Targeting Siglec Receptors to Reverse Immune Dysfunction Mediated By Plasmacytoid Dendritic Cells in Multiple Myeloma

In multiple myeloma (MM), plasmacytoid dendritic cells (pDCs) promote the growth of cancerous plasma cells and contribute to the immunosuppressive microenvironment. However, the mechanisms of this and how to target them are not well understood. Therefore, we characterized the role of pDCs in MM pathogenesis using single-cell RNA sequencing of bone marrow cells from relapsed/refractory MM patients (n=3) and healthy donors (n=3). One of the most striking perturbations we observed in pDCs from patients vs healthy donors was in the sialylation pathway.

Sialylation, which is the addition of sialic acid to glycoproteins, is a post-translational modification found on proteins on the cell surface. Hypersialylation is common in a variety of cancers, including multiple myeloma (MM), and it contributes to trafficking, drug resistance and immune escape of tumor cells. The Siglec family of receptors recognizes sialylated glycans on the surface of cells and, in response, transmits inhibitory signals that reduce the activation and proliferation of immune cells. Most of the SIGLEC genes are upregulated in the MM bone marrow microenvironment with SIGLEC6/CD327 being especially highly expressed and also upregulated in MM-pDCs vs normal pDCs (Log2FC: 1.291; adj p: 0.0001), as validated both by qPCR and flow cytometry (p < 0.05). Conversely, we found insignificant SIGLEC6/CD327 expression on myeloma cells. Several alpha-2-3 and alpha-2-6-sialyltransferases were also upregulated in MM bone marrow, of which ST3GAL6 was highly expressed in MM pDCs (Log2FC: 3.124 vs norm-pDC; adj p: 0.0009). In addition, the Reactome gene set ‘SIGLEC2/CD22-mediated B cell receptor regulation’ was especially upregulated in MM pDC (NES = 1.81). Therefore, we hypothesized that the Siglec receptors/Sialic Acid immune checkpoint could be targeted to counter MM-associated immunosuppression.

MM patient total BM-MNCs were treated with different concentrations of a sialyltransferase inhibitor, 3Fax-Neu5Ac, for 3 days. This treatment upregulated the activation markers CD83/CD86 on MM pDCs (1.5-fold ±SD; treated vs untreated, p < 0.05). It also triggered the activation of autologous T cells, as shown by the upregulation of CD69 on CD3⁺T cells and an increased population of CD3⁺/CD69⁺ T cells (MFI: 1.93-fold ±SD; %T cell proliferation: 1.77-fold ±SD; treated vs untreated, p < 0.05). Treatment of patient BM-MNCs with Siglec-6 antibody also upregulated CD83/CD86/HLA-DR on MM pDCs, indicating activation (1.25-1.75-fold ±SD; p < 0.05). Finally, we found that 3FaxNeu5Ac or anti-Siglec-6 Ab treatment induced autologous MM tumor cell killing in patient BM-MNCs (Viability: 55%, treated vs untreated, N = 5, p = 0.0233), and blockade of the Siglec/Sialic acid axis triggered MM NK cell-mediated lysis of target K562 cells. (Viability: 60%, treated vs untreated, N = 3, p < 0.05).

Conclusions Our results provide a comprehensive understanding of the role of Siglec6/CD327 in pDC-mediated immune suppression in MM and show that targeting the Siglec-Sialic Acid immune checkpoint may provide a novel therapy to enhance anti-MM immunity.

Anderson:Genentech: Consultancy; Pfizer: Consultancy; Janssen: Consultancy; Window: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; Amgen: Consultancy; Starton Therapeutics: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Dynamic Cell Therapies: Membership on an entity's Board of Directors or advisory committees.