Siglec-15 Expression Research Articles

Abstract 3151: Quantitative measurement of Siglec-15 expression in non-small cell lung cancer and its association with PD-L1, B7-H4 and tumor infiltrating lymphocytes

Abstract Introduction: Siglecs are sialic acid-binding transmembrane receptors that regulate the functions of innate and adaptive immune system through the recognition of glycan ligands. Siglec-15 has been recently described as an immune suppressive molecule that shares molecular structure with PD-L1, but its role in NSCLC is still unknown. In this study, we determined Siglec-15 expression in three retrospective NSCLC cohorts and evaluated its association with mutation status, major clinicopathologic characteristics and survival. Experimental procedures: We used multiplexed automated quantitative immunofluorescence (QIF) to develop a validated assay for Siglec-15 measurement and used it to assess Siglec-15 expression and its association to major clinicopathologic variables and survival in three NSCLC cohorts of over 600 patients. Additionally, we investigated the correlation of Siglec-15 expression with tumor-infiltrating lymphocytes, PD-L1, B7-H3 and B7-H4 tumor expression. Results: In our study, Siglec-15 tumor positivity was found in 12.5%, 13.7% and 22.8% of NSCLC cohort patients. Siglec-15 expression was higher in EGFR mutant and EGFR/KRAS wild type tumors compared to KRAS mutants but was not associated with any other major clinicopathological characteristics. Siglec-15 followed a mutually exclusive pattern of expression with PD-L1, B7-H3 and B7-H4 (co-expression in 3.1%, 13.2% and 7.7% of the patients respectively), while high levels were not correlated with lymphocyte infiltration or T-cell activation, suggesting a different upregulation mechanism not mediated by IFN-gamma. Conclusions: Siglec-15 is a protein expressed with high frequency in NSCLC. Co-expression of Siglec-15 with PD-L1, B7-H3 and B7-H4 is relatively rare, suggesting that Siglec-15 has distinct and non-redundant features compared to other B7 family members. Citation Format: Maria Toki, Jon Zugazagoitia, Mehmet Altan, Linda Liu, Nikita Mani, Yuting Liu, Konstantinos Syrigos, Lieping Chen, Solomon Langermann, Roy Herbst, David Rimm. Quantitative measurement of Siglec-15 expression in non-small cell lung cancer and its association with PD-L1, B7-H4 and tumor infiltrating lymphocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3151.

Abstract 4151: Siglec-15 is a novel immunomodulatory effector of adaptive immune evasion in acute lymphoblastic leukemia

Abstract Despite advances in therapy for acute lymphoblastic leukemia (ALL), it remains a leading cause of illness-related death in children and has poor long term disease-free survival and prognosis in recurrence in adults. Recent developments in immune-based therapies, such as CAR T cells, have shown great promise, but immune evasion mechanisms of leukemia remain poorly understood, and alternative strategies to engage the immune system are needed. Our group has characterized a role for calcineurin (CN) as a driver of a potent immune escape mechanism in an aggressive murine model of BCR-ABL+ ALL. We performed RNA-seq analysis of control and CN-deficient ALL cells to identify downstream mediators of this effect. Siglec-15 emerged as one of the most differentially expressed genes, being preferentially downregulated in the more immunogenic CN-deficient cells. The role of this Siglec-family immunomodulator is yet unstudied in the context of leukemia and could represent a novel mechanism for immune evasion. We first validated lower expression of Siglec-15 in CN-deficient leukemia cells by RT-qPCR. We next mined publicly available databases, which revealed higher expression of Siglec-15 in primary B-ALL and AML leukemias as compared to normal PBMCs, as well as in post-chemotherapy MRD samples as compared to at-diagnosis samples. Western blot analysis of human leukemia cell lines demonstrated upregulation of Siglec-15 expression in B-ALL, some AML, as well as DLBCL, as compared to normal PBMCs. To determine if Siglec-15 mediates immune evasion in vivo, we knocked down Siglec-15 in the murine model of BCR-ABL+ ALL, which resulted in rapid suppression of the leukemia following engraftment in immunocompetent mice, an effect lost in immunocompromised counterparts. Preliminary studies with cells in which Siglec-15 was knocked-out using CRISPR/Cas9 revealed similar results. Importantly, Siglec-15-deficient cells do not have any changes in proliferation or susceptibility to DNA damage-induced apoptosis. To address the capacity for therapeutic targeting of Siglec-15, mice with parental leukemia were treated with a monoclonal blocking antibody against Siglec-15, yielding an overall reduction in leukemic burden and prolonged survival. Taken together, these results suggest that Siglec-15 is a novel immunomodulatory effector and has a therapeutically exploitable role in immune evasion of ALL. Citation Format: Claire Pillsbury, Jairo A. Fonseca, Jodi Dougan, Lori Gardner, Jennifer Rabe, Christy Gearheart, Chris C. Porter. Siglec-15 is a novel immunomodulatory effector of adaptive immune evasion in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4151.

