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Glycosylation is a stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process, especially increased sialylation, have been associated with malignant transformation and metastasis. The role of altered sialylation in multiple myeloma (MM) cell trafficking has not been previously investigated. In the present study we identified high expression of beta-galactoside alpha-2,3-sialyltransferase, ST3GAL6, in MM cell lines and patients. This gene plays a key role in selectin ligand synthesis in humans through the generation of functional sialyl Lewis X. In MRC IX patients, high expression of this gene is associated with inferior overall survival. In this study we demonstrate that knockdown of ST3GAL6 results in a significant reduction in levels of alpha-2,3-linked sialic acid on the surface of MM cells with an associated significant reduction in adhesion to MM bone marrow stromal cells and fibronectin along with reduced transendothelial migration in vitro. In support of our in vitro findings, we demonstrate significantly reduced homing and engraftment of ST3GAL6 knockdown MM cells to the bone marrow niche in vivo, along with decreased tumor burden and prolonged survival. This study points to the importance of altered glycosylation, particularly sialylation, in MM cell adhesion and migration.

Background Glycosylation is a post translational modification which results in the addition of carbohydrate determinants to proteins and lipids. This has a major influence on the function of molecules known to be important in myeloma cell adhesion and trafficking such as integrins and selectins. We previously reported that ST3GAL6, a sialyltransferase involved in the synthesis of functional selectin ligands in humans, is over expressed in myeloma cells and associated with inferior survival in the MRC Myeloma IX study (MRC IX). We now extend our analysis of the MRC IX dataset to evaluate the potential prognostic significance of other glycosylation genes that are differentially regulated between normal and malignant plasma cells. Methods Analyzing publically available microarray transcriptomic datasets (Mayo Clinic GSE6477, University of Arkansas (UAMS) GSE24080, GSE2658) we first identified dysregulated glycosylation genes in MM. The prognostic significance of these candidate genes in MRCIX (singly and in combination) was analyzed using Kaplan Meier survival estimates for progression free survival and overall survival (PFS, OS). These results were validated in independent datasets (TT2 and TT3). Any genes significantly associated with survival were correlated with FISH abnormalities and ISS and multivariate analysis was performed to determine their independence as prognostic factors. QPCR was performed in cell lines to validate GEP findings. Matching SNP-based mapping array and methylation array data were analyzed. Membrane protein extracts from MM cell lines were applied directly to lectin microarrays following fluorescent labeling to generate cell surface glycan profiles. Immunohistochemistry for glycosylation enzymes and glycans was performed on patient bone marrow samples. Results High expression (top quartile as cut-off) of the sialyltransferase gene ST3GAL1 showed a trend towards inferior OS (median survival 35 mos .v. 45 mos, p=0.07) with significantly reduced PFS (19 .v. 14 mos, p=0.015). Increased expression of ST3GAL1 correlated with the presence of t(4;14) (p=0.009), del13q (p=0.001), +1q (p=0.01) and hypodiploidy (p=0.00001). Low expression (lower quartile by GEP) of the gene FUCA1, which encodes tissue alpha-L-fucosidase, was linked to inferior outcome (median OS 44 .v. 38 mos, p=0.025). On multivariate analysis low FUCA1 expression was independent of other important prognostic factors (HR=1.61, p=0.017). Patients with t(11;14) by FISH were more likely to have low FUCA1 than t(11;14) negative patients. There was no significant association between ISS and ST3GAL6, ST3GAL1 or FUCA1. We verified the effect of low FUCA1 in the GSE24080 dataset from TT2 and TT3 combined. Patients with low FUCA1 had significantly worse OS (p=0.0002). SNP mapping array and methylation array analysis did not show any evidence of correlation between copy number alterations or hypermethylation of FUCA1 and its low expression level. Since FUCA1 participates in N-glycan degradation with removal of fucose residues, this raises the possibility that a reduction in FUCA1 may lead to excessive fucosylation of cancer related glycans involved in adhesion and trafficking, such as sLex. Indeed, lectin arrays of MM cell lines revealed high levels of a-1,3/a-1,6 linked fucose, as determined by binding of Aleuria Aurantia Lectin (AAL). The HECA-452 antibody recognizes a functional trisaccharide domain shared by sialyl Lewis a and sLex and known to bind to E-selectin. Preliminary immunohistochemistry revealed increased HECA452 staining in bone marrow samples, which had both strong ST3GAL6 and low FUCA1 staining. Combining gene expression data showed that reduced expression of FUCA1 with increased expression of either ST3GAL6 or ST3GAL1 or both, identified a subgroup of patients (18%) in MRC Myeloma IX with particularly poor outcome (figure 1). Similar results were found in TT2 and TT3 with 17% of patients affected. Conclusions Altered glycosylation gene expression patterns may identify patients at high risk of disease progression and early death. Our data implicates sialyltransferases and selectin ligands as potential therapeutic targets in MM. Disclosures: Morgan: Celgene: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Millenium: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Johnson and Johnson: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees. Ghobrial:Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

