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.

该提议的长期目标是定义改变表观遗传模式的贡献和 急性髓样白血病的发病机理的基因组组织。急性髓样白血病（AML）是 由造血干/祖细胞中的体细胞突变引发的毁灭性癌症。 AML细胞是 也以DNA甲基化的变化和基因表达模式改变了，但关系 在AML突变，DNA甲基化和AML中的转录活性之间，人们了解得很糟糕。我们有 进行了全面的表观遗传分析，以研究控制表达的调节机制 AML细胞中HOX基因位点的编码转录因子，这些转录因子维持正常的造血茎 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远程三 - HOXA基因座的尺寸（3D）基因组相互作用在AML中增加与正常造血 干细胞。进一步的分析表明，这些相互作用所涉及的基因座具有AML特异性表观遗传学 变化表明它们可能是增强剂。我们已经通过进行全基因组来扩展这些研究 基准AML样品中DNA甲基化和3D基因组结构的分析。这表明AMLS IDH1或IDH2中的规范突变在形成直接的增强子处具有局灶性高甲基化 与与AML发病机理相关的基因的相互作用，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会导致 导致AML发病机理的基因失调。在这里，我们建议通过 对原代AML样品和AML进行增强子和基因调节的详细机械研究 单元线模型。在AIM 1中，我们将使用Capture-HIC对主要AML中HOXA基因座进行深入研究 样品和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，我们将使用大量平行的记者分析，CRISPR/CAS9介导的基因组 在体外和体内进行编辑和功能研究，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AIM 2中，我们将使用原位来定义3D基因组组织 在IDH1和IDH2中具有突变的初级AML样品在增强子处具有局灶性DNA高甲基化。我们 将将这些数据与DNA甲基化，染色质分析和基因表达相结合，以确定DNA如何 甲基化会影响AML细胞中增强子促销相互作用和基因调节。在一起，这些 研究将提供有关HOX基因调节的机械见解，以指导治疗 接近针对AML细胞中HOX自我更新途径的方法，并确定 DNA甲基化通过改变调节剂的功能来促进白血病表型。