Selectin Ligands Research Articles

Acyclic tethers mimicking subunits of polysaccharide ligands: selectin antagonists.

We report on the design and synthesis of molecules having E- and P-selectins blocking activity both in vitro and in vivo. The GlcNAc component of the selectin ligand sialyl Lewis(X) was replaced by an acyclic tether that links two saccharide units. The minimization of intramolecular dipole-dipole interactions and the gauche effect would be at the origin of the conformational bias imposed by this acyclic tether. The stereoselective synthesis of these molecules, their biochemical and biological evaluations using surface plasmon resonance spectroscopy (SPR), and in vivo assays are described. Because the structure of our analogues differs from the most potent E-selectin antagonists reported, our acyclic analogues offer new opportunities for chemical diversity.

B4GALNT2 gene expression controls the biosynthesis of Sda and sialyl Lewis X antigens in healthy and cancer human gastrointestinal tract

The histo blood group carbohydrate Sda antigen and its cognate biosynthetic enzyme B4GALNT2 show the highest level of expression in normal colon. Their dramatic down regulation previously observed in colon cancer tissues could play a role in the concomitant elevation of the selectin ligand sLex, involved in metastasis. However, down regulation of sLex expression by B4GALNT2 has been so far demonstrated in vitro, but not in tissues. The human B4GALNT2 gene specifies at least two transcripts, diverging in the first exon, never studied in normal and cancer tissues. The long form contains a 253 nt exon 1L; the short form contains a 38 nt exon 1S. Using qPCR, we showed that cell lines and normal or cancerous colon, expressed almost exclusively the short form, while the long form was mainly expressed by the embryonic colon fibroblast cell line CCD112CoN. Immunochemistry approaches using colon cancer cells permanently expressing either B4GALNT2 cDNAs as controls, led to the observation of several protein isoforms in human normal and cancerous colon, and cell lines. We showed that tissues expressing B4GALNT2 protein isoforms were able to induce Sda and to inhibit sLex expression; both of which are expressed mainly on PNGase F-insensitive carbohydrate chains. Concomitant expression of B4GALNT2 and siRNA-mediated inhibition of FUT6, the major fucosyltransferase involved in sLex synthesis in colon, resulted in a cumulative inhibition of sLex. In normal colon samples a significant relationship between sLex expression and the ratio between FUT6/B4GALNT2 activities exists, demonstrating for the first time a role for B4GALNT2 in sLex inhibition in vivo.

Lack of functional selectin ligand interactions enhances inherent neutrophil function leading to increased resistance to systemic Listeria monocytogenes infection (INC7P.408)

Abstract Leukocyte recruitment towards sites of infection involves coordinated ligand-receptor interactions between leukocytes and the vascular wall. Selectins transiently bind to selectin ligands to enable leukocyte tethering and rolling on the vascular wall prior to leukocyte adhesion and transmigration into tissues. These interactions occur following post-translational modifications of selectin ligands, including terminal fucosylation by Fucosyltransferase-IV and -VII. In mice, deficiency in both enzymes (FtDKO) leads to loss of leukocyte tethering and rolling, increased circulating neutrophils and impaired T cell trafficking. However, the role of functional selectin ligand interactions in modulating immunity to bacterial infections remains unclear. We analyzed the kinetics of infection and modulation of immune responses in FtDKO mice infected with Listeria monocytogenes, an intracellular bacterium. FtDKO mice controlled systemic infection more rapidly relative to wild-type mice. This was correlated to increased number and functionality of neutrophils. Adoptive transfer of bone marrow cells from FtDKO mice into irradiated wild-type mice resulted in expansion of neutrophil populations and increased resistance to infection in a neutrophil-dependent manner. Functional selectin ligand deficiency on neutrophils was correlated to increased resistance to cell death. Thus, functional selectin ligand expression is a potential target for enhancing innate immunity against bacterial infection.

