RESUMO
B-cell antigen receptor (BCR)-mediated signaling plays a critical role in chronic lymphocytic leukemia (CLL) pathogenesis and gives an in vitro survival advantage to B cells isolated from patients with unfavorable prognostic factors. In this study, we undertook to elucidate the signaling intermediates responsible for this biologic alteration. In responding cells only, in vitro BCR engagement triggers global phosphorylation of Syk, activation of phospholipase Cγ2, and intracellular calcium mobilization, reflecting competency of BCR signaling. The calcium-calcineurin-dependent transcription factor NFAT2 is up-regulated and to some extent constitutively activated in all CLL B cells. In contrast, its DNA-binding capacity is enhanced on IgM stimulation in responding cells only. NFAT inhibition using the VIVIT peptide prevents induction of CD23 target gene and IgM-induced survival, converting responding cells to unresponsive status. At the opposite, ionomycin-induced NFAT activity allows survival of nonresponding cells. These results demonstrate that the functional heterogeneity relies on variability of protein levels establishing BCR-dependent thresholds and NFAT-dependent activation. Finally, status of the BCR-NFAT pathway for each patient reveals its relevance for CLL clinical outcome and points out to BCR-NFAT intermediates as promising functional therapeutic targets.
Assuntos
Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Sinalização do Cálcio , Sobrevivência Celular , Humanos , Imunoglobulina M/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/terapia , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/genética , Oligopeptídeos/farmacologia , Fosfolipase C gama/metabolismo , Fosforilação , Prognóstico , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Quinase Syk , Regulação para Cima , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
Despite very similar gene expression profiles, the clinical course of B-cell chronic lymphocytic leukemia (B-CLL) is heterogeneous. Immunoglobulin VH (IgVH) mutational status and expression of B-cell receptor (BCR) signaling mediators have been associated with disease progression. However, the consequences of BCR engagement on cell survival and evolution of the disease remain unclear. We show here that B-CLL cell survival is dependent on the threshold of BCR stimulation induced by immobilized antibody, in contrast to soluble anti-mu F(ab)'2 antibody, which leads to apoptosis. Measurement of metabolic activity and apoptotic response discriminated two subgroups. "Nonresponders" showed low metabolic activity and unmodified apoptotic response upon BCR stimulation. In contrast, "responders" exhibited increased metabolic activity and inhibition of spontaneous apoptosis. This survival advantage was associated to a BCR-dependent activation profile leading to induction of cyclin D2/cyclin-dependent kinase 4 (cdk4) expression and G1 cell cycle progression. The ability to respond to BCR ligation correlated with an unfavorable clinical course and allowed to define an additional group of patients among IgVH-mutated cases exhibiting a risk of progression. Remarkably, we show that Zap70 expression was neither mandatory nor sufficient to generate downstream survival signals and cyclin D2/cdk4 up-regulation. In conclusion, BCR engagement has a significant effect on B-CLL cell survival, activation, and G1 progression. Furthermore, our results provide new insights in the physiopathology of progressive IgVH-mutated cases.