2.55 CLL Cell Survival Depends on B Cell Receptor Signaling-Induced NFAT2 Activation

B cell receptor (BCR) signaling plays an important role in Chronic Lymphocytic Leukemia (CLL) pathogenesis. Although CLL cells demonstrate survival capacity and accumulate in vivo, they rapidly become apoptotic in vitro. We previously demonstrated that CLL B cells from some patients bypass this in vitro apoptotic process by increasing their survival response to BCR stimulation. This IgM-induced differential cell survival allows the distinction between Responder and non Responder cases. Cell survival is under the control of a BCR-mediated signaling complex comprising Syk family kinases and phospholipase C gamma 2 (PLCγ2). The latter is responsible for mobilization of intracellular pools of calcium. In lymphocytes, sustained calcium flux provokes activation of the NFAT (Nuclear Factor Activated T cells) family of transcription factors, which promote transcriptional regulation of survival genes. Analysis of the molecular mechanisms underlying differential IgM induced B cell survival between CLL cases should give insight into the pathophysiology of CLL, and allow the specific use of inhibitors in development. In agreement with our previous results, measurement of CLL cell survival upon in vitro BCR stimulation led to the distinction between responder (R) and non responder (NR) groups of patients. As expected, only the responder group showed short progression-free survival and overall survival. To further explain this functional heterogeneity between the two groups of patients, our studies revealed that, despite an overall low membrane expression in CLL B lymphocytes, IgM levels were higher in cells from R cases compared to NR ones. In contrast, no significant heterogeneity was observed for BCR co-receptors between NR and R groups, with higher levels of CD5 and lower levels of CD20 as compared to normal B cells. Our results also demonstrated that Syk protein levels were more abundant in CLL B cells compared to normal B lymphocytes and in responding than in non responding B cells. Over-expression of Syk was not due to genetic amplification. Analysis of global tyrosine phosphorylation of Syk showed a constitutive activation in all CLL B cells. However, upon antigenic stimulation, differential patterns of Syk phosphorylation were observed between NR and R B cells. Moreover, higher phosphorylation rates of both Syk and its effector PLCγ2 were found in samples from the R group compared to the NR group. As expected, IgM triggering did not allow significant release of calcium in non responding cells in contrast with an efficient uptake in responding cells. Furthermore, our data revealed an overexpression of the endogenous calcium/calcineurin-dependent transcription factor NFAT2 (Nuclear Factor of Activated T cells) in all CLL B cells; this up-regulation was not caused by a genetic amplification. A partial constitutive nuclear localization of NFAT2 was observed in all CLL B cells, but this transcription factor showed a higher capacity to bind to DNA after BCR stimulation in the responding cells only. Furthermore, inhibition of NFAT activity by VIVIT peptide prevented IgM-induced transcriptional regulation of endogenous CD23 and B cell survival, in responding cells only. Altogether, our results indicate that membrane expression levels of BCR and its early effectors are responsible for a threshold level in antigenic stimulation. Over-passing this threshold allows differential phosphorylation of PLCγ2, calcium uptake and activation of NFAT. These molecular mechanisms contribute to cell survival advantage in responding cells and will inform the therapeutic use of specific inhibitors targeting these intermediates in the BCR signaling pathway.