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

该项目的长期目标是确定与急性髓系白血病复发/耐药相关的基因变化 化疗。这项工作需要配对的初治和复发的AML细胞样本，这些细胞几乎 成分均一，组织培养和小鼠模型验证功能结果 这些基因变化。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调查AML复发/耐药：具体目标1：我们将 确定在选择进行化疗的小鼠APL细胞系中发生的基因变化 体外抗药性。我们已经建立了10个“亲代”小鼠APL肿瘤细胞系。我们将生成一个总计 10个对柔红霉素(DNR)具有耐药性的小鼠APL肿瘤细胞系和配对亚克隆，和/或 Ara-C在体外和体内均有表达。使用这些定义明确的克隆细胞群体，我们将执行基因 表达谱分析，我们将定义获得的微缺失和扩增使用基于阵列 用NimbleGen 210万小鼠低聚物阵列进行比较基因组杂交(CGH)。这些基因是 在DNR或Ara-C抗性亚克隆中持续失调、缺失或扩增的情况将得到验证 定量聚合酶链式反应方法。通过这些基于阵列的基因组筛选确定的选定基因将被重新测序 来定义更微妙的基因变化。将使用强制过度表达执行功能验证 和shRNAi推倒方法。具体目标2：我们将定义 通过比较初诊时获得的AML细胞基因组有助于AML复发 与首次复发的对比。因为大多数复发样本与成对的从头样本不是很匹配 在细胞组成方面，我们将通过从CTE新生的AML细胞中分离“BLAST GATE”AML细胞来纯化AML细胞 以及至少20名AML患者的复发样本对。使用RNA和DMA从这些配对的、丰富的 示例，我们将使用 来自NimbleGen的210万个人类低聚物阵列。在AIMS 1和2中发现的改变的基因将被用来 在所描述的小鼠APL模型中选择靶基因的子集进行重测序和生物验证 在目标1.具体目标3：我们将评估骨髓微环境在急性髓系白血病中的作用 抵抗和复发。我们将使用一种独特的小鼠模型，在该模型中，基因标记的小鼠APL细胞 在小鼠骨髓(BM)中生长和扩增。我们将确定是否会中断 保护性AML细胞-间质相互作用(使用SDF-1-CXCR4和VCAM-1-VLA-4轴的抑制剂) 在体内可使APL细胞对化疗增敏。最后，我们设计了一个临床试验，我们将在其中测试 抢救前给予CXCR4-SDF-1轴小分子抑制剂的作用 化疗可提高复发AML患者的缓解率和总存活率。

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.