Amarsaikhan, Nansalmaa

Abstract Immunoglobulin (Ig) secretion by terminally differentiated B-cells is important in the immune response to pathogens. Its over production is a defining characteristic of many B-cell malignancies, including Waldenström Macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Thus, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for these diseases. Here, we define a novel pathway involving the oncogenic transcription factor, GLI2, modulating IgM secretion via regulation of IL-6 receptor alpha subunit (IL6Rα) expression in WM cells. We have previously identified components of the Hedgehog (HH)-GLI pathway as modulators of Ig secretion. Pharmacological (GANT61) and genetic (shRNA) inhibition of GLI proteins resulted in no significant effect on cell proliferation or viability, while there was a significant reduction in IgM secretion (60%) in BCWM.1 (p<0.0001), (55%) in MWCL-1 (p<0.0001) and (50%) in RPCI-WM1 (p<0.0001) cells. Interestingly, this occurred independent of HH signaling as manipulation of HH had no effect on IgM secretion, cell growth or viability. Screening for a mechanism identified IL6Rα as a downstream direct target of GLI activity. GANT61 treatment significantly reduces IL6Rα promoter activity in BCWM.1 (p=0.0002), MWCL-1 (P=0.0003) and RPCI-WM1 (p<0.0001) cells. GLI2 knockdown using two independent shGLI2 constructs resulted in reduced IL6Rα mRNA expression (p<0.0001) determined by qPCR, and IL6Rα protein expression examined by FACS analysis. Similarly, GLI2 knockdown diminished IL6Rα promoter activity in BCWM.1 (p=0.0218), MWCL-1 (p=0.0008) and RPCI-WM1 (p=0.0002) cells. Using bioinformatics analysis, we identified three candidate GLI2 binding sites in the IL6Rα promoter region. Using chromatin immunoprecipitation assay (ChIP) followed by qPCR, we confirmed that GLI2 directly binds to the IL6Rα promoter region (-1294 / -1065). Finally, we were able to rescue the reduction in IgM secretion in GLI2 knockdown group by overexpressing IL6Rα (p=0.0142). Taken together, these data reveal a novel mechanism where GLI2 modulates IgM secretion in WM through the regulation of IL6Rα expression. Given the poor prognosis associated with elevated IgM in WM patients, components of this new signaling axis could be important therapeutic targets. Disclosures No relevant conflicts of interest to declare.

Abstract Ig secretion by terminally differentiated B cells is an important component of the immune response to foreign pathogens. Its overproduction is a defining characteristic of several B cell malignancies, including Waldenström macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Therefore, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for this disease. In this study, we define a novel pathway involving the oncogenic transcription factor GLI2 modulating IgM secretion by WM malignant cells. Pharmacological and genetic inhibition of GLI2 in WM malignant cells resulted in a reduction in IgM secretion. Screening for a mechanism identified the IL-6Rα (gp80) subunit as a downstream target of GLI2 mediating the regulation of IgM secretion. Using a combination of expression, luciferase, and chromatin immunoprecipitation assays we demonstrate that GLI2 binds to the IL-6Rα promoter and regulates its activity as well as the expression of this receptor. Additionally, we were able to rescue the reduction in IgM secretion in the GLI2 knockdown group by overexpressing IL-6Rα, thus defining the functional significance of this receptor in GLI2-mediated regulation of IgM secretion. Interestingly, this occurred independent of Hedgehog signaling, a known regulator of GLI2, as manipulation of Hedgehog had no effect on IgM secretion. Given the poor prognosis associated with elevated IgM in WM patients, components of this new signaling axis could be important therapeutic targets.