scholarly journals CST6 Is a Small Autocrine Molecule That Targets Myeloma Growth and Bone Destruction

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Dongzheng Gai ◽  
Stewart JP ◽  
Xuxing Shen ◽  
Jin-Ran Chen ◽  
Can Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Cell Growth ◽  
Bone Resorption ◽  
Bone Formation ◽  
Bone Disease ◽  
Bone Destruction ◽  
Cathepsin K ◽  
Bone Marrow Biopsies ◽  
Lysosomal Protease

Bone destruction is a major complication of multiple myeloma (MM). Healthy bone is constantly remodeled through bone resorption by osteoclasts and bone formation by osteoblasts. New bone formation in MM is virtually non-existent, because differentiation of osteoblasts is inhibited by DKK1, a Wnt-β-catenin signaling inhibitor secreted by MM cells, reported by our group in NEJM, 2003. MM in its early stages is totally dependent on its microenvironment and for the hyperdiploid type MM this dependence is perpetual. Based on concordant gene expression signatures, predominantly driven by recurrent translocations and hyperdiploidy, we have classified MM into 7 distinct molecular entities. One subgroup, with significantly less bone disease and superior event-free and overall survival following high-dose therapy and stem cell transplantation than the other subgroups, defined as the Low Bone (LB) disease subgroup. Consistent with the LB phenotype, we have observed a strong inverse correlation between DKK1 and CST6 expression and by analyzing gene expression profiling (GEP) and RNA-sequencing data of more than 1,000 myeloma patients, we identified CST6 as the most upregulated gene in the LB subgroup. The aim of the present study was to determine the role of Cystatin E/M (CST6) in MM biology and to apply this knowledge to prevent both bone disease and MM cell growth. CST6, a 14-17 kD secretory protein, is a lysosomal protease inhibitor and suggested tumor suppressor gene. We showed that overexpression of CST6 in human MM cell lines prevents MM cell growth in vitro and in vivo. Also, purified CST6 protein from conditioned media of CST6-overexpressing MM cells significantly inhibits MM cell growth (p<0.01) and RANKL-induced osteoclast differentiation (p<0.01), decreases MM cell-induced bone destruction (p<0.05), and extends MM mouse survival (p<0.01). Mechanistic studies indicate that CST6 abrogates the alternative NF-kB signaling pathway as evidenced by a decrease in nuclear p52 protein in CST6-treated osteoclast precursors. Cathepsin K (CTSK), an osteoclast specific cysteine protease involved in bone resorption, was inhibited by CST6. GEP studies of whole bone marrow biopsies (WBMBx) across a spectrum of samples show higher expression of CTSK in WBMBx relative to purified plasma cells, while levels in MM remission WBMBx were higher than seen in healthy adult donors, MGUS/SMM, and newly diagnosed MM. Importantly, CTSK levels where not significantly different between remission and relapsed MM WBBx. These data show that Cathepsin K levels, and therefore osteoclasts, are elevated in the bone marrow of MM in remission and that these levels are similar to that seen in relapsed MM. Based on GEP data and experimental confirmation, we conclude that CST6, secreted by MM cells could be used clinically to target MM cells and prevent bone damage in MM. Inhibiting CTSK by CST6 in MM remission may aid in the prevention of MM relapse. Disclosures van Rhee: CDCN: Consultancy; Takeda: Consultancy; Karyopharm: Consultancy; Adaptive Biotech: Consultancy; EUSA: Consultancy.

Bone Marrow Monocyte / Macrophage Derived Activin A Mediates the Osteoclastogenic Effects of IL-3 in Myeloma,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3933-3933 ◽  
Author(s):  
Rebecca Silbermann ◽  
Marina Bolzoni ◽  
Paola Storti ◽  
Benedetta Dalla Palma ◽  
Sabrina Bonomini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Bone Disease ◽  
Healthy Subjects ◽  
Research Funding ◽  
Bone Destruction ◽  
Activin A ◽  
Negative Regulator ◽  
Normal Donor ◽  
Myeloma Bone Disease

