Defining the role of Siglec-6-DOCK8 axis in cell adhesion and migration in Chronic Lymphocytic Leukemia

Abstract Siglec-6 is a sialic acid-binding immunoglobulin-like lectin that is involved in diverse functions in trophoblasts. We recently identified expression of Siglec-6 on B cells from chronic lymphocytic leukemia (CLL) patients (B-CLL cells) but not normal donors. To determine the role of Siglec-6 in B-CLL cells, we used the CRISPR-Cas9 technique to knock-out (KO) Siglec-6 in a human CLL cell line, MEC1. We then evaluated the migratory role of Siglec-6+MEC1 cells towards sialyl-Tn (sTn), a known Siglec-6 ligand, or sTn+ CLL patient derived bone marrow stromal cells (BMSCs). Compared to Siglec-6+ MEC1 cells, Siglec-6− MEC1 cells exhibited significant reduction in adhesion to and migration towards sTn or sTn+ CLL-BMSCs in cell adhesion and trans-well migration assays. Interestingly, sTn is overexpressed on CLL-BMSCs compared to normal donor derived BMSCs. Importantly, a Siglec-6 targeted antibody inhibited homing of Siglec-6+ MEC1 cells to the spleen and bone marrow in NSG mice. Mass spectrometry and co-immunoprecipitation analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6+ MEC1 cells with sTn resulted in Cdc42 activation, a downstream target of DOCK8, and actin polymerization which was compromised in Siglec-6 or DOCK8 KO MEC1 cells. In summary our studies have identified a novel role for Siglec-6 and its ligand in CLL through promotion of Siglec-6 and DOCK8 dependent activation of Cdc42, actin polymerization, cell migration, and subsequent attachment of B-CLL cells to CLL-BMSCs. Ongoing studies are focused on molecular mechanisms of Siglec-6 mediated regulation of actin polymerization and CLL-BMSC interactions in cell migration and adhesion.