Abstract
Neuroblastoma cells highly express the disialoganglioside GD2, a tumor-associated carbohydrate antigen, which is only sparsely expressed on healthy tissue. GD2 is a primary target for the development of immunotherapy for neuroblastoma. Immunotherapy with monoclonal anti-GD2 antibodies has proven safety and efficacy in clinical trials and is included in the standard treatment for children with high-risk neuroblastoma. Strategies to modulate GD2 expression in neuroblastoma could further improve anti-GD2-targeted immunotherapy. Here, we report that the cellular sialylation pathway, as well as epigenetic reprogramming, strongly modulates GD2 expression in human and mouse neuroblastoma cell lines. Recognition of GD2 by the 14G2a antibody is sialic acid-dependent and was blocked with the fluorinated sialic acid mimetic Ac53FaxNeu5Ac. Interestingly, sialic acid supplementation using a cell-permeable sialic acid analogue (Ac5Neu5Ac) boosted GD2 expression without or with minor alterations in overall cell surface sialylation. Furthermore, sialic acid supplementation with Ac5Neu5Ac combined with various histone deacetylase (HDAC) inhibitors, including vorinostat, enhanced GD2 expression in neuroblastoma cells beyond their individual effects. Mechanistic studies revealed that Ac5Neu5Ac supplementation increased intracellular CMP-Neu5Ac concentrations, thereby providing higher substrate levels for sialyltransferases. Furthermore, HDAC inhibitor treatment increased mRNA expression of the sialyltransferases GM3 synthase (ST3GAL5) and GD3 synthase (ST8SIA1), both of which are involved in GD2 biosynthesis. Our findings reveal that sialic acid analogues and HDAC inhibitors enhance GD2 expression and could potentially be employed to boost anti-GD2 targeted immunotherapy in neuroblastoma patients.
Highlights
Neuroblastoma cells highly express the disialoganglioside GD2, a tumor-associated carbohydrate antigen, which is only sparsely expressed on healthy tissue
Our findings reveal that sialic acid analogues and histone deacetylase (HDAC) inhibitors enhance GD2 expression and could potentially be employed to boost anti-GD2 targeted immunotherapy in neuroblastoma patients
Human IMR-32 cells were treated with the sialic acid inhibitor Ac53FaxNeu5Ac (100 M) for 3 days or with Clostridium perfringens sialidase for 1 h after which membrane GD2 expression was measured by flow cytometry using the anti-GD2 antibody 14G2a
Summary
The tumor antigen GD2 carries two sialic acid residues, one that is linked via an ␣2,3 linkage to galactose and a second one that is linked to the sialic acid via an ␣2,8 linkage (Fig. 1A). The enhanced GD2 expression induced by vorinostat resulted in improved antiGD2 antibody–mediated ADCC and reduced tumor growth [27]; the vorinostat-increased GD2 expression in 9464D neuroblastoma cells was caused by increased GD2 synthase protein but not mRNA levels These findings prompted us to assess the combined effect of sialic acid analogues and HDAC inhibitors on GD2 expression in murine 9464D and the human IMR-32 and SK-N-AS cell lines. Expression of GD1b synthase (B3GALT4), which converts GD2 to GD1b, was not detected in IMR-32 cells (data not shown) These findings show that the combination of increased intracellular CMP–sialic acid levels and increased ST3GAL5 and ST8SIA1 expression induced by Ac5Neu5Ac and HDAC inhibition, respectively, results in increased expression of GD2 on neuroblastoma cells
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