Siglec-15 dependent alteration of the leukemia immune microenvironment

Abstract

Abstract Introduction: Relapsed and refractory hematologic malignancies remain a significant clinical challenge despite integration of newer therapeutic strategies engaging the immune system as standard of care. As such, identifying and characterizing specific defects in the anti-tumor immune response will be key to the development of newer therapies that improve survival. We have previously reported that the immune checkpoint Siglec-15, a member of the sialic acid binding immunoglobulin-like lectin family of proteins, is overexpressed in hematologic malignancies and circulates at high levels in the plasma of children in leukemia. Importantly, inhibition of Siglec-15 restored local anti-tumor immune responses and abrogated tumor progression highlighting a crucial role for Siglec-15 in immune evasion in murine models of leukemia and lymphoma. However, the mechanism(s) by which Siglec-15 promotes immune escape and leukemogenesis has not yet been defined. Here we investigate how Siglec-15 creates an immunosuppressive immune microenvironment through aberrant hematopoiesis in a murine model of leukemia. Methods: Using a well-established model of murine B-cell acute lymphoblastic leukemia (B-ALL), control (Cas9) or CRISPR knockout Siglec-15 BCR-ABL1+cells wereinjected into un-irradiated, immune competent (wild type, WT) or immune deficient (Rag1-/-) C57BL/6 mice and single cell RNA sequencing (scRNA seq) performed on paired bone marrow (N=4/group) and spleen (N=4/group) samples. To validate scRNA findings, multiparameter flow cytometry was performed to determine impact of knockout of Siglec-15 on hematopoiesis and innate immune subsets. Results: scRNA seq suggests that knockout of Siglec-15 in B-ALL restores circulation of mature neutrophils that are associated with an anti-tumoral, pro-inflammatory phenotype as compared to control where there are predominantly more immature neutrophils that have a pro-tumoral and immunosuppressive gene signature. With Siglec-15 KO, there is increased expression of genes such as Egr1, Ccl6, Klf6 that are known to promote neutrophil differentiation, pro-inflammatory cytokine production and recruitment to tumor sites. By flow cytometry, there is an increase in pluripotent stem cells in the bone marrow of Siglec-15 knockout mice (p<0.01, one-way ANOVA, n=10 mice/group) compared to control. Further, there is increased skewing to myeloid primed multipotent progenitors, MPP2/3 and FCyR+ MPP3, with Siglec-15 knockout (p<0.01, one-way ANOVA, n=10 mice/group). Interestingly, there is no expansion of the immediate myeloid precursors, common myeloid precursors and granulocyte -monocyte precursors, in control mice, there is a significant decrease with knockout of Siglec-15 (p<0.001). However, we observe an increase in innate and adaptive effector immune cells, including neutrophils, B and T cells, in the bone marrow and spleen of knockout mice. Conclusions: We have found that there are significant Siglec-15 dependent changes in the bone marrow and spleen in B-ALL by scRNA sequencing. Preliminary data suggests that Siglec-15 may promote aberrant myelopoiesis in murine model of B-ALL at early time points. Ongoing experiments aim to examine the extent to which B-ALL promotes aberrant myelopoiesis in children. Together, these data implicate Sig-15 as an immune checkpoint that may be inhibited therapeutically to promote an immune response to leukemia cells.