The characterization of chromosomal translocations specific to malignant disease has led to the identification of a number of genes that play critical roles in normal cellular development or proliferation, and that produce abnormal growth when altered. Approximately 10% of all non- Hodgkin s lymphomas possess a t(2;5)(p23;q35) chromosomal rearrangement. Positional cloning studies performed in the applicants laboratory have revealed that the t(2;5) produces a fusion gene which encodes a chimeric protein consisting of the amino-terminal portion of the nucleolar phosphoprotein nucleophosmin (NPM) linked to the catalytic domain of a novel receptor tyrosine kinase of the insulin receptor subfamily, anaplastic lymphoma kinase (ALK). The ligand that interacts with ALK and the normal function(s) of this receptor are unknown. As a consequence of NPM-ALK fusion, the ALK kinase becomes constitutively activated and is expressed ectopically in lymphoid cells. In addition, the NPM residues of the chimera bind to wild-type NPM, targeting a portion of the fusion protein to the nucleus. It is presently unknown whether the nuclear localization of NPM-ALK is required for the complete expression of its biological activity. The long-range goals of this proposal are to determine the mechanisms by which NPM-ALK contributes to lymphomagenesis, and to determine the role of the ALK receptor kinase in normal cell growth and development. The experimental plan is to identify the structural motifs required for activation of the ALK catalytic domain and NPM-ALK nuclear targeting and to determine the biological consequences of alterations of these motifs by using in vitro transformation assays, as well as assays based on the ability of the NPM-ALK chimera to render hematopoietic cells growth factor-independent, to analyze constructs containing mutations of the NPM portion of the fusion. The oncogenicity of the chimeric protein under physiological conditions will be tested by generating transgenic mice that express the NPM-ALK gene under the control of promoters specific for different stages of lymphoid cell development. The role of ALK in embryonic development and its normal function in specific tissues will be addressed by three complementary approaches. First, the pattern of ALK expression will be determined in murine embryos at different developmental stages, and in selected adult tissues, by in situ hybridization and immunocytochemical staining. Second, the in vivo phenotypic effects of inactivation of the ALK receptor will be assessed by targeted disruption of the gene in mice. Third, expression cloning strategies will be used to identify and characterize the cognate ligand of ALK -- the on-off switch that determines when ALK signaling normally occurs. Taken together, these studies should provide a mechanistic framework for how activation of ALK by the t(2;5) contributes to the genesis of non-Hodgkin s lymphoma.
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