Abstract Abstract 3933 Multiple myeloma (MM) is characterized by bone destruction with suppressed new bone formation that is mediated by multiple factors including MIP-1α (CCL3), RANKL, IL-3, DKK1 and recently, Activin A (ActA). IL-3 is a bifunctional cytokine that indirectly increases osteoclastogenesis and suppresses osteoblastogenesis via CD14+ bone marrow monocytes (BMM). Additionally, IL-3 levels are elevated in the BM plasma of MM patients compared with normals, however the mediators of IL-3's effects on myeloma bone disease are unknown. To investigate this we performed gene expression profiling using Affymetrix GeneChip® analysis of IL-3 treated BMM from MM patients and found ActA gene expression was increased 180-fold and confirmed this finding at the protein level by ELISA. ActA is a negative regulator of bone mass that promotes osteoclastogenesis and is overproduced in MM patients. Interestingly, treatment of MM cells or bone marrow stromal cells (BMSC) with IL-3 did not induce secretion of ActA. We found that ActA was produced by MM and MGUS patient CD14+ cells treated with IL-3 to a significantly higher degree compared to healthy subjects. (Median ActA levels for MM and MGUS ActA levels were increased 66.57 and 51.6 fold respectively over untreated cells, while IL-3 treatment of normal cells increased ActA 8.5 fold.) ActA levels were also increased in freshly isolated marrow plasma of a cohort of patients with active MM as compared to patients with smoldering MM (SMM), MGUS, or healthy subjects (median ActA levels: active MM 453 pg/ml, SMM 328 pg/ml, MGUS 332 pg/ml, normal 286 pg/ml). ActA levels in MM patients with and without bone disease were not significantly different. (Median value with bone disease 463pg/ml vs. 407 pg/ml without bone disease.) ActA has also been reported to have a role in the differentiation and polarization of CD14+ tumor associated macrophages (TAMs), which are osteoclast precursors and can block MM cell apoptosis. Therefore, we examined the potential role of ActA in IL-3 mediated osteoclast (OCL) formation. Culture of normal BMM with ActA or IL-3 significantly enhanced osteoclastogenesis compared with control (mean number of OCL / 1×105 normal marrow non-adherent cells plated in IL-3 (100pg/ml) treated cultures 73; ActA (1ng/ml) treated cultures 123; cultures with vehicle alone, 8), and ActA enhanced RANKL-induced osteoclastogenesis. Osteoprotegerin treatment of normal donor BMM stimulated with ActA failed to block the osteoclastogenic effects of ActA, demonstrating that ActA's osteoclastogenic effects were RANKL independent. Importantly, the osteoclastogenic effect of IL-3 was dose-dependently inhibited by anti-ActA, and IL-3 induced ActA expression by BMM decreased during OCL differentiation. In support of early OCL precursors as the source of IL-3 induced ActA, we did not identify IL-3 receptors on mature OCL by flow cytometry. These results demonstrate that IL-3 induction of osteoclastogenesis is mediated by ActA produced by CD14+ BMM and is RANKL independent. Thus, we hypothesize that therapies targeting the ActA receptor, such as the recently developed ActA receptor antagonist, should block both IL-3 and ActA, and thereby significantly impact MM bone disease via their effects on TAMs. Disclosures: Bolzoni: Celgene Italy: Research Funding. Roodman:Millennium: Consultancy; Amgen: Consultancy. Giuliani:Celgene: Research Funding; Novartis: Research Funding.

scholarly journals Continuous PTH in Male Mice Causes Bone Loss Because It Induces Serum Amyloid A

Endocrinology ◽  
2018 ◽  
Vol 159 (7) ◽  
pp. 2759-2776 ◽  
Author(s):  
Shilpa Choudhary ◽  
Elizabeth Santone ◽  
Sui-Pok Yee ◽  
Joseph Lorenzo ◽  
Douglas J Adams ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Bone Resorption ◽  
Bone Formation ◽  
Serum Amyloid A ◽  
Serum Amyloid ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Dependent Manner ◽  
Amyloid A

