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.
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