Constitutive Activation of STAT1 and STAT3 Proteins on Serine and Tyrosine Residues Were Found in Majority of CLL Patients.

Background: Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in the world. CLL is currently an incurable disease, characterized by the gradual accumulation of mature B-lymphocytes arrested in the G0 phase of the cell cycle. STAT (signal transducers and activators of transcription) proteins are involved in normal cellular events, such as differentiation, proliferation, cell survival, apoptosis and hematopoiesis. STAT proteins are constitutively activated in hematopoetic cells transformed by oncogenic tyrosine kinases and also in a variety of leukemias and lymphomas. The tyrosine phosphorylation of the STATs allows formation of dimmers, translocation to the nucleus and bindind of specific DNA sequences in the promoters of target genes, leading to activation of transcription. Serine phosphorylation is not sufficient to activate the STAT, providing a degree of modulation of the gene activation induced by the tyrosine phosphorylation. Serine phosphorylation of STAT1 and STAT3 has been described in untreated B-CLL cells, but no constitutive tyrosine phosphorylation has been detected in B-CLL cells.

Aims: Aim of our study was to examine defects in STAT1 and STAT3 pathways in our CLL patients.

Methods: We analyzed defects in STAT1 and STAT3 pathways after stimulation of cells with 10 ng/ml IFNγ and 5000 IU/ml IFNα in 42 primary cultures derived from CLL patients. STAT1 and STAT3 inducibilities of their activated phospho-isoforms were detected by Western-blotting analysis using specific polyclonal and monoclonal antibodies.

Results: Constitutive phosphorylation of STAT1 and STAT3 proteins was detected in majority of patients (STAT1 PS727 95.2%, STAT3 PS727 90,6 %, STAT1 PY701 85,7% and STAT3 PY705 79,4%). The IFNα was shown to be a more effective inductor in comparison with IFNγ. The inducibility after IFNα with constitutive activation of both STAT proteins on serine and tyrosine was the most frequently detected. Inducibility after IFNα with constitutive activation at STAT1 PY701 was found in 61,9 % of samples, with 21,4% having the inducibility after both IFNs. Inducibility after IFNα at STAT3 PY705 was detected in 88,2% of cases, with 70,6% having a constitutive activation and 20,5% having not. Inducibility after IFNα with constitutive activation at both serine residues is the most commonly observed pattern (STAT1 PS727 40.5%, STAT3 PS727 70,6 %). A high proportion of patients had constitutive activation with no inducibility at serine (STAT1 PS727 47,6%, STAT3 PS727 28,1 %) compared to that at tyrozine (STAT1 PY701 19%, STAT3 PY705 2,9). The inducibility after IFNγ did not exceed 10 % in all cases.

Conclusions: Our results show constitutive phosphorylation of STAT1 and STAT3 proteins on both serine and tyrosine residues in most of our CLL patients. More effective inductor is the IFNα. Approximately a half of patients have defect of inducibility in serine phosphorylation of STAT1 protein.