Abstract Increased bone resorption is considered to explain why intermittent PTH is anabolic for bone but continuous PTH is catabolic. However, when cyclooxygenase-2 (COX2) is absent in mice, continuous PTH becomes anabolic without decreased resorption. In murine bone marrow stromal cells (BMSCs), serum amyloid A (SAA)3, induced in the hematopoietic lineage by the combination of COX2-produced prostaglandin and receptor activator of nuclear factor κB ligand (RANKL), suppresses PTH-stimulated osteoblast differentiation. To determine whether SAA3 inhibits the anabolic effects of PTH in vivo, wild-type (WT) and SAA3 knockout (KO) mice were infused with PTH. In WT mice, continuous PTH induced SAA3 and was catabolic for bone. In KO mice, PTH was anabolic, increasing trabecular bone, serum markers of bone formation, and osteogenic gene expression. In contrast, PTH increased all measurements associated with bone resorption, as well as COX2 gene expression, similarly in KO and WT mice. SAA1 and SAA2 in humans are likely to have analogous functions to SAA3 in mice. RANKL induced both SAA1 and SAA2 in human bone marrow macrophages in a COX2-dependent manner. PTH stimulated osteogenesis in human BMSCs only when COX2 or RANKL was inhibited. Addition of recombinant SAA1 or SAA2 blocked PTH-stimulated osteogenesis. In summary, SAA3 suppresses the bone formation responses but not the bone resorption responses to PTH in mice, and in the absence of SAA3, continuous PTH is anabolic. In vitro studies in human bone marrow suggest that SAA may be a target for enhancing the therapeutic effects of PTH in treating osteoporosis.

Osteoclast Gene Expression Profiling in Multiple Myeloma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2735-2735
Author(s):  
Jerome Moreaux ◽  
Dirk Hose ◽  
Thierry Rème ◽  
Philippe Moine ◽  
Karène Mahtouk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Resorption ◽  
Bone Disease ◽  
Plasma Cells ◽  
Osteoclastic Bone Resorption ◽  
Myeloma Cells ◽  
Myeloma Bone Disease ◽  
Osteolytic Bone Disease

Abstract Multiple myeloma (MM) is a fatal hematologic malignancy associated with clonal expansion of malignant plasma cells within the bone marrow and the development of a destructive osteolytic bone disease. The principal cellular mechanisms involved in the development of myeloma bone disease are an increase in osteoclastic bone resorption, and a reduction in bone formation. Myeloma cells (MMC) are found in close association with sites of active bone resorption, and the interactions between myeloma cells and other cells within the specialized bone marrow microenvironment are essential, both for tumor growth and the development of myeloma bone disease. In order to investigate the gene expression profile (GEP) of osteoclastic cells, we compare GEP of osteoclastic cells (7 samples) with normal B cells (7 samples), normal bone marrow plasma cells (7 samples), bone marrow stromal cells (5 samples), bone marrow CD3 cells (5 samples), CD14 cells (7 samples), CD15 cells (7 samples), CD34 cells (7 samples) and primary MMC (123 samples). Using SAM analysis, a set of 552 genes was overexpressed in osteoclasts compared to others cell subpopulations with a FDR ≤ 1% and a ratio ≥ 2. Osteoclasts specifically overexpressed genes coding for chemokines (CCL2, CCL7, CCL8, CCL13, CCL18, CXCL5 and CCL23) and MMC growth factors (IGF-1, APRIL and IL-10). Anti- IGF-1 receptor and TACI-Fc inhibit MMC growth induced by osteoclasts. Among the chemokines overexpressed by osteoclasts, the majority of them have a common receptor: CCR2 expressed by MMC. Anti-CCR2 MoAb inhibits migration of the CCR2+ HMCL in response to osteoclasts. Expression data of purified MMC were analyzed by supervised clustering of group with higher (CCR2high) versus lower (CCR2low) CCR2 expression level. Patients in the CCR2high group are characterized by a higher bone disease. A set of 176 genes was differentially expressed between CCR2high and CCR2low MMC. CCR2high displayed a gene signature linked to the dependency of MMC on the interactions with the BM osteoclastic subpopulation and the osteoclastic bone resorption. Taken together, our findings suggest addition of chemokine antagonists to current treatment regimens for MM should result in better therapeutic responses because of the loss of both the protective effect of the bone marrow environment on the MMC and the osteoclastic cells activity.