Selectins Mediate Small Cell Lung Cancer Systemic Metastasis

Metastasis formation is the major reason for the extremely poor prognosis in small cell lung cancer (SCLC) patients. The molecular interaction partners regulating metastasis formation in SCLC are largely unidentified, however, from other tumor entities it is known that tumor cells use the adhesion molecules of the leukocyte adhesion cascade to attach to the endothelium at the site of the future metastasis. Using the human OH-1 SCLC line as a model, we found that these cells expressed E- and P-selectin binding sites, which could be in part attributed to the selectin binding carbohydrate motif sialyl Lewis A. In addition, protein backbones known to carry these glycotopes in other cell lines including PSGL-1, CD44 and CEA could be detected in in vitro and in vivo grown OH1 SCLC cells. By intravital microscopy of murine mesenterial vasculature we could capture SCLC cells while rolling along vessel walls demonstrating that SCLC cells mimic leukocyte rolling behavior in terms of selectin and selectin ligand interaction in vivo indicating that this mechanism might indeed be important for SCLC cells to seed distant metastases. Accordingly, formation of spontaneous distant metastases was reduced by 50% when OH-1 cells were xenografted into E-/P-selectin-deficient mice compared with wild type mice (p = 0.0181). However, as metastasis formation was not completely abrogated in selectin deficient mice, we concluded that this adhesion cascade is redundant and that other molecules of this cascade mediate metastasis formation as well. Using several of these adhesion molecules as interaction partners presumably make SCLC cells so highly metastatic.

Regulatory T cells dynamically regulate selectin ligand function during multiple challenge contact hypersensitivity (146.12)

Regulatory T cells (Tregs) play critical roles in regulation of T cell‐mediated inflammation, and in organs such as the skin, this is dependent on their expression of selectin ligands required for rolling in peripheral microvessels. However, whether there are differences in the molecules used by Tregs and pro‐inflammatory T cells to undergo rolling remains unclear. Here we used spinning disk confocal microscopy of Foxp3‐GFP mice to visualize rolling of endogenous Tregs in dermal postcapillary venules. Tregs were observed to undergo infrequent but consistent rolling interactions under resting and inflamed conditions. Despite this, during inflammation, Tregs were able to adhere efficiently, comprising 40% of the total adherent CD4+ T cell population at the peak of the response. In a multiple challenge model of contact sensitivity, rolling of both Tregs and conventional CD4+ T cells was mostly dependent on overlapping contributions of P‐ and E‐selectin. However, shortly after a second challenge, rolling of Tregs but not conventional T cells became P‐selectin‐independent, an effect associated with a reduction in their capacity to bind P‐selectin in vitro. These findings demonstrate that Treg selectin‐binding capacity and the molecular basis of Treg rolling in the peripheral microvasculature can be regulated dynamically in a multiple challenge model of inflammation.

Tissue resident macrophages regulate IL‐23‐dependent granulopoiesis (146.7)

Mice deficient in α(1,3)‐fucosyltransferases 4 and 7 (FUT‐/‐) lack selectin ligand activity and selectin‐dependent leukocyte trafficking, resulting in altered IL‐23‐dependent granulopoiesis. It is not clear if this granulopoiesis is regulated by bone marrow‐derived macrophages (BMMC) or a distinct population of tissue resident macrophages (TRM). We hypothesized that the loss of FUT‐dependent trafficking reduces the number of neutrophils able to infiltrate tissues, undergo apoptosis, and be phagocytosed by TRMs, altering IL‐23 production. Primary Kupffer cells (pKC) and BMMC were isolated from FUT‐/‐ and WT mice, stimulated in vitro with LPS, and assessed for IL‐23 production. WT mice were irradiated and their hematopoietic system was reconstituted with 1x106 bone marrow cells isolated from WT or FUT‐/‐ mice. Six weeks after reconstitution, circulating WBC counts were assessed. There was no difference in gene expression levels of IL‐23 between WT and FUT‐/‐ pKCs or BMMCs, indicating that IL‐23 production from FUT‐/‐ macrophages is intact. WT mice reconstituted with FUT‐/‐ bone marrow show a pronounced neutrophilia similar to that seen in FUT‐/‐ mice. These results confirm that FUT‐dependent neutrophil trafficking is required for the regulation of granulopoiesis and suggest that the TRM populations are responsible for clearing apoptotic neutrophils and regulating IL‐23 production in granulopoiesis.Grant Funding Source: R01‐HL090823, T32‐HL069768

Aberrant Presentation of HPA-Reactive Carbohydrates Implies Selectin-Independent Metastasis Formation in Human Prostate Cancer

