Bcl-6 May Be a Survival Factor in Pre-B Cell Ph+ Blast Crisis Cell Lines.

The constitutively active tyrosine kinase Bcr-Abl fusion protein is essential for the development of chronic myeloid leukaemia. Inhibitors of its tyrosine kinase activity e.g. Imatinib, are highly effective in treating chronic phase disease but are only transiently useful in blast crisis, especially lymphoid blast crisis. Bcl-6 is a transcriptional repressor that is required for the formation and maintenance of germinal centre B-cells. Following reports that Imatinib increases expression of Bcl-6 in Ph+ cell lines representative of lymphoid blast crisis we have investigated the regulation of this molecule and its functional importance. We utilised BV173 and Z119. Basal Bcl-6 protein expression was detectable in Z119, but not BV173. As anticipated Imatinib increased Bcl-6 expression in both cell lines. STAT5 is an important target of Bcr-Abl and has been implicated as both a positive and negative regulator of Bcl-6 transcription. Transient transfection with a mammalian expression plasmid bearing a dominant negative STAT5 reduced Bcl-6 expression in Z119 implying that activated STAT5 promotes Bcl-6 expression in this cell line, but also that STAT5 cannot be the main target of Bcr-Abl because Imatinib and dominant negative STAT5 have opposing effects. Next we considered the possibility that phosphorylation status of the transcription factor FoxO3a, which has been shown to bind to the Bcl-6 promoter, correlated with Bcl-6 expression. We found that decreased phosphorylation of Foxo3a, activating this transcription factor, was highly associated with increased Bcl-6 expression suggesting that it is an significant Bcr-Abl target. Next we wished to find out the functional implications of increased Bcl-6 expression. We found that a low concentration of Imatinib (0.25μM) was sufficient to induce Bcl-6 in BV173 and Z119, and caused death of these cell lines over several days. Next we combined Imatinib with a previously reported peptide antagonist of the Bcl-6/SMRT co-repressor interaction. Neither this peptide nor a mutated negative control peptide had an effect on cell numbers or apoptosis over 48 hours of culture when used alone. However, when combined with Imatinib the wild-type peptide, but not the control, reduced cell growth in Z119. We conclude that Bcl-6 may be a survival factor in Ph+ lymphoid blast crisis cell lines following treatment with Imatinib and that specific treatments to abrogate Bcl-6 function may find a place in the treatment of lymphoid blast crisis of CML.