DNMT3A MUTATIONS IN ACUTE MYELOID LEUKEMIA

描述（由申请人提供）：本提案的长期目标是确定DNMT 3A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对抗这些突变。DNMT 3A（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%，中等风险的34%）。大多数突变是错义突变，其中最常见的（占所有病例的60%）发生在氨基酸R882。然而，许多AML基因组在DNMT 3A中具有功能丧失突变，这让人想起经典肿瘤抑制因子中的失活突变。所有DNMT 3A突变都与不良总生存期相关。了解常见的DMNT 3A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。我们将评估目标1中DNMT 3A突变的生化后果，以及目标2和3中的体内后果，如下所示：具体目标1：我们将定义AML相关突变如何改变DNMT 3A体外功能。具有R882（和其它）突变的重组DNMT 3A蛋白将在E.大肠杆菌，并测试了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会导致功能丧失或功能获得特性（和/或具有显性负活性），并使用此信息选择其他突变进行体内研究。具体目标2：我们将确定DNMT 3A R882 H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WT和突变体R882 H DNMT 3A cDNA。将在集落测定和小鼠中评价转导的细胞，以评估单独的DNMT 3A突变或与通常伴随DNMT 3A突变发生的其他突变（例如FLT 3 ITD、IDH 1 R132 H、NPMc）组合的致白血病潜力。使用ES细胞中的同源重组，我们将创建在小鼠Dnmt 3a基因（R878 H）的相同位置具有R882 H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Dnmt 3a单倍不足对AML发病机制的影响。Dnmt 3a单倍型不足的小鼠不具有可测量的造血表型。我们将使用Dnmt 3a单倍不足的小鼠进行肿瘤观察，我们将使用WT与Dnmt 3a单倍不足的骨髓细胞比较FLT 3 ITD、NPMc和IDH 1/2突变的致白血病性。

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.