Page 224 - 2020_02-Haematologica-web

A. Natoni et al.
ity in MM include matrix metalloproteinase and integrin α4β1-dependent adhesion on fibronectin and Vascular cell adhesion molecule 1 (VCAM1).10-12 Recently, E-selectin has also been shown to play a role in homing and retention of MM cells in the BM.13,14 In particular, we have shown that sialofucosylated structures recognized by E-selectin, such as Sialyl Lewisa/x (SLea/x), enable MM cells to escape the cytotoxic effects of bortezomib in vivo most likely by hid- ing in the BM.14 Indeed, MM cells enriched for E-selectin ligands recognized by the monoclonal antibody Heca452, were resistant to bortezomib treatment in vivo and this resistance was reversed by a small glycomimetic molecule GMI-1271, which inhibits the interaction between E-selectin and E-selectin ligands.14 Thus, SDF1α and E-selectin may act co-operatively to allow extravasation of MM cells into the BM niche where they can proliferate and evade drug treatments.
Post-translational glycosylation of proteins and lipids plays many physiological and pathophysiological roles. There is a growing appreciation that aberrant glycosyla- tion is considered a hallmark of cancer,15,16 with one of the most prominent changes being a role for hypersialylation as a driver of tumor progression, metastasis and inva- sion.17,18 Hypersialylation is largely the result of overex- pression of sialyltransferases (STs), which catalyze the attachment of sialic acids via different glycosidic linkages (α2-3, α2-6, or α2-8) to the underlying glycan chain.17,19 We have previously established an important role for aberrant sialylation in homing and survival in MM.20 Specifically, high expression of the ST3 β-galactoside α2-3-sialyltrans- ferase 6 (ST3GAL6) in MM cell lines and patient samples is associated with inferior outcomes. Knocking down ST3GAL6 reduces sialic acid expression on MM cells, decreasing their ability to home to the BM. Since ST3GAL6 participates in the generation of SLea/x struc- tures, which forms the minimal E-selectin ligand, and may also be involved in sialylation of other structures impor- tant in MM homing and adhesion,21-23 we sought to inves- tigate if we could therapeutically target sialylation on MM cells, and whether this would affect BM homing and sur- vival in mice.
Here we show that pre-treatment of MM cells enriched for E-selectin ligands with 3Fax-Neu5Ac, a global inhibitor of the ST family,24 significantly reduces cell surface sialyla- tion of these cells, prolongs survival in xenograft mice and enhances their in vivo sensitivity to bortezomib. In vitro, 3Fax-Neu5Ac impairs the interaction between MM cells and E-selectin under shear stress and, surprisingly, also greatly reduces their interaction with VCAM1 and MAD- CAM1 under similar conditions. In this respect, we show that 3Fax-Neu5Ac alters the post-translational modifica- tion of integrin α4 on MM cells. This implies a dual effect on homing, whereby blockade of selectin ligands and inte- grin-mediated interactions with BM endothelial cells pre- vents extravasation of MM cells in the BM. Our results suggest great potential for improved patient outcomes by targeting sialylation on MM cells, especially when used in combination with other active MM agents.
Methods
Selection of E-selectin ligand-enriched cells
The E-selectin ligand-enriched MM1S cell line (MM1SHeca452) was generated from GFP+/Luc+ MM1S and
parental MM1S cell lines by two rounds of fluorescently- activated cell sorting (FACS) using the fluorescent Heca452 antibody (Biolegend; San Diego, CA, USA). Cells were maintained in RPMI-1640 (VWR; Radnor, PA, USA) containing L-glutamine, 10% heat inactivated fetal bovine serum (HI-FBS, VWR), and 1X antibiotic-antimycotic (Corning; Kennebunk, ME, USA).
Animal experiments
All experimental studies and procedures involving mice were performed in accordance with protocols approved by the governing Institutional Animal Care and Use Committee (IACUC) and all state and federal laws. In the toxicity study, 8-week old male and female C57BL/6J mice (n=8) received 0, 6.25, 12.5 or 25 mg/kg body weight doses of 3Fax-Neu5Ac (EMD Millipore; Burlington, USA) delivered intraperitoneally (i.p.) once daily for seven days. The drug was dissolved in dimethyl sulfoxide (DMSO) (VWR) and subsequently diluted 2-fold in PEG-300 (Sigma Aldrich; St. Louis, MO, USA). Mice were monitored daily for signs of discomfort, especially at the site of injection. In the homing study, 6-week old female Fox Chase SCID-Beige mice (Charles River Laboratory; Wilmington, MA, USA) (n=9 or 10) were inoculated via tail vein injections with 5x106 Heca452-enriched GFP+/Luc+ MM1S cells, which had been pre-treated with either vehicle or 300 μM 3Fax-Neu5Ac for seven days in culture before inoculation. Starting one day post inoculation, mice received either vehicle (PBS) or bortezomib (Selleck; Houston, TX, USA) injections intraperitoneally twice weekly. To monitor toxicity, mice were weighed twice weekly. Mice were frequently moni- tored for clinical signs of treatment-related side effects. Survival end points were mouse death or euthanasia as required by the IACUC (a single observation of >30% body weight loss, 3 consecutive measurements of >25% body weight loss, or impaired hind limb use). Survival differences were analyzed by the Kaplan-Meier method.
Bioluminescent imaging
Starting on day 7, and biweekly until day 30, tumor bur- den was assessed with bioluminescence imaging (BLI) in an IVIS® Lumina LT (Perkin Elmer Inc.; Waltham, MA, USA) equipped with a CCD camera (cooled at -90°C), mounted on a light-tight specimen chamber. Mice were injected with 150 mg/kg i.p. filter-sterilized D-luciferin substrate (VivoGlo, Promega; Madison, WI, USA) and imaged after 10 minutes. Data were acquired and ana- lyzed using LivingImage software 4.5.1. (PerkinElmer). BLI flux equaling the radiance (photons/s) in each pixel inte- grated over the region of interest (ROI) area (cm2), where the ROI was the whole mouse, was used to quantify tumor burden. BLI and mouse weight data were graphed and analyzed only for days in which all mice remained in the study to avoid artifacts due to mouse death.
Statistical analysis
All data are expressed as mean±standard error of the mean (SEM), unless otherwise noted. Student’s t-test, ordi- nary one-way or two-way ANOVA tests were used to determine significance, using P<0.05 as the cut-off, with Tukey’s multiple comparison post-hoc testing unless other- wise noted. **** P<0.0001; *** P<0.001; ** P<0.01; * P<0.05. GraphPad Prism 6.02 software (La Jolla, CA, USA) was used to compute all statistical calculations unless oth- erwise noted.
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