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Megakaryocytes (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. Our studies of Src family kinases demonstrate that Lyn and Fyn kinases are activated by thrombopoietin (TPO) and have an inhibitory effect on cellular proliferation. In fact, adding PP1 to murine bone marrow cells in culture resulted in a higher percentage of polyploid cells. In the present report, we examined the effect of SU6656 on TPO-induced growth and differentiation. SU6656 is reported to be more specific than previous inhibitors for the Src family of tyrosine kinases. Remarkably, when SU6656 (2.5 mM) was added to a megakaryocytic cell line, UT-7/TPO, the cells underwent dramatic increase in cell size and polyploidization. By 24 h, 8N cells were present. After 48 h a 16N population was identified by FACS, and continued growth in SU6656 resulted in higher ploidy states (32N and 64N). This was accompanied by cessation of cellular proliferation (i.e. cell number remained constant), increase in CD41 and CD61 expression, and was notable for the absence of apoptosis. Similarly, polyploidization was observed when SU6656 was added to expanded human bone marrow progenitors with partial MK differentiation. The ability to induce differentiation was also seen using bone marrow from two 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. 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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