Tonic B-Cell Receptor Signaling Promotes the Survival of Diffuse Large B-Cell Lymphomas: Identification of SYK as a Rational Treatment Target.

The role of BCR-mediated survival signals in diffuse large B-cell lymphoma (DLBCL) remains undefined. B-cell antigen receptor (BCR) signaling induces receptor oligomerization and Ig alpha/beta ITAM phosphorylation. Thereafter, the protein tyrosine kinase (PTK) SYK is recruited and activated, initiating downstream events and amplifying the original BCR signal. Recent studies indicate that BCRs also transmit low-level survival signals in the absence of receptor engagement and highlight the critical role of SYK in this “tonic” BCR signaling. We recently found that SYK is a major substrate of the protein tyrosine phosphatase, PTPROt. PTPROt-mediated dephosphorylation and inactivation of SYK inhibited DLBCL proliferation and induced apoptosis in the absence of BCR cross-linking. These observations suggested that tonic BCR signaling may be an important survival mechanism in certain DLBCLs and identified SYK as a possible rational therapeutic target. For these reasons, we assessed the efficacy of a recently developed ATP-competitive inhibitor of SYK, R406, in a panel of 8 DLBCL cell lines. R406 inhibited the proliferation of 6 DLBCL cell lines at IC50s of 0.9–1.8 micromolar. The SYK inhibitor was cytotoxic (30–85% apoptotic cells) at low micromolar doses in 6 of these lines. To delineate the relationship between tonic BCR signaling, associated SYK activity and R406 responsiveness, we assessed SYK phosphorylation (pTYR 352 and 525/526) in the DLBCL cell line panel. Upon BCR signaling, SYK is phosphorylated at TYR 352; thereafter, the kinase undergoes autophosphorylation at TYR 525/526, which is required for SYK activity. In sensitive DLBCL cell lines, R406 specifically inhibited TYR 525/526 phosphorylation and additional downstream signals (p-ERK). In addition, all 6 R406-sensitive DLBCL cell lines exhibited basal SYK 352 phosphorylation whereas the 2 R406-resistant lines lacked pTyr 352 phosphorylation. Basal SYK pTYR 352 could be detected by immunoblotting total SYK immunoprecipitates with a phospho-specific SYK 352 antibody or using the same pTYR 352 antibody for intracellular phospho-specific flow cytometry. We conclude that :

1. tonic BCR signaling is an important survival mechanism in many DLBCLs;

2. SYK is a rational therapeutic target in these tumors;

3. R406 is a promising SYK inhibitor; and

4. SYK pTYR 352 is an excellent indicator of tonic BCR signaling and predictor of R406 sensitivity in DLBCL.