The long-term objectives of this project address the molecular mechanisms by which perturbations of Hox regulatory pathways in hematopoietic cells contribute to the pathogenesis of acute leukemias. Hox proteins comprise a distinctive group of transcriptional regulators that share a characteristic DNA binding motif known as the homeodomain. They make important contributions to normal hematopoietic cell differentiation and are targets for mutations in human and murine leukemias. In addition, the genes for Pbx1 and Meis1, that belong to the TALE (three amino acid loop extension) superclass of divergent homeodomain proteins, are also mutated in subsets of acute leukemias. Furthermore, Pbx forms stable complexes with Meis proteins and serves as DNA binding partners for Hox as well as Meis proteins. Therefore, mutations of Pbx, Hox or Meis may represent alternative mechanisms for disruption of common genetic pathways in hematopoietic cells by subverting the functions of molecular complexes containing two or more of these components. The studies proposed in Aim number 1 of this application will address the hypothesis that Hox proteins normally function as higher order trimeric complexes with members of the TALE superclass of homeodomain proteins that includes Pbx1 and Meis1. The formation and function of tertiary Meis/Pbx/Hox complexes will be investigated in vitro and correlated with expression studies in transgenic embryos utilizing reporter genes containing HOX enhancer elements. Studies in Aim number 2 will employ transgenic and in vitro transformation assays to determine if the oncogenic properties of chimeric Pbx and Hox proteins result from subversions of normal trimeric constraints on the transcriptional functions of the respective wild type homeodomain proteins. In a third specific aim, microarray techniques will be employed to identify direct target genes that are subordinate to Pbx fusion proteins in lymphoid cells and their oncogenic contributions will be determined using gene transfer techniques. The studies in a fourth specific aim will employ biochemical and genetic approaches to identify and characterize heterologous proteins that affect Meis/Pbx/Hox hetero-trimer function or DNA binding specificity. These studies will help define a molecular pathway by which Hox developmental regulators contribute to neoplastic transformation and will yield novel insights into the biologic features of leukemic cell growth. They may also provide a new reagents for the improved diagnosis and longitudinal monitoring of patients with leukemia and establish animal models for evaluation of newer generations of anti-neoplastic therapies.
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