Modulation and exploitation of Siglec-6 function to broadly impede mast cell activation - PROJECT SUMMARY Mast cells are essential cellular effectors of a number of allergic and non-allergic diseases and conditions, ranging from anaphylaxis to pseudoallergic drug responses and chronic spontaneous urticaria to systemic mastocytosis. Therapeutic options for human mast cell-driven diseases and conditions generally focus on ameliorating symptoms rather than disease modification or cure, and many remain incompletely effective. In addition, because a number of different receptors may induce deleterious mast cell activation, a broadly applicable inhibitory strategy would offer far-reaching clinical benefits. Sialic acid-binding immunoglobulin-like lectin (Siglec)-6, a member of a receptor family characterized by binding to glycan chains that terminate with sialic acid, is highly, uniformly, and selectively present on human mast cell populations. Many Siglecs, including Siglec-6, possess cytoplasmic ITIMs and ITIM-like motifs and exhibit inhibitory activity when engaged. Indeed, we have shown that Siglec-6 can reduce mast cell activation through the IgE receptor, Mrgprx2, and C5aR upon ligation, presumably through the recruitment of inhibitory signaling proteins like protein tyrosine phosphatases to its cytoplasmic ITIM. Generally, however, ITIM-bearing receptors usually do not prevent cellular activation without co-associating with the relevant activating receptor. In addition, the abundant and selective expression of the receptor on human mast cells makes it an ideal target through which to deliver therapeutic cargo into mast cells. The overall goal of this proposal is to exploit the biology of Siglec-6 to prevent or treat mast cell-driven diseases or conditions. This concept will be explored in studies dedicated to understanding the signaling pathways initiated by ligation or co-ligation of Siglec-6 as well as the functional consequences of Siglec-6 upregulation on mast cells (Aim 1), understanding the endocytic pathway utilized by Siglec-6 and exploiting this pathway to deliver toxic cargo to malignant or disease-promoting mast cells (Aim 2), and exploiting Siglec-6 antibody engagement or co-engagement with activating receptors to prevent or treat harmful mast cell responses (Aim 3). A unique toolkit has been amassed to complete this study, including primary skin-derived human mast cells, human CD34+ cell-derived mast cells, human mast cell lines (HMC-1.2, LAD2, and ROSA KITD816V), cell lines transfected with Siglec-6 or mutants lacking an intact ITIM and/or ITSM, mice engineered to express human Siglec-6 in the mast cell compartment (driven by the Mcpt5 or Cpa3 promoters), liposomes bearing a neoglycolipid ligand of Siglec-6, and streptavidin complexes bearing anti- Siglec-6 mAb as well as agents targeted to various activating receptors found on mast cells. Experiments deploying these unique assets will provide mechanistic insight into the biology, signaling, pharmacology and immunology of the Siglec-6 receptor as a selective human mast cell target.