Impaired osteoclast differentiation and function and mild osteopetrosis development in Siglec-15-deficient mice

Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is a cell surface receptor for sialylated glycan ligands. Recent in vitro studies revealed upregulated Siglec-15 expression in differentiated osteoclasts and inhibition of osteoclast differentiation by anti-Siglec-15 polyclonal antibody, demonstrating Siglec-15 involvement in osteoclastogenesis. To discern the physiological role of Siglec-15 in skeletal development and osteoclast formation and/or function in vivo, we generated Siglec-15-deficient (siglec-15−/−) mice and analyzed their phenotype. The siglec-15−/− mice developed without physical abnormalities other than increased trabecular bone mass in lumbar vertebrae and metaphyseal regions of the femur and tibia, causing mild osteopetrosis. Histological analyses demonstrated that the number of osteoclasts present on the femoral trabecular bone of the mutant mice was comparable to that of the wild-type mice. However, urinary deoxypyridinoline, a systemic bone resorption marker, decreased in the siglec-15−/− mice, indicating that impaired osteoclast function was responsible for increased bone mass in the mutant mice. In addition, the ability of bone marrow-derived monocytes/macrophages from the siglec-15−/− mice to differentiate into osteoclasts was impaired, as determined in vitro by cellular tartrate-resistant acid phosphatase activity in response to the receptor activator of nuclear factor-κB ligand or tumor necrosis factor-α. These results reveal the importance of Siglec-15 in the regulation of osteoclast formation and/or function in vivo, providing new insights into osteoclast biology.

Siglec-15 Protein Regulates Formation of Functional Osteoclasts in Concert with DNAX-activating Protein of 12 kDa (DAP12)

Osteoclasts are multinucleated giant cells that reside in osseous tissues and resorb bone. Signaling mediated by receptor activator of nuclear factor (NF)-κB (RANK) and its ligand leads to the nuclear factor of activated T cells 2/c1 (NFAT2 or NFATc1) expression, a critical step in the formation of functional osteoclasts. In addition, adaptor proteins harboring immunoreceptor tyrosine-based activation motifs, such as DNAX-activating protein of 12 kDa (DAP12), play essential roles. In this study, we identified the gene encoding the lectin Siglec-15 as NFAT2-inducible, and we found that the protein product links RANK ligand-RANK-NFAT2 and DAP12 signaling in mouse osteoclasts. Both the recognition of sialylated glycans by the Siglec-15 V-set domain and the association with DAP12 through its Lys-272 are essential for its function. When Siglec-15 expression was knocked down, fewer multinucleated cells developed, and those that did were morphologically contracted with disordered actin-ring structures. These changes were accompanied by significantly reduced bone resorption. Siglec-15 formed complexes with Syk through DAP12 in response to vitronectin. Furthermore, chimeric molecules consisting of the extracellular and transmembrane regions of Siglec-15 with a K272A mutation and the cytoplasmic region of DAP12 significantly restored bone resorption in cells with knocked down Siglec-15 expression. Together, these results suggested that the Siglec-15-DAP12-Syk-signaling cascade plays a critical role in functional osteoclast formation.

Aspirin Plain and Buffered

Siglec-15 is a conserved sialic acid–binding Ig-like lectin, which is expressed on osteoclasts. Deficiency of Siglec-15 leads to an impaired osteoclast development, resulting in a mild osteopetrotic phenotype. The role of Siglec-15 in arthritis is still largely unclear. To address this, we generated Siglec-15 knockout mice and analyzed them in a mouse arthritis model. We could show that Siglec-15 is directly involved in pathologic bone erosion in the K/BxN serum-transfer arthritis model. Histological analyses of joint destruction provided evidence for a significant reduction in bone erosion area and osteoclast numbers in Siglec-15^−/− mice, whereas the inflammation area and cartilage destruction was comparable to wild-type mice. Thus, Siglec-15 on osteoclasts has a crucial function for bone erosion during arthritis. In addition, we generated a new monoclonal anti–Siglec-15 Ab to clarify its expression pattern on immune cells. Whereas this Ab demonstrated an almost exclusive Siglec-15 expression on murine osteoclasts and hardly any other expression on various other immune cell types, human Siglec-15 was more broadly expressed on human myeloid cells, including human osteoclasts. Taken together, our findings show a role of Siglec-15 as a regulator of pathologic bone resorption in arthritis and highlight its potential as a target for future therapies, as Siglec-15 blocking Abs are available.