Activation of NFAT and NFkB Transcription Factors upon B Cell Receptor (BCR) Stimulation Is Restricted to Evolutive Chronic Lymphocytic Leukemia (CLL) Cases.

Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease in which BCR signaling capacity plays a central role in disease progression. Previously, we have shown that BCR engagement allows the identification of two groups of patients with a strong correlation between the in vitro cell survival response and the biological criteria (IgVH somatic hypermutations, ZAP 70 expression ) or the clinical stage. One group, corresponding to non evolutive patients, showed absence of a substantial BCR signaling (Non responder). In contrast, BCR signaling promoted in vitro cell survival in the group of Responder cases corresponding to evolutive patients. In the latter only, response to BCR ligation was associated with induction of NFkB and NFAT regulated genes, such as Cyclin D2 and CD23.

Since NFAT and NFkB transcription factors, regardless of the patients clinical status, have been described as constitutively nuclear in B-CLL cells, the aim of our study was to investigate whether the functional implication of these factors was different between our two groups. First, we confirmed by confocal microscopy and band shift analysis that localization of NFAT and NFkB was uniformely nuclear independently of the CLL status and the BCR response capacity. By RQ-PCR , we also investigated the expression of the various isoforms of NFAT (NFAT1, 2, 3 and 4) and showed that their expression profile was similar to normal B cells. Despite the nuclear localization of NFkB and NFAT, DNA binding capacity was only enhanced in cases responsive to BCR stimulation. Finally, using specific Tat-peptide inhibitors of NFAT and NFkB nuclear transport upregulation of cyclin D2 and CD23 was abrogated and survival advantage was lost in responder cases thereby confirming the crucial role of these factors upon BCR stimulation. However, in non responder cases in which no CD23 or cyclin D2 induction was present, this inhibition had no impact on cell survival upon BCR stimulation . Nonetheless, these cells retained the ability to respond to ionomycin or PMA reflecting functional PKC/calcium-calcineurin signaling pathways. Altogether, our results showed that NFAT and NFkB were activated in responder cells only despite their homogenous expression in all cases. In conclusion, the inability of the cells from stable B-CLL cases to activate the NFAT and NFkB pathways upon BCR signaling is not related to a functional defect or abnormal expression of these factors but likely reflects an early signaling defect. Moreover, our results show that the use of peptide inhibitors may restore a stable profile in B-CLL cells from evolutive patients.