Research ArticleBone BiologyHematology Open Access | 10.1172/JCI159527

Dongzheng Gai,1,2 Jin-Ran Chen,3 James P. Stewart,1 Intawat Nookaew,4 Hasem Habelhah,5 Cody Ashby,1,4 Fumou Sun,1 Yan Cheng,1 Can Li,1,2 Hongwei Xu,1 Bailu Peng,1 Tarun K. Garg,1 Carolina Schinke,1 Sharmilan Thanendrarajan,1 Maurizio Zangari,1 Fangping Chen,2 Bart Barlogie,1 Frits van Rhee,1 Guido Tricot,1 John D. Shaughnessy Jr.,1 and Fenghuang Zhan1

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Gai, D. in: JCI | PubMed | Google Scholar

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Chen, J. in: JCI | PubMed | Google Scholar

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Stewart, J. in: JCI | PubMed | Google Scholar |

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Nookaew, I. in: JCI | PubMed | Google Scholar |

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Habelhah, H. in: JCI | PubMed | Google Scholar

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Ashby, C. in: JCI | PubMed | Google Scholar |

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Sun, F. in: JCI | PubMed | Google Scholar |

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Cheng, Y. in: JCI | PubMed | Google Scholar |

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Li, C. in: JCI | PubMed | Google Scholar |

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Xu, H. in: JCI | PubMed | Google Scholar

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Peng, B. in: JCI | PubMed | Google Scholar

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Garg, T. in: JCI | PubMed | Google Scholar |

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Schinke, C. in: JCI | PubMed | Google Scholar |

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Thanendrarajan, S. in: JCI | PubMed | Google Scholar

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Zangari, M. in: JCI | PubMed | Google Scholar

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Chen, F. in: JCI | PubMed | Google Scholar |

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Barlogie, B. in: JCI | PubMed | Google Scholar

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by van Rhee, F. in: JCI | PubMed | Google Scholar

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Tricot, G. in: JCI | PubMed | Google Scholar

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Shaughnessy, J. in: JCI | PubMed | Google Scholar

1Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

2Department of Hematology, Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Arkansas Children’s Nutrition Center and

4Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

5Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: Fenghuang Zhan or John D. Shaughnessy Jr., Myeloma Center, Winthrop P. Rockefeller Cancer Institute, Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot # 508, Little Rock, Arizona 72205, USA. Phone: 501.526.6000 ext. 25228; Email: FZhan@uams.edu (FZ); JDShaughnessy@uams.edu (JDS).

Authorship note: DG and JRC contributed equally to this work.

Find articles by Zhan, F. in: JCI | PubMed | Google Scholar

Authorship note: DG and JRC contributed equally to this work.

Published July 26, 2022 - More info

Published in Volume 132, Issue 18 on September 15, 2022
J Clin Invest. 2022;132(18):e159527. https://doi.org/10.1172/JCI159527.
© 2022 Gai et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published July 26, 2022 - Version history
Received: February 21, 2022; Accepted: July 21, 2022 View PDF 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.

Introduction

Osteolytic lesions (OLs) of the axial skeleton are a hallmark of multiple myeloma (MM), a malignancy of antibody-secreting plasma cells (PCs). While bone metastases are seen in many cancers, the presence of OLs is one of the diagnostic criteria for MM. Osteolysis in MM is linked to both suppressed osteoblastogenesis and increased osteoclastogenesis (1). New bone formation is suppressed, at least in part, via Dickkopf-1 (DKK-1) mediated inhibition of Wnt/β-catenin signaling, which is essential for osteoblast differentiation (2). DKK1 also increases osteoclast numbers by increasing the RANKL/osteoprotegerin (OPG) ratios in the BM microenvironment (3–5).

Using global gene expression profiling (GEP), we and others have created a molecular classification of MM (6–8). Correlation of clinical parameters with molecular subtypes revealed a statistically significant lower incidence of bone disease in a subtype of disease we referred to as the low-bone (LB) disease subtype (6). The existence of this subgroup was independently verified (6–8). These data strongly suggest that MM lacking bone disease represents a distinct pathologic entity.

