Genomics of Acute Myelogenous Leukemia (AML): Relapse and Resistance Factors

急性髓性白血病 (AML) 的基因组学：复发和耐药因素

• 批准号: 8375660
• 负责人: John F. Dipersio
• 金额: $ 33.53万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8375660
• 关键词: Acute Myelocytic Leukemia; Ara-C; Biology; Blast Cell; Bone Marrow; CXCR4 gene; Candidate Disease Gene; Cell Line; Cells; Chromosome abnormality; Clinical Trials; DNA; DNA Resequencing; Daunorubicin; Disease remission; Disease-Free Survival; Epigenetic Process; Event; Gene Expression Profiling; Gene Targeting; Genes; Genetic; Genome; Genomics; Goals; Home environment; Human; In Vitro; Integrin alpha4beta1; Interruption; Methods; Modeling; Molecular Profiling; Mus; Mutation; PTPRC gene; Patients; Population; Principal Investigator; RNA; Recurrence; Relapse; Research Infrastructure; Resistance; Resolution; Risk; Role; Sampling; Sampling Studies; Side; Sorting - Cell Movement; Stromal Cell-Derived Factor 1; Stromal Cells; Survival Rate; Techniques; Testing; Translating; Tumor Cell Line; Validation; Vascular Cell Adhesion Molecule-1; Work; base; chemotherapy; comparative genomic hybridization; in vivo; inhibitor/antagonist; knock-down; leukemia; microdeletion; mouse model; outcome forecast; overexpression; programs; resistance factors; small molecule; tissue culture

The long term goal of this project is to define the genetic changes associated with AML relapse/resistance to chemotherapy. This work requires paired samples of de novo and relapsed AML cells that are nearly homogeneous in composition, and tissue culture and mouse models to validate the functional consequences of these genetic changes. The Genomics of AML PPG provides the appropriate infrastructure for this project. We will investigate AML relapse/resistance via the following Specific Aims: Specific Aim 1: We will define the genetic changes that occur in murine APL cell lines selected for chemotherapeutic resistance in vitro. We have generated 10 "parental" murine APL tumor cell lines. We will generate a total of 10 murine APL tumor cell lines and paired subclones that are resistant to daunorubicin (DNR), and/or Ara-C both in vitro and in vivo. Using these well-defined clonal populations of cells, we will perform gene expression profiling and we will define acquired microdeletions and amplifications using array-based comparative genomic hybridization (CGH) with the NimbleGen 2.1M murine oligomer array. Genes that are consistently dysregulated, deleted, or amplified in DNR or Ara-C resistant subclones will be validated with qPCR approaches. Selected genes identified with these array-based genomic screens will be resequenced to define more subtle genetic changes. Functional validation will be performed using forced overexpression and shRNAi knock-down approaches. Specific Aim 2: We will define the genetic changes that contribute to AML relapse by comparing the genomes of AML cells obtained at initial presentation vs. first relapse. Because most relapsed samples are not well matched to the paired de novo samples in terms of cellular composition, we will purify AML blasts by sorting "blast gate" AML cells from the cte novo and relapsed sample pairs for at least 20 AML patients. Using RNA and DMA from these paired, enriched samples, we will perform array based expression profiling and high resolution array based CGH using the 2.1M human oligomer arrays from NimbleGen. The altered genes identified in Aims 1 and 2 will be used to select a subset of target genes for resequencing and biologic validation in the mouse APL model described in Aim 1. Specific Aim 3: We will assess the role of the bone marrow microenvironment on AML resistance and relapse. We will use a unique mouse model in which genetically-marked murine APL cells home to and expand in the mouse bone marrow (BM). We will determine whether interruption of the protective AML cell-stromal interaction (using inhibitors of the SDF-1-CXCR4 and the VCAM-1-VLA-4 axes) can sensitize APL cells to chemotherapy in vivo. Finally, we have devised a clinical trial in which we will test the role of a small molecule inhibitor of the CXCR4-SDF-1 axis given immediately prior to salvage chemotherapy in patients with relapsed AML to enhance remission rates and overall survival.

该项目的长期目标是确定与AML复发/耐药相关的遗传变化， 化疗这项工作需要新生和复发AML细胞的配对样本， 组成均匀，组织培养和小鼠模型，以验证功能结果 这些基因变化的影响。AML PPG的基因组学为此提供了适当的基础设施 项目我们将通过以下具体目标研究AML复发/耐药性：具体目标1：我们将 定义选择用于化疗的鼠APL细胞系中发生的遗传变化 体外抗性。我们已经产生了10个“亲本”小鼠APL肿瘤细胞系。我们将生成一个 10种对柔红霉素（DNR）具有抗性的鼠APL肿瘤细胞系和配对亚克隆，和/或 Ara-C在体外和体内。使用这些明确定义的细胞克隆群体，我们将进行基因转染。 表达谱分析，我们将使用基于阵列的方法定义获得的微缺失和扩增 使用NimbleGen 2.1M鼠寡聚体阵列进行比较基因组杂交（CGH）。的基因 DNR或Ara-C耐药亚克隆中持续失调、缺失或扩增的基因将通过以下方法进行验证： qPCR方法。通过这些基于阵列的基因组筛选鉴定的选定基因将被重新测序 来定义更微妙的基因变化。将使用强制过表达进行功能验证 和shRNAi敲低方法。具体目标2：我们将定义遗传变化， 通过比较初始表现时获得的AML细胞的基因组， vs.第一次复发因为大多数复发样本与配对的新生样本不匹配， 就细胞组成而言，我们将通过从新生AML细胞中分选“母细胞门”AML细胞来纯化AML母细胞。 和至少20名AML患者的复发样品对。使用RNA和DNA从这些配对的，富集的 样品，我们将进行基于阵列的表达谱分析和高分辨率阵列的CGH使用 来自NimbleGen的2.1M人寡聚体阵列。在目标1和2中鉴定的改变的基因将用于 选择靶基因的子集用于在所述小鼠APL模型中进行重新测序和生物学验证 目标1。具体目标3：我们将评估骨髓微环境在AML中的作用。 抵抗和复发。我们将使用一种独特的小鼠模型，其中遗传标记的小鼠APL细胞 归巢并在小鼠骨髓（BM）中扩展。我们将确定是否中断 保护性AML细胞-基质相互作用（使用SDF-1-CXCR 4和VCAM-1-VLA-4轴抑制剂） 可使APL细胞对体内化疗敏感。最后，我们设计了一个临床试验， 补救治疗前即刻给予的CXCR 4-SDF-1轴小分子抑制剂的作用 复发性AML患者的化疗，以提高缓解率和总生存率。