Epigenetic Regulation of Differentially Expressed Genes in Cutaneous T Cell Lymphoma

基因组不稳定和失调的表观遗传控制现在都被认为是癌症的特征， 尽管人们对相关机制知之甚少。两者都会导致基因表达异常，并且 能够促进肿瘤发生的功能的得失。肿瘤抑制基因经常被抑制 CpG位点的高甲基化。癌症基因组也经常在全球范围内发生低甲基化。 基因调控元件的低甲基化，如转录因子结合位点和增强子，可能 导致谱系--不适当的“异位”基因表达，以及致癌途径的激活。高度 癌细胞特异表达的基因是吸引人的阳性生物标记物，可能在诊断中有用 测试，或治疗的新靶点。在癌症中因基因表达缺失而导致的阴性生物标志物有 由于它们的间接性和非特异性而有问题。与超甲基化在人类基因组中的作用不同 对于癌症，低甲基化的影响研究得很少。Sezary综合征(SS)，一种侵袭性白血病 变异型皮肤T细胞淋巴瘤(CTCL)是研究癌症相关疾病的良好模型 低甲基化是因为它是最严重的低甲基化癌症之一。SS，它有6-8倍高 在退伍军人群体中的发病率，以表观遗传调节因子的频繁突变为标志，包括 参与DNA甲基化和去甲基化的酶。我们最近发表了一份基因表达图谱 用高分辨率芯片研究SS中的恶性T细胞，并鉴定了一些高水平的 高表达的SS-T细胞特异性基因(SS-HEG)，可作为阳性生物标志物。我们有 还发表了我们的发现，启动子低甲基化与PLS3的过度表达有关， GATA6和Twist1基因在SS T细胞中的表达，DNA甲基化可以调节PLS3基因的表达。至 更全面地探索DNA甲基化变化可能导致SS中异位基因表达的影响，我们 最近获得了SS T细胞的全基因组DNA甲基化图谱。我们的初步数据显示， 在SS T细胞中，额外的SS-HEG显著低甲基化，这表明DNA低甲基化可能 导致这些基因的过度表达。额外的初步数据表明，DNA甲基化可能 也有助于真菌样肉芽肿(MF)的异位基因表达。中粮占集装箱货柜的一半以上。 4例，但恶性T细胞仅局限于皮肤。对于几个协调过度表达和 淋洗液中T细胞SS、CpG的低甲基化基因也明显增加。 基于这些观察，我们假设改变的DNA甲基化支持致病性和异位性 基因在早期和进展期MF/SS中的表达将以下列具体目标来解决这一问题。 目的1.确定SS异位基因表达的主要表观遗传学驱动因素。成对的转录组测序和 全基因组DNA甲基化分析将定位SS T细胞中差异甲基化和基因表达 同一受试者识别晚期疾病的生物标记物。目标2.确定有用的SS-HEG MF中疾病严重程度的生物标志物。SS和MF肿瘤洗脱T细胞的DNA甲基化图谱将是 比较发现SS-HEG在两个谱系中存在差异甲基化。选定的SS-HEG将在 皮损T细胞从早期和晚期MF中洗脱出来，以识别与分期相关的生物标记物。目标3.确定SS-HEG 推动了MF/SS的进步。SS-HEG将在进展者和非进展者中进行纵向检查。一个 我们方法的新方面将是对静止和激活的T细胞进行RNA测序，因此DNA 甲基化可能与基础基因的表达和激活的允许性有关。这个 拟议实验的结论将产生有用的基因表达和DNA甲基化生物标记物 这可能使退伍军人早期诊断CTCL成为可能。这可以防止多年的不适当治疗 患有CTCL的退伍军人。研究计划还将产生对CTCL的机械性见解 发病机制，并为皮肤来源的MF T细胞的研究建立实验模型。

Genomic instability and dysregulated epigenetic control are now both recognized as hallmarks of cancer, although the mechanisms responsible are poorly understood. Both lead to abnormal gene expression, and gains and losses of function that can promote oncogenesis. Tumor suppressor genes are often repressed by hypermethylation of CpG sites. Cancer genomes are also frequently globally hypomethylated. Hypomethylation of gene-regulatory elements, such as transcription factor binding sites and enhancers, may cause lineage-inappropriate “ectopic” gene expression, and activation of oncogenic pathways. Highly expressed genes specific to cancer cells make attractive positive biomarkers that may be useful in diagnostic tests, or new targets for therapy. Negative biomarkers resulting from loss of gene expression in cancer are problematic due to their indirect nature and nonspecificity. In contrast to the role of hypermethylation in cancer, the impact of hypomethylation is very understudied. Sezary syndrome (SS), an aggressive, leukemic variant of cutaneous T cell lymphoma (CTCL) is a good model for studies of cancer-associated hypomethylation because it is one of the most heavily hypomethylated cancers. SS, which has 6-8 fold higher incidence in the Veteran population, is marked by frequent mutations in epigenetic modulators, including enzymes involved in methyation and demethylation of DNA. We recently published a gene expression profiling study of malignant T cells in SS using high resolution microarrays, and identified a number of highly overexpressed genes specific to SS T cells (SS-HEG), that could function as positive biomarkers. We have also published our discovery that promoter hypomethylation is associated with overexpression of PLS3, GATA6, and TWIST1 genes in SS T cells, and that DNA methylation can regulate PLS3 gene expression. To more fully explore the effect of DNA methylation changes that may drive ectopic gene expression in SS, we have recently obtained genome wide DNA methylation profiles for SS T cells. Our preliminary data shows that additional SS-HEG are significantly hypomethylated in SS T cells, suggesting that DNA hypomethylation may contribute to overexpression of these genes. Additional preliminary data indicates that DNA methylation may also contribute to ectopic gene expression in mycosis fungoides (MF). MF represents more than half of CTCL cases, but malignant T cells in MF are limited to the skin. For several coordinately overexpressed and hypomethylated genes in SS, CpG hypomethylation was also increased in T cells eluted from MF tumors. Based on these observations, we hypothesize that altered DNA methylation supports pathogenic and ectopic gene expression in early and progressing MF/SS. This will be addressed with the following specific aims. Aim 1. Identify key epigenetic drivers of ectopic gene expression in SS. Paired transcriptome sequencing and genome-wide DNA methylation assays will map differential methylation and gene expression in SS T cells from the same subject to identify biomarkers of advanced disease. Aim 2. Identify SS-HEG that are useful biomarkers of disease severity in MF. DNA methylation profiles of SS and MF tumor-eluted T cells will be compared to identify SS-HEG differentially methylated in both lineages. Selected SS-HEG will be examined in lesional T cells eluted from early and late MF to identify stage-associated biomarkers. Aim 3. Identify SS-HEG that drive MF/SS progression. SS-HEG will be examined longitudinally in progressors and non-progressors. A novel facet of our approach will be to conduct RNA sequencing on both resting and activated T cells, so DNA methylation may be correlated to both basal gene expression and the permissiveness of activation. The conclusion of the proposed experiments will yield useful gene expression and DNA methylation biomarkers that could enable earlier diagnosis of CTCL in Veterans. This could prevent years of inappropriate therapy for Veterans suffering from CTCL. The research plan will also produce mechanistic insight into CTCL pathogenesis, and generate an experimental model for studies of skin-derived MF T cells.