BCL6 and PLZF, two members of the POK (POZ and Kruppel) family of transcription factors, have been directly implicated in the pathogenesis of Non-Hodgkin's lymphoma (NHL) and Acute Promyelocytic Leukemia (APL), respectively. POK proteins can homodimerize and heterodimerize and these interactions are critical in modulating their respective DNA binding ability and specificity as well as their transcriptional activity. We have isolated a new proto-oncogenic member of this family, POKEMON that physically interacts with BCL6 and PLZF-RARalpha and is co-expressed with these proteins in the relevant hemopoietic compartment. Our preliminary analysis in Pokemon knock-out (KO; -/-) mice and cells demonstrates that this protein plays a key and pleiotropic role in controlling cellular differentiation and tumorigenesis, acting as a proto-oncogene essential for neoplastic transformation. We hypothesize that these key functions of POKEMON are deregulated in NHLs, in APL and, more in general, in hemopoietic malignancies. We propose to test this hypothesis with the following Specific Aims: 1. To define, in Pokemon-/- embryos, in Pokemon-/- ES cells and by conditional hemopoietic tissue specific inactivation of the gene, the role of Pokemon in lymphoid and myeloid differentiation. Preliminary analysis indicates that Pokemon inactivation results in embryonic lethality because of severe anemia due to a block in erythroid differentiation and in a profound impairment of B-cell and myeloid differentiation. We have also generated Pokemon[floxed] mutants for conditional inactivation of the gene. Using Pokemon[floxed] mutants, mice lacking Pokemon specifically in the B-cell or the myeloid compartments will be generated by a Cre-loxP mediated approach. Pokemon-/- ES cells will also be generated. Pokemon's role in hemopoiesis will be studied in vivo and in vitro in these mutants/cells. 2. To determine the molecular basis underlying Pokemon oncogenic activity. Our more recent analysis indicates that Pokemon displays a potent oncogenic activity. Furthermore and strikingly, Pokemon-/- cells are totally refractory to neoplastic transformation by key oncogenic pathways (see preliminary results). We will determine the molecular mechanisms underlying Pokemon's oncogenic potential. 3. To establish the role of POKEMON in hemopoietic malignancies. We have generated transgenic mice (TM) that overexpress Pokemon in the lymphoid and myeloid compartment, respectively. The oncogenic role of Pokemon will be studied in vivo in these mutants. In parallel, we will study the status of the POKEMON gene, mRNA and protein in human hemopoietic malignancies employing a multifaceted high throughput approach by FISH, tumor tissue arrays (TTMs) and mutation analysis. 4. To determine POKEMON and BCL6 functional interactions in lymphopoiesis and cooperative oncogenesis, i) In vivo in double mutants generated by crossing Bcl6-/- with Pokemon+/- and Pokemon B-cell conditional mutants, whose phenotype will be characterized. Pokemon and Pokemon/Bcl6 target genes in purified B-cell populations from our single or double mouse mutants will be identified by microarray and chromatin immunoprecipitation (CHIP) analysis, ii) In vitro, by studying the reciprocal transcriptional interactions of POKEMON with BCL6 on their responsive elements/target genes, as well as on gene regulatory regions that share BCL6/POKEMON DNA binding elements, iii) In vivo, by crossing lymphoma prone BCL6 Knock-in mutants with Pokemon KO mice and TM in which Pokemon is expressed in the lymphoid compartment, iv) In vitro, by studying BCL6 and POKEMON cooperative tumorigenesis in classical transformation assays in WT, Pokemon-/- and Bcl6-/- MEFs.
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