The Function And Mechanism Study Of CST6 In Multiple Myeloma Bone Disease

Background and Aims: Multiple myeloma bone disease(MBD)is one of the hallmark of Multiple myeloma(MM),almost 80% of the newly diagnosed MM patients present with osteolytic lesions(OLs).However,a fraction of newly diagnosed MM patients does not develop osteolytic bone disease,the molecular explanation for the absence of bone disease manifestations remains largely unknown.After analyzing the PET-CT and plasma cell gene expressing profling in newly diagnosed MM patiens,we found that Cystatin 6(CST6)mainly expressed in the patients without bone osteolytic lesions.CST6 protein is a cysteine protease inhibitor coded by CST6 gene,which mainly regulates the activities of lysosomal cysteine protease and aspartate endopeptidase,participates in the regulation of the activity of extracellular protein kinases,osteoclast function and cancer metastasis.However,the function of CST6 in the MBD is still not fully elucidated,we aim to decipher the function and molecular mechanism of CST6 in MBD.Methods1.In this study,we included 512 newly diagnosed MM(NDMM)patients in UAMS from 2005-2012,all the NDMM patients’ diagnosis refers to the International Myeloma Working Group Guidelines.2.We reviewed the PET-CT of 512 NDMM subjects and separated the 512 patients into Low Bone disease group and Bone disease group according to the osteolytic lesions(0 or ≥1)in the PET-CT reports.3.Evaluating the differences of gene expression profiling between the Low Bone disease group and Bone disease group via analyzing the microarray data of bone marrow CD138 positive plasma cells in 512 patients.4.Detecting the CST6 protein level in the bone marrow serum or plasma by Enzyme-linked Immunosorbent Assay(ELISA)and analyzing the correlation of CST6 protein and CD138 positive plasma cell CST6 m RNA.5.Purifying the recombinant CST6 protein through the chromatography system;detecting the inhibition effects of CST6 protein on the Cathepsin K(CTSK)activity;analyzing the effects of CST6 protein on the osteoclast differentiation and function via tartrate resistant acid phosphatase(TRAP)staining and bone resorption assay.6.Evaluating the effects of CST6 protein on the myeloma bone disease by utilizing the 5TGM1-Ka Lw Rij mice model.7.In the 5TGM1-Ka Lw Rij MM mice model,utilizing the single cell RNA sequencing(sc RNA-seq)to elucidate the influence of CST6 treatment on the bone marrow microenvironment.8.Detecting the osteoclast differentiation genes after treatment with CST6 protein by Western Blots and RNA-sequencing(RNA-seq);furthermore,detecting the osteoclast differentiation signaling pathways influenced by CST6.9.Utilizing in-vitro kinase cleavage and activity assay to detect the inhibition effect of CST6 on Cathepsin L(CTSL).10.Immunofluorescence detects the distribution of CST6 protein in macrophages.Results1.We studied the PET-CT of 512 MM subjects,178 patients without osteolytic lesions and 334 patients with osteolytic lesions.2.The expression of 54 genes distinguished these two groups through analyzing the gene expression profiling of CD138 positive bone marrow plasma cells(P < 0.0001).The most significant gene was CST6 in low bone disease group(P < 0.0001).3.An enzyme-linked immunosorbent assay detected the CST6 protein level in the bone marrow serum of 75 MM patients.The result revealed a strong correlationship between CST6 level in bone marrow serum and CST6 m RNA expression(r = 0.6,P < 0.0001).4.The in-vitro kinase assay showed that recombinant CST6 protein significantly inhibited the activity of cathepsin K in a dose dependent manner with a 90% inhibition rate at dose of 2.5 n M.5.TRAP stainning and bone resorption assay revealed that compared with the control group recombinant CST6 protein blocked osteoclast differentiation and function in a dose dependent manner(P < 0.05).6.In the 5TGM1-Ka Lw Rij MM mice model,micro-CT showed that compared with PBS group 50 μg/kg recombinant CST6 protein inhibited osteolytic lesions on the tibia,increased the trabecular bone number,bone mineral density and decreased the trabecular bone separation(P < 0.01);meanwhile,TRAP staining demonstrated that recombinant CST6 protein dcreased the osteoclasts on the surface of trabecular bone(P < 0.001);furthermore,recombinant CST6 protein decreased the bone resorption marker Type 1 Collagen Cross-Linked C-Telopeptide(CTX-1)(P <0.001).7.Single cell RNA-sequencing identified that CST6 decreased the proportion of macrophages in the mice bone marrow(P < 0.01);meanwhile,the osteoclast precursor cell specific marker genes Csf1r、Cx3cr1 、 c-Fos and Jun were downregulated in the CST6 treatment group.8.Recombinant CST6 protein inhibited receptor activator of nuclear factor kappa-B ligand(RANKL)induced activation of osteoclast specific genes and protein expression,such as CTSK and TRAP.Western Blot showed that CST6 prevented tumor necrosis factor receptor-associated factor 3(TRAF3)and nuclear factor kappa-B/p100(NF-κB/p100)degradation caused by RANKL to inhibit the non-canonical NF-κB signaling pathway.8.In vitro kinase cleavage assay showed that CST6 protein blocked osteoclast differentiation by suppressing cathepsin L-mediated cleavage of TRAF3 and NF-κB/p100 following RANKL stimulation.9.Immunofluorescence demonstrated that CST6 can be endocytosed into macrophages and co-localized with lysosome and Cathepsin L.Conclusions1.CST6 was the most significantly differentially expressed gene in the plasma cell of MM patients without osteolytic lesions.2.Recombinant CST6 protein suppressed osteolytic bone disease caused by MM cells by blocking osteoclast differentiation and function.3.Recombinant CST6 protein attenuated polarization of monocytes to osteoclast precursors in MM bone marrow microenvironment.4.CST6 could block osteoclast differentiation by suppressing cathepsin L-mediated cleavage of TRAF3 and NF-κB/p100 following RANKL stimulation.Figure 27;Table 30;Reference 114.