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: 9070740
• 负责人: Mingjiang Xu
• 金额: $ 31.85万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 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 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9070740
• 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; Health; Hematopoiesis; Hematopoietic; Histones; Homeostasis; Human; In Vitro; Knock-in; Knockout Mice; Lead; LoxP-flanked allele; Maintenance; Maps; Mediating; Methylation; Methyltransferase; Mitosis; Molecular; 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 methylation; genome-wide; histone demethylase; histone modification; homeodomain; knock-down; leukemia; leukemogenesis; loss of function; molecular targeted therapies; mouse model; mutant; novel; outcome forecast; 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）：Asxl 1基因在多种形式的骨髓恶性肿瘤（包括CMML、MDS、MPN和AML）中突变和/或缺失频率较高。这些患者中的大多数Asxl 1突变是杂合的，导致无义/移码，表明功能丧失。ASXL 1突变与预后不良相关。因此，ASXL 1被推测为一个假定的肿瘤抑制基因，与骨髓恶性肿瘤的发病机制密切相关。该项目的目的是确定ASXL 1的生理功能及其在骨髓恶性肿瘤发病机制中的作用。我们产生了几种Asxl 1靶向的小鼠模型。Asxl 1（+/-）的单倍不足导致小鼠中MDS的发展，其可随着年龄的增长而进展为MDS/MPN和白血病。因此，Asxl 1靶向小鼠模型重现了骨髓恶性肿瘤患者的病理情况，从而使我们能够及时获得有关Asxl 1生物学的所需信息。缺失Asxl 1导致骨髓细胞的凋亡和有丝分裂增加，这是MDS的特征性细胞特征。竞争性重建实验表明，Asxl 1-/-造血干/祖细胞（HSC/HPC）的造血重建能力下降。因此，我们假设Asxl 1通过改变HSC/HPC的行为在骨髓生成中起肿瘤抑制剂的作用。我们将在3个具体目标中检验这一假设：目标1：通过表征各种Asxl 1缺陷小鼠（包括MxCre或Vav 1Cre介导的Asxl 1条件性敲除小鼠）的表型，确定Asxl 1是否在骨髓生成中充当肿瘤抑制因子。此外，我们将评估Asxl 1单倍不足与其他遗传改变（如Nf 1单倍不足）合作触发骨髓恶性肿瘤的作用。目的2：确定小鼠Asxl 1功能丧失导致骨髓恶性肿瘤的细胞机制。具体而言，我们将研究Asxl 1缺失/单倍不足对HSC/HPC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来确定Asxl 1缺失/单倍不足对HSC/HPC的自我更新和分化潜能的影响。目的3：阐明Asxl 1调控正常造血和骨髓细胞增殖的分子机制。我们将通过ChIP-Seq分别在Asxl 1：Tag和WT或Asxl 1-/- HSC/HPC中绘制Asxl 1及其相互作用的组蛋白修饰酶的基因组分布来鉴定Asxl 1靶基因。此外，我们将用ChIP-Seq分析Asxl 1缺失诱导的HSC/HPC中H3甲基化和H2 A单泛素化的失调，并与基因表达谱相关。 这些研究的完成使我们能够揭示Asxl 1在正常造血和骨髓恶性肿瘤的多步骤发病机制中的作用，这可能导致识别用于治疗骨髓恶性肿瘤患者的新分子靶点。