To investigate the impact of prostate cancer cell surface glycosylation as part of the tumor cell-endothelial cell interaction in prostate cancer metastasis. Glycosyltransferase expression was profiled in metastasis-derived prostate cancer cell lines and compared with primary epithelium. Prostate cancer cells were examined for HPA- and selectin-binding and adhesion to endothelium. Spontaneous metastasis xenograft models were established to test the lectin HPA-binding sites as a marker of metastatic competence and to evaluate E-selectin-binding sites in vivo. The importance of selectins for metastasis formation was analyzed using Sele(-/-)/Selp(-/-) mice. The clinical relevance of HPA- and E-selectin-binding sites in prostate cancer was determined. Glycosyltransferases involved in the synthesis of common HPA-binding sites are downregulated in prostate cancer cells. An absence of HPA-reactive carbohydrates specifically indicates spontaneous metastatic spread of prostate cancer xenografts in vivo and a poor prognosis of patients with prostate cancer. HPA-binding sites decrease in lymph node metastases compared with corresponding primary tumors. Common selectin ligands are absent on prostate cancer cells, which do not adhere to recombinant selectins or endothelium under shear stress in vitro. Spontaneous metastasis formation is largely independent of selectins in vivo. E-selectin-binding sites are detectable in only 2% of patients with prostate cancer without prognostic significance. Prostate cancer is characterized by an inverse functional and prognostic importance of HPA-binding sites compared with other adenocarcinomas. Accordingly, this study surprisingly shows that the selectin-selectin ligand axis, which is essential for extravasation and thus metastasis formation in several malignancies, can be circumvented in prostate cancer.

Deficiency of the Sialyltransferase St3Gal4 Reduces Ccl5-Mediated Myeloid Cell Recruitment and Arrest

Rationale: Sialylation by α2,3-sialyltransferases has been shown to be a crucial glycosylation step in the generation of functional selectin ligands. Recent evidence suggests that sialylation also affects the binding of chemokines to their corresponding receptor. Objective: Because the chemokine receptors for Ccl5 and Ccl2 are important in atherogenic recruitment of neutrophils and monocytes, we here investigated the role of α2,3-sialyltransferase IV (ST3Gal-IV) in Ccl5- and Ccl2-mediated myeloid cell arrest and further studied its relevance in a mouse model of atherosclerosis. Methods and Results: St3Gal4 -deficient myeloid cells showed a reduced binding of Ccl5 and an impaired Ccl5-triggered integrin activation. Correspondingly, Ccl5-induced arrest on tumor necrosis factor-α–stimulated endothelium was almost completely abrogated, as observed in flow chamber adhesion assays and during ex vivo perfusion or intravital microscopy of carotid arteries. Moreover, Ccl5-triggered neutrophil and monocyte extravasation into the peritoneal cavity was severely reduced in St3Gal4 −/− mice. In contrast, St3Gal4 deficiency did not significantly affect Ccl2 binding and only marginally decreased Ccl2-induced flow arrest of myeloid cells. In agreement with the crucial role of leukocyte accumulation in atherogenesis, and the importance of Ccl5 chemokine receptors mediating myeloid cell recruitment to atherosclerotic vessels, St3Gal4 deficiency drastically reduced the size, stage, and inflammatory cell content of atherosclerotic lesions in Apoe −/− mice on high-fat diet. Conclusions: In summary, these findings identify ST3Gal-IV as a promising target to reduce inflammatory leukocyte recruitment and arrest.

Defining the Functional Boundaries of the Murine α1,3-Fucosyltransferase Fut7 Reveals a Remarkably Compact Locus

