The Role of CST6 in Immunosuppression in the Multiple Myeloma Microenvironment
Authors: Zhang Zijun , Cheng Yan , Mery David E. , Xu Hongwei , Koss Brian , Karbassi Behjatolah Monzavi , Shaughnessy, Jr John D , Zhan Fenghuang 
Published: December 2024

Introduction
Multiple myeloma (MM) is the second most prevalent hematologic cancer in the US and causes over 12000 deaths every year. The development of MM involves the remodeling of the bone marrow (BM) microenvironment by tumor cells, which is crucial for early-stage MM progression. Osteolytic lesions (OLs) are one of the hallmarks of MM. The formation of OLs is frequently seen in MM cases, caused by a disturbance of the BM microenvironment, specifically, the dysregulation of osteoclastogenesis and osteoblastogenesis in the BM. The development of OLs contributes to a more serious tumor progression and destructive bone degradation, which further leads to severe pain and immobility. CST6 is a small intercellular protease inhibitor secreted by MM tumor cells in approximately 20% of newly diagnosed MM patients. Intriguingly, these patients rarely develop OLs. We have previously shown that CST6 suppresses the differentiation of BM osteoclasts in multiple stages of osteoclast maturation; thereby, preventing bone resorption and the development of lytic bone disease in multiple myeloma. However, based on the fact that CST6 is largely expressed in MM cells of this specific group of patients, we suspect that tumor cells secrete CST6 for their growth advantage. To progress CST6 as a potential therapy for MM-induced bone disease, this study focused on uncovering any negative effects that CST6 may have on the bone marrow microenvironment.
Methods and Materials
GEP data from 3852 MM patients in the UAMS Myeloma Center Database were retrieved for the estimated overall survival (OS) and event-free survival (EFS) analysis. The CIBERSORT method was applied to whole biopsy data from 167 MM patients to estimate the BM cell populations. Tumor-associated macrophages (TAMs) were cultured in vitro from murine BM mononuclear cells isolated from C57BL/6 mice femurs and tibias. These TAMs were lysed and analyzed by qPCR and western blot. To evaluate the immunosuppressive effect of TAMs, they were co-cultured with murine CD8+ T-cell isolated from C57BL/6 mice spleens. CD8+ T-cell proliferation was evaluated by CTV dilution. All recombinant proteins used in this study were purified from the transfected HEK293T cell conditional media. All the mutagenesis was done with the NEB Q5 site-directed mutagenesis kit.
Results
Our analysis of MM patient GEP data revealed that CST6-high patients do not have significantly better therapeutic outcomes compared with CST6-low patients. CIBERSORT analysis also showed that high expression of CST6 shifts the BM cell population towards a more immunocompromised stage. We have also found that CST6-treated murine TAMs upregulate the expression of PD-L1 on both RNA and protein levels. In addition, CST6-treated TAMs also exhibit a higher inhibitory effect on CD8+ T-cell proliferation in vitro. This suppressive effect could be partially reversed by treating the T-cell & TAM co-culture system with PD-L1 antibody. This indicates that CST6 induces immunosuppression in the BM microenvironment by enhancing TAM's immunosuppressive activity. To study which structural region(s) on CST6 confer this immunosuppressive function, we generated several CST6 mutants. Among all the mutants, CST6N137D, which cannot undergo N-linked glycosylation, retained similar osteoclastogenesis inhibition to CST6wt, but its immunosuppressive effects were strongly reduced compared to CST6wtin vitro. Our result showed that CST6N137D-treated TAMs do not have enhanced PD-L1 expression, nor further suppress CD8+ T-cell proliferation in vitro. This suggests that the immunosuppressive function of CST6wt is directly linked to N-linked glycosylation (N137).
Conclusion
We have shown evidence that both CST6wt and CST6N137D prevent the differentiation of BM osteoclasts, but CST6N137D lacks strong immunosuppressive effects, making it a more favorable therapeutic candidate for MM-induced bone disease.