SB431542, a TGF-Beta Receptor Kinase Inhibitor, Restores Bone Formation Which Ameliorates Myeloma-Induced Microenvironment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3479-3479 ◽  
Author(s):  
Kyoko Takeuchi ◽  
Masahiro Abe ◽  
Asuka Oda ◽  
Hiroe Amou ◽  
Masahiro Hiasa ◽  
...  
Keyword(s):  
Cell Growth ◽  
Bone Resorption ◽  
Bone Formation ◽  
Bone Destruction ◽  
Bone Lesions ◽  
Nodule Formation ◽  
Type I ◽  
Receptor Kinase ◽  
Tgf Beta ◽  
The Impact

Abstract Multiple myeloma (MM) cells stimulate bone resorption and concomitantly suppress bone formation, leading to devastating bone destruction. TGF-beta, a potent inhibitor for terminal osteoblasts (OB) maturation and mineralization, is abundantly produced and released from bone tissues through enhanced bone resorption, and activated by osteoclasts (OC)-derived acids and MMPs in MM bone lesions. In the present study we investigated the impact of TGF-beta inhibition on induction of bone formation in MM as well as the effects of matured OB on MM growth. TGF-beta completely suppressed BMP-2-induced mineralized nodule formation by OB cultured in osteogenic media supplemented with beta-glycerophosphate and vitamin C. SB431542, an inhibitor of TGF-beta type I receptor kinase, potently suppressed induction of Smad6 by TGF-beta, which inhibits BMP-2 signaling. SB431542 along with BMP-2 abolished such TGF-beta actions and enhanced mineralized nodule formation more than BMP-2 alone. Notably, addition of SB431542 antagonized the inhibitory effects of conditioned media from MM cell lines (RPMI8226 and U266) and bone marrow plasma from patients with MM, resulting in restoration of the BMP-induced mineralized nodule formation. Furthermore, MC3T3-E1 cells matured by BMP-2 enough to exhibit mineralized nodules suppressed the proliferation of 5TGM1 MM cells in sharp contrast to stromal cells as well as untreated or TGF-beta-treated undifferentiated MC3T3-E1 cells that promote MM cell growth and survival. Interestingly, 5TGM1 MM cell growth was also potently suppressed by MC3T3-E1 cells cultured with BMP-2 in the absence of beta-glycerophosphate to achieve OB maturation without formation of mineralized nodules, suggesting a responsible role for OB-derived non-mineralized factors associated with terminal differentiation of OB. In addition, the induction of OB maturation down-regulated the production by OB of IL-6 and up-regulated osteoprotegerin, an inhibitor for osteoclastogenesis. Taken together, blockade of TGF-beta actions release OB from the differentiational arrest in MM bone disease and thus can be a good therapeutic maneuver restoring bone formation as well as suppressing osteoclastogenesis to ameliorate bone destruction and at the same time suppressing MM expansion by disrupting the MM-induced microenvironment which can be called as MM niche.

Prospective evaluation of spatial heterogeneity at single cell resolution in multiple myeloma.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 8524-8524
Author(s):  
Maximilian Merz ◽  
Almuth Maria Anni Merz ◽  
Jie Wang ◽  
Lei Wei ◽  
Qiang Hu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Single Cell ◽  
Bone Disease ◽  
Bone Destruction ◽  
The Body ◽  
Response To Treatment ◽  
Risk Scores ◽  
Specific Gene

