Dysregulated genome architecture in acute myeloid leukemia

急性髓系白血病基因组结构失调

• 批准号: 10555303
• 负责人: David H Spencer
• 金额: $ 35.23万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10555303
• 关键词: 3-Dimensional; Acute Myelocytic Leukemia; Affect; Architecture; Biological Assay; CRISPR/Cas technology; Cell Line; Cells; Chromatin; DNA; DNA Integration; DNA Methylation; Data; Development; ETV6 gene; Enhancers; Epigenetic Process; Evolution; Exhibits; Gene Cluster; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Enhancer Element; Genetic Transcription; Genome; Genomic Segment; Goals; Hematopoietic; Hematopoietic stem cells; Homeobox Genes; Hypermethylation; Impairment; In Situ; In Vitro; Individual; Link; Malignant Neoplasms; Mediating; Methylation; Modeling; Modification; Mus; Mutation; Pathogenesis; Pathway interactions; Pattern; Phenotype; Preleukemia; Regulatory Element; Reporter; Role; Sampling; Somatic Mutation; Testing; Therapeutic; Xenograft Model; Xenograft procedure; acute myeloid leukemia cell; data integration; defined contribution; genome editing; genome-wide; genome-wide analysis; genomic locus; improved; in vivo; insight; leukemia; methylation pattern; mutant; new therapeutic target; novel strategies; prevent; promoter; self-renewal; transcription factor

The long-term goal of this proposal is to define the contribution of altered epigenetic patterns and genome organization to the pathogenesis of acute myeloid leukemia. Acute myeloid leukemia (AML) is a devastating cancer that is initiated by somatic mutations in hematopoietic stem/progenitor cells. AML cells are also characterized by DNA methylation changes and altered gene expression patterns, but the relationships between AML mutations, DNA methylation, and transcriptional activity in AML are poorly understood. We have performed comprehensive epigenetic analysis to investigate the regulatory mechanisms that control expression of the HOX gene loci in AML cells, which encode transcription factors that maintain normal hematopoietic stem cell identity and promote self-renewal in AML. These studies have identified specific long-range three- dimensional (3D) genome interactions at the HOXA locus that are increased in AML vs. normal hematopoietic stem cells. Further analysis has showed that the loci involved in these interactions have AML-specific epigenetic changes suggesting they may be enhancers. We have extended these studies by performing a genome-wide analysis of DNA methylation and 3D genome architecture in primary AML samples. This demonstrated that AMLs with canonical mutations in either IDH1 or IDH2 have focal hypermethylation at enhancers that form direct interactions with genes relevant for AML pathogenesis, including MYC and ETV6. Based on these findings, we hypothesize that epigenetic changes at specific regulatory enhancers in AML cells can cause the dysregulation of genes that contribute to AML pathogenesis. Here we propose to test this hypothesis by performing detailed, mechanistic studies of enhancers and gene regulation in primary AML samples and AML cell line models. In Aim 1, we will use capture-HiC to perform in-depth studies of the HOXA locus in primary AML samples and AML cell lines that will define the relationships between AML mutations, enhancer interactions, and HOXA gene expression. We will then use massively parallel reporter assays, CRISPR/Cas9 mediated genome editing, and functional studies in vitro and in vivo to identify the specific enhancers and epigenetic pathways that regulate expression of HOXA genes. In Aim 2, we will use in situ HiC to define the 3D genome organization of primary AML samples with mutations in IDH1 and IDH2 that have focal DNA hypermethylation at enhancers. We will integrate these data with DNA methylation, chromatin profiling, and gene expression to determine how DNA methylation influences enhancer-promoter interactions and gene regulation in AML cells. Together, these studies will provide mechanistic insights into HOX gene regulation that may guide therapeutic approaches that target the HOX self-renewal pathway in AML cells, and determine the extent to which DNA methylation contributes to the leukemia phenotype by altering the function of regulatory enhancers.

该提案的长期目标是定义改变的表观遗传模式的贡献和 基因组组织对急性髓系白血病发病机制的影响。急性髓系白血病 (AML) 是一种 由造血干细胞/祖细胞的体细胞突变引发的毁灭性癌症。 AML 细胞是 也以 DNA 甲基化变化和基因表达模式改变为特征，但这些关系 人们对 AML 突变、DNA 甲基化和转录活性之间的关系知之甚少。我们有 进行全面的表观遗传学分析以研究控制表达的调控机制 AML 细胞中的 HOX 基因位点，编码维持正常造血干的转录因子 细胞身份并促进 AML 的自我更新。这些研究已经确定了特定的长期三 与正常造血系统相比，AML 中 HOXA 位点的三维 (3D) 基因组相互作用增加 干细胞。进一步分析表明，参与这些相互作用的位点具有 AML 特异性表观遗传 变化表明它们可能是增强剂。我们通过进行全基因组研究扩展了这些研究 分析原发性 AML 样本中的 DNA 甲基化和 3D 基因组结构。这表明 AML IDH1 或 IDH2 的典型突变在增强子处具有局灶性高甲基化，从而形成直接的 与 AML 发病机制相关基因的相互作用，包括 MYC 和 ETV6。基于这些发现， 我们假设 AML 细胞中特定调节增强子的表观遗传变化可能导致 导致 AML 发病机制的基因失调。在这里，我们建议通过以下方式检验这一假设： 对原发性 AML 样本和 AML 中的增强子和基因调控进行详细的机制研究 细胞系模型。在目标1中，我们将使用capture-HiC对原发性AML中的HOXA位点进行深入研究 样本和 AML 细胞系将​​定义 AML 突变、增强子相互作用和 HOXA 基因表达。然后我们将使用大规模并行报告分析、CRISPR/Cas9 介导的基因组 体外和体内的编辑和功能研究，以确定特定的增强子和表观遗传途径 调节 HOXA 基因的表达。在目标 2 中，我们将使用原位 HiC 来定义 3D 基因组组织 具有 IDH1 和 IDH2 突变的原发性 AML 样本，这些样本在增强子处具有局灶性 DNA 高甲基化。我们 将这些数据与 DNA 甲基化、染色质分析和基因表达相结合，以确定 DNA 如何 甲基化影响 AML 细胞中的增强子-启动子相互作用和基因调控。在一起，这些 研究将为 HOX 基因调控提供机制见解，从而指导治疗 针对 AML 细胞中 HOX 自我更新途径的方法，并确定其程度 DNA 甲基化通过改变调节增强子的功能而导致白血病表型。