APL is associated with reciprocal translocations always involving the RAR[1] 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-RAR[1] and RAR[1]-X fusion proteins. We hypothesize that the various X/RAR[1] fusion proteins disrupt the genetic events synergistically cooperate with X/RAR[1] leading to APL. We will test this hypothesis in vivo, by a direct genetic approach, with the following Specific Aims: 1) To define the leukemogenic potential of the fusion proteins of APL in transgenic mice (TM). Hemopoiesis, leukemogenesis, and response to RA and As2O3 treatments will be analyzed in X/RAR[1] and RAR[1]/X TM in which the expression of the transgene is restricted to the myeloid/promyelocytic hemopoietic compartment through the use of a human Cathepsin-G (hCG) minigene expression vector. hCG-X-RAR[1] and RAR[1]-X lines will be intercrossed to elucidate the possible cooperative roles of these molecules in leukemogenesis. 2) To establish the role of X and RAR[1] proteins in APL promotion and progression. We will test whether the reduction to heterozygosity of X and/or RAR[1] in APL, or their functional inactivation by the fusion products is a crucial event in APL leukemogenesis. To this end, we will interbreed hCG-X/RAR[1] and RAR[1]/X TM with PML, PLZF or RAR[1] Knock-Out mice to reproduce in vivo the genetic complexity observed in the APL blast, and study hemopoiesis and leukemogenesis in the resulting transgene combinations. 3) To define the mechanisms of leukemogenesis and transcriptional repression in APL. We will study in vivo, in TM, as well as in vitro, in transcriptional and cell culture assays, the biochemical and oncogenic activities of X/RAR[1] and RAR[1] proteins harboring mutations in ligand binding, repressive and activating domains. 4) To study leukemogenesis and the concept of dominance/interference by X-RAR[1] on X function utilizing an in vivo """"""""Knock-in"""""""" approach. We will introduce the PLZF-RAR[1] fusion gene in the PLZF locus in mouse ES cells by a Knock-in approach. We will study the resulting phenotype and leukemogenesis by the PLZF-RAR[1] fusion protein in vivo, in mice and/or chimeras generated from these ES cells, as well as in vitro, in ES cells differentiation experiments. 5) To identify target genes of the oncogenic activity the various X-RAR[1] and RAR[1]-X fusion proteins. We will utilize purified promyelocytic cell populations from our TM to identify, on a comparative basis, target genes relevant for their oncogenic functions utilizing microarray and chromatin immunoprecipitation techniques. 6) To identify additional genetic events contributing to APL pathogenesis. Retroviral insertional mutagenesis in PML-RAR[1] and PLZF-RAR[1] TM, and Spectral Karyotyping (SKY) will be utilized to identify additional genetic events in APL leukemogenesis.
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