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Desialylation of MM overcomes bortezomib resistance in vivo
the aggressive nature of these cells. Indeed, 3Fax-Neu5Ac treatment reduced tumor burden, increased bortezomib sensitivity and, most importantly, improved survival, suggesting that sialylation contributes to the aggressive phenotype of the MM1SHeca452 cells and inhibit- ing it could represent a valuable treatment in MM. Currently, systemic administration of 3Fax-Neu5Ac is not feasible due to irreversible nephrotoxicity. While it is pos- sible that a different dose and schedule could reveal a ther- apeutic window, clearly the potential for off-target toxici- ty is a major obstacle to clinical development. This is par- ticularly relevant in MM where the kidney is one of the organs whose function is greatly impaired by the disease. However, alternative approaches could be explored to address this issue, including the development of more selective sialyltransferase inhibitors or the use of a target- ed delivery system, which would release 3Fax-Neu5Ac selectively into the BM microenvironment or the MM cells. Indeed, Bull et al. have previously reported that tar- geted delivery of antibody-labeled nanoparticles contain- ing 3Fax-Neu5Ac into melanoma cells facilitates long-term sialic acid blockade and, importantly, reduces lung metas- tasis in vivo.33 A similar strategy could be employed using antibodies specific to MM antigens (such as CD38 and BCMA) or by incorporating bisphosphonates into the nanoparticles to target the BM.34 Achievement of suffi- ciently high local BM concentrations of 3Fax-Neu5Ac should result in sialylation inhibition on MM cells, with- out off target toxicity. Inhibiting sialylation using these approaches could also target the tumor microenvironment including the immune environment. For instance, it has been recently reported that sialic acid blockade via intra- tumoral injection of 3Fax-Neu5Ac could suppress tumor growth by enhancing T-cell-mediated tumor immunity.35 In addition to a reduction in sialic acid expression by tumor cells, sialyltransferase inhibition converted the immune suppressive tumor microenvironment to an immune promoting one with significantly higher numbers of activated effector immune cells, including CD8+ T cells and natural killer (NK) cells, along with a reduction in reg- ulatory T cells (Tregs).35 Sialyltransferase inhibition also
led to anti-tumor effects, which were mediated by CD8+ effector cells as well as potential activation of stimulated dendritic cells (DC).35
A number of different mechanisms could account for the 3Fax-Neu5Ac-mediated increased-sensitization of the MM1SHeca452 cells to bortezomib in vivo. First, we explored the possibility that 3Fax-Neu5Ac could directly inhibit BM- mediated bortezomib resistance in vitro. HS5, patient- derived BMSC and the BM endothelial cell line BMEC-60 showed significant inhibition of bortezomib-induced cell death in MM1SHeca452. However, we observed that 3Fax- Neu5Ac induced only a partial re-sensitization to borte- zomib on HS5 and patient-derived BMSC, suggesting that desialylation plays a minor role in blocking BM-mediated drug resistance. The BM microenvironment can induce drug resistance through cell adhesion-mediated drug resistance (CAM-DR) and soluble factors.1,36-39 It is possible that in our in vitro model system, inhibition of sialylation is not enough to inactivate all the pathways responsible for the BM-mediated bortezomib resistance.1,39,40 Therefore, it is conceivable that the increased-sensitiza- tion to bortezomib observed in vivo may be predominantly due to mechanisms other than blockade of BM-mediated drug resistance. Nonetheless, an important future direc- tion will be to test 3Fax-Neu5Ac in in vitro models that more faithfully reproduce the tumor microenvironment to bet- ter understand the effects of 3Fax-Neu5Ac on the microen- vironment-mediated drug resistance.
Previously, we showed that in the same model system, the small molecule glycomimetic GMI-1271, which inhibits interactions between E-selectin and E-selectin lig- ands, could increase the number of MM cell in circulation, where they are more susceptible to bortezomib.14 In a sim- ilar way, we hypothesized that inhibition of sialylation by 3Fax-Neu5Ac could also inhibit homing and retention of MM cells in the BM. To this end, we examined the inter- action of vehicle or 3Fax-Neu5A-treated MM1SHeca452 with E-selectin under shear stress. We found that 3Fax-Neu5Ac treatment, by reducing SLea/x, effectively inhibited the interaction between the MM1SHeca452 cells and E-selectin, confirming previous observations.24 However, we rea-
A
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Figure 7. Post-translational modification of integrin α4 is altered by 3Fax-Neu5Ac treatment. Whole cell extracts from MM1SHeca452 cells treated for seven days with 300 μM 3Fax-Neu5Ac or dimethyl sul- foxide (DMSO) (vehicle control) were subjected to SDS PAGE, transferred to nitrocellulose membrane and blotted for integrin α4 (A), β1 (B), and β7 (C).
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