STAT3, Constitutively Activated In ABC-Like DLBCL, Regulates Expression of the Prognostic Factor Cyclin D2

Abstract 705

As the most common subtype of non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL) remains clinically heterogeneous. Genome-scale expression profiles have defined two distinct molecular subtypes of DLBCL: Germinal Center B-Cell like (GCB-like) and Activated B-Cell like (ABC-like), with separate postulated tumor cells of origin and different pathogenesis, e.g. activation of NF-kB and Signal Transducer and Activator of Transcription 3 (STAT3) in the ABC-like DLBCLs. These subtypes are also characterized by distinct clinical outcomes. Since global gene expression is not practical for routine prognosis determination, we established an IPI independent 6-gene model based on the expression of BCL6, LMO2, FN1, BCL2, SCYA3 and cyclin D2 (CCND2) genes, which was capable of predicting patients' survival. While some of these genes may merely reflect different postulated tumor cells of origin, others may represent distinct pathogenetic mechanisms. Herein we demonstrate that CCND2, whose expression correlates with shorter survival, is positively regulated by the STAT3 signaling pathway, activated in the ABC-like DLBCL. Immunohistochemical staining of phosphorylated STAT3 (pSTAT3) demonstrated strong positive pSTAT3 staining (greater than 30% of nuclei) in 85 of 204 (41.7%) primary DLBCLs. Hierarchical clustering analysis of 143 DLBCL specimens stained for GCB and ABC markers demonstrated that expression of the pSTAT3 protein correlated with the expression patterns of non-GCB markers MUM1/IRF4 and BCL2 but not with GCB-specific markers HGAL, BCL6, and CD10. However, pSTAT3 expression was not different between the GCB-like and non-GCB-like DLBCLs defined by Han's model (Hans et al, Blood 2004). Examination of pSTAT3 levels in GCB-like DLBCL cell lines (OCI-LY7, OCI-LY19, and Sud-HL6) and the ABC-like cell lines (OCI-LY3 and OCI-LY10) by Western blotting demonstrated its expression in OCI-LY3 and OCI-LY10 but not in other cell lines. pSTAT3 expression in these cells was confirmed by phospho-flow cytometry. Interestingly, pSTAT3 expressing cell lines also expressed high levels of Cyclin D2, while it was not detected by Western blotting in the DLBCL cell lines not expressing pSTAT3. Analysis of the CCND2 promoter showed a single potential STAT3 binding site. Chromatin immunoprecipitation (ChIP) assay confirmed specific binding of STAT3 to this site in the CCND2 promoter. BCL6 is reported to negatively regulate expression of CCND2 and to competitively bind to STAT3 binding sites. Consequently, using quantitative ChIP assay we measured BCL6 and STAT3 binding to the CCND2 promoter in Sud-HL6, Oci-LY7 and Oci-LY19 GCB-like and in Oci-LY3 and Oci-LY10 ABC-like DLBCL cell lines. Levels of the bound STAT3 and BCL6 proteins were significantly higher in the Oci-LY3 and Oci-LY10 cell lines compared to their GCB counterparts. Treatment with BCL6 peptide inhibitor (BPI) slightly increased CCND2 mRNA expression in both GCB-like and ABC-like DLBCL, confirming the inhibitory effect of BCL6 on the CCND2 expression. To examine the effect of pSTAT3 on Cyclin D2 in the physiological context, we have knocked-down expression of STAT3 and pSTAT3 by specific siRNA in the OCI-LY3 cell line. A 37% decrease in the total levels of STAT3 led to a 52% decrease in pSTAT3 and a 55% reduction in the Cyclin D2 protein levels and was associated with a statistically significant decrease in cell proliferation as measured by the MTT assay. Overall our results demonstrate that a constitutively active STAT3 signaling pathway contributes to the Cyclin D2 expression and proliferation of the ABC-like DLBCLs. These findings elucidate the mechanism regulating the expression of prognostic factor Cyclin D2 and link it to the STAT3 signaling pathway constitutively activated in ABC-like DLBCL.