Fut7 encodes an α1,3-fucosyltransferase critical for biosynthesis of glycan ligands for all three selectins. Consistent with this function, Fut7 expression is limited to hematopoietic cells and high endothelial cells which express selectin ligands. Mechanisms that govern Fut7 expression are poorly defined. To begin to understand the molecular genetic basis for transcriptional regulation of Fut7, a transgenic, gain-of-function, genetic complementation approach in mice was used to define the "functional boundaries" of the murine Fut7 locus, defined here as any uninterupted stretch of genomic DNA that contains all cis-acting genetic elements essential for accurate physiologic expression. A 12.7-kb contiguous genomic interval, which lies completely between the highly conserved flanking Npdc1 and Abca2 loci on chromosome 2 and which contains the complete transcriptional unit plus ∼7.4 kb upstream of the transcriptional start site and ∼2 kb downstream of the transcriptional termination and polyadenylation sites, was used as a transgene (Tg) on a Fut7 null background. Tg+ mice exhibited restoration of Fut7 gene expression and physiologic levels of selectin ligand expression and function on neutrophils, activated T cells, and high endothelial cells and corrected the functional defects in these cells found in Fut7 null mice without leading to detectable expression of Fut7 in normally non-expressing tissues. These results demonstrate that all genetic information essential for appropriate and selective expression of Fut7 in diverse cell types and in response to distinct developmental signals is contained within this comparatively small genetic region.

Selectins and their ligands are required for homing and engraftment of BCR-ABL1+ leukemic stem cells in the bone marrow niche

AbstractWe investigated adhesion pathways that contribute to engraftment of breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL1)–induced chronic myelogenous leukemia (CML)-like myeloproliferative neoplasia in a mouse retroviral transduction/transplantation model. Compared with normal stem/progenitor cells, BCR-ABL1+ progenitors had similar expression of very late antigen-4 (VLA4), VLA5, leukocyte functional antigen-1, and CXCR4 but lower expression of P-selectin glycoprotein ligand-1 (PSGL-1) and of L-selectin. Whereas vascular cell adhesion molecule-1 and P-selectin were not required, deficiency of E-selectin in the recipient bone marrow endothelium significantly reduced engraftment by BCR-ABL1–expressing stem cells following intravenous injection, with leukemogenesis restored by direct intrafemoral injection. BCR-ABL1–expressing cells deficient for PSGL-1 or the selectin ligand-synthesizing enzymes core-2 β1,6-N-acetylglucosaminyltransferase or fucosyltransferases IV/VII were impaired for engraftment, and destruction of selectin ligands on leukemic progenitors by neuraminidase reduced engraftment. BCR-ABL1–expressing L-selectin–deficient progenitors were also defective in homing and engraftment, with leukemogenesis rescued by coexpression of chimeric E/L-selectin. Antibody to L-selectin decreased the engraftment of BCR-ABL1–transduced stem cells. These results establish that BCR-ABL1+ leukemic stem cells rely to a greater extent on selectins and their ligands for homing and engraftment than do normal stem cells. Selectin blockade is a novel strategy to exploit differences between normal and leukemic stem cells that may be beneficial in autologous transplantation for CML and perhaps other leukemias.

Metastatic Growth Progression Caused by PSGL-1–Mediated Recruitment of Monocytes to Metastatic Sites

Tumor cell-derived selectin ligands mediate contact to the endothelium, platelets, and leukocytes through binding to selectins that facilitates metastasis. Here, we describe the mechanism of how endogenous (non-tumor derived) selectin ligands contribute to metastasis using α(1,3)fucosyltransferase 7 (Fuc-TVII(-/-))-deficient mice. Experimental metastasis of MC-38GFP and Lewis lung (3LL) carcinoma cells was attenuated in Fuc-TVII(-/-) mice, which express minimal amount of selectin ligands. We show that metastasis is dependent on selectin ligands carried on hematopoietic cells. P-selectin glycoprotein ligand-1 (PSGL-1) was identified as the major ligand facilitating monocyte accumulation at metastatic sites. Reduced recruitment of monocytes to metastasizing tumor cells in Fuc-TVII(-/-) mice correlated with attenuated metastasis. Adoptive transfer of Fuc-T7(+) monocytes rescued metastasis in Fuc-TVII(-/-) mice, indicating that selectin ligand-dependent recruitment of monocytes is required for cancer progression. Cytokine analysis in metastatic lungs revealed high expression of CCL2 in C57BL/6 mice that was significantly lower in Fuc-TVII(-/-) mice. The absence of monocyte recruitment in Fuc-TVII(-/-) mice correlated with increased apoptosis of tumor cells. Thus, the recruitment of monocytes to metastasizing tumor cells is facilitated by endogenous selectin ligands on monocytes that enable efficient tumor cell survival, extravasation, and metastasis.

