NF-κB Is Active in Mcl-1 Promoter Regulation in Human CLL.

The NF-κB family of transcription factors is linked to neoplasia due to its role in increasing cell proliferation as well as inhibiting apoptosis. NF-κB has been reported to be constitutively active in chronic lymphocytic leukemia (CLL), but the mechanism underlying this this activation is not fully understood and the critical target genes involved have not been identified. To further define the mechanism(s) by which NF-κB promotes survival in CLL, we investigated the role of this transcription factor in the regulation of the myeloid cell leukemia 1 (Mcl-1) gene. The promoter region of Mcl-1 contains a putative NF-κB binding site, and increased expression of the Mcl-1 protein has been related to rapid disease progression and resistance to apoptosis in CLL. However, a direct link between NF-κB and Mcl-1 transcriptional regulation has not yet been established in B-cells or human CLL. We demonstrate here that NF-κB binding is a critical event in the transcriptional regulation of Mcl-1, as deletion of the NF-κB binding site in the promoter results in decreased activity of an Mcl-1-luciferase reporter construct. In addition, pharmacological inhibition of NF-κB reduced p65 nuclear localization and binding of NF-κB to the Mcl-1 promoter. Interestingly, when CLL patient samples were exposed to an IKK inhibitor, Bay-11, the extent of Mcl-1 inhibition varied in patients. We therefore examined whether the effect on Mcl-1 correlated with in vitro cell survival, a result that might be expected given that NF-κB expression has been previously reported to correlate with CLL cell survival. We found that CLL patient cells more sensitive to Bay-11-induced apoptosis also showed a larger decrease in Mcl-1 mRNA, suggesting that Mcl-1 message level may be a useful diagnostic to predict patients that will respond to NF-κB targeted therapy. Furthermore, different inducers of the NF-κB signaling pathway (CD40L, immune stimulatory CpG-ODN and TNF-α), promote differential effects on Mcl-1 regulation in CLL patient cells. Although all three treatments increase NF-κB nuclear localization and DNA binding, Mcl-1 RNA and protein increased with CpG and CD40L treatment, but not TNFα. This result provides evidence of stimulus-specific regulation of Mcl-1 by NF-κB. Based on these observations, we hypothesize that there is a direct link between NF-κB DNA binding and transcriptional control of Mcl-1, and that this gene could serve as a pharmacodynamic endpoint to monitor the efficacy of NF-κB inhibitors in CLL cells. Additionally, a direct link of NF-κB activity to Mcl-1 expression and cell survival provide further justification for targeting this transcription factor for treatment in CLL.