeting in Non-Hodgkin's Lymphoma

描述(申请人提供)：启动子CpG岛的高甲基化在癌症中起着重要作用。随着组蛋白乙酰化/甲基化的改变，这种表观遗传事件建立了一种抑制染色质结构，导致关键的癌症相关基因沉默。基因组中DNA甲基化的发生不是随机的，而是产生特定于基因和肿瘤类型的甲基化模式。DNA甲基化模式是如何建立的仍然知之甚少。由于在体内发现了与DNA甲基转移酶(DNMT)相关的各种转录因子或调节因子，我们假设：1)致癌转录因子可以招募DNMT作为靶基因启动子，并在肿瘤细胞中定义独特的表观遗传特征；2)解剖这种复杂的表观遗传层次结构将为诊断、预后和治疗干预寻找新的分子靶点。为了验证我们的假设，我们开发了一种高通量技术，用于全基因组范围内与特定蛋白质(如组蛋白、转录因子或任何DNA结合蛋白)相关的DNA甲基化分析。这种名为ChlP-Chop-DMH的新方法将结合全基因组定位分析(也称为ChlP-on-Chip)和差异甲基化杂交(DMH)分析，这两种新兴技术用于表观遗传学研究。与目前的方法相比，该方法具有明显的优势：第一，该方法直接检查特定蛋白质与整个基因组中甲基化DNA的体内相互作用；第二，该方法可能揭示转录因子的新的生物学特性；第三，该方法可用于发现与肿瘤发生相关的新的表观遗传生物标记物。在初步研究中，我们已经验证了这种方法的实用性，在人类癌细胞中，组蛋白H3在赖氨酸9位和赖氨酸4位甲基化。在R21阶段，我们将继续对方法进行细微的改进，并追求三个目标：1)改进和优化ChlP-Chop-DMH方法，以分析全基因组范围内DNA甲基化与组蛋白修饰的关联；2)利用所提出的方法研究DNA甲基化与染色质重塑因子的关系：3)使用阵列检测原发性非霍奇金淋巴瘤(NHL)的概念验证。在这一开发阶段，我们将重点研究所建议方法的灵敏度、重复性和准确性。在R33阶段，我们的目标是利用这项技术来测试生物学假说。我们将全面实施该方法，并追求以下目标：1)发现与已知致癌转录因子c-Myc和bcl6相关的表观遗传学靶基因：2)验证已识别的表观遗传学靶点，并研究相关致癌转录因子的调控作用。这种系统化的方法将为未来的机制研究和癌症诊断提供一个强有力的工具。

DESCRIPTION (provided by applicant): Hypermethylation of promoter CpG islands plays a prominent role in cancer. In partnership with alterations in histone acetylation/methylation, this epigenetic event establishes a repressive chromatin structure that leads to silencing of key cancer-related genes. The occurrence of DNA methylation within the genome is not random, but rather patterns of methylation are generated that are gene and tumor type specific. How DNA methylation patterns are established is still poorly understood. Since various transcriptional factors or regulators are found in association with DNA methyltransferases (DNMTs) in vivo, we hypothesize that: 1) Oncogenic transcription factors can recruit DNMTs to target gene promoters and define a unique epigenetic signature in tumor cells; 2) Dissecting such complex epigenetic hierarchy will identify novel molecular targets for diagnosis, prognosis and therapeutic intervention. To test our hypothesis, we developed a high throughput technique for genome wide analysis of DNA methylation associated with specific proteins such as histones, transcription factors or any DNA binding proteins. The new approach named ChlP-Chop-DMH will combine both genome wide location analysis (also known as ChlP-on-Chip) and Differential Methylation Hybridization (DMH) analysis, two emerging technologies used in epigenetic research. The proposed method has distinct advantages over current protocols: first, this method directly examines the in vivo interaction of specific proteins with methylated DNA throughout the genome; second, this method may uncover novel biological properties of transcription factors; third, this method can be applied to discover novel epigenetic biomarkers relevant to tumorigenesis. In preliminary studies, we have verified the utility of this method with methylated histone H3 at lysine 9 and lysine 4 in human cancer cells. In the R21 phase, we will continue minor refinement of the method and pursue three aims: 1) Improve and optimize the ChlP-Chop-DMH method for analyzing genome wide association of DNA methylation with histone modification; 2) Utilize the proposed method to investigate the association of DNA methylation with chromatin remodeling factors: 3) Show proof-of-concept using the array to examine primary non-Hodgkin's lymphomas (NHLs). In this development phase we will focus on the sensitivity, reproducibility and accuracy of the proposed method. In the R33 phase, our goal is to utilize the technology to test biological hypotheses. We will fully implement the method and pursue these aims: 1) Discover epigenetic target genes associated with known oncogenic transcription factors c-Myc and BCL6: 2) Validate the identified epigenetic targets and investigate the regulatory role of the associated oncogenic transcription factors. This systematic approach will provide a powerful tool for future mechanistic studies as well as cancer diagnosis.