Targeting liposomes loaded with melphalan prodrug to tumour vasculature via the Sialyl Lewis X selectin ligand

Earlier we showed that liposome formulation of DL-melphalan lipophilic prodrug bearing tetrasaccharide Sialyl Lewis X (SiaLeX) caused prolonged therapeutic effect on mammary cancer in mice. Here, we compare antivascular effect of SiaLeX-liposomes loaded with diglyceride ester of melphalan (Mlph) against SiaLeX-free formulation in Lewis lung carcinoma model.Methods: Liposomes of egg phosphatidylcholine/yeast phosphatidylinositol/1,2-dioleoyl glycerol (DOG) conjugate of Mlph/±SiaLeX-PEG8–15-DOG, 8:1:1:0.2 by mol, were prepared by standard extrusion. After two intravenous injections with Mlph or liposomes under either standard or delayed treatment protocols, vascular-disrupting effects of the preparations were evaluated basing on tumour section histomorphology, lectin perfusion assay and immunohistochemistry (anti-CD31 staining) data. Also, untreated mice were administered with fluorescently-labelled liposomes to assess their distribution in tumour sections with confocal laser scanning microscopy.Results: Two injections of SiaLeX-liposomes reproducibly caused severe injuries of tumour vessels. SiaLeX-liposomes co-localized with CD31 marker on vascular endothelium while the non-targeted formulation extravasated into tumour.Discussion: Cytotoxic SiaLeX-liposomes exhibit superior vascular-disrupting properties compared to non-targeted liposomes, yet the effect starts to transform into gain in tumour growth inhibition only under delayed treatment regimen.Conclusion: SiaLeX-ligand provides targeting of cytotoxic liposomes to tumour endothelium and subsequent antivascular effect.

BCR-ABL1+ Leukemic Stem Cells Are Dependent On Selectin-Ligand Interactions For Engraftment In The Bone Marrow Niche

Leukemic progenitors from patients with chronic myelogenous leukemia (CML) have defects in the adhesive function of β1 integrins and in their response to the chemokine CXCL12 (Stem Cells 2002;3:259), pathways that are critical for engraftment and maintenance of normal hematopoietic stem cells (HSC) in the bone marrow (BM) niche. Previous studies in a mouse BCR-ABL1 retroviral transduction/transplantation model of CML demonstrated that BCR-ABL1+ leukemic stem cells, but not normal murine HSC, are dependent on expression of the CD44 adhesion molecule for BM homing and engraftment (Nat Med 2006;12:1175). To investigate further other adhesion molecules required for stable engraftment of CML stem cells, we employed donor and recipient mice with targeted mutations in genes encoding selectins and those required for expression of selectin ligands in the mouse retroviral CML model. Neither the β1 integrin ligand VCAM-1 nor P-selectin was required in the BM endothelium of recipient mice for efficient engraftment and induction of CML-like myeloproliferative neoplasia (MPN) by BCR-ABL1-expressing stem cells. By contrast, loss of recipient E-selectin significantly impaired engraftment of BCR-ABL1+ stem/progenitor cells, as demonstrated by decreased frequency of BM proviral leukemia-initiating clones, a defect overcome by direct intrafemoral injection of the leukemic cells. We also demonstrated a requirement for expression of selectin ligands on the leukemic stem cells, as BCR-ABL1-expressing stem cells lacking enzymes contributing to biosynthesis of selectin ligands (Core2 or Fucosyltransferases IV and VII) exhibited decreased efficiency of engraftment, resulting in attenuated disease. Further, donor cells deficient in P-selectin glycoprotein ligand-1 (PSGL-1) or in both PSGL-1 and CD44, a glycoform of which can function as a selectin ligand on hematopoietic stem cells (J. Biol Chem 2001;278:47623), displayed intermediate or profound impairment in engraftment of CML-like leukemia, respectively. Removal of selectin ligands by neuraminidase treatment of BCR-ABL1-transduced HSC blocked engraftment of leukemic stem cells and resulted in long-term survival of transplant recipients. Together, these results demonstrate that BCR-ABL1-expressing stem cells are dependent on selectin ligands, expressed on PSGL-1 and possibly on CD44, and their interaction with BM E-selectin for engraftment and retention in the BM microenvironment. Disrupting these niche interactions could represent new avenues for targeting CML stem cells that are resistant to ABL kinase inhibitors like imatinib. Disclosures:Van Etten:Bristol Myers Squibb: Consultancy; Deciphera Pharmaceuticals: Consultancy; TEVA Pharmaceuticals: Consultancy, Research Funding.

