1046 - Unveiling the Role of Siglec-6 in Regulating B Cell Functions: Implications for Chronic Lymphocytic Leukemia Therapeutics

B cell Chronic lymphocytic leukemia (B-CLL) is a malignancy of CD5+CD23+CD19+ B lymphocytes. It is the most prevalent adult leukemia in the western world, accounting for more than 4000 deaths in the United States every year. It is characterized by activated B cell receptor (BCR) signaling that in turn promotes several downstream cell survival pathways and migration to pro-survival niches such as the bone marrow, lymph nodes and spleen tumor microenvironment (TME). The only curative therapy for CLL is allogeneic hematopoietic stem cell transplantation which is associated with undesirable risks for the elderly patients. BCR signaling inhibitors are effective agents for CLL treatment, but therapeutic resistance and relapse remain problematic, necessitating the identification of new CLL targets that can bypass current disease resistance. Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are a family of cell surface glycoproteins that were shown to play a role in tumor immunosurveillance and immunosuppression, thereby making them attractive anti-cancer targets. Our study focuses on evaluating a novel Siglec member, Siglec-6, which was recently found to be upregulated on B cells from CLL patients but not on healthy donor B cells. To study the biological role of Siglec-6, we generated a novel human (hu) Siglec-6 transgenic mouse model that expresses huSiglec-6 on B cells. We observed a selective decrease in antigen specific immunoglobulin responses in response to T independent and T dependent antigens in the huSiglec-6 mice. Interestingly, decreased proliferation of B cells in response to B cell activation was observed in huSiglec-6 mice, pointing to a role for Siglec-6 as a negative regulator of B cell immune response and proliferation. The molecular signals that direct B-CLL trafficking to the TME is an ongoing field of study, and interfering with B-CLL cell migration to or adhesion within these organs serves to target and eliminate survival of B-CLL cells. We show for the first time that CLL patient derived bone marrow stromal cells (BMSCs) express the ligand of Siglec-6, sialyl Tn (sTn). Mass spectrometry analysis revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. We utilized the CRISPR-Cas9 system to generate novel MEC1-002 Siglec-6 and DOCK8 knock-out cell lines to explore the signaling pathways involved in Siglec-6 functions. Genetic ablation or antibody blocking revealed a novel role for Siglec-6 in migration and adhesion of B-CLL cells to CLL-BMSCs in vitro and compromised migration to bone marrow and spleen in vivo. Stimulation of MEC1-002 CLL cells with sTn resulted in Cdc42 activation, WASP protein recruitment and F-actin polymerization, which are all associated with cell migration. To evaluate the therapeutic potential of Siglec-6, we crossed our huSiglec-6 transgenic mouse model with the TCL1 CLL mouse model to obtain Siglec-6 x TCL1 mice which develop Siglec-6+ leukemia. An immunocompetent mouse model using humanized CD3 mice engrafted with Siglec-6+ leukemia showed improved overall survival when treated with a huSiglec-6/huCD3-bispecific T-cell-recruiting antibody (T-biAb). This T-biAb also induced rapid killing of CLL cells in a CLL patient-derived xenograft model.
Taken together, our studies uncover a novel role for Siglec-6 in regulating the BCR signaling pathway, proliferation of B cells, and homing of B-CLL cells to pro-survival niches. As therapies that target Siglecs become more common for the treatment of various types of cancer, our need to understand the biological role of these Siglecs and our search for new drug targets intensify. Our ability to adapt current treatment regimens to newly discovered targets is paramount to effective treatment of CLL patients, and understanding how these new disease targets regulate CLL function is thus a driving factor in taking these therapies to clinical trials. Our findings thus suggest that Siglec-6 is an effective therapeutic target for the treatment of CLL patients and warrants further exploration into the exact mechanism by which Siglec-6+ CLL cells interact with the TME, and the biological role of Siglec-6 in other leukemias.