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

基本信息

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
项目类别：
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

项目摘要

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（申请人提供）：Asxl1 基因在多种形式的骨髓恶性肿瘤（包括 CMML、MDS、MPN 和 AML）中高频率突变和/或缺失。这些患者的大多数 Asxl1 突变是杂合的，导致无义/移码，表明功能丧失。 ASXL1 突变与不良预后相关。因此，ASXL1 被推测是一种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 ASXL1 的生理功能及其在骨髓恶性肿瘤发病机制中的作用。我们生成了多个 Asxl1 靶向小鼠模型。 Asxl1 (+/-) 的单倍体不足会导致小鼠患上 MDS，随着年龄的增长，可能会发展为 MDS/MPN 和白血病。因此，Asxl1靶向小鼠模型概括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Asxl1生物学的所需信息。删除 Asxl1 会导致骨髓细胞凋亡和有丝分裂增加，这是 MDS 的特征性细胞特征。竞争性重建测定显示 Asxl1-/- 造血干/祖细胞 (HSC/HPC) 的造血再生能力下降。因此，我们假设 Asxl1 通过改变 HSC/HPC 的行为来充当骨髓细胞生成中的肿瘤抑制因子。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 Asxl1 缺陷小鼠（包括 MxCre 或 Vav1Cre 介导的 Asxl1 条件敲除小鼠）的表型，确定 Asxl1 是否在骨髓细胞生成中充当肿瘤抑制因子。此外，我们将评估 Asxl1 单倍体不足与其他遗传改变（例如 Nf1 单倍体不足）在触发骨髓恶性肿瘤中的作用。目标 2：明确小鼠 Asxl1 功能丧失导致骨髓恶性肿瘤的细胞机制。具体来说，我们将检查Asxl1缺失/单倍体不足对HSC/HPC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/单倍体不足对HSC/HPC自我更新和分化潜力的影响。目标3：明确Asxl1调节正常造血并在骨髓生成中发挥肿瘤抑制功能的分子机制。我们将通过 ChIP-Seq 分别在 Asxl1:Tag 和 WT 或 Asxl1-/- HSC/HPC 中绘制 Asxl1 及其相互作用的组蛋白修饰酶的基因组分布图来鉴定 Asxl1 靶基因。此外，我们将通过 ChIP-Seq 剖析 Asxl1 缺失诱导的 HSC/HPC 中 H3 甲基化和 H2A 单泛素化的错误调节，并与基因表达谱相关联。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的多步发病机制中的作用，这可能有助于确定治疗髓系恶性肿瘤患者的新分子靶点。