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）：虽然大多数急性早幼粒细胞白血病（APL、FAB M3 AML）患者预后良好，但仍有 15% 的患者死于疾病复发。这项工作的长期目标是了解导致 APL 发病机制的遗传事件，以便为复发患者开发更好的治疗方法。我们和其他人已经确定，PML-RARA 可以在小鼠中引发 APL，产生 ATRA 、砷和化疗反应性 APL 样疾病，该疾病具有长潜伏期和不适当的骨髓分化，表明该模型仍然缺少重要的遗传元件这“塑造”了在 APL 患者中观察到的骨髓成熟停滞。为了更好地了解控制 APL 起始和进展的关键遗传事件，我们提出以下具体目标： 具体目标 1：我们将定义 RARA 单倍体不足在 APL 发病机制中的作用。 RARA 单倍体不足是 t(15;17) 的结果，但其在 APL 发病机制中的作用尚未确定。我们开发了一种新的 APL 小鼠模型，通过将条件性 PML-RARA bcr1 融合 cDNA 靶向 B6 小鼠中小鼠 PML 基因的 5' 非翻译区，产生 PML 单倍体不足以及 PML-RARA 的适当表达水平。通过将这些小鼠与 RARA 单倍体不足的小鼠杂交，我们创造了具有由 t(15;17) 引起的四个潜在相关体细胞事件中的三个的动物。这些动物的 APL 表型（有或没有 RARA 单倍体不足）将被完全定义。具体目标2：我们将鉴定PML-RARA的基因组DNA结合位点及其直接调节的基因。我们开发了一种功能性 3x-FLAG 标记的 PML-RARA 蛋白 (FLAG-PR)，它能够与 RXRA 相互作用，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 Chip-Seq 和并行 ChIP-on-chip 研究来解析。 APL 患者的表达数据将用于鉴定可能受 PML-RARA 直接调控的基因，并对候选基因进行验证。具体目标 3：我们将定义有助于 APL 发病机制的进展突变。使用 Illumina 平台，我们将对源自 C57Bl/6 小鼠的 15 个 APL 肿瘤的整个基因组进行测序，这些小鼠在鼠组织蛋白酶 G 基因座的控制下表达 PML-RARA，并最终对目标 1 中描述的新型小鼠进行测序。在近交小鼠中，只需每个基因组 10 倍的覆盖率（即约 270 亿个碱基对的序列）即可发现所有获得性突变。将在小鼠和人类 APL/AML 样本中评估已验证突变的频率，并将研究复发突变的体外和体内功能后果。