DNMT3A MUTATIONS IN ACUTE MYELOID LEUKEMIA

急性髓系白血病中的 DNMT3A 突变

• 批准号: 8465202
• 负责人: TIMOTHY J. LEY
• 金额: $ 41.15万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 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/8465202
• 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

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（由申请人提供）：该提案的长期目标是定义DNMT3A突变在急性髓样白血病（AML）的发病机理中的作用，以便可以设计出合理的靶向疗法来抵消这些突变。 DNMT3A（编码从头DNA甲基转移酶）是AML中最常见的突变基因之一（所有患者的22％，占中间风险的34％）。大多数突变是错义的，其中最常见的（60％的情况）发生在氨基酸R882。但是，许多AML基因组在DNMT3A中具有功能丧失突变，这让人联想到经典肿瘤抑制子中失活的突变。所有DNMT3A突变都与不利的总体生存有关。重要的是要了解常见的DMNT3A突变（尤其是在R882）是功能障碍或功能丧失等位基因，因为这些信息将影响目标疗法的方法。我们将评估AIM 1中DNMT3A突变的生化后果，以及AIM 2和3中的体内后果，如下：特定目标1：我们将定义与AML相关的突变如何改变DNMT3A的体外功能。具有R882（和其他）突变的重组DNMT3A蛋白将在大肠杆菌中产生，并测试了胞质甲基酶活性和特异性的改变，并与其他细胞蛋白结合。我们将确定特定的突变是否导致功能丧失或功能获得的特性（和/或具有显性负面活动），并使用此信息选择其他在体内进行研究的突变。具体目标2：我们将确定DNMT3A R882H突变是否可以导致体内AML发病机理。使用带有荧光标签的逆转录病毒载体，我们将在鼠骨髓细胞中表达WT和突变的R882H DNMT3A cDNA。转导的细胞将在菌落测定和小鼠中评估，以评估单独或与DNMT3A突变通常发生的其他突变（例如FLT3 ITD，IDH1 R132H，npmc）相结合的其他突变的白血病潜力。使用ES细胞中的同源重组，我们将在小鼠DNMT3A基因（R878H）中相同位置的R882H突变创建小鼠，并评估发育，造血和癌症的敏感性。具体目标3：我们将评估DNMT3A单倍性对AML发病机理的影响。 DNMT3A的单倍弹性不足的小鼠没有可测量的造血表型。我们将使用对DNMT3A的单倍弹性的小鼠进行肿瘤手表，并将比较使用WT与DNMT3A单倍氨基调式骨骨髓细胞的FLT3 ITD，NPMC和IDH1/2突变的白血病性。