The long-term objective of this project is to obtain a better understanding of the control mechanisms involved in regulating the processes of self-renewal, proliferation, and differentiation in normal and malignant hematopoietic stem-progenitor cells. This understanding is necessary in order to rationally manipulate these cells for optimal use in transplantation and to develop new methods for treating transformed cells. Our premise is that the study for STK-1 (FLT3/FLK-2) is likely to further our understanding of both normal and abnormal hematopoiesis. We were the first to clone and study the human receptor. We found it to be restricted in expression to the CD34+ fraction of human bone marrow though more recently we have also found it to be expressed on CD34- monocytic precursors (CD15+). We showed that antisense inhibition of STK-1 expression by CD34+ cells in the long-term bone marrow culture assays (which is reflective of stem-progenitor cell activity) suppressed hematopoietic colony formation. We then showed that STK-1 was over- expressed at both the RNA and protein level by leukemia cells in patients with B-lineage ALL (100%), AML (90%), and subsets of CML (blast crisis- 30%), T-ALL (30%), and CLL (67%). FL, the ligand for this receptor has undergone a profusion of studies over the last 2 years. The ligand shows promise for potential clinical use to increase the mobilization of stem- progenitor cells for harvest, to expand stem/progenitor cells, and to facilitate retroviral transduction of stem/progenitor cells for gene therapy. Our proposal is a natural extension of our ongoing work on STK-1. Although its ligand is being intensively studied, little is known of the regulation of the receptor. In addition, the receptor is aberrantly expressed in leukemia and nothing is known of the reasons for this alteration. Thus, our work will concentrate on the mechanism(s) for both the normal and abnormal control of receptor expression. We propose an initial scrutiny of the approximately 900 bp upstream of the transcription start site. We will delineate both cis elements and trans acting factors important for STK-1's expression and regulation. We also propose to investigate the role that STK-1 expression may play in leukemia. We will determine if it gives leukemic cells a survival and/or proliferative advantage. We will also model the effects of an activated receptor by generating a fusion protein that results in constitutive activation of the tyrosine kinase domain.
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