8524 Background: Osteolytic lesions (OL) characterize symptomatic multiple myeloma (MM). It is still unclear why plasma cells (PC) cause OL in certain regions of the body while other areas show no signs of bone destruction despite significant bone marrow infiltration. We conducted the first study of single cell RNA sequencing (scRNA-seq) and whole-exome sequencing (WES) of PC obtained from random bone marrow samples (RS) and paired OL. Methods: As part of a prospective clinical trial, patients consented to an imaging-guided biopsy of new OL identified by PET/CT in addition to the RS from the iliac crest. Both samples were acquired in the same session. On the same day PC were isolated using a CD138 positive selection kit and single cell gene expression libraries were generated for scRNA-seq. Frozen PC were subjected to DNA extraction and WES. Results: We sequenced 93569 purified, viable PC from paired samples from 15 different locations in the first 7 consecutive patients (median PC from location: 7203; range 1121-10279). Quality assessment of scRNA-seq data revealed no differences between PC in OL and RS. Based on scRNA-seq, 9-24 different subpopulations of PC in individual patients were identified. Over 90% of clusters found in the RS were also present in corresponding OL suggesting a common ancestor. This was true for patients with overlapping as well as divergent mutational profiles in RS and OL as shown by WES. In each patient we found PC clusters that were predominantly present in OL. Respective clusters were characterized by expression of Wnt-signaling inhibitors like DKK-1, Frzb and sFRP-2 and other genes linked to MM bone disease (HGF, CXCL-12, CCL3). Lysosome-associated membrane protein-like molecule 5 (LAMP5) and J-chain were overexpressed in OL clusters. Analysis of genes (IKZF1 and IKZF3) associated with response to treatment and outcome revealed vast heterogeneity and differences in risk scores (UAMS70 and IFM15) on a single cell level from different locations in individual patients. Conclusions: Our study provides the first evidence that PC from OL have distinct transcriptomic profiles that link site-specific gene expression to development of bone disease and adverse outcome.

scholarly journals Development of an in vivo model of human multiple myeloma bone disease

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1495-1501 ◽  
Author(s):  
M Alsina ◽  
B Boyce ◽  
RD Devlin ◽  
JL Anderson ◽  
F Craig ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Resorption ◽  
Bone Disease ◽  
Bone Destruction ◽  
Long Bone ◽  
X Rays ◽  
Myeloma Bone Disease ◽  
Human Multiple Myeloma

Osteolytic bone destruction and its complications, bone pain, pathologic fractures, and hypercalcemia, are a major source of morbidity and mortality in patients with multiple myeloma. The bone destruction in multiple myeloma is due to increased osteoclast (OCL) activity and decreased bone formation in areas of bone adjacent to myeloma cells. The mechanisms underlying osteolysis in multiple myeloma in vivo are unclear. We used a human plasma cell leukemia cell line, ARH-77, that has disseminated growth in mice with severe combined immunodeficiency (SCID) and expresses IgG kappa, as a model for human multiple myeloma, SCID mice were irradiated with 400 rads and mice were injected either with 10(6) ARH-77 cells intravenously (ARH-77 mice) or vehicle 24 hours after irradiation. Development of bone disease was assessed by blood ionized calcium levels, x-rays, and histology. All ARH-77, but none of control mice that survived irradiation, developed hind limb paralysis 28 to 35 days after injection and developed hypercalcemia (1.35 to 1.46 mmol/L) a mean of 5 days after becoming paraplegic. Lytic bone lesions were detected using x-rays in all the hypercalcemic mice examined. No lytic lesions or hypercalcemia developed in the controls. Controls or ARH-77 mice, after developing hypercalcemia, were then killed and bone marrow plasma from the long bones were obtained, concentrated, and assayed for bone-resorbing activity. Bone marrow plasma from ARH-77 mice induced significant bone resorption in the fetal rat long bone resorption assay when compared with controls (percentage of total 45Ca released = 35% +/- 4% v 11% +/- 1%). Histologic examination of tissues from the ARH-77 mice showed infiltration of myeloma cells in the liver and spleen and marked infiltration in vertebrae and long bones, with loss of bony trabeculae and increased OCL numbers. Interestingly, cultures of ARH-77 mouse bone marrow for early OCL precursors (colony-forming unit-granulocyte- macrophage [CFU-GM]) showed a threefold increase in CFU-GM from ARH-77 marrow versus controls (185 +/- 32 v 40 +/- 3 per 2 x 10(5) cell plated). Bone-resorbing human and murine cytokines such as interleukin- 6 (IL-6), IL-1 alpha or beta, TGF-alpha, lymphotoxin, and TNF alpha were not significantly increased in ARH-77 mouse sera or marrow plasma, compared with control mice, although ARH-77 cells produce IL-6 and lymphotoxin in vitro. Conditioned media from ARH-77 cells induced significant bone resorption in the fetal rat long bone resorption assay when compared with untreated media (percentage of total 45Ca released = 22% +/- 2% v 11% +/- 1%). This effect was not blocked by anti-IL-6 or antilymphotoxin (percentage of total 45Ca released = 19% +/- 1% and 22% +/- 1%, respectively). Thus, we have developed a model of human multiple myeloma bone disease that should be very useful to dissect the pathogenesis of the bone destruction in multiple myeloma.

