During normal development, as many as 90 percent of lymphocyte precursors undergo nonproductive antigen receptor gene rearrangements are selected for apoptotic death through an orderly, genetically controlled program consisting of three distinct stages: cell fate decisions, execution of death signals and the induction of caspase- mediated proteolysis. The E2A-HLF chimeric transcription factor, found in leukemic pro-B lymphocytes with the chromosomal translocation t(17;19)(q22;p13), is postulated to activate expression of SLUG, a mammalian ortholog of the ces-1 survival gene of C. elegans, whose increased transcriptional repressor activity blocks the expression of downstream cell death effectors. Thus, lymphocyte precursors that normally would be slated for destruction (e.g, due to defective antigen receptor gene rearrangements, the absence of exogenous survival signals or to DNA damage with p53 activation) continue to survive, with some cells acquiring additional mutations that promote a leukemic phenotype.
Aim 1 of this proposal seeks to identify the cell fate genes downstream from the E2A-HLF binding site that drive leukemogenesis and mediate cell survival decisions during normal lymphopoiesis. The experiments will exploit inducible gene expression systems, representational difference analysis (RDA) and other emerging technologies to assess mRNAs transcribed in the presence and absence of DNA binding by the chimeric protein. Genetically altered mice will be examined to define the normal developmental roles of mammalian ces-1- like genes (e.g., the Snail/Slug family) and Nfil3/E4bp4, a cytokine- regulated mammalian ortholog of the C. elegans ces-2 gene. Apart form classical gene transactivation, mediated by a conserved bZIP domain, E2A-HLF mutants with disabled bZIP DNA-binding domains can protect cells from apoptosis through the activity of the AD1 and AD2 transactivator domains within the amino-terminus of the E2A molecule. Thus, Aim 2 tests leading to expression of a Groucho-like protein that blocks a second downstream genetic program required for apoptosis. Current insights into the apoptotic pathways operating in mammalian lymphopoiesis have profited enormously from comparisons with the ces/ced pathway in nematodes (C. elegans). The proposed research will extend this knowledge by revealing many of the still-elusive intermediate steps between the initiating events of apoptosis and activation of the caspase-mediated proteolytic cascade. An understanding of how E2A-HLF subverts the cell death program in leukemic lymphoblasts may ultimately suggest a new basis for drug resistance in leukemic cells and could reveal attractive targets for improving clinical outcomes after exposure to cytotoxic agents.
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