To Catch a Killer: Delineating the Mechanistic Role of Siglec-6, a Novel Glycoprotein Target, for the Treatment of Chronic Lymphocytic Leukemia

Introduction: Sialic-acid-binding immunoglobulin-like lectins (Siglec) are a family of cell surface glycoproteins characterized by their cell-type specific expression and ability to modulate receptor signaling. We and others have shown overexpression of Siglec-6, a member of Siglec family, on B cells from patients with chronic lymphocytic leukemia (CLL) compared to healthy donor derived B cells. While placental expression of Siglec-6 has been shown to regulate invasion of trophoblast cells, the biochemical role of Siglec-6 in B cells in CLL patients is not known. We describe here for the first time the functional and therapeutic relevance of Siglec-6 in cell adhesion and migration in CLL. Biochemical mechanisms of Siglec-6 mediated cell adhesion and migration through Siglec-6 ligand (sialyl Tn) mediated DOCK8 dependent activation of Cdc42 associated with actin polymerization are presented. To evaluate the therapeutic potential of Siglec-6, we generated a human (hu) Siglec-6 transgenic mouse model and crossed it with the well-known TCL1 CLL mouse model to obtain Siglec-6 x TCL1 mice which develop Siglec-6+ leukemia. An in-vivo study with anti-Siglec-6/CD3 targeted bispecific antibody (BsAb) showed a survival benefit in humanized CD3 (huCD3) mice engrafted with Siglec-6+ leukemic cells. These studies thus elucidate the biological role of Siglec-6 in malignant CLL B cells and demonstrate therapeutic opportunities targeting Siglec-6 in CLL.

Methods: Flow cytometry was used to analyze cell surface expression of Siglec-6 and sialyl Tn (sTn). Transwell migration assay was used to assess in-vitro migratory role of Siglec-6. We performed mass spectrometry analysis to identify Siglec-6 interacting proteins. CRISPR/Cas9 technique was used to generate knock-out (KO) cell lines for mechanistic studies. Phalloidin staining followed by confocal imaging was used to examine actin polymerization. For in-vivo migration assays, CLL cells were blocked with an isotype antibody or Siglec-6 targeted antibody and injected into the tail vein of NSG mice. Spleens and bone marrow were processed 24 hrs later to determine the number of CLL cells that have migrated. For in-vivo BsAb treatment experiments, huCD3 mice were engrafted with 5 million Siglec-6+ leukemic cells isolated from Siglec-6 x TCL1 mouse splenocytes. Weekly treatment (i.v) was started after mice display 5% circulating leukemia.

Results: We confirmed Siglec-6 overexpression on B cells and sTn expression on bone marrow stromal cells (BMSCs) from CLL patients when compared to normal donor derived B cells and BMSCs respectively. Compared to Siglec-6⁺ CLL cells, Siglec-6^- CLL cells exhibited significant reduction in adhesion to (~50%) and migration towards (~50%) CLL-BMSCs. Importantly, a Siglec-6 targeted antibody inhibited homing of Siglec-6⁺ MEC1 cells and primary CLL cells to the spleen and bone marrow in NSG mice (~35%). 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 and WASP protein recruitment, followed by increased actin polymerization, that were compromised in Siglec-6 or DOCK8 KO MEC1 cells. At 9 months, Siglec-6 x TCL1 mice have ~25% more circulating leukemic cells when compared to TCL1 mice (n=4), suggesting that Siglec-6 may accelerate leukemogenesis in CLL. Finally, Siglec-6+ leukemia engrafted huCD3 mice treated with anti-Siglec-6/CD3 BsAb has so far demonstrated a survival benefit with median survival of 18 weeks versus a median survival of 8 weeks in the control group that received a non-targeting BsAb (n=3/group).

Conclusions: In summary, we have for the first time shown Siglec-6 dependent recruitment of DOCK8 leading to migration and adhesion of B-CLL cells. Siglec-6 signals via DOCK8 to mediate sTn ligand dependent actin polymerization via Cdc42 activation and WASP protein recruitment. Moreover, all these effects were prevented by CRISPR/Cas9 mediated knock out of Siglec-6 or DOCK8 in MEC1 CLL cell line. Siglec-6 x TCL1 mice demonstrate accelerated disease progression, and an anti-Siglec-6/CD3 BsAb provides a survival benefit against Siglec-6+ leukemia. Ongoing studies are focused on studying the complex interactions between Siglec-6+ CLL cells and the bone marrow niche.

Byrd:Janssen: Consultancy; Zencor: Research Funding; Kura: Consultancy; Vincerx: Consultancy, Current equity holder in private company, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company; Pharmacyclics: Research Funding; AstraZeneca: Consultancy; AbbVie: Consultancy; Newave: Consultancy; Ohio State University: Patents & Royalties; Trillium: Consultancy; Syndax: Consultancy; Novartis: Consultancy. Rader:T-CURX: Current equity holder in publicly-traded company; RiverVest Ventures: Membership on an entity's Board of Directors or advisory committees; Pyxis Oncology: Membership on an entity's Board of Directors or advisory committees; Alnylam Pharmaceuticals: Research Funding.