bms345541 and Leukemia--Lymphocytic--Chronic--B-Cell

Chronic lymphocytic leukaemia (CLL) is a disease with a highly variable prognosis. The clinical course can however be predicted thanks to prognostic markers. Poor prognosis is associated with expression of a B cell receptor (BCR) from unmutated immunoglobulin variable heavy-chain genes (IGHV) and expression of zeta-associated protein of 70 kDa (ZAP70). The reason why ZAP70 expression is associated with poor prognosis and whether the protein has a direct pathogenic function is at present unknown. By transfer of ZAP70 to CLL cells, we show here that expression of ZAP70 in CLL cells leads to increased expression of the nuclear factor (NF)-κB target genes interleukin-1β (IL1B), IL6 and IL8 upon BCR triggering. This could be blocked by inhibition of NF-κB signalling through inhibition of IκB kinases (IKK). Transcriptome analysis identified a NF-κB RELA signature imposed by ZAP70 expression in BCR-stimulated CLL cells. We conclude that ZAP70 acts directly as an amplifier of NF-κB signalling in CLL cells which could be an underlying mechanism for its association with poor prognosis and which may represent a therapeutic target.

Topics: Adult; Aged; Calcium Signaling; Electroporation; Female; Gene Expression Regulation, Leukemic; Humans; I-kappa B Kinase; Imidazoles; Immunoglobulin Heavy Chains; Immunoglobulin M; Immunoglobulin Variable Region; Interleukin-1beta; Interleukin-6; Interleukin-8; Interleukins; Jurkat Cells; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Neoplasm Proteins; NF-kappa B; Prognosis; Quinoxalines; Receptors, Antigen, B-Cell; Recombinant Fusion Proteins; RNA, Messenger; Transcription Factor RelA; Transcriptome; Tumor Cells, Cultured; ZAP-70 Protein-Tyrosine Kinase

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.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; I-kappa B Kinase; Imidazoles; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; NF-kappa B; Quinoxalines; Transcription Factor RelA; ZAP-70 Protein-Tyrosine Kinase