Myeloma bone disease and proteasome inhibition therapies

Blood ◽  
2007 ◽  
Vol 110 (4) ◽  
pp. 1098-1104 ◽  
Author(s):  
Evangelos Terpos ◽  
Orhan Sezer ◽  
Peter Croucher ◽  
Meletios-Athanassios Dimopoulos
Keyword(s):  
Bone Resorption ◽  
Bone Formation ◽  
Bone Disease ◽  
Proteasome Inhibitors ◽  
Bone Destruction ◽  
Osteoclast Formation ◽  
Bone Specific Alkaline Phosphatase ◽  
Myeloma Bone Disease

AbstractBone disease is one of the most debilitating manifestations of multiple myeloma. A complex interdependence exists between myeloma bone disease and tumor growth, creating a vicious circle of extensive bone destruction and myeloma progression. Proteasome inhibitors have recently been shown to promote bone formation in vitro and in vivo. Preclinical studies have demonstrated that proteasome inhibitors, including bortezomib, which is the first-in-class such agent, stimulate osteoblast differentiation while inhibiting osteoclast formation and bone resorption. Clinical studies are confirming these observations. Bortezomib counteracts the abnormal balance of osteoclast regulators (receptor activator of nuclear factor-κB ligand and osteoprotegerin), leading to osteoclast inhibition and decreased bone destruction, as measured by a reduction in markers of bone resorption. In addition, bortezomib stimulates osteoblast function, possibly through the reduction of dickkopf-1, leading to increased bone formation, as indicated by the elevation in bone-specific alkaline phosphatase and osteocalcin. The effect of bortezomib on bone disease is thought to be direct and not only a consequence of the agent's antimyeloma properties, making it an attractive agent for further investigation, as it may combine potent antimyeloma activity with beneficial effects on bone. However, the clinical implication of these effects requires prospective studies with specific clinical end points.

Heparanase Expression Correlates with Bone Destruction in the Patients with Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1813-1813 ◽  
Author(s):  
Shi Wei ◽  
Racquel Innis-Shelton ◽  
Li Nan ◽  
Jian Ruan ◽  
Rebecca S Sollie ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Disease ◽  
Bone Marrow Cells ◽  
Bone Destruction ◽  
Bone Lesions ◽  
Bone Marrow Biopsies ◽  
Myeloma Cells ◽  
Lytic Bone Lesions ◽  
Marrow Cells

Abstract Abstract 1813 Multiple myeloma is an incurable malignancy, and excessive bone destruction is a major cause of morbidity in myeloma patients. However, the biologic mechanisms involved in the pathogenesis of myeloma-induced bone disease are poorly understood. Heparanase, an enzyme that cleaves the heparan sulfate chains of proteoglycans, is upregulated in a variety of human tumors, including myeloma. In the present study, bone marrow biopsies from 40 myeloma patients were stained with antibodies raised against heparanase, RANKL (an osteoclastogenic cytokine), OPG (a decoy receptor for RANKL), TRAP (a marker of osteoclastogenesis) and osteocalcin (a marker of osteoblastogenesis). The radiologic studies for bone lesions of these patients were also recorded. We analyzed the correlations between heparanase expression in bone marrow myeloma cells with (1) the numbers of TRAP positive osteoclasts, (2) RANKL and OPG expression in myeloma cells and osteoblastic cells, (3) the numbers of osteocalcin positive osteoblasts in bone marrow, and (4) the presence/absence of lytic bone lesions. We found a positive correlation between heparanase expression and RANKL expression as well as the numbers of TRAP positive osteoclasts in myeloma and bone marrow cells, but no correlation was found between the expressions of heparanase and OPG in bone marrow cells (myeloma cells do not express OPG). In contrast, heparanase expression was negatively correlated with the numbers of osteocalcin positive osteoblasts. Taken together, these data suggest that heparanase expression by myeloma cells promotes osteoclastogenesis and at same time inhibits osteoblastogenesis. Clinical data show that 92% of patients with high level of heparanase had one or more lytic bone lesions, while only 63% of patients with median∼ low levels of heparanase had bone lesions (p<0.0001). In summary, enhanced heparanase expression in myeloma cells promotes bone resorption and inhibits bone formation; these events contribute to the uncontrolled bone destruction that is characteristic of myeloma. These data provide novel insight into the mechanisms driving myeloma bone disease and suggest that heparanase inhibitors are valid therapeutic targets for the treatment of multiple myeloma. Disclosures: No relevant conflicts of interest to declare.

