Bcr-Abl regulates Mcl-1 expression in patients with CML
aH. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL, United States, bJames A. Haley Veterans’ Administration Hospital, Tampa, FL, United States, cPenn State College of Medicine, Penn State Cancer Institute, Hershey, PA, United States
AIMS: The aim of this study was to investigate the signaling pathways that control survival in chronic myelogenous leukemia (CML). CML, a hematopoietic disorder, begins as a chronic myeloproliferative disease with accumulation of myeloid cells due to dysregulated anti-apoptotic signaling. Chronic phase eventually progresses to a more aggressive phase called blast crisis and 100% of cases will develop into acute leukemia. A hallmark of the disease is the reciprocal translocation t(9:22) which creates the Bcr-Abl fusion gene that encodes a highly active tyrosine kinase with dysregulated activity. The Bcr-Abl gene product has been directly linked to the oncogenesis of CML. We hypothesize that Mcl-1 is an antiapoptotic molecule in CML that is regulated by Bcr-Abl. METHODS: We examined the effect of Bcr-Abl inhibitors on survival, Mcl-1 expression, and Mcl-1 transcription in cell lines that overexpressed Bcr-Abl. Bcr-Abl tyrosine kinase activity was blocked with two different inhibitors, imatinib meyslate (Gleevacä) and PD180970. The cell lines used for these studies were HL-60 parental cells that are Bcr-Abl negative, HL-60 cells stably transfected with Bcr-Abl (p185, HL60-Bcr-Abl) and K562, which endogenously expresses Bcr-Abl. We used transient transfection assays with a mcl-1-luciferase reporter (p-204/+10-luc) to examine the mechanism of Bcr-Abl regulation. We also examined Mcl-1 expression by RNase protection assays and Western blot analysis in the presence and absence of Bcr-Abl inhibitors in patient derived CML blasts. RESULTS: Imatinib meyslate and PD180970 treatment induced apoptosis in both HL-60 -Bcr-Abl and K562 cells but not in HL-60 parental cells. These inhibitors also induced apoptosis in purified primary leukemic (CD34+) blasts from patients with CML in chronic phase and blast crisis. In Bcr-Abl inhibitor-treated cells, there was a reduction in Mcl-1 mRNA and protein expression that correlated with apoptosis. Stable overexpression of Bcr-Abl (p185) in HL-60 cells resulted in increased expression of Mcl-1. These data are consistent with Bcr-Abl induction of Mcl-1 expression. An important signaling event that contributes to CML survival is activation of STAT5. The anti-apoptotic protein Bcl-XL is transcriptionally regulated by STAT5 in a Bcr-Abl-dependent fashion. Transient transfection experiments were performed with a Mcl-1-luciferase construct in HL60-Bcr-Abl and K562 cells. We found that Bcr-Abl-dependent STAT5 activation regulates Mcl-1 expression. CONCLUSIONS: These findings demonstrate that Mcl-1 is a novel anti-apoptotic molecule increased in CML that is transcriptionally regulated by a Bcr-Abl-dependent STAT5 signaling pathway. Because of the important role that Bcr-Abl and STAT5 activation plays in CML survival, anti-apoptotic proteins regulated by these pathways have a high probability of contributing to disease pathogenesis.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in poster session 798 (Growth factors & signalling).