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Desialylation of MM overcomes bortezomib resistance in vivo
Additional information concerning materials and meth- ods can be found in the Online Supplementary Appendix.
Results
Treatment of mice with 3Fax-Neu5Ac causes a dose dependent decrease in sialoside expression on multiple organs systemically
Building upon the previous 3Fax-Neu5Ac in vivo experi- ence,25,26 we first studied the effects of global desialylation in vivo after systemic administration of 3Fax-Neu5Ac. Mice were treated with 6.25, 12.5, and 25 mg/kg of 3Fax-Neu5Ac daily for seven consecutive days. Sialylation was monitored using two different lectins after seven days of treatment: the Sambucus nigra lectin (SNA), which binds α2-6 linked sialic acids, and the Peanut agglutinin lectin (PNA), which binds to desialylated T antigen. In mice treated with 25 mg/kg 3Fax-Neu5Ac, there was a clear decrease in SNA staining in the kidney, spleen and liver (Online Supplementary Figure S1A-C), consistent with a reduction in α2-6 linked sialic acid expression. Moreover, at the same dose, there was a contemporary increase in PNA staining (Online Supplementary Figure S2A-C) consis- tent with decreased sialic acid expression leading to expo- sure of terminal galactose residues, such as the T antigen (Galβ1-3GalNAcαSer/Thr). To determine the effects of 3Fax-Neu5Ac treatment on sialylation of cells of the immune system, peripheral blood B cells were used as rep- resentative immune cells. The median fluorescent intensi- ty (MFI) values for SNA and PNA positive staining were determined on the seventh day of treatment and the sev- enth day after the final treatment. Similar to what was observed in histology sections, 3Fax-Neu5Ac treatment induced a decrease in the SNA MFI with the highest dose (25 mg/kg) having a significant fold change compared to the control at the seventh day of dosing (Figure 1A). As expected, the PNA lectin MFI significantly increased with 3Fax-Neu5Ac treatment by the final day of treatment as well (Figure 1C). Sialic acid expression remained low after seven days of recovery after the 25 mg/kg dose, as partic- ularly evident in cells stained with PNA, although a trend was also observed in the SNA-stained cells (Figure 1B and D), suggesting that recovery takes longer than seven days. Because sialylation is crucial to kidney filtration, we also determined the effects of the dose regimen in the kidneys. H&E staining showed no obvious histological changes in the kidney after seven days of recovery (Figure 1E). However, mice that received the highest dose, 25 mg/kg, did experience edema in the peritoneal cavity, as previous- ly reported.27 These data demonstrate that 3Fax-Neu5Ac can successfully inhibit the expression of sialic acid sys- temically, but that local BM- or myeloma-specific delivery may be necessary to overcome effects on other organs in future studies.
Treatment of MM1SHeca452 with 3Fax-Neu5Ac decreases sialylation in vitro
We next examined whether 3Fax-Neu5Ac could signifi- cantly reduce the expression of sialic acid on MM1SHeca452 cells. These cells are enriched for E-selectin ligand expres- sion compared to the MM1S parental line and in vivo gen- erate a very aggressive disease which displays resistance to bortezomib.14 The MM1SHeca452 cell line has been exten- sively characterized; their sensitivity to bortezomib,
clonogenic potential, and proliferation in vitro are identical to the parental line and their aggressive phenotype becomes evident only in vivo.14 Sialylation was monitored using the Heca452 and CD15s antibodies, which recog- nize the sialofucosylated structure SLea/x, the Maackia Amurensis Lectin II (MAL II), which preferentially binds to α2-3 linked sialic acid, and SNA. Over a seven day span in culture, 300 μM of 3Fax-Neu5Ac significantly decreased the Heca452 staining in a time-dependent manner (Online Supplementary Figure S3). After seven days of treatment, 3Fax-Neu5Ac decreased the MFI and the total number of the Heca452, CD15s, MALII and SNA positive cells (Figure 2). Importantly, 3Fax-Neu5Ac treatment did not induce a significant change in the sensitivity to borte- zomib in vitro (Figure 4). Based on these data, we chose to pre-treat the MM1SHeca452 cells with 300 μM of 3Fax-Neu5Ac for seven days to significantly reduce sialylation on the cell surface.
In vivo, pre-treatment of MM1SHeca452 cells with 3Fax-Neu5Ac reduces tumor burden and increases survival, and co-treatment with bortezomib further enhances these outcomes
To study the impact of global sialylation inhibition specifically on MM cells and to avoid kidney toxicity relat- ed to 3Fax-Neu5Ac treatment, we pre-treated the MM1SHeca452 cells with 300 μM of 3Fax-Neu5Ac or vehicle for seven days, inoculated these cells into immunocom- promised mice, and followed tumor burden using biolu- minescence imaging. Mice inoculated with 3Fax-Neu5Ac- pre-treated MM1SHeca452 (3Fax-Neu5Ac MM1SHeca452 mice) showed reduced tumor burden compared to mice inocu- lated with vehicle-pre-treated MM1SHeca452 (vehicle MM1SHeca452 mice) (Figure 3A and B). Two cohorts in this study in addition received bortezomib treatment, which decreased the tumor burden in the vehicle and 3Fax-Neu5Ac MM1SHeca452 mice (Figure 3A and B). The 3Fax-Neu5Ac MM1SHeca452 mice that received bortezomib treatment had the least tumor burden throughout the study compared to the other groups (Figure 3B). Importantly, even in the absence of bortezomib, pre-treat- ment of MM1SHeca452 cells with 3Fax-Neu5Ac reduced tumor burden compared to vehicle MM1SHeca452 mice, suggesting that 3Fax-Neu5Ac pre-treatment is beneficial even without the addition of chemotherapy. We also observed that the 3Fax-Neu5Ac MM1SHeca452 mice survived significantly longer than the vehicle MM1SHeca452 mice (Figure 3C). Notably, bortezomib treatment did not prolong survival of the vehicle MM1SHeca452 mice, confirming our previous observation that in this in vivo model, these MM cells are more refractory to bortezomib treatment (Figure 3C).14 Above all, pre-treatment of MM1SHeca452 cells with 3Fax-Neu5Ac in combination with bortezomib led to longer survival compared to the other groups, suggesting a synergistic therapeutic effect. No significant difference was observed in change in body weight between the treatment groups (Figure 3D). Overall, we demonstrate that 3Fax-Neu5Ac reduces tumor burden and increases sur- vival, and that additional treatment with bortezomib has a synergistic effect with 3Fax-Neu5Ac.
3Fax-Neu5Ac treatment partially reverts stroma but not endothelial-induced bortezomib resistance in vitro
To gain insight into the mechanism(s) of increased bortezomib sensitivity in response to 3Fax-Neu5Ac treat-
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