DNMT3A MUTATIONS IN ACUTE MYELOID LEUKEMIA

描述（由申请人提供）：本申请的长期目标是确定DNMT3A突变在急性髓性白血病（AML）发病机制中的作用，以便设计合理的靶向治疗来抵消这些突变。DNMT3A（编码一种从头开始的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%，中等风险的34%）。大多数突变是错义的，其中最常见的（占所有病例的60%）发生在氨基酸R882上。然而，许多AML基因组在DNMT3A中具有功能丧失突变，这让人想起经典肿瘤抑制基因中的失活突变。所有DNMT3A突变都与不良的总生存率相关。了解常见的DMNT3A突变（特别是R882）是否作为功能获得或功能丧失等位基因是至关重要的，因为这些信息将影响靶向治疗的方法。我们将在Aims 1中评估DNMT3A突变的生化后果，以及Aims 2和Aims 3中的体内后果，具体内容如下：特异性Aims 1：我们将定义aml相关突变如何在体外改变DNMT3A功能。将在大肠杆菌中产生R882（和其他）突变的重组DNMT3A蛋白，并测试胞嘧啶甲基化酶活性和特异性的变化，以及与其他细胞蛋白的结合。我们将确定特定突变是否会导致功能丧失或功能获得特性（和/或具有显性负活性），并利用这些信息选择其他突变进行体内研究。特异性目的2：我们将确定DNMT3A R882H突变是否有助于体内AML发病。利用带有荧光标记的逆转录病毒载体，我们将在小鼠骨髓细胞中表达WT和突变的R882H DNMT3A cdna。将在集落试验和小鼠中评估转导的细胞，以评估DNMT3A突变单独或与DNMT3A突变（例如FLT3 ITD， IDH1 R132H, NPMc）共同发生的其他突变的白血病发生潜力。利用胚胎干细胞的同源重组，我们将在小鼠Dnmt3a基因（R878H）的相同位置产生R882H突变的小鼠，并评估发育、造血和癌症易感性。特异性目的3：我们将评估Dnmt3a单倍不足在AML发病机制中的作用。缺乏Dnmt3a基因单倍体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，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突变的白血病发生性。

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.