SLP76 Is Ectopically Expressed in Chronic Lymphocytic Leukemia Cells and Involves in B-Cell Receptor Signaling

In the last decade, the B-cell receptor (BCR) has emerged as a pivotal stimulus in CLL pathogenesis. The BCR responsiveness in CLL cells is heterogeneous among patients and correlates with disease aggressiveness. Here we show for the first time, that SLP76 a key scaffold protein in T-cell receptor (TCR) signaling, is ectopically expressed in CD19+ purified CLL cells (purity >95%) in the majority of patients, and is co-expressed with other components of the TCR pathway, including LCK and ZAP70.  SLP76 mRNA levels correlated with its protein expression and SLP76 protein levels were higher in unmutated IGHV and ZAP70⁺ CLL cells. SLP76 was found to be functionally active in CLL cells, as it becomes phosphorylated in response to BCR engagement in a time dependent manner. Activation with anti-IgM antibody results in phosphorylation of SLP76 on the positive regulatory tyrosine residue Y128 residue with increased physical association with Btk, peaking after 15 minutes. The negative regulatory residue S376 became phosphorylated only after 45', concomitantly with downregulation of the tyrosine phosphorylation. SLP76 phosphorylation in response to BCR engagement did not correlate with total ZAP70 expression. Pre-incubation of CLL cells with the LCK kinase inhibitor LCKi and with the SYK inhibitor R406, inhibited SLP76 phosphorylation in response to BCR activation, while the BTK inhibitor-ibrutinib had no effect. These suggest that LCK and SYK, but not ZAP70, play a central role in the upstream signaling involved in SLP76 activation in CLL cells.  Knockdown of SLP76 in CLL cells resulted in decreased induction of BTK and PLCγ2 phosphorylation after BCR activation with anti-IgM.  Consistent with our findings that SLP76 in involved in BCR signaling in CLL cells, we found that high total SLP76 expression was associated with a shorter time to first progression or need for treatment. In conclusion, SLP76 is ectopically expressed in CLL cells and plays a role in BCR signaling in those cells.