Roles of TET2-dependent DNA demethylation intermediates in hematological malignancies

TET2依赖性DNA去甲基化中间体在血液恶性肿瘤中的作用

基本信息

批准号：
10063734
负责人：
Mingjiang Xu
金额：
$ 55.76万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-15 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10063734
关键词：
Adult Age-Years B-Lymphocytes Binding Biological Biology Cell Lineage Cell physiology Cells Cytosine DNA Data Analyses Data Set Development Dioxygenases Elderly Event Family Gene Expression Gene Expression Regulation Gene Mutation Genes Genome Genomic DNA Genomics Goals Hematologic Neoplasms Hematology Hematopoiesis Hematopoietic Hematopoietic stem cells Human Impairment Individual Interphase Cell Knock-in Mouse Maintenance Malignant - descriptor Maps Mediating Modification Molecular Mus Mutate Mutation Myeloid Cells Naegleria Non-Malignant Oxides Pathogenesis Patients Phenotype Physiological Play Proteins Proto-Oncogene Protein c-kit Regulation Reporting Role Site Surveys T-Lymphocyte Testing Therapeutic Time Transcription Alteration Variant alpha ketoglutarate demethylation differential expression exome sequencing genome-wide hematopoietic stem cell self-renewal in vivo loss of function member mouse model mutant mutant mouse model novel novel therapeutics oxidation tool transcriptome sequencing tumor

项目摘要

Abstract TET2 is one of the most commonly mutated/deleted genes in adult hematological malignancies. TET2 mutations are also prevalent in healthy elderly individuals with clonal hematopoiesis. Thus, TET2 mutations are an ancestral mutational event that drives non-malignant clonal outgrowth and facilitates hematological malignancy transformation. Indeed, Tet2 loss in mice leads to increased HSC self-renewal and the development of various hematological malignancies. However, the underlying molecular mechanisms remain largely unknown. TET2 is a dioxygenase that catalyzes the stepwise conversion of 5mC to 5hmC, 5fC and 5caC, initial steps of active DNA demethylation. The oxidation and demethylation of 5mC in the genome are regulated in a sophisticated manner. It has been shown that 5hmC and 5fC are present as relatively stable cytosine modifications in genomic DNA of both dividing and nondividing cells. TET2 likely requires its catalytic activity to exert tumor suppressive function in HSC/HPCs. We recently showed that Tet2 loss leads to hypermutagenicity in HSC/HPCs, preferentially at genomic sites that gained 5hmC and TET2 normally binds to. TET2 loss would naturally remove part of, but also creates a new set of, stable 5hmC and 5fC marks in genomic DNA for an extended period in HSC/HPCs. However, the physiological significance of the TET2 loss-mediated stalling of 5hmC/5fC formation in HSC/HPC regulation and pathogenesis of hematological malignancies remains to be elucidated. We have created two novel Tet2 5hmC stalling (T1285E, Tet2E/+) and Tet2 catalytic-inactive (H1295Y/D1297A, Tet2YA/+) mutant knock-in mouse models, which provide us unique tools to elucidate the specific biological role of TET2 catalytic activity and TET2-dependent 5hmC5fC conversion in HSC/HPC regulation and hematological malignancies. In Aim 1, we will elucidate the biological role of Tet2 loss-associated stalling of 5hmC and 5fC/5caC formation in HSC/HPC regulation and hematological malignancies using the catalytic-inactive and 5hmC stalling Tet2 mutant mouse models. In Aim 2, we will determine the effects of TET2 enzymatic activity and specific TET2-dependent DNA demethylation intermediates on gene expression regulation and genomic mutagenicity in HSC/HPCs. Using WT, Tet2-/-, Tet2YA/YA and Tet2E/E HSC/HPCs, we will: (1) perform RNA-seq to identify the differentially expressed genes (DEGs); (2) map genome-wide 5mC/5hmC/5fC/5caC marks; and (3) perform whole-exome sequencing to identify spontaneous mutations. Integrational analysis of these data sets will allow us to determine whether the DEGs and mutations caused by loss of TET2 catalytic activity or 5hmC5fC conversion correlate with specific 5mC/5hmC/5fC/5caC alterations in HSC/HPCs upon Tet2 loss. These studies could unveil potential roles of specific TET2-dependent cytosine species in: (1) gene regulation and genomic mutagenicity in HSC/HPCs; (2) TET2 loss-mediated HSC/HPC dysregulation and hematological malignancies. These findings will greatly impact on the identification of novel therapeutic strategies for TET2-mutated hematological malignancies.

抽象的 TET2 是成人血液恶性肿瘤中最常见的突变/缺失基因之一。 TET2 突变在具有克隆造血功能的健康老年人中也很常见。一种祖先突变事件，可驱动非恶性克隆生长并促进血液学事实上，小鼠中 Tet2 的缺失会导致 HSC 自我更新的增加和然而，潜在的分子机制仍然存在。 TET2 是一种双加氧酶，可催化 5mC 逐步转化为 5hmC、5fC 和 5caC，主动DNA去甲基化的初始步骤是基因组中5mC的氧化和去甲基化。已表明 5hmC 和 5fC 的存在相对稳定。分裂细胞和非分裂细胞的基因组 DNA 中的胞嘧啶修饰可能需要其催化。我们最近发现 Tet2 缺失会导致 HSC/HPC 发挥肿瘤抑制功能。 HSC/HPC 中的超致突变性，优先在正常获得 5hmC 和 TET2 的基因组位点 TET2 损失自然会去除一部分，但也会创建一组新的稳定的 5hmC 和 5fC 标记。然而，TET2 的生理意义在 HSC/HPC 中存在较长时间。 HSC/HPC 调节和血液学发病机制中丢失介导的 5hmC/5fC 形成停滞我们已经创建了两种新型 Tet2 5hmC 停滞（T1285E、Tet2E/+）和 Tet2催化失活（H1295Y/D1297A、Tet2YA/+）突变体敲入小鼠模型，为我们提供了独特的阐明 TET2 催化活性和 TET2 依赖的 5hmC5fC 的特定生物学作用的工具 HSC/HPC 调节和血液恶性肿瘤中的转化在目标 1 中，我们将阐明其生物学作用。 Tet2 丢失相关的 5hmC 和 5fC/5caC 形成停滞在 HSC/HPC 调节和使用催化失活和 5hmC 失速 Tet2 突变小鼠模型研究血液恶性肿瘤。 2、我们将确定TET2酶活性和特定TET2依赖性DNA去甲基化的影响使用 WT、Tet2-/-、HSC/HPC 中基因表达调控和基因组致突变性的中间体。 Tet2YA/YA和Tet2E/E HSC/HPC，我们将：（1）进行RNA-seq来鉴定差异表达基因 (DEG)；(2) 绘制全基因组 5mC/5hmC/5fC/5caC 标记；(3) 进行全外显子组测序；对这些数据集的整合分析将使我们能够确定是否存在自发突变。由 TET2 催化活性丧失或 5hmC→5fC 转化引起的 DEG 和突变与特异性相关 Tet2 丢失后 HSC/HPC 中 5mC/5hmC/5fC/5caC 的变化可能揭示了其潜在作用。特定 TET2 依赖性胞嘧啶种类：(1) HSC/HPC 中的基因调控和基因组致突变性；(2) TET2 缺失介导的 HSC/HPC 失调和血液恶性肿瘤。对 TET2 突变血液恶性肿瘤新治疗策略的确定产生影响。