BCL6 Regulates Tonic BCR Signaling in Diffuse Large B-Cell Lymphomas by Repressing the SYK Phosphatase, PTPROt

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease in which subsets of tumors likely rely upon different survival pathways. We previously identified a subset of primary DLBCLs with increased abundance of multiple components of the BCR signaling cascade including the spleen tyrosine kinase, SYK (“BCR-type” tumors). In related studies, we found that BCR-type DLBCL cell lines and primary tumors exhibited tonic B-cell receptor signaling. Since BCR-associated SYK activation initiates downstream events and amplifies the original BCR signal, we previously evaluated the efficacy of targeted SYK inhibition. Inhibition of SYK-dependent tonic BCR signaling induced apoptosis in the majority of “BCR-type” cell lines and primary DLBCLs. Furthermore, an oral SYK inhibitor (FosD) had promising activity in a subset of relapsed/refractory DLBCLs in a recently completed phase I/II clinical trial. “BCR-type” tumors also have more abundant expression of the transcriptional repressor, BCL6, and more frequent BCL6 translocations, greater repression of BCL6 target genes and increased sensitivity to BCL6 inhibitors. Since the same subset of primary DLBCLs relies upon SYK-dependent BCR signaling and BCL6-mediated transcriptional repression, we evaluated potential connections between BCL6 and SYK. SYK is a major substrate of the tissue-specific and developmentally regulated protein tyrosine phosphatase, PTPROt. For this reason, we first assessed the relative expression of BCL6 and PTPROt in two large independent series of transcriptionally profiled primary DLBCLs and two independent groups of highly purified normal B-cell subpopulations (naïve, germinal center, memory). There was a highly significant reciprocal pattern of expression of PTPROt and BCL6 in both normal B cells and primary DLBCLs, prompting speculation that PTPROt might be a target of BCL6-mediated transcriptional repression. We next analyzed the PTPROt promoter region in silico and identified 3 candidate BCL6 binding sites. Using chromatin immunoprecipitation assays in BCR-type DLBCL cell lines, we demonstrated that BCL6 binding sites in the PTPROt promoter were occupied in vivo by the transcription factor. To assess the role of BCL6 in regulating PTPROt transcription, we cotransfected a luciferase reporter vector driven by the PTPROt promoter with constructs encoding either wild-type BCL6 or one of two inactive BCL6 mutants. Wild-type BCL6 suppressed PTPROt-driven luciferase activity in a dose-dependent manner whereas neither BCL6 mutant altered luciferase levels. Consistent with these observations, individual or combined mutations of the BCL6 binding sites in the PTPROt promoter reduced or abolished the response to BCL6. Next, we asked whether PTPROt was a physiologic target of BCL6 in normal B cells. Forced overexpression of BCL6 in normal naïve B cells was associated with a marked reduction in PTPROt transcript abundance. Since PTPROt functions as a SYK phosphatase, we then assessed the consequences of BCL6 depletion on SYK Y352 phosphorylation and BCR signaling in BCR-type DLBCL cell lines. BCL6 siRNA increased PTPROt transcript abundance 10-fold whereas other components of the BCR signaling pathway were unchanged. In cells transduced with BCL6 siRNA, tonic and anti-Ig associated phosphorylation of SYK Y352 and BLNK Y84 were significantly lower than in parental or mock-transduced cells. Taken together, these studies indicate that BCL6 represses the expression of the SYK phosphatase, PTPROt, and augments SYK-dependent tonic BCR signaling. Since BCL6 and SYK are both promising rational therapeutic targets in many DLBCLs, combined inhibition of these functionally related pathways warrants further study.