Role of Asxl1 in normal hematopoiesis and pathogenesis of myeloid malignancies

Asxl1在正常造血和骨髓恶性肿瘤发病机制中的作用

• 批准号: 8628269
• 负责人: Mingjiang Xu
• 金额: $ 32.37万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8628269
• 关键词: Acute Myelocytic Leukemia; Age; Apoptosis; Behavior; Biological Assay; Biology; Bone Marrow Cells; C-terminal; CD34 gene; Cell Cycle; Cell Differentiation process; Cell physiology; Cells; ChIP-seq; Characteristics; Chromatin; Chromatin Structure; Chromosome Mapping; Chromosomes; Comb animal structure; Complex; Data; Defect; Development; Dysmyelopoietic Syndromes; Enzymes; Exhibits; Frequencies; Gene Expression; Gene Expression Profiling; Gene Targeting; Genes; Genomics; Goals; Hematopoiesis; Hematopoietic; Histones; Homeostasis; Human; In Vitro; Knock-in Mouse; Knockout Mice; Lead; Maintenance; Maps; Mediating; Methylation; Methyltransferase; Mitosis; Modeling; Molecular; Molecular Target; Monoubiquitination; Mus; Mutant Strains Mice; Mutate; Mutation; Myelofibrosis; Myelogenous; Myeloid Cells; Myelopoiesis; Myeloproliferative disease; N-terminal; NF1 gene; Other Genetics; Pathogenesis; Pathologic; Patients; Phenotype; Physiological; Plants; Protein Truncation; Regulation; Reporting; Role; Stem cells; Testing; Transplantation; Tumor Suppressor Genes; Tumor Suppressor Proteins; genome-wide; histone modification; homeodomain; leukemia; leukemogenesis; loss of function; mouse model; mutant; novel; outcome forecast; public health relevance; reconstitution; self-renewal; sex; small hairpin RNA; stem

DESCRIPTION (provided by applicant): Asxl1 gene is mutated and/or deleted with high frequencies in multiple forms of myeloid malignancies including CMML, MDS, MPN and AML. The majority of the Asxl1 mutations in these patients are heterozygous, leading to nonsense/frameshift, suggesting loss of function. Mutations in ASXL1 are associated with poor prognosis. Therefore, ASXL1 has been speculated to be a putative tumor suppressor gene that is strongly implicated in the pathogenesis of myeloid malignancies. The objective of this project is to define the physiological function of ASXL1 and its role in the pathogenesis of myeloid malignancies. We generated several Asxl1-targeted murine models. Haploinsufficiency of Asxl1 (+/-) leads to the development of MDS in mice, which can progress to MDS/MPN and leukemia as they age. The Asxl1-targeted mouse model, therefore, recapitulates the pathologic situation of patients with myeloid malignancy, thus allows us to gain the needed information about Asxl1 biology in a timely fashion. Deleting Asxl1 leads to increased apoptosis and mitosis of bone marrow cells, characteristic cellular feature of MDS. A competitive reconstitution assay showed that Asxl1-/- hematopoietic stem/progenitor cells (HSC/HPCs) had a decreased hematopoietic repopulating capacity. We, therefore, hypothesize that Asxl1 acts as a tumor suppressor in myelopoiesis by altering the behavior of HSC/HPCs. We will test this hypothesis in 3 specific aims: Aim 1: To determine if Asxl1 acts as a tumor suppressor in myelopoiesis by characterizing the phenotype of various Asxl1-deficient mice, including MxCre or Vav1Cre mediated Asxl1 conditional knock-out mice. In addition, we will evaluate the role of Asxl1 haploinsufficiency in cooperation with other genetic alterations, such as Nf1 haploinsufficiency, for triggering myeloid malignancies. Aim 2: To define the cellular mechanisms by which loss of Asxl1 function in mice leads to myeloid malignancies. Specifically, we will examine the effects of Asxl1 deletion/haploinsufficiency on the proliferation, differentiation, apoptosis and cell cycle o HSC/HPCs. In addition, we will determine the effect of Asxl1 deletion/haploinsufficiency on self-renewal and differentiation potential of HSC/HPCs by serial transplantation. Aim 3: To define the molecular mechanisms by which Asxl1 regulates normal hematopoiesis and exerts its tumor suppressor function in myelopoiesis. We will identify Asxl1-target genes by mapping the genomic distribution of Asxl1 and its interacting histone modifying enzymes by ChIP-Seq in Asxl1:Tag and WT or Asxl1-/- HSC/HPCs, respectively. Furthermore, we will dissect Asxl1- deletion induced misregulation of H3 methylation and H2A monoubiquitination in HSC/HPCs with ChIP-Seq and correlate with the gene expression profiling. Accomplishment of these studies allows us to uncover the role of Asxl1 in normal hematopoiesis and in the multiple-step pathogenesis of myeloid malignancies, which may lead to the identification of novel molecular targets for the treatment of patients with myeloid malignancies.

描述（由申请人提供）：Asxl1基因在多种髓系恶性肿瘤（包括CMML、MDS、MPN和AML）中高频突变和/或缺失。这些患者中大多数Asxl1突变是杂合的，导致无义/移码，表明功能丧失。ASXL1基因突变与不良预后相关。因此，ASXL1被推测为一种推定的肿瘤抑制基因，与髓系恶性肿瘤的发病机制密切相关。本项目的目的是明确ASXL1的生理功能及其在髓系恶性肿瘤发病机制中的作用。我们生成了几个asx11靶向小鼠模型。Asxl1（+/-）的单倍性不足导致小鼠MDS的发展，随着年龄的增长可发展为MDS/MPN和白血病。因此，Asxl1靶向小鼠模型概括了髓系恶性肿瘤患者的病理情况，从而使我们能够及时获得所需的Asxl1生物学信息。删除Asxl1导致骨髓细胞凋亡和有丝分裂增加，这是MDS的典型细胞特征。竞争性重组实验显示Asxl1-/-造血干细胞/祖细胞（HSC/HPCs）造血再生能力下降。因此，我们假设Asxl1通过改变HSC/HPCs的行为在骨髓形成中起肿瘤抑制作用。我们将在三个特定目标中验证这一假设：目的1：通过表征各种Asxl1缺陷小鼠（包括mxre或Vav1Cre介导的Asxl1条件敲除小鼠）的表型，确定Asxl1是否在骨髓形成中起肿瘤抑制作用。此外，我们将评估Asxl1单倍不全与其他遗传改变（如Nf1单倍不全）在触发髓系恶性肿瘤中的作用。目的2：明确小鼠中Asxl1功能缺失导致髓系恶性肿瘤的细胞机制。具体来说，我们将研究Asxl1缺失/单倍不足对HSC/HPCs的增殖、分化、凋亡和细胞周期的影响。此外，我们将通过连续移植确定Asxl1缺失/单倍不足对HSC/HPCs自我更新和分化潜力的影响。目的3：明确Asxl1调控正常造血并在骨髓生成中发挥抑瘤功能的分子机制。我们将通过ChIP-Seq分别在Asxl1:Tag和WT或Asxl1-/- HSC/HPCs中绘制Asxl1及其相互作用的组蛋白修饰酶的基因组分布来鉴定Asxl1靶基因。此外，我们将通过ChIP-Seq分析Asxl1缺失导致HSC/HPCs中H3甲基化和H2A单泛素化的错误调控，并分析其与基因表达谱的相关性。这些研究的完成使我们能够揭示Asxl1在正常造血和髓系恶性肿瘤多步骤发病机制中的作用，这可能会导致髓系恶性肿瘤患者治疗的新分子靶点的鉴定。