Runx2 Transcription Factor Regulates Heparanase-Induced Bone Resorption in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 567-567
Author(s):  
Li Nan ◽  
Haiyan Chen ◽  
Jian Ruan ◽  
Rebecca S Sollie ◽  
Cara Schafer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Resorption ◽  
Bone Disease ◽  
Bone Destruction ◽  
Chip Assay ◽  
Myeloma Cells ◽  
Myeloma Bone Disease ◽  
D Cells ◽  
High Level

Abstract Abstract 567 Background. Despite advances in treatment strategies, myeloma remains incurable, and bone destruction is a major cause of morbidity in myeloma patients. We have documented in earlier studies that heparanase enzyme is preferentially expressed in myeloma cells and induces severe bone destruction in myeloma. We also discovered that heparanase increases the production of two major bone resorbing factors named Receptor Activator of NF-κB Ligand (RANKL) and Matrix Metalloproteinase 9 (MMP-9) by myeloma cells. Runx2, a member of the runt-related gene family, is a bone-specific transcription factor. Runx2 regulates osteoblast differentiation and is essential for bone tissue development. Interestingly, Runx2 also controls expression of RANKL and MMP-9 genes in osteoblasts. Recent evidence indicates that ectopic induction and overexpression of Runx2 in breast, uterine and prostate cancer cells is associated with bone-metastasis, and osteolytic bone disease in these cancers. However, very little is known about the function of Runx2 in myeloma cells. Here we report for the first time that heparanase engages the Runx2 pathway to promote expression of RANKL and MMP-9 in myeloma cells. Methods. Molecular, biochemical, cellular and in vivo approaches were used to assess the role of Runx2 in heparanase-induced expression of RANKL and MMP-9. These included: (1) Real-time PCR and Western blot analysis to monitor Runx2 levels in CAG myeloma cells expressing high level of heparanase (HPSE-high cells) or with knockdown of endogenous heparanase (HPSE k/d cells), and the corresponding control cells. (2) Chromatin Immunoprecipitation (ChIP) assay to determine in vivo occupancy of the RANKL and MMP-9 gene promoters in myeloma cells by Runx2. (3) Zymography to determine MMP-9 activity in both human and murine myeloma cells. (4) Real-time PCR to determine changes in RANKL and MMP-9 gene expression in Runx2 knockdown MM1.S and 5TGM1 myeloma cells. (5) Assessment of tumor growth/burden and bone resorption in the 5TGM1 syngenic model of murine myeloma. 5TGM1 cells with specific knockdown of Runx2 or non-target shRNA were injected into C57BL/KaLwRij mice through the tail vein and levels of IgG2b and TRAP5b were monitored in the sera of the mice by ELISA. Results. We find Runx2 expression is significantly increased in CAG myeloma cells expressing a high level of heparanase and dramatically reduced in HPSE k/d cells. Increased Runx2 in HPSE-high myeloma cells results in an increase in expression of RANKL and MMP-9 mRNA as well as MMP-9 activity. In sharp contrast, knockdown of Runx2 in human myeloma MM1.S and murine myeloma 5TGM1 cells results in a significant reduction of RANKL, MMP-9 gene expression and MMP-9 activity. Thus heparanase promotes RANKL and MMP-9 expression via Runx2. Analysis of initial 1-kb sequences of RANKL and MMP-9 gene promoter in human and mice, revealed presence of multiple high affinity Runx2-binding sites. ChIP assay confirmed that Runx2 mediates induction of RANKL and MMP-9 gene transcription in the both human and murine myeloma cells by direct association with the proximal promoter of these genes. Together these results demonstrate that Runx2 is a positive regulator of RANKL and MMP-9 gene expression in myeloma cells. Finally, in the syngenic model, knockdown of Runx2 in 5TGM1 murine myeloma cells remarkably inhibits the growth of these cells in vivo and bone resorption. Conclusions. Runx2 is a central regulator of heparanase induced myeloma bone disease. Our discoveries provide new insight into the mechanism of myeloma-induced bone disease and identify Runx2 as a novel target to block myeloma bone disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The p62-ZZ Domain Inhibitor XRK3F2 Alters Myeloma-Induced Suppression of Osteoblast Differentiation and Is Highly Cytotoxic to Myeloma Cells in Combination with Bortezomib

