The Immune Checkpoint Siglec-15 in Promoting Immune Dysregulation in Non-Hodgkin's Lymphomas

Abstract

Outcomes for patients with relapsed or refractory (r/r) hematologic malignancies remain dismal despite therapeutic advances over the last several years. This is particularly striking in patients with r/r aggressive mature B-cell lymphomas such as Diffuse Large B-cell Lymphoma (DLBCL) and Burkitt's Lymphoma (BL). With use of intensive multiagent chemo-immunotherapy, DLBCL, which accounts for approximately 10-20% of pediatric Non-Hodgkin's Lymphoma (NHL) cases and is the most common subtype in adults, is highly curable with 5-year event free survival >75%. However, approximately 30-40% of adults diagnosed with DLBCL will become refractory to or relapse after first-line treatment. Yet even with intensive salvage regimens, r/r DLBCL remains a clinical challenge. There remains an unmet need to identify specific defects in the immune response, among other pathways, that drive lymphomagenesis that will in turn inform the development of novel therapeutics to improve survival in this population of patients. Siglec-15 (Sig-15), a member of the sialic acid binding immunoglobulin-like lectin family of proteins, has recently been identified as a critical immune suppressor that is highly expressed in human cancers and intra-tumoral myeloid cells. Importantly, recent reports have demonstrated inhibition of Sig-15, has a restorative effect on local anti-tumor immune responses and abrogated tumor progression. While reported in solid malignancies and acute leukemias, a role for Siglec-15 in promoting disease progression in aggressive B-cell lymphomas has not yet been described. We have found higher expression of SIGLEC15 in DLBCL cell lines compared to normal B cells at the RNA level, through analyses of the publicly available ONCOMINE database. This was confirmed at the protein level by western blot demonstrating higher Sig-15 expression in various lymphoma cell lines (RAJI, SUDHL6, OCI-LY10, HBL-1) compared to healthy donor PBMCs. Further, immunohistochemistry evaluating Sig-15 expression was performed on a tumor microarray consisting of 139 cases of DLBCL from adult patients along with validation samples from 15 primary pediatric lymphoma cases (DLBCL, BL and Anaplastic Large Cell Lymphoma (ALCL)). Sig-15 was found to be highly expressed, with histologic scores ranging from weak (1+) to strong (3+) in 124 out of the 139 adult cases (Figure 1A) and in all pediatric lymphoma samples but with distinct staining patterns observed in the aggressive B-cell lymphomas. Specifically, Sig-15 appears to be highly expressed and associated with the cell membrane in most DLBCL and Burkitt's lymphoma. Expression is more variable in ALCL, primarily elevated in the cytoplasm at low levels and/or in macrophages (Figure 1A). There is heterogeneity in Sig-15 expression with preliminary analysis of RNA-seq data from a cohort of 693 DLBCL patient samples demonstrates higher levels in the germinal center B-cell (GCB) versus activated B-cell molecular (ABC) subtype of DLBCL when compared to healthy controls (Kruskal-Wallis, *p = 0.041). Finally, using a well-established murine lymphoma model, A20 cells were stably transduced with control, non-silencing shRNA (shNS) or shRNA against Sig-15 and injected into un-irradiated immune competent (wild type, WT) or immune deficient ( Rag1 -/-) BALB/C mice and monitored for signs of lymphoma development. Knockdown of Sig-15 in A20 cells abrogates disease progression in WT but not immune deficient mice (Log-rank, **p <0.0001, n=10 mice/group), consistent with a role for Sig-15 in immune evasion in lymphoma (Figure 1B). Together, these data implicate Sig-15 as an immune checkpoint that may be inhibited therapeutically to promote an immune response to lymphoma cells.