Proteostasis or protein homeostasis is a process that regulates intracellular proteins to maintain a balanced, functional proteome (9). Protease-mediated hydrolysis plays a key role in maintaining proteostasis. Several important proteases have been identified in different organelles; the proteasome, cathepsins, human caseinolytic protease p (hCIpP), and metallopeptidases (MMPs) are in the cytoplasm, lysosome, mitochondria, and extracellular environment, respectively. The intracellular proteases include the cytoplasmic ubiquitin-proteasome system (UPS) and autophagy lysosomal system (ALS), which regulate intracellular protein degradation and also osteoclast differentiation and function (10). Osteoclasts are multinucleated cells formed by the fusion of mononuclear progenitors of the monocyte/macrophage lineage (11). RANKL interacts with its cognate receptor RANK in the presence of macrophage CSF (M-CSF) to promote osteoclast differentiation and maturation via the NF-κB signaling pathway. Both canonical and noncanonical NF-κB pathways are regulated by the UPS during osteoclastogenesis (12). The canonical signaling pathway is activated within a short period of time, leading to IκBα degradation and translocation of p65/p50 heterodimers into the nucleus (13). In the noncanonical signal pathway, RANKL stimulation induces TRAF3 degradation, resulting in the stabilization of NF-κB–inducing kinase (NIK). NIK activates IκB kinase α (IKKα), which promotes p100 processing to p52 and the subsequent nuclear translocation of RelB/p52 complexes (12, 13). It is known that p100 is a suppressor of the noncanonical NF-κB pathway in osteoclastogenesis (14). The p100 can be removed from the cytosol either through processing or complete degradation triggered by the NIK/IKK1 complex (15). However, it is unknown whether ALS or other proteases are involved in p100 protein processing.

A small but significant fraction (~20%) of patients with MM present without OL at diagnosis. The molecular basis for the absence of OL in MM is currently not understood. PET-CT is recommended by the International Myeloma Working Group (IMWG) to ascertain the presence of MM focal bone lesions (16). In the current study, we combined PET-CT with global GEP of CD138-selected PCs from the BM of 512 patients with newly diagnosed MM (NDMM) to identify secreted molecules that might account for the absence of OL in MM. This analysis identified a marked link between the absence of PET-CT–defined OL and elevated expression of the soluble protease inhibitor CST6. CST6, a 14 to 17 kDa secretory protein, is a member of the family of type 2 cystatins, cysteine proteinase inhibitors that regulate lysosomal cysteine proteases and the asparaginyl endopeptidase legumain (LGMN). We have demonstrated that both purified CST6 and BM serum from patients with high CST6 expression suppress osteoclast function and differentiation in a CST6-dependent fashion and that recombinant CST6 inhibits bone disease in an in vivo myeloma mouse model. Mechanistic studies reveal that CST6 regulates osteoclastogenesis through at least 3 different mechanisms: depolarization of osteoclast precursors (OCPs), stabilization of p100 and TRAF3, and inhibition of the extracellular environmental protease cathepsin K (CTSK).

Results

Elevated expression of CST6 is linked to absence of MM bone disease. We correlated global mRNA expression levels in CD138-selected BM PCs from 512 NDMM patients with the presence or absence of PET-CT–defined focal OL (Figure 1A). Of these, 178 had no evidence of PET-CT bone lesions, while 334 cases showed 1 or more focal lesions. Supervised cluster analysis showed the expression levels of 54 genes that were significantly differentially expressed (greater than 1.4-fold and P < 0.0001) between these 2 groups (Figure 1B). CST6, coding for cystatin M/E, a soluble inhibitor of cysteine proteases, was the most significantly differentially expressed gene in the analysis (P < 0.0001) and was significantly higher in the group with no PET-CT lesions (Figure 1C). Genes associated with cell proliferation were expressed at significantly higher levels in cases with 1 or more PET-CT lesions (Figure 1B). Table 1 shows that, among clinical variables, the absence of PET-CT lesions was associated with a higher incidence of normal albumin levels, a lower incidence of a GEP70 high-risk gene signature (17), higher frequency of gain of chromosome 1q21, and a lower incidence of chromosome 1p deletion and chromosome 5 gain. We found that 67% of the previously defined LB molecular subtype had no PET-CT lesions, while 90% of the proliferation (PR) subtype had 1 or more PET-CT lesions (Table 1 and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI159527DS1). CST6 was virtually undetectable in PCs isolated from healthy subjects and patients with Waldenstrom’s macroglobulinemia (WM), a BM PC dyscrasia lacking OL (Figure 1C). CST6 was expressed in a subset of patients with monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM) (Figure 1C). We have previously shown an inverse relationship between DKK1 and CST6 and a strong correlation between DKK1 and the presence of MRI-defined bone lesions in MM (2). We divided the 512 cases into those in which MM tumor cells expressed either CST6 or DKK1 above 5000 relative fluorescence intensity (RFI). CST6 was more than 5000 in 33 and DKK1 more than 5000 RFI in 161 (Supplemental Figure 1B). Only one of the 161 cases expressing high DKK1 also had high CST6. None of the 33 cases with high CST6 had high DKK1 (Supplemental Figure 1B). These data indicate that elevated DKK1 and CST6 define 2 separate subtypes of MM, one with and one without OL bone disease.

