The long term goal of this project is to define the genetic changes associated with AML relapse/resistance tochemotherapy. This work requires paired samples of de novo and relapsed AML cells that are nearlyhomogeneous in composition, and tissue culture and mouse models to validate the functional consequencesof these genetic changes. The Genomics of AML PPG provides the appropriate infrastructure for thisproject. We will investigate AML relapse/resistance via the following Specific Aims:
Specific Aim 1 : We willdefine the genetic changes that occur in murine APL cell lines selected for chemotherapeuticresistance in vitro. We have generated 10 'parental' murine APL tumor cell lines. We will generate a totalof 10 murine APL tumor cell lines and paired subclones that are resistant to daunorubicin (DNR), and/orAra-C both in vitro and in vivo. Using these well-defined clonal populations of cells, we will perform geneexpression profiling and we will define acquired microdeletions and amplifications using array-basedcomparative genomic hybridization (CGH) with the NimbleGen 2.1M murine oligomer array. Genes that areconsistently dysregulated, deleted, or amplified in DNR or Ara-C resistant subclones will be validated withqPCR approaches. Selected genes identified with these array-based genomic screens will be resequencedto define more subtle genetic changes. Functional validation will be performed using forced overexpressionand shRNAi knock-down approaches.
Specific Aim 2 : We will define the genetic changes thatcontribute to AML relapse by comparing the genomes of AML cells obtained at initial presentationvs. first relapse. Because most relapsed samples are not well matched to the paired de novo samples interms of cellular composition, we will purify AML blasts by sorting 'blast gate' AML cells from the cte novoand relapsed sample pairs for at least 20 AML patients. Using RNA and DMA from these paired, enrichedsamples, we will perform array based expression profiling and high resolution array based CGH using the2.1M human oligomer arrays from NimbleGen. The altered genes identified in Aims 1 and 2 will be used toselect a subset of target genes for resequencing and biologic validation in the mouse APL model describedin Aim 1.
Specific Aim 3 : We will assess the role of the bone marrow microenvironment on AMLresistance and relapse. We will use a unique mouse model in which genetically-marked murine APL cellshome to and expand in the mouse bone marrow (BM). We will determine whether interruption of theprotective 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 testthe role of a small molecule inhibitor of the CXCR4-SDF-1 axis given immediately prior to salvagechemotherapy in patients with relapsed AML to enhance remission rates and overall survival.
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