Abstract
Primary mediastinal B-cell lymphoma (PMBL) is characterized by aberrant activation of JAK/STAT-signaling resulting in constitutive presence of phosphorylated STAT6 (pSTAT6). In primary PMBL samples pSTAT6 is only expressed in a sub-population of lymphoma cells in a pattern that is reminiscent of that of the BCL6 oncogene. Double-fluorescence staining was carried out to determine the association between these two proteins in ten primary PMBL cases and three available PMBL cell line models. Surprisingly, only a minute fraction of double-positive nuclei was observed, while each sample contained considerable fractions of single-positive pSTAT6 and BCL6 nuclei. The intratumoral coexistence of BCL6+/pSTAT6- and BCL6-/pSTAT6+ subpopulations suggests a negative interaction between these factors. In silico screening of the STAT6 /BCL6 promoters for DNA consensus binding sites identified five STAT-binding-sites in the BCL6 promoter. We confirmed STAT6 binding to the BCL6 promoter in vitro and in vivo by band shift / super shift assays and chromatin immunoprecipitations. Using BCL6 luciferase reporter assays, depletion of STAT6 by siRNA, and ectopic overexpression of a constitutive active STAT6 mutant, we proved that pSTAT6 is sufficient to transcriptionally repress BCL6. Recently developed small molecule inhibitors 79-6 and TG101348 that increases BCL6 target gene expression and decreases pSTAT6 levels, respectively, demonstrate that a combined targeting results in additive efficacy regarding their negative effect on cell viability. The delineated pSTAT6-mediated molecular repression mechanism links JAK/STAT to BCL6-signaling in PMBL and may carry therapeutic potential.
Highlights
Primary mediastinal B-cell lymphoma (PMBL) is a distinct subtype of diffuse, large B-cell lymphoma (DLBCL) that arises in the thymus [reviewed in 1]
We quantitatively examined ten primary PMBL tumor samples by double IF for phosphorylated STAT6 (pSTAT6) and BCL6 and verified staining in tumor cells using the B-cell surface marker CD19 (Figure 1C, left cutouts)
Averages of BCL6+/pSTAT6- and BCL6-/pSTAT6+ fractions surmount to ~46% of the tumor cells in primary PMBL cases leaving ~54% CD19+ BCL6-/pSTAT6- cells. (Figure 1D)
Summary
Primary mediastinal B-cell lymphoma (PMBL) is a distinct subtype of diffuse, large B-cell lymphoma (DLBCL) that arises in the thymus [reviewed in 1]. While the gene expression signature of PMBL is distinct and differs from other DLBCL subtypes [2;3], morphology and immunophenotype, are less characteristic. PMBL does not harbor JAK2 activating mutations [6]. JAK2 signalling may be activated either due to gene-dosage effect of JAK2 [7] or other molecular aberrations which take place in PMBL. According to Guiter [10], nuclear pSTAT6 is a characteristic finding in PMBL. The extent of nuclear staining is heterogeneous within each PMBL case, and the number of positively labeling neoplastic cells ranges from 10-50% [10]. We delineated a novel molecular repression mechanism that links activated JAK/STAT signaling to BCL6 expression. In conjunction with the additive efficacy of small molecule inhibitors on cell viability our findings may carry therapeutic potential
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