Low Expression Of The FUCA1 Gene Is An Adverse Prognostic Factor In Myeloma and Combined With High Sialyltransferase Gene Expression Identifies Patients At Increased Risk Of Early Disease Progression and Death

BackgroundGlycosylation 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. MethodsAnalyzing 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. ResultsHigh 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. [Display omitted] ConclusionsAltered 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.

Silencing The Sialyltransferase Gene ST3GAL6 Inhibits Adhesion and Migration Of Myeloma Cells In Vitro and Reduces The Homing and Proliferation Of Tumor Cells In Vivo

BackgroundGlycosylation 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. MethodsMM 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). ResultsKnockdown 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. ConclusionsshRNA 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.

Peptide Vaccination in Montanide Adjuvant Induces and GM-CSF Increases CXCR3 and Cutaneous Lymphocyte Antigen Expression by Tumor Antigen–Specific CD8 T Cells

T cell infiltration of melanoma is associated with enhanced clinical efficacy and is a desirable endpoint of immunotherapeutic vaccination. Infiltration is regulated, in part, by chemokine receptors and selectin ligands on the surface of tumor-specific lymphocytes. Therefore, we investigated the expression of two homing molecules--CXCR3 and CLA - on vaccine-induced CD8 T cells, in the context of a clinical trial of a melanoma-specific peptide vaccine. Both CXCR3 and CLA have been associated with T cell infiltration of melanoma. We demonstrate that a single subcutaneous/intradermal administration of peptide vaccine in Montanide adjuvant induces tumor-specific CD8 T cells that are predominantly positive for CXCR3, with a subpopulation of CXCR3(+)CLA(+) cells. Addition of GM-CSF significantly enhances CXCR3 expression and increases the proportion of CLA-expressing cells. Concurrent with CXCR3 and CLA expression, vaccine-induced CD8 cells express high levels of Tbet, IFN-γ, and IL-12Rβ1. Collectively, these studies demonstrate that peptide vaccination in adjuvant induces CD8 T cells with a phenotype that may support infiltration of melanoma.

Human multipotent adult progenitor cells transcriptionally regulate fucosyltransferase VII

Background aimsTargeted recruitment of leukocytes to sites of inflammation is a crucial event in normal host defense against pathogens, and attachment to and rolling on activated endothelial cells is a prerequisite first step for eventual leukocyte extravasation into sites of inflammation. These key events are mediated by interactions between glycosylated ligands expressed on leukocytes and selectins expressed on activated endothelium. Cell surface expression of selectin ligands on leukocytes is regulated by the rate-limiting enzyme fucosyltransferase VII (Fut7), and in its absence extravasation of leukocytes is severely inhibited. Multipotent adult progenitor cells (MAPCs) are an adherent cell population isolated from adult bone marrow. Intravenous administration of MAPCs provided functional improvement in multiple pre-clinical models of injury or disease, but the mechanisms by which these outcomes were achieved remain poorly understood. MethodsIn vitro cell analysis studies including fluorescence-activated cell sorting, messenger RNA analysis, T-cell proliferation assays and endothelial cell binding assays were performed. ResultsThe in vitro cell analysis studies characterized the ability of MAPCs to secrete factors that transcriptionally attenuate expression of Fut7 in T cells, blocking the terminal fucosylation event in the biosynthesis of selectin ligands and reducing T-cell binding to endothelial cells. ConclusionsThis study presents the first example of a distinct regulatory mechanism involving transcriptional down-regulation of Fut7 by MAPCs that could modulate the trafficking behavior of T cells in vivo.

