Glycosylation-Related Gene Expression Is Dysregulated in Multiple Myeloma and Overexpression of the Sialyltransferase ST3GAL6 Is Associated with Inferior Survival.

Alterations in glycosylation are associated with malignant transformation, with growing evidence implicating the dysregulation of glycosylation genes (glyco-genes) in multiple myeloma (MM). P selectin glycoprotein ligand-1 (PSGL1), recently reported to be important in MM cell adhesion and trafficking is over expressed by MM cells. Importantly, the generation of functional selectin ligands requires post-translational modification of scaffold proteins by glycosyltransferases and sialyltransferases generating selectin ligands such as Sialyl Lewis X (sLex). This background led us to undertake a comprehensive evaluation of the role glyco-genes in MM disease progression.

Analyzing publically available microarray transcriptomic datasets (Mayo Clinic GSE6477, University of Arkansas (UAMS) GSE24080, GSE2658) we sought to identify dysregulated glyco-genes in MM. The most significantly dysregulated genes were validated using real time quantitative PCR (RT_PCR) in the MM cell lines RPMI8226 and MMIR and in primary MM patient CD138 positive cells. The prognostic significance of any dysregulated genes was analyzed using Kaplan Meier survival estimates. Membrane protein extracts from MM cell lines were applied directly to lectin microarrays following fluorescent labeling to generate cell surface glycan profiles. Protein expression was assessed by immunohistochemistry (IHC) on primary MM bone marrow sections from MM patients and healthy controls. Lectin based flow cytometry was used to assess for the binding of sialic acid lectins to MM cell line RPMI8226.

Gene expression microarray dataset analysis confirmed the over-expression of genes related to the process of glycosylation in MM. Seventy-three genes were differentially expressed (> 2 fold change in MM from combined datasets). Nineteen genes were dysregulated in smoldering MM, 12 of which were common to MM. Thirteen were differentially regulated in MGUS, 6 of which were common to both MM and MGUS. One of these genes, the sialyltransferase ST3GAL6 (ST3 beta-galactosidase, alpha-2,3-sialyltransferase 6), which plays a critical role in generation of functional selectin ligands such as sLex, was upregulated in MM (fold change = 2.67) and smoldering MM (fold change = 2.22) but was absent in MGUS. Patients from GSE24080 (n=509) were stratified into two groups based on their expression intensities for ST3GAL6. Patients with higher normalized intensity values corresponding to probeset ID 210942_s_at (ST3GAL6) were found to have a lower median overall survival compared to their lower expressing counterparts. The difference in overall survival observed was 5.13 months (p <0.001 CI 1.3, 8.9). The association with reduced survival was independently verified in the MRC Myeloma IX microarray dataset (n=260). In this dataset using the median expression of ST3GAL6 as a cutoff there was a statistically significant reduction on overall survival with higher expression of ST3GAL6 (median OS 35.7 vs. 48 months, log rank test p=0.04)

RT-PCR analysis validated the over expression of glycogenes, including ST3GAL6 in MM cell lines and primary samples compared to healthy controls. We observed a trend for higher fold changes for ST3GAL6 in samples from patients with relapsed/refractory disease compared to those with responsive disease. IHC for ST3GAL6 on primary bone marrow sections from MM patients (n=57), demonstrated specific Golgi staining compared to controls. Consistent with the over expression of ST3GAL6 lectin microarray analysis of membrane protein extracts from MM cell lines RPMI8226 and MMIR showed binding to sialic acid-specific lectins Maackia amurensis agglutinin (MAA), which is specific for a2–3 linked sialic acids, and Sambucus nigra(SNA) which is specific for a2–6 linked sialic acids. This pattern was confirmed using biotinylated lectin based flow cytometry, which demonstrated a shift in allophycocyanin (APC) median fluorescence intensity for these lectins on RPMI8226 cells.

Glyco-genes, including the sialyltransferase ST3GAL6, are differentially regulated in all stages of MM with potential effects on MM biology and survival. Upregulation of ST3GAL6 may play an important role in MM cell trafficking and in this analysis is associated with inferior survival. Studies are ongoing to address the roles of ST3GAL6 over expression and altered sialylation in MM cell adhesion and trafficking.

Morgan:Novartis: Consultancy; Celgene: Consultancy; J&J: Consultancy.