CST6 suppresses osteolytic bone disease in multiple myeloma by blocking osteoclast differentiation

Abstract

Osteolytic bone disease is a hallmark of multiple myeloma (MM). A significant fraction (~20%) of MM patients do not develop osteolytic lesions (OL). The molecular basis for the absence of bone disease in MM is not understood. We combined PET-CT and gene expression profiling (GEP) of purified bone marrow (BM) CD138 + MM cells from 512 newly diagnosed MM patients to reveal that elevated expression of cystatin M/E (CST6) was significantly associated with the absence of OL in MM. An enzyme-linked immunosorbent assay revealed a strong correlation between CST6 levels in BM serum/plasma and CST6 mRNA expression. Both recombinant CST6 protein and BM serum from patients with high CST6 significantly inhibited the activity of the osteoclast-specific protease cathepsin K, and blocked osteoclast differentiation and function. Recombinant CST6 inhibited bone destruction in ex vivo and in vivo myeloma models. Single cell RNA-sequencing identified that CST6 attenuates polarization of monocytes to osteoclast precursors. Furthermore, CST6 protein blocks osteoclast differentiation by suppressing cathepsin-mediated cleavage of NF-B/p100 and TRAF3 following RANKL stimulation. Secretion by MM cells of CST6, an inhibitor of osteoclast differentiation and function, suppresses osteolytic bone disease in MM and probably other diseases associated with osteoclast-mediated bone loss. * Fisher's exact test was used. † The chi-square test was used.**Cytogenetic abnormalities were acquired by gene expression profiling (56).Significant differences in patients' characteristics according to their bone-disease status were evaluated with the use of either Fisher's exact test or chi-square test.

Cystatin M/E ameliorates bone resorption through increasing osteoclastic cell estrogen influx

“…M0 macrophages are undifferentiated macrophages with potential to polarize into different macrophage sub-types, including osteoclasts 19 . The specific role of M4 macrophages is currently unknown, however this subtype has previously been shown to have increased expression of osteoclast differentiation genes 16 . As with the decrease in total BM macrophage cell percentage the decrease in M0 and M4 macrophage subtypes following rmCst6 treatment can potentially be explained as estrogen preventing monocyte polarization to macrophage cell types and macrophage polarization into osteoclasts 16,[31][32][33][34] .…”

“…The specific role of M4 macrophages is currently unknown, however this subtype has previously been shown to have increased expression of osteoclast differentiation genes 16 . As with the decrease in total BM macrophage cell percentage the decrease in M0 and M4 macrophage subtypes following rmCst6 treatment can potentially be explained as estrogen preventing monocyte polarization to macrophage cell types and macrophage polarization into osteoclasts 16,[31][32][33][34] . For ZA treatment, decrease in M0 and M4 macrophages may be from known apoptotic effects of ZA 22 .…”

“…Since it is known that macrophages are osteoclast precursors, the change in percentage of previously identified macrophage subtypes was also investigated using UMAP plots of BM macrophages following MM mouse treatment with either PBS, rmCst6 or ZA 16 . rmCst6 and ZA treatment was found to decrease percentage of M0 and M4 macrophages, identified as early precursors of osteoclasts and tumor associated macrophages with high expression of osteoclast differentiation regulators (Jun and c-Fos) 16,19 . rmCst6 and ZA also decreased the percentage of M5 macrophages, while ZA alone decreased percentage of M3 macrophages (Supplemental Figure 1).…”

“…Effects of rmCST6 and ZA on the viability and differentiation of osteoclasts, osteoblasts and chondrocytes rmCst6 has previously been shown to suppress MM induced osteolytic bone disease through interfering with osteoclast differentiation and function but not viability 16 . On the other hand, ZA was shown to suppress skeletal related events in MM induced osteolytic bone disease through inactivation and apoptosis of osteoclasts 1 .…”

“…Recombinant CST6 protein inhibits the activity of the osteoclast-specific protease cathepsin K, blocks osteoclast differentiation and function, and inhibits bone destruction in ex vivo and in vivo myeloma models. Suppression of cathepsin L blocks cleavage of p100 to p52 as well as degradation of TRAF3, suppressing the alternative NF-B pathway 16 . Furthermore, Li et al reported that CST6 and CST6 peptides (containing the conserved QLVAG residues) inhibit breast cancer bone metastasis by suppressing cathepsin B activity 17 .…”