Mechanism of Bcr/abl-dependent stat-protein activation

Bcr/Abl is a chimeric oncogene generated by a reciprocal translocation between chromosomes 9 and 22, and fusion proteins of either 210 kD (p210) or 190 kD (p190) are generated. Both Bcr/Abl proteins display transforming properties, and activation of Ras- and STAT-pathways seem to be involved in the mechanism of malignant transformation. In the present study, we have shown that STAT5 and STAT6 are constitutively tyrosine phosphorylated in Bcr/Abl expressing cell lines, and that STAT5 and STAT6 physically bind to p190- and p210-Bcr/Abl. Various Bcr/Abl mutants (n=8) have been analyzed in an effort to determine specific motifs of Bcr/Abl needed for binding of STAT5 and STAT6 to Bcr/Abl. In particular, we were able to show that STAT5-phosphorylation and binding to Bcr/Abl is abrogated in a kinase dead mutation of Bcr/Abl. Another focus is to determine the effects of a dexamethasone-inducible dominant negative form of STAT5 (dnSTAT5) on malignant transformation in Ba/F3- p210 cells. Specifically, we could show that Ba/F3-p210 cells transfected with the dnSTAT5 express the truncated form of STAT5, and that STAT5-reporter gene activity is inhibited upon induction with dexamethasone. We are currently addressing the question, whether (i) expression of the dnSTATS protein in Ba/F3-p210 cells Ieads to inhibition of transformation, (ii) Bcr/Abl itself is a kinase for and directly phosphorylates STATs, and (iii) regulation of Bcr/Abl kinase activity (either through the Bcr/Abl inhibitor CGP57148B, or by the use of a doxycycline inducible Bcr/Abl expression system) is associated with STAT activation. Moreover, (iv) a constitutive active STAT5 gene will be introduced in embryonic stem cells (ES) and, the effects on hemopoiesis will be analyzed in a transgenic mouse system.