Figure 1

High expression of CST6 is linked to the absence of bone lesions in MM. (A) Workflow of the study. BMMC, BM mononuclear cells. (B) Heatmap showing that 54 genes were significantly differentially expressed in MM cells from patients with no (n = 178) or 1 or more focal lesions (n = 334) on PET-CT (P < 0.0001). Shown are 17 genes with elevated levels of expression in MM cells from patients with no lesions on PET-CT and ranked from top to bottom by significance; and 37 genes with significantly upregulated expression in tumor cells with 1 or more lesions on PET-CT and ranked from bottom to top by significance. Gene symbols are listed on the right. (C) Affymetrix MAS5.0 normalized mRNA expression signal is indicated on the y axis. Expression level of CST6 in each sample is indicated by the height of the bar. Samples are ordered from the lowest to highest level of expression of CST6 from left to right on the x axis. P value was obtained using 2-tailed, unpaired Student’s t test. (D) Dot plot shows the correlation between CST6 mRNA in purified MM tumor cells and BM serum CST6 levels. The level of expression of CST6 mRNA was quantified by microarray analysis, and CST6 protein was measured by ELISA in 75 NDMM patients. Each spot indicates the relative relation of CST6 mRNA and protein expression levels for a patient. There was a significant correlation between the level of CST6 mRNA in MM cells and the level of CST6 protein in MM BM serum/plasma (r = 0.60, P < 0.0001). P value was obtained by Pearson’s correlation and a linear regression analysis.

Table 1

Patient characteristics for 512 patients with MM in the presence or absence of bone lesions by PET-CT

Since CST6 codes for a secreted protein, we developed an ELISA and standard curve for CST6 using recombinant CST6 protein. CST6 was detected in serum isolated from the BM aspirates from which the CD138-purified MM cells were obtained for mRNA microarray studies; serum protein and mRNA levels were correlated (Figure 1D). The mean (±SD) level of CST6 protein in the BM serum/plasma from 75 patients with NDMM for whom gene expression data were also available was 673.0 ± 1076.1 ng/mL. In contrast, the CST6 level was 13.2 ± 19.4 ng/mL in 10 control subjects. These data indicate that the CST6 protein is significantly elevated in MM BM from patients whose tumor cells express high levels of CST6 mRNA.

CST6 protein inhibits MM cell–induced bone resorption in vivo. We utilized the 5TGM1-KaLwRij murine MM model (18) to investigate whether recombinant mouse Cst6 protein (rmCst6) could inhibit bone disease in vivo. One million 5TGM1 cells were inoculated into C57BL/KaLwRij mice via the tail vein, and mice were treated with purified rmCst6 (Figure 2A). Intraperitoneal injection of purified rmCst6 protein (50 μg/kg, once per day) significantly decreased OLs in MM-bearing mice (Figure 2, B and C). Micro-computed tomography (μCT) reconstruction of mouse tibiae showed that rmCst6 protein increased trabecular bone volume over total volume (BV/TV), trabecular number (Tb.N), and bone mineral density (BMD) and was accompanied by a decrease in trabecular separation (Tb.Sp) in treated versus control mice (Figure 2, B and C). Histomorphometric analyses demonstrated that rmCst6 administration significantly reduced osteoclast numbers as well as the proportion of bone surface occupied by osteoclasts in MM-bearing mice (Figure 2, D and E). ELISA analyses showed that collagen type 1 (CTX-1), which is a marker of osteoclast activity, was significantly reduced in mice treated with rmCst6 protein (Figure 2F). Serum procollagen type I N-propeptide (PINP), a marker of bone formation, did not show any difference between rmCst6-treated and untreated mice (Figure 2G), suggesting that rmCst6 does not alter osteoblast function. Our in vitro study further confirmed that CST6 protein did not influence osteoblast differentiation (Supplemental Figure 2). Serum measurement of the tumor-specific M protein IgG2b after 25 days by ELISA in MM-bearing mice with or without rmCst6 treatment showed no difference between control and rmCst6-treated groups (Figure 2H). We also did not find any evidence that CST6 influenced MM cell proliferation or survival in vitro (Supplemental Figure 3). These data suggest that rmCst6 prevents tumor-induced osteolysis by acting directly on osteoclasts.