THE BIOLOGY OF PROMYELOCYTIC LEUKEMIA IN A MOUSE MODEL

小鼠模型中早幼粒细胞白血病的生物学

• 批准号: 8196753
• 负责人: TIMOTHY J. LEY
• 金额: $ 39.14万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8196753
• 关键词: 5' Untranslated Regions; A Mouse; Acute Myelocytic Leukemia; Acute Promyelocytic Leukemia; Alleles; Animals; Arsenic; Base Pairing; Binding; Binding Sites; Biological Assay; Biology; Candidate Disease Gene; Cathepsin G; Cells; ChIP-on-chip; ChIP-seq; Chimeric Proteins; Chromatin; Complementary DNA; DNA; DNA Binding; DNA Methylation; DNA Sequence; Data; Disease; Disease Progression; Epigenetic Process; Event; Exhibits; FLT3 gene; Flow Cytometry; Frequencies; Gene Expression; Genes; Genetic; Genome; Genomics; Goals; Grant; Health; Human; In Vitro; Inbred Mouse; MicroRNAs; Modeling; Mus; Mutation; Myelogenous; Outcome; PML gene; Pathogenesis; Patients; Phenotype; Population; Progressive Disease; Proteins; RARA gene; RXRA gene; Recurrence; Recurrent disease; Relapse; Reproducibility; Role; Sampling; Shapes; Tretinoin; Tumor-Derived; U937 Cells; Validation; Work; Zinc; base; chromatin immunoprecipitation; comparative genomic hybridization; effective therapy; fusion gene; genetic element; genome sequencing; histone modification; in vivo; insight; mouse model; novel; overexpression; progenitor; tumor

DESCRIPTION (provided by applicant): Although most patients with Acute Promyelocytic Leukemia (APL, FAB M3 AML) have good outcomes, 15% still die from relapsed disease. The long-term goal of this work is to understand the genetic events that contribute to the pathogenesis of APL, so that better therapies can be developed for patients who relapse. We and others have established that PML-RARA can initiate APL in mice, yielding an ATRA-, arsenic-, and chemo-responsive APL-like disease that has long latency and inappropriate myeloid differentiation, suggesting that this model is still missing important genetic elements that 'shape' the myeloid maturation arrest observed in APL patients. To better understand the key genetic events controlling APL initiation and progression, we propose the following Specific Aims: Specific Aim 1: We will define the role of RARA haploinsufficiency for APL pathogenesis. RARA haploinsufficiency is a consequence of t(15;17), but its role has not yet been defined for APL pathogenesis. We have developed a new mouse model of APL by targeting a conditional PML-RARA bcr1 fusion cDNA to the 5' untranslated region of the mouse PML gene in B6 mice, creating haploinsufficiency for PML, and appropriate expression levels of PML-RARA. By crossing these mice with RARA haploinsufficient mice, we have created animals with three of the four potentially relevant somatic events caused by t(15;17). The APL phenotype of these animals (with and without RARA haploinsufficiency) will be fully defined. Specific Aim 2: We will identify the genomic DNA binding sites of PML-RARA and the genes that it directly regulates. We have developed a functional 3x-FLAG-tagged PML-RARA protein (FLAG-PR) that is capable of interacting with RXRA, and binding to DNA sequences that contain known RARA binding sites. FLAG-PR will be used as the 'bait' for chromatin immunoprecipitation of DNA; PML-RARA binding sites in the chromatin of PR-9 cells and APL cells will be resolved by Chip-Seq and parallel ChIP-on-chip studies. Expression data from APL patients will be used to identify possible genes that are directly regulated by PML- RARA, and candidate genes will be validated. Specific Aim 3: We will define progression mutations that contribute to APL pathogenesis. Using the Illumina platform, we will sequence the entire genomes of 15 APL tumors derived from C57Bl/6 mice that express PML-RARA under control of the murine cathepsin G locus, and ultimately, the novel mice described in Aim 1. Since these tumors arose in inbred mice, only 10x coverage of each genome (i.e. ~27 billion base pairs of sequence) should be required to discover all acquired mutations. The frequency of validated mutations will assessed in both mouse and human APL/AML samples, and recurrent mutations will be studied for functional consequences in vitro and in vivo.

描述（由申请人提供）：尽管大多数患有急性临床前白血病（APL，Fab M3 AML）的患者的结果良好，但仍有15％的患者死于复发性疾病。这项工作的长期目标是了解有助于APL发病机理的遗传事件，以便为复发的患者开发更好的疗法。我们和其他人已经确定，PML-RARA可以在小鼠中启动APL，从而产生ATRA，砷和化学反应性APL样疾病，具有长期潜伏期和不适当的髓样差异化，这表明该模型仍然缺少在APL患者中“塑造”基因成熟的重要遗传元素。为了更好地理解控制APL启动和进展的关键遗传事件，我们提出了以下特定目的：具体目标1：我们将定义RARA单倍弥倍不足在APL发病机理中的作用。 RARA单倍不足是T（15; 17）的结果，但尚未定义为APL发病机理的作用。我们通过将有条件的PML-RARA BCR1融合cDNA靶向到B6小鼠中小鼠PML基因的5'未翻译区域，从而开发了一种新的APL小鼠模型，从而为PML创造了单倍氨基和PML-RARA的表达水平。通过将这些小鼠与RARA单倍弹性小鼠跨越，我们创造了动物，其中四个可能由T（15; 17）引起的四个潜在相关的体细胞事件。这些动物的APL表型（具有和不具有RARA单倍宽度）将被充分定义。具体目标2：我们将确定PML-RARA的基因组DNA结合位点及其直接调节的基因。我们已经开发了能够与RXRA相互作用的功能性3X-FLAG标记的PML-RARA蛋白（FLAG-PR），并与包含已知RARA结合位点的DNA序列结合。 Flag-pr将用作DNA染色质免疫沉淀的“诱饵”。 PR-9细胞和APL细胞染色质中的PML-RARA结合位点将通过芯片序列和平行芯片研究来解决。来自APL患者的表达数据将用于识别受PML-RARA直接调节的可能基因，并将验证候选基因。特定目标3：我们将定义有助于APL发病机理的进展突变。使用Illumina平台，我们将对源自鼠calter蛋白蛋白酶基因蛋白蛋白酶基因座的C57BL/6小鼠的15个APL肿瘤的整个基因组进行测序，最终在AIM 1中描述的新小鼠。由于这些肿瘤在INBRED小鼠中出现了，因此只有10x的Genotiations（I. 27账单）是繁殖的。。。。。。。。。。在小鼠和人APL/AML样品中都将评估经过验证的突变的频率，并将研究复发突变以在体外和体内进行功能后果。