Prospective analysis of NF-kappaB as a superior prognostic marker in chronic lymphocytic leukaemia

In this study, I characterized basal NF-kappaB DNA binding in CLL samples and investigated the value of NF-kappaB as a prognostic marker and therapeutic target in CLL. In contrast to the previous studies, I demonstrated wide heterogeneity in basal NF-kappaB DNA binding among patients which was associated with in vitro survival (P = 0.01) with high white cell count (P = 0.01) and shorter lymphocyte doubling time (P = 0.01). Subunit analysis revealed that in primary CLL cells the principal components were p50, Rel A, and c-Rel. I next investigated the cytotoxicity of a putative NF-kappaB inhibitor, LC-1, and elucidated its mechanism of action in CLL patient samples. LC-1 induced apoptosis with a mean LD50 of 2.9muM after 24 hours normal B and T-cells were significantly more resistant to its apoptotic effects (P <0.001). Apoptosis was associated with caspase-3 activation that was mediated via the upstream activation of both caspase-8 and caspsase-9. Apoptosis was preceded by a reduction of nuclear Rel A DNA binding and down regulation of the anti-apoptotic NF-kappaB target genes CFLAR, BIRC5 and BCL2. LC-1 was highly synergistic with fludarabine (mean combination index 0.26). Rel A DNA binding was strongly associated with advanced Binet stage (P<0.0001) but did not correlate with lgVH mutation status (P = 0.25), CD38 expression (P = 0.87) or ZAP-70 expression (P = 0.55). In addition, it was predictive of time to first treatment (P = 0.02) and time to subsequent treatment (P = 0.0001). Indeed, Rel A was the most predictive marker of survival both from date of diagnosis (hazard ratio 9.1, P = 0.01) and date of entry into the study (hazard ratio 3.9, P = 0.05). Taken together, the data suggests that NF-kB is a promising therapeutic target and prognostic marker in CLL. Prospective clinical trials designed to evaluate these conclusions are clearly now warranted