Mechanism of Bcr/abl-dependent STAT activation

Bcr/Abl is a chimeric oncoprotein generated by a reciprocal translocation between chromosome 9 and 22 and implicated in the pathogenesis of Philadelphia chromosome (Ph+) positive human leukemias. Fusion proteins are generated and are associated with chronic myelogenous leukemia (CML). The Bcr/Abl protein exhibits increased tyrosine kinase activity and transforming properties compared with the normal c-Abl protein. Recently, a signal transduction pathway that involves Janus kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs) has been shown to be regulated by several hematopoietic cytokine receptors, including the L-3 receptor and the GM-CSF receptor. It has recently been shown that Bcr/Abl induces activation of STAT1 and STAT5 without activation of JAK kinases -1, -2, -3 and Tyk-2. This observation suggests that STATs are either activated by Bcr/Abl itself without activation of JAK kinases or by the use of signaling elements other than JAK. Moreover, the mechanism of p210Bcr/Abl induced STAT1- and STAT5-activation remains unknown. It is also not known whether the activation of the various STAT proteins is required for abnormal proliferation and malignant transformation in cells expressing the p2l0Bcr/Abl. Aim of the present study is to further clarify the mechanism of constitutive STAT1- and STAT5-activation in Bcr/Abl positive cell lines. Using purified STAT- and Bcr/Abl-proteins it should therefore be clarified, whether Bcr/Abl protein itself is able to phosphorylate STATs. Another way to address this question is to analyze insect cells transfected with both, the STAT5- and Bcr/Abl-genes, in a baculovirus system. The question of the biological significance of STAT activation in leukemic cells in patients with CML should also be addressed. We want to investigate whether activation of the various STAT proteins is required for abnormal proliferation and malignant transformation in CML. Experiments using anti-STAT-antisense oligonucleotide probes will be done. Transfection of Bcr/Abl positive cell lines with dominant. negative STAT mutations is also planned. STAT activation patterns in primary, leukemic cells from CML patients at various stages of their disease will also be investigated. We want to correlate stages of disease with the phosphorylation status of various STATs. The analysis of the Bcr/Abl positive KU812 cell line, which exhibits a marked, spontaneous basophilic differentiation is also planned.