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.

这项提案的长期目标是确定改变的表观遗传模式的贡献， 急性髓细胞白血病的发病机制。急性髓细胞白血病（AML）是一种 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 也以DNA甲基化改变和基因表达模式改变为特征，但 AML突变之间的关系，DNA甲基化和AML中的转录活性知之甚少。我们有 进行了全面的表观遗传分析，以调查控制表达的调节机制， AML细胞中的HOX基因位点，其编码维持正常造血干细胞的转录因子， 细胞特性并促进AML的自我更新。这些研究已经确定了特定的远程三- 与正常造血细胞相比，在AML中增加的HOXA基因座处的三维（3D）基因组相互作用 干细胞进一步的分析表明，参与这些相互作用的基因座具有AML特异性表观遗传学特征。 这些变化表明它们可能是增强剂。我们通过进行全基因组的 分析原发性AML样本中的DNA甲基化和3D基因组架构。这表明AML IDH 1或IDH 2中的典型突变在增强子处具有局灶性超甲基化， 与与AML发病机制相关的基因（包括MYC和ETV 6）的相互作用。根据这些发现， 我们假设AML细胞中特异性调节增强子的表观遗传学变化可以导致AML细胞的凋亡。 导致AML发病机制的基因失调。在这里，我们建议测试这个假设， 在原发性AML样本和AML中进行增强子和基因调控的详细机制研究 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基因座进行深入研究 样本和AML细胞系，将定义AML突变，增强子相互作用， HOXA基因表达。然后，我们将使用大规模平行的报告基因测定，CRISPR/Cas9介导的基因组， 编辑和功能研究，以鉴定特异性增强子和表观遗传途径， 调控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， 在IDH 1和IDH 2中具有突变的原发性AML样本在增强子处具有局灶性DNA超甲基化。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 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。所有这些 研究将提供HOX基因调控的机制见解， 靶向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增强子的功能而促成白血病表型。