HISTONE H3 METHYLATION AND EPIGENOMICS IN ACUTE LEUKEMIA

急性白血病中的组蛋白 H3 甲基化和表观基因组学

• 批准号: 8581843
• 负责人: Lukas D Wartman
• 金额: $ 15.37万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8581843
• 关键词: 5' Untranslated Regions; Acute Myelocytic Leukemia; Acute Promyelocytic Leukemia; Acute leukemia; Address; Alleles; Animals; Antibodies; Biological Assay; Bone Marrow Cells; Cathepsin G; Cells; ChIP-seq; Complementary DNA; Control Animal; Data; Development; Engineering; Epigenetic Process; Event; Exons; Gene Expression; Genetic Recombination; Genomics; Goals; Health; Hematopoiesis; Hematopoietic; Histone H3; Histones; Human; Journals; Knock-out; Knockout Mice; Lead; Lentivirus Vector; Link; Malignant - descriptor; Malignant Neoplasms; Marrow; Mediating; Mentors; Methodology; Methylation; Modeling; Mus; Mutate; Mutation; Pancytopenia; Penetrance; Phenotype; Physicians; Population; Publishing; RARA gene; RNA; Research; Research Personnel; Role; Sampling; Scientist; Site; Somatic Mutation; Stem cells; Sum; Therapeutic; Training; Training Support; Transgenic Mice; X Chromosome; base; career; cell growth; epigenomics; exome sequencing; genome sequencing; in vivo; leukemia; leukemogenesis; male; mouse model; progenitor; protein expression; research study; self-renewal; skills; stem; transcriptome sequencing; tumor; tumor progression

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to provide the training and support for the applicant to become an independent physician-scientist with a career focused on studying the genomics and epigenomics of acute leukemia. The goals for the applicant include: obtaining practical and didactic training in scientific methodology, refining technical skills, and publishing the outlined research in top-tier scientific journals. The long-tem goal of the research is to define the role of KDM6A in normal and leukemic hematopoiesis. KDM6A encodes a H3K27 histone demethylase on the X chromosome that is mutated in a wide range of human cancers, including acute myeloid leukemia. Inactivating mutations of Kdm6a are the most common acquired progression events in our mouse model of acute promyelocytic leukemia (APL). To define the functional consequences of Kdm6a inactivation, we propose the following aims: Specific Aim 1: We will analyze a Kdm6a conditional knockout mouse to define the role of Kdm6a in normal and leukemic hematopoiesis. We will cross Kdm6a conditional knockout mice (with LoxP sites flanking the 3rd exon) with Vav-Cre transgenic mice to selectively delete Kdm6a in the hematopoietic lineage. We will characterize normal hematopoiesis using competitive repopulation assays, flow-based analysis of hematopoietic progenitors, and serial replating experiments. For these experiments, we will analyze 6-8 week old male Vav-Cre mice hemizygous for the floxed Kdm6a allele, along with the appropriate control mice. We will establish a tumor watch to determine if Kdm6a inactivation is associated with spontaneous leukemia development or bone marrow failure. We will intercross Kdm6a conditional knockout mice (with the Vav-Cre allele) with Ctsg-PML-RARA mice to define changes in cell growth, differentiation, and self renewal, and we will determine whether Kdm6a deficiency alters APL latency, penetrance, or phenotype in a tumor watch study. Specific Aim 2: We will define the epigenetic consequences of Kdm6a inactivation using RNA-Seq and ChIP-Seq. We will perform RNA-Seq on mouse APL tumors with and without spontaneous Kdm6a deletions or mutations, and on RNA derived from bone marrow cells from young, male Vav-Cre mice hemizygous for the Kdm6a conditional allele, along with the appropriate control animals. We will perform ChIP-Seq on mouse APL tumors with and without spontaneous Kdm6a deletions or mutations. We will use an anti-H3(tri-methyl K27) antibody to determine if changes in H3K27 methylation occur, and also with an anti-H3(tri-methyl K4) antibody, since Kdm6a inactivation may alter H3K4 methylation. Finally, we will perform RNA-Seq and ChIP-Seq on selected cell populations and leukemias (if they develop) in the male Vav-Cre mice hemizygous for the Kdm6a conditional allele, and APL tumors that arise in Ctsg-PML-RARA mice with and without the engineered deletion of Kdm6a. We will therefore comprehensively identify and analyze the epigenetic and transcriptional alterations associated with Kdm6a inactivation in wild type vs. PML-RARA expressing hematopoietic and leukemia cells.

描述（由申请人提供）：该提案的长期目标是为申请人提供培训和支持，使其成为一名独立的医师科学家，其职业重点是研究急性白血病的基因组学和表观基因组学。申请人的目标包括：获得科学方法方面的实践和教学培训，提高技术技能，并在顶级科学期刊上发表概述的研究。该研究的长期目标是明确 KDM6A 在正常和白血病造血中的作用。 KDM6A 编码 X 染色体上的 H3K27 组蛋白去甲基化酶，该酶在多种人类癌症中发生突变，包括急性髓系白血病。 Kdm6a 失活突变是我们的急性早幼粒细胞白血病 (APL) 小鼠模型中最常见的获得性进展事件。为了定义 Kdm6a 失活的功能后果，我们提出以下目标： 具体目标 1：我们将分析 Kdm6a 条件敲除小鼠，以确定 Kdm6a 在正常和白血病造血中的作用。我们将 Kdm6a 条件敲除小鼠（LoxP 位点位于第三外显子两侧）与 Vav-Cre 转基因小鼠杂交，以选择性删除造血谱系中的 Kdm6a。我们将使用竞争性再增殖测定、造血祖细胞的流式分析和系列再铺板实验来表征正常造血功能。对于这些实验，我们将分析 6-8 周大的具有 floxed Kdm6a 等位基因的半合子雄性 Vav-Cre 小鼠以及相应的对照小鼠。我们将建立肿瘤监测以确定 Kdm6a 失活是否与自发性白血病发展或骨髓衰竭相关。我们将 Kdm6a 条件敲除小鼠（带有 Vav-Cre 等位基因）与 Ctsg-PML-RARA 小鼠杂交，以确定细胞生长、分化和自我更新的变化，并且我们将确定 Kdm6a 缺陷是否会改变 APL 潜伏期、外显率或表型在一项肿瘤观察研究中。具体目标 2：我们将使用 RNA-Seq 和 ChIP-Seq 定义 Kdm6a 失活的表观遗传后果。我们将对具有或不具有自发 Kdm6a 缺失或突变的小鼠 APL 肿瘤以及来自 Kdm6a 条件等位基因半合子的年轻雄性 Vav-Cre 小鼠的骨髓细胞衍生的 RNA 以及适当的对照动物进行 RNA 测序。我们将对有或没有自发 Kdm6a 缺失或突变的小鼠 APL 肿瘤进行 ChIP-Seq。我们将使用抗 H3（三甲基 K27）抗体来确定 H3K27 甲基化是否发生变化，并且还会使用抗 H3（三甲基 K4）抗体，因为 Kdm6a 失活可能会改变 H3K4 甲基化。最后，我们将对 Kdm6a 条件等位基因半合子的雄性 Vav-Cre 小鼠中选定的细胞群和白血病（如果它们发生）以及 Ctsg-PML-RARA 小鼠中出现的 APL 肿瘤进行 RNA 测序和 ChIP 测序。并且没有 Kdm6a 的工程删除。因此，我们将全面鉴定和分析野生型与表达 PML-RARA 的造血细胞和白血病细胞中与 Kdm6a 失活相关的表观遗传和转录改变。