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

• 批准号: 10320391
• 负责人: Mingjiang Xu
• 金额: $ 56.12万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-15 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10320391
• 关键词: Adult; Age-Years; B-Lymphocytes; Binding; Biological; Biology; Cell Lineage; 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 therapeutic intervention; oxidation; stem cell function; tool; transcriptome sequencing; tumor

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.

摘要 TET2是成人血液系统恶性肿瘤中最常见的突变/缺失基因之一。TET2 基因突变在有克隆性造血的健康老年人中也很常见。因此，TET2突变是 一种祖先突变事件，驱动非恶性克隆性生长并促进血液学 恶变。事实上，小鼠体内TET2的缺失会导致HSC自我更新的增加，并且 各种血液系统恶性肿瘤的发展。然而，潜在的分子机制仍然存在。 很大程度上是未知的。TET2是一种双加氧酶，催化5mC逐步转化为5hmC，5fC和 5caC，活性DNA脱甲基化的起始步骤。基因组中5mC的氧化和去甲基化是 以一种复杂的方式进行管理。已经表明，5hmC和5fC是相对稳定的 分裂细胞和未分裂细胞基因组DNA中的胞嘧啶修饰。TET2可能需要它的催化剂 HSC/HPC发挥肿瘤抑制功能的活性。我们最近表明，TET2的缺失会导致 HSC/HPC的超致突变性，优先于正常获得5hmC和TET2的基因组位置 绑定到。TET2的损失自然会移除部分，但也会创建一组新的、稳定的5hmC和5fC标记 在HSC/HPC中存在较长时期的基因组DNA。然而，TET2的生理意义 HSC/HPC调控中5hmC/5fC形成失控与血液学发病机制 恶性肿瘤仍有待阐明。我们创造了两个新颖的TET2 5 hmC失速(T1285E，Tet2E/+)和 TET2催化失活(H1295Y/D1297A，Tet2YA/+)突变敲入小鼠模型，为我们提供了独特的 阐明TET2催化活性和依赖于TET2的5hmC5fC的特定生物学作用的工具 HSC/HPC调节的转换与血液系统恶性肿瘤。在目标1中，我们将阐明生物 TET2损失相关的5hmC和5fC/5caC形成失速在HSC/HPC调节中的作用 恶性血液病采用催化失活和5hmC停滞的TET2突变小鼠模型。在AIM 2，我们将确定TET2酶活性和依赖于TET2的特异性DNA去甲基化的影响 HSC/HPC基因表达调控和基因组致突变性的中间体。使用WT、TET2-/-、 Tet2YA/YA和Tet2E/E HSC/HPC，我们将：(1)进行RNA-SEQ以鉴定差异表达基因 (2)绘制全基因组5mC/5hmC/5fC/5caC标记图；(3)进行全外显子组测序 识别自发突变。对这些数据集的综合分析将使我们能够确定 TET2催化活性丧失或5hmC5fC转换引起的DEGS和突变与特定的 TET2丢失后HSC/HPC的5mC/5hmC/5fC/5caC变化。这些研究可能会揭示这些潜在的作用 依赖TET2的特异性胞嘧啶：(1)HSC/HPC的基因调控和基因组突变；(2) TET2缺失介导的HSC/HPC失衡与血液系统恶性肿瘤这些发现将极大地 对TET2突变血液系统恶性肿瘤新治疗策略识别的影响。