p65 activity and ZAP-70 status predict the sensitivity of chronic lymphocytic leukemia cells to the selective IkappaB kinase inhibitor BMS-345541.

Abstract

Constitutive nuclear factor-kappaB (NF-kappaB) activation has been implicated in the pathogenesis of chronic lymphocytic leukemia (CLL). Our purpose was to characterize the molecular mechanisms underlying for the selective IkappaB kinase inhibitor BMS-345541 in CLL cells together with the analysis of its combination with several antineoplasic drugs. Primary cells from 34 CLL patients were incubated with different doses of BMS-345541. NF-kappaB DNA-binding activity was analyzed by ELISA-based kits and the characterization of the apoptotic pathway was done by flow cytometry, immunoblotting, quantitative reverse transcription-PCR, and immunofluorescence techniques. BMS-345541 selectively induced apoptosis in CLL cells in the low micromolar range irrespective of p53 status. Noteworthy, the high ZAP-70 group was significantly more sensitive to BMS-345541 than the low ZAP-70 group, in correlation with high levels of p65 phosphorylation and DNA-binding activity. Following NF-kappaB inhibition, BMS-345541 led to induction of the mitochondrial apoptotic pathway and activation of both caspase-dependent and caspase-independent factors. Moreover, BMS-345541-induced apoptosis was accompanied by down-regulation of several antiapoptotic NF-kappaB-target genes, including both BCL2 family members and apoptotic endogenous inhibitors. In addition, we showed a strong synergism between BMS-345541 and conventional chemotherapeutics such as mitoxantrone and dexamethasone as well as with new promising drugs such as the BH3-mimetic GX15-070/Obatoclax or the anti-TRAIL-R1 monoclonal antibody mapatumumab. These data confirm that NF-kappaB is a relevant target in CLL and indicate that inhibitors of IkappaB kinase, alone or in combination, represent a novel therapeutic strategy for the treatment of CLL patients, especially for the group with high ZAP-70.