APL is associated with reciprocal tranlocations always involving the RARa locus which variably fuses to the PML, PLZF, NPM or NuMA genes (referred to as X genes). Two aberrant fusion genes are thus generated which encode X-RARa and RARa-X fusion proteins. They hypothesize that the X-RARa and RARa-X fusion proteins disrupt/interfere with the normal functions of RARa and the various X proteins and that the latter play a critical role in controlling proliferation, survival and differentiation in the hemopoietic compartment. They propose to test the above hypothesis by a direct genetic approach with the following Specific Aims: 1) To define, in KO mice, the role of X proteins in ontogenesis and hemopoiesis. They have disrupted the PML and the PLZF gene in the mouse and null (-/-) mice have been generated and are being characterized. They will generate and characterize NPM and NuMA-/- mice. They will investigate the role of X proteins in development and hemopoiesis. They will utilize X-/- cells from various histological origins in order to identify X target genes possibly deregulated in APL, utilizing microarray techniques. 2) To establish, in KO mice or derived null cells, the role of X proteins in oncogenesis. PML-/- cells display a marked growth advantage and are resistant to multiple apoptotic stimuli. As a result PML-/- mice are tumor prone. They will examine spontaneous and/or induced tumorigenesis in X-/- mutants; the proliferative properties and susceptibility to transformation by oncogenes or dominant negative tumor suppressor genes of X-/- cells; the apoptotic response in X-/- cells and mice. The will determine if loss of X gene functions cooperates in tumorigenesis with known ocogenes or the lack of tumor suppressor gene function in vivo by crossing relevant mutants with X-/- mice. 3) To determine the molecular mechanisms by which PML exerts its tumor suppressive activity. They have defined an essential role for PML in mediating the function of proteins important for tumor suppression (p53 and BLM), apoptosis (p53 and DAXX) and the differentiation/proliferation of hemopoietic cells (RARa). They will determine the role of PML in the p53, BLM, RARa and Daxx pathways, also by intercrossing PML-/- mice with Daxx+/-, BLM+/-, p53+/- and RARa+/- mutants. 4) To study leukemogenesis in double mutants lacking PML and RARa functions. They will test genetically whether the interference of the PML-RARs oncoprotein with the functions of PML and RARa is sufficient for leukemogensis. To this end, they will interbreed PML-/- and RARa+/- mutants to and study hemopoiesis and leukemogenesis in compound mutants. 5) To test, genetically the hypothesis that the pathways controlled by the various X genes may converge. They have generated PML-/-/PLZF-/- mutants. The preliminary characterization of these mutants demonstrates a striking genetic interaction between these two molecules. The biological nature and the molecular basis underlying these genetic interactions will be analyzed in PML-/-/PLZF-/- mice and cells.
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