P65 activity and ZAP-70 status predict the sensitivity of chronic lymphocytic leukemia cells to the selective lκB kinase inhibitor BMS-345541

Abstract

Purpose: Constitutive nuclear factor-κB(NF-κB) activation has been implicatedin the pathogenesis of chronic lymphocytic leukemia (CLL). Our purpose was to characterize the molecular mechanisms underlying for the selective IκB kinase inhibitor BMS-345541 in CLL cells together with the analysis of its combination with several antineoplasic drugs. Experimental Design: Primary cells from 34 CLL patients were incubated with different doses of BMS-345541. NF-κB 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. Results: 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 andDNA-binding activity. Following NF-κB inhibition, BMS-345541 ledto 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-κB-target genes, including both BCL2 family members andapoptotic endogenous inhibitors. In addition, we showeda strong synergism between BMS-345541 andconventional 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. Conclusions: These data confirm that NF-κB is a relevant target in CLL andindicate that inhibitors of IκB kinase, alone or in combination, represent a novel therapeutic strategy for the treatment of CLL patients, especially for the group with high ZAP-70. © 2009 American Association for Cancer Research.