Transcription Factor NF-kB as a Biomarker for Disease Progression and Target for Treatment in Chronic Lymphocytic Leukaemia.

NF-kB is part of a multi-component signalling pathway that regulates the expression of hundreds of genes involved in the control of cell proliferation, cell survival, stress responses, innate immunity and inflammation. The principal constituents of mammalian NF-kB are p65, p50, p52, c-Rel and Rel-B and in their inactive state they are bound in the cytoplasm to the inhibitor protein IKB. Phosphorylation of IKB by IKB kinase has been shown to result in the translocation of NF-kB into the nucleus where it can bind to kB responsive elements. It has been reported that NF-kB is constitutively activated in chronic lymphocytic leukaemia (CLL) cells. However, no previous studies have prospectively quantified the subunits of NF-kB in primary CLL cells or examined their relative expression between patient samples. Therefore, the aim of our study was to perform quantitative subunit analysis of NF-κB in freshly isolated CLL cells and examine their relationship with some of the well established clinical and molecular prognostic markers. NF-κB was evaluated using EMSA (n= 48), supershift assays and p65 subunit-specific ELISA (n=100). Apoptosis was measured at 24h and 48h using annexin V/propidium iodide labelling. We demonstrated a wide variation in NF-κB expression in CLL samples and p50, p65 and c-rel were consistently shown to be the predominant NF-kB subunits. Importantly, we showed a negative correlation between in vitro apoptosis and nuclear p65 NF-kB expression (r² = −0.22) suggesting that high levels of p65 confer a cytoprotective effect on primary CLL cells. Furthermore, we found that p65 has a positive correlation (r² = 0.3) with total white cell count (WBC) and CLL samples with a lymphocyte doubling time below 12 months have significantly higher p65 DNA binding (p= 0.02) implicating this NF-κB subunit in disease progression. Finally we have also demonstrated that the NF-κB inhibitor, LC-1, has potent in vitro apoptosis-inducing effects on primary CLL cells. Together, this work supports the use of specific inhibitors of NF-kB, particularly targeting p65, for treatment of CLL.