Temporal Proteomic Analysis of CLL B Cell Response to Antigen Receptor Stimulation.

Unraveling the molecular basis for the different functional consequences of BCR cross-linking is essential to understand the leukemogenesis process of CLL. There is increasing evidence that an antigen driven process is crucial in CLL proliferation, based on restriction of the IgVH repertory as well as shared antigen-binding motifs used by mutated or unmutated CLL B-cells. BCR activation leads to a signaling cascade reinforced in the more aggressive CLL form by the ZAP70 protein tyrosine kinase and constitutive phosphorylation of HS1 protein. We have previously shown, using gene expression profiling over time, that crosslinking of the BCR induces a specific temporal gene expression program in the leukemic cells, leading to a complex balance disorder between proliferation and cell death. These results demonstrated a core BCR gene expression shared among all B cell (and across species). Furthermore, each cell category (healthy B-cells, indolent and aggressive CLL cells) also showed a specific temporal gene expression after BCR cross-linking. This analysis has revealed complex expression disorder of multiple genes coding proteins involved in proliferation and death regulation (Vallat et al. Blood 07). To further investigate the functional consequences of BCR cross-linking we also examined modulation of the BCR proteome, since a dynamic description of the BCR functional consequences in CLL cells would not be complete without parallel temporal proteomic analysis. We now have cross-linked the BCR in freshly isolated B-cells from CLL patients. We have isolated total proteins at 2 early, one intermediate and a later time point 30 min later than the time points selected in the previous mRNA experiments. At each analysis time point both stimulated and control unstimulated cells were examined. A 2D electrophoresis analysis (pH 3–10, 150–20 kDa) revealed between 1100 and 1800 (mean 1400) polypetidic spots at each time point after BCR engagement. A total of 600 different polypeptides show a specific pattern of expression over time after stimulation, up- (450 polypeptides) or down-regulated (150 polypeptides). We are currently identifying these different proteins of interest by mass spectrometry (Maldi-TOF and LC-MS/MS) and learning these results in parallel of the previous genomic results. Preliminary results show the complementarity of the two analyses. Many of the proteins identified correspond to changes in mRNA belonging to the temporal gene program previously described after BCR engagement, thereby validating the gene expression data at the protein level, but some proteins show a specific temporal pattern of expression whereas their corresponding coding mRNA were not present in the temporal gene expression. Such complementarities in this dynamical approach allow us to refine the molecular basis for the functional BCR cross-linking consequences in CLL and to identify putative therapeutic targets for intervention in this disease. Ongoing experiments are examining the consequences of intervention to alter expression of critical components within the functional consequences of BCR cross-linking in CLL, and potentially the consequences of antigen driven leukemogenesis.