CST6 suppresses osteolytic bone disease in multiple myeloma by blocking osteoclast differentiation
Dongzheng Gai, Jin-Ran Chen, James P. Stewart, Intawat Nookaew, Hasem Habelhah, Cody Ashby, Fumou Sun, Yan Cheng, Can Li, Hongwei Xu, Bailu Peng, Tarun K. Garg, Carolina Schinke, Sharmilan Thanendrarajan, Maurizio Zangari, Fangping Chen, Bart Barlogie, Frits van Rhee, Guido Tricot, John D. Shaughnessy Jr., Fenghuang Zhan
Dongzheng Gai, Jin-Ran Chen, James P. Stewart, Intawat Nookaew, Hasem Habelhah, Cody Ashby, Fumou Sun, Yan Cheng, Can Li, Hongwei Xu, Bailu Peng, Tarun K. Garg, Carolina Schinke, Sharmilan Thanendrarajan, Maurizio Zangari, Fangping Chen, Bart Barlogie, Frits van Rhee, Guido Tricot, John D. Shaughnessy Jr., Fenghuang Zhan
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Research Article Bone biology Hematology

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 (OLs). 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 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-Seq showed that CST6 attenuates polarization of monocytes to osteoclast precursors. Furthermore, CST6 protein blocks osteoclast differentiation by suppressing cathepsin-mediated cleavage of NF-κB/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.

Authors

Dongzheng Gai, Jin-Ran Chen, James P. Stewart, Intawat Nookaew, Hasem Habelhah, Cody Ashby, Fumou Sun, Yan Cheng, Can Li, Hongwei Xu, Bailu Peng, Tarun K. Garg, Carolina Schinke, Sharmilan Thanendrarajan, Maurizio Zangari, Fangping Chen, Bart Barlogie, Frits van Rhee, Guido Tricot, John D. Shaughnessy Jr., Fenghuang Zhan

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Figure 3

CST6 protein inhibits osteoclast differentiation and function.

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CST6 protein inhibits osteoclast differentiation and function. (A) Human...
(A) Human OCPs were induced to differentiate into osteoclasts by addition of M-CSF and RANKL. 200 ng/mL rhCST6, 4 μg/mL anti-CST6 antibody, or nonspecific mouse IgG was added to the culture media (upper panels). BM serum from healthy donors and MM patients was added to the culture media with indicated CST6 concentrations (lower panels). On day 7, half of these wells in each group were stained with TRAP solution and the remaining wells were quantified resorption areas. Culture media containing high CST6 protein (final concentration 200 ng/mL) from patient 5 (P5) showed significant decreased TRAP+ osteoclasts and bone resorption, while culture media containing low CST6 protein from a healthy donor and patient 1 (P1) with low levels of CST6 did not show these effects. Anti-CST6 antibody or nonspecific mouse IgG (4 μg/mL) was also added to the culture media during human osteoclast differentiation. The CST6 level in each BM serum was determined by ELISA, as described in Supplemental Table 1. Control is represented for RANKL only and was the same as in Supplemental Figure 5 (n = 3). Scale bars: 500 μm. (B) Bar plots present quantifications of TRAP+ osteoclasts and bone-resorption areas. Numbers in parentheses represent CST6 concentrations in patient serum detected by ELISA. Data are represented as mean ± SEM and were analyzed by 2-way ANOVA (B). *P < 0.05, **P < 0.01, ***P < 0.001.