DNMT3A MUTATIONS IN ACUTE MYELOID LEUKEMIA

急性髓系白血病中的 DNMT3A 突变

基本信息

批准号：
8309966
负责人：
TIMOTHY J. LEY
金额：
$ 43.9万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8309966
关键词：
Acute Myelocytic Leukemia Age Alleles Amino Acids Animals BRCA1 gene Baculoviruses Binding Biochemical Biological Assay Bone Marrow Cells Complementary DNA CpG Islands Cytosine DNA DNA Methyltransferase DNA Modification Methylases Data Development Dominant-Negative Mutation Escherichia coli FLT3 gene Gene Expression Profile Genes Genome Goals Hematopoiesis Hematopoietic Human In Vitro Malignant Neoplasms Marrow Measurable Methionine Methylation Methyltransferase Missense Mutation Mus Mutate Mutation NPM1 gene Nonsense Mutation Outcome Pathogenesis Patients Pattern Phenotype Positioning Attribute Predisposition Property Proteins RNA Instability Recombinants Recurrence Retroviral Vector Retroviridae Risk Role Sampling Somatic Mutation Specificity System TP53 gene Techniques Testing Transplantation Tumor Suppressor Proteins adverse outcome cellular transduction design embryonic stem cell fusion gene gain of function genome-wide homologous recombination in vivo insertion/deletion mutation leukemia loss of function loss of function mutation mouse development mouse model mutant promoter tumor

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to define the role of DNMT3A mutations for the pathogenesis of acute myeloid leukemia (AML), so that rational targeted therapies can be designed to counteract these mutations. DNMT3A (which encodes a de novo DNA methyltransferase) is among the most commonly mutated genes in AML (22% of all patients, and 34% of intermediate risk). Most mutations are missense, and the most common of these (60% of all cases) occurs at amino acid R882. However, many AML genomes have loss-of-function mutations in DNMT3A that are reminiscent of inactivating mutations in classical tumor suppressors. All DNMT3A mutations are associated with adverse overall survival. It is critical to understand whether the common DMNT3A mutations (especially at R882) are acting as gain-of-function or loss-of-function alleles, since this information will influence approaches to targeted therapies. We will assess the biochemical consequences of DNMT3A mutations in Aim 1, and the in vivo consequences in Aims 2 and 3, as follows: Specific Aim 1: We will define how AML-associated mutations alter DNMT3A functions in vitro. Recombinant DNMT3A protein with R882 (and other) mutations will be produced in E. coli, and tested for alterations in cytosine methylase activity and specificity, and binding to other cellular proteins. We will determine whether specific mutations cause loss-of-function or gain-of-function properties (and/or have dominant negative activities), and use this information to select additional mutations for study in vivo. Specific Aim 2: We will determine whether the DNMT3A R882H mutation can contribute to AML pathogenesis in vivo. Using retroviral vectors with fluorescent tags, we will express WT and mutant R882H DNMT3A cDNAs in murine bone marrow cells. Transduced cells will be evaluated in colony assays and mice to assess the leukemogenic potential of DNMT3A mutations alone or in combination with other mutations that commonly occur with DNMT3A mutations (e.g. FLT3 ITD, IDH1 R132H, NPMc). Using homologous recombination in ES cells, we will create mice with the R882H mutation at the identical position in the mouse Dnmt3a gene (R878H), and assess development, hematopoiesis, and cancer susceptibility. Specific Aim 3: We will assess the effect of Dnmt3a haploinsufficiency on AML pathogenesis. Mice haploinsufficient for Dnmt3a do not have a measurable hematopoietic phenotype. We will perform tumor watches using mice that are haploinsufficient for Dnmt3a, and we will compare the leukemogenicity of FLT3 ITD, NPMc, and IDH1/2 mutations using WT vs. Dnmt3a haploinsufficient bone marrow cells.

描述（申请人提供）：本提案的长期目标是明确DNMT3A突变在急性髓系白血病（AML）发病机制中的作用，以便设计合理的靶向治疗来对抗这些突变。 DNMT3A（编码从头 DNA 甲基转移酶）是 AML 中最常见的突变基因之一（占所有患者的 22%，中等风险患者的 34%）。大多数突变都是错义的，其中最常见的突变（占所有病例的 60%）发生在氨基酸 R882 处。然而，许多 AML 基因组在 DNMT3A 中存在功能丧失突变，这让人想起经典肿瘤抑制因子的失活突变。所有 DNMT3A 突变都与不良的总生存期相关。了解常见的 DMNT3A 突变（尤其是 R882）是否充当功能获得或功能丧失等位基因至关重要，因为这些信息将影响靶向治疗的方法。我们将评估目标 1 中 DNMT3A 突变的生化后果，以及目标 2 和 3 中的体内后果，如下：具体目标 1：我们将定义 AML 相关突变如何改变 DNMT3A 的体外功能。带有 R882（和其他）突变的重组 DNMT3A 蛋白将在大肠杆菌中生产，并测试胞嘧啶甲基化酶活性和特异性的改变，以及与其他细胞蛋白的结合。我们将确定特定突变是否导致功能丧失或功能获得特性（和/或具有显性负活性），并使用此信息来选择其他突变用于体内研究。具体目标 2：我们将确定 DNMT3A R882H 突变是否有助于体内 AML 发病机制。使用带有荧光标签的逆转录病毒载体，我们将在小鼠骨髓细胞中表达野生型和突变型 R882H DNMT3A cDNA。转导的细胞将在集落测定和小鼠中进行评估，以评估 DNMT3A 突变单独或与 DNMT3A 突变常见的其他突变（例如 FLT3 ITD、IDH1 R132H、NPMc）组合的致白血病潜力。利用 ES 细胞中的同源重组，我们将创建在小鼠 Dnmt3a 基因 (R878H) 的相同位置具有 R882H 突变的小鼠，并评估发育、造血和癌症易感性。具体目标 3：我们将评估 Dnmt3a 单倍体不足对 AML 发病机制的影响。 Dnmt3a 单倍体不足的小鼠没有可测量的造血表型。我们将使用 Dnmt3a 单倍体不足的小鼠进行肿瘤观察，并且我们将使用 WT 与 Dnmt3a 单倍体不足的骨髓细胞比较 FLT3 ITD、NPMc 和 IDH1/2 突变的致白血病性。