Background Glycosylation is a stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process, especially increased sialylation, have been associated with malignant transformation and metastasis. The adhesion and trafficking of multiple myeloma (MM) cells is strongly influenced by glycosylation and multiple myeloma cells express a variety of adhesion molecules, including selectin ligands and integrins, which are typically dependent on glycosylation for their function. We have previously reported that the sialyltransferase ST3GAL6 is up-regulated in plasma cells from MM patients and that increased expression is associated with inferior overall survival (OS) in MM gene expression profiling (GEP) datasets. The functional significance of increased sialylation of MM cells has not previously been reported. Methods MM cell lines MM1s and RPMI-8226 were confirmed to have high expression levels of ST3GAL6 at the gene and protein level compared to healthy controls. Knockdown of ST3GAL6 was confirmed in MM cell lines RPMI-8226 and MM1s using lentiviral shRNAs targeting different regions in the ST3GAL6 mRNA. Specific ST3GAL6 knockdown was confirmed by reduced ST3GAL6 mRNA and protein expression in comparison to a scrambled control. In a calcein-AM fluorescence based adhesion assay we next evaluated the effects of ST3GAL6 knockdown on MM-cell adhesion to bone marrow stromal cells (BMSC’s) and fibronectin coated plates. Migration to 30nM SDF1-α was assessed using transwell plates comparing ST3GAL6 knockdown cells to scrambled controls. The commercially available sialyltransferase inhibitor 3Fax-Neu5Ac was used to pre-treat MM cells in vitro prior to assessment of apoptosis by flow cytometry. shST3GAL6 MM1s cells positive for green fluorescent protein and luciferin (GFP-Luc+) were injected into tail veins of SCID-Bg mice (5x106 cells, n=5/group) and mice were followed weekly using bioluminescent imaging (BLI) for tumor development. Bone marrow homing of tumor cells was assessed using in vivoconfocal imaging of the skull vasculature (n=3/group). Results Knockdown of ST3GAL6 in MM cell lines resulted in a 50% reduction in cell surface staining with the monoclonal antibody HECA-452. This indicated reduced expression of cutaneous lymphocyte associated antigen (CLA), a carbohydrate domain shared by sialyl Lewis X (sLex) and sialyl Lewis a (sLea) antigens, confirming suppression of ST3GAL6 activity. There was a significant reduction in the ability of knockdown cells to adhere to BMSC’s and fibronectin in-vitro compared to scrambled controls (P=0.016, 0.032 respectively). Migration ability of these cells in response to SDF1-α was also reduced (P=0.01). In vivo in a xenograft SCID-Bg mouse model shST3GAL6 cells demonstrated a reduced tumor burden as assessed by weekly BLI (P=0.017 at week 4). A consolidated map of the skull bone marrow niche in mice injected with shST3GAL6 MM1s GFP-Luc+ cells revealed a reduced homing ability of these cells in comparison to mice injected with scrambled control cells. Treatment of the MM cell lines MM1s and RPMI-8226 with a sialyltransferase inhibitor 3Fax-Neu5Ac resulted in almost complete elimination of cell surface sLex and/or sLea expression as determined by HECA-452 staining. Following pre-treatment with 3Fax-Neu5Ac, MM1S cells grown in co-culture with BMSC’s cells showed increased sensitivity to Bortezomib compared to cells treated with bortezomib alone. Conclusions shRNA knockdown of ST3GAL6 in MM cells significantly inhibits adhesion and migration in vitro with reduced homing and proliferation potential in vivo. In conjunction with the results of enzymatic inhibition this indicates that sialylation may play an important role in the malignant behavior of MM cells. Studies are ongoing to address the potential role of altered glycosylation in MM. Disclosures: Ghobrial: Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

Abstract 2931 Background 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. Methods 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. Results 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 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. Conclusions 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. Disclosures: Morgan: Novartis: Consultancy; Celgene: Consultancy; J&J: Consultancy.