α(1,3) Fucosyltransferases IV and VII Are Essential for the Initial Recruitment of Basophils in Chronic Allergic Inflammation

Basophils act as initiator cells for the development of IgE-mediated chronic allergic inflammation (IgE-CAI). However, detailed mechanisms of initial recruitment of basophils into the skin have yet to be clarified. Selectins mediate leukocyte capture and rolling on the vascular endothelium for extravasation. Counter-receptor activity of selectins is regulated by α(1, 3) fucosyltransferases (FTs) IV and VII. To clarify the contribution of selectin ligands regulated by FTs for initial basophil recruitment, IgE-CAI was induced in mice deficient in FT-IV and/or FT-VII genes. Although FT-IV(-/-) and FT-VII(-/-) mice exhibited comparable skin responses to wild-type mice, the FT-IV(-/-)/FT-VII(-/-) mice showed significantly impaired inflammation. Although the transfer of basophils to FcRγ(-/-) mice induced IgE-CAI, this induction was completely absent when basophils from FT-IV(-/-)/FT-VII(-/-) mice were transferred. L-selectin, but not P- and E-selectin, blocking Abs inhibited skin inflammation in vivo. P-selectin glycoprotein-1 (PSGL-1) antibody also ameliorated skin inflammation, and basophils were bound to L-selectin in a PSGL-1-dependent manner, which was regulated by FT-IV/VII. Functional PSGL-1 generated by basophil FT-IV/VII and its subsequent binding to L-selectin could be one of the essential steps required for initial basophil recruitment and the development of IgE-CAI in mice.

Fucosyltransferase VII improves the function of selectin ligands on cord blood hematopoietic stem cells

Selectins and their carbohydrate ligands mediate the homing of hematopoietic stem/progenitor cells (HSPCs) to the bone marrow. We have previously shown that ex vivo fucosylation of selectin ligands on HSPCs by α1,3 fucosyltransferase VI (FUT6) leads to improved human cord blood (CB)-HSPC engraftment in non-obese diabetic (NOD)/severe combined immune deficient (SCID) mice. In the present study, we determined whether surface fucosylation with α1,3 fucosyltransferase VII (FUT7), which is primarily expressed by hematopoietic cells, improves the function of selectin ligands on CB-HSPCs in comparison with FUT6. A saturating amount of either FUT6 or FUT7, which generates comparable levels of expression of fucosylated epitopes on CB CD34(+) cells, was used for these experiments. In vitro, FUT7-treated CB CD34(+) cells exhibited greater binding to P- or E-selectin than that of FUT6-treated CB CD34(+) cells under static or physiological flow conditions. In vivo, FUT7 treatment, like FUT6, improved the early engraftment of CB CD34(+) cells in the bone marrow of sublethally irradiated NOD/SCID interleukin (IL)-2Rγ(null) (NSG) mice. FUT7 also exhibited marginally-yet statistically significant-increased engraftment at 4 and 6 weeks after transplantation. In addition, FUT7-treated CB CD34(+) cells exhibited increased homing to the bone marrow of irradiated NSG mice relative to sham-treated cells. These data indicate that FUT7 is effective at improving the function of selectin ligands on CB-HSPCs in vitro and enhancing early engraftment of treated CB-HSPCs in the bone marrow of recipients.

E- and P-Selectins Are Essential for Repopulation of Chronic Myelogenous and Chronic Eosinophilic Leukemias in a Scid Mouse Xenograft Model

In chronic myelogenous (CML) and chronic eosinophilic leukemia (CEL), neoplastic cells spread via the circulation into various extramedullary organs. As E- and P-selectin constitute the starting point for the leucocyte adhesion/invasion cascade, and CEL and CML cells share many properties with normal granulocytes, we investigated the role of these selectins in CEL and CML cell expansion and organ invasion in a xenotransplantation model using scid mice. Using two human leukemic cell lines (EOL-1 and K562), we were able to show that E- and P-selectins mediate leukemia cell tethering and adherence in a laminar flow assay. While E-selectin binding depended on sialylated carbohydrate moieties, P-selectin binding was completely (K562) or partially (EOL-1) independent of these carbohydrates indicating the involvement of non-canonical selectin ligands. In a xenograft model in scid mice, both cell lines invaded the bone marrow and other organs, formed chloromas, and ultimately produced an overt leukemia. In contrast, in E- and P-selectin knockout scid mice, the cells failed to show engraftment in 8 out of 10 animals and even if they did engraft, they produced only little organ invasion and chloroma formation. Together, these data suggest that E- and P-selectins play an important role in leukemic dissemination in CML and CEL.