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My research is focused on interrelated projects pertinent to the mechanisms involved in the development, proliferation, and function of bone marrow cells, particularly of the megakaryocytic lineage. The long-term objectives are to gain an understanding of the molecular events controlling megakaryocytopoiesis by studying the genetic and signaling factors that control lineage commitment and the regulation of cell cycle during this process.
More specifically, my current laboratory interests fall into two main categories. One is the role of Src family kinases (SFKs) in megakaryocyte development, and the other is identifying key elements of molecular regulation of cell cycle control during polyploidization. Megakaryocytes (MK) arise in the bone marrow and produce platelets, the cells that mediate primary hemostasis. Precise regulation of platelet production is critical over the life of an organism and can be abnormal during disease states. The TPO/Mpl signaling pathway is essential both for optimal growth of hematopoietic progenitors and megakaryocytopoiesis. MKs undergo successive rounds of endomitosis during differentiation, resulting in polyploidy (typically, 16-64N). Previous studies have demonstrated that this occurs through an interruption of normal cell cycle progression during anaphase. However, the molecular mechanism(s) controlling this unique process is undefined. We have shown that Mpl stimulation results in the activation of both Fyn and Lyn kinases in primary MKs, and inhibition of these kinases results in enhanced proliferation and differentiation. Furthermore, we have demonstrated that pharmacologic inhibitors of Src kinases induce rapid MK differentiation and increased nuclear ploidy in leukemic cell lines as well as in primary bone marrow cells. This increase in maturation correlates with increased intensity and duration of Erk1/2 activity. Future directions are geared towards understanding how Fyn and Lyn mediate these phenotypic effects. The aims to be addressed include: 1) analyze the phenotype of MKs and marrow progenitors in lyn-deficient, fyn-deficient, and lyn/fyn double-knockout mice; 2) determine whether or not the effect of SFKs is primarily mediated through modifying MAPK activity and identify the mechanism(s) through which SFKs inhibit Erk1/2 activity; and 3) investigate additional signaling molecules (PI3 kinase, Protein kinase C, and Jak2) to determine how convergent signaling pathways affect SFKs and Erk1/2 activity during normal and abnormal megakaryocytopoiesis.
A second area of active study is identifying key elements of molecular regulation of MK polyploidization and terminal differentiation. We have shown that hematopoietic cell lines and primary cells become highly polyploidy and undergo spontaneous fragmentation in the presence of rhTPO and the specific Src kinase inhibitor, SU6656. This was accompanied by cessation of cellular proliferation, increase in CD41 and CD61 expression, and was notable for the absence of apoptosis. The ability to induce differentiation was also seen using bone marrow from patients with myelodysplastic syndrome and thrombocytopenia, suggesting that SU6656 might be useful as a differentiation-inducing agent for MKs. Although SU6656 is clearly a potent inhibitor of the Src family kinases, we have undertaken studies to determine if an additional target can be identified. We found no affect on Jak2, STAT3, and STAT5 tyrosine phosphorylation. However, the activity of Aurora kinase B was inhibited in vitro by as little as 50 nM SU6656. To begin understanding the molecular basis for these effects we will focus on the changes in cell cycle regulatory proteins during terminal differentiation. We will examine changes in expression and activity of critical cell cycle regulators (cyclins, cyclin-dependent kinases, cyclin-dependent kinase inhibitors, and checkpoint proteins such as p53 and Rb) in response to TPO and SU6656. We propose that SU6656 may be an important tool for understanding the molecular basis of MK endomitosis and may also have therapeutic potential for individuals with MDS, thrombocytopenia, or other disorders resulting from incomplete megakaryocyte maturation. |
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