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2083-2083 ◽  
Author(s):  
Rebecca Silbermann ◽  
Dan Zhou ◽  
Jumpei Teramachi ◽  
Xiang-Qun Xie ◽  
G. David Roodman ◽  
...  
Keyword(s):  
Cell Growth ◽  
Bone Formation ◽  
Cell Lines ◽  
Bone Disease ◽  
Stromal Cells ◽  
Research Funding ◽  
Bone Destruction ◽  
Tumor Burden ◽  
Cell Cytotoxicity ◽  
Sequestosome 1

Abstract Multiple myeloma (MM) bone disease results in increased osteoclastic bone destruction and irreversible osteoblast (OB) suppression. Cell-cell interactions between MM cells and bone marrow stromal cells (BMSC), which are OB precursors, activate signaling pathways in BMSC that result in enhanced MM cell growth, osteoclast formation, and inhibition of OB differentiation. OB inhibition results from suppression of key OB transcription factors, such as RUNX2, and induction of Gfi1, a transcriptional repressor of RUNX2. We reported that the ZZ domain of p62 (sequestosome-1) is required for BMSC support of MM cell growth and acts as a signaling hub for the formation of BMSC signaling complexes activated by MM cells and TNFα, resulting in NFκB, p38MAPK, and PI3K activation. We developed a small molecule inhibitor targeting the ZZ domain, XRK3F2 (previously CMPD3) that blocks p62-ZZ domain-mediated protein interactions, including TNFα-mediated signaling in BMSC. Further, we found that XRK3F2 was directly cytoxic to MM cells at high concentrations (5TGM1 murine MM cell IC50 4.35μM). Recently, we reported that administration of XRK3F2 two weeks after intratibial injection of 5TGM1 MM cells induces dramatic new cortical bone formation adjacent to MM cells in an immunocompetent in vivo model of MM bone disease. Additionally, bones from XRK3F2 treated animals not inoculated with tumor did not demonstrate new bone formation. This suggests that XRK3F2 changes the local effect of MM on bone at concentrations that were not sufficient for MM cell death. We now report that XRK3F2 alters MM-induced suppression of OB differentiation, and that the combination of XRK3F2 and bortezomib increases in vitroMM cell cytotoxicity. To determine if the dramatic bone formation induced by XRK3F2 resulted from reduced production of OB inhibitors by MM cells, enhanced BMSC differentiation to OB, or alteration of MM cell-BMSC interactions, we treated 5TGM1 cells, MC4 (murine stromal) cells, and 5TGM1-MC4 co-cultures with XRK3F2 and evaluated expression of RUNX2, Gfi1, and other key OB differentiation genes by qPCR. XRK3F2 treatment of 5TGM1 cells did not change 5TGM1 production of the OB inhibitors IL-7 or TNFα, and did not alter osteogenic differentiation of MC4 cells. However, XRK3F2 treatment of 5TGM1-MC4 co-cultures performed with a transwell insert increased MC4-derived RUNX2 expression 3-fold, as compared with vehicle treated co-cultures, and fully blocked the induction of Gfi1 in MC4 cells by MM cells (MC4-5TGM1 co-culture resulted in 12.4-fold increase in MC4 cell Gfi1 expression as compared with MC4 expression of Gfi1 without MM cell co-culture.) XRK3F2 treatment of MC4 cells also blocked TNFα-induced upregulation of Gfi1 (TNFα treatment increased MC4 cell Gfi1 expression 16.4-fold compared with control.) These results suggest that XRK3F2 blocks the TNFα-induced suppression of OB differentiation observed in MM cell-BMSC co-cultures through it’s effects on TNFα-mediated signaling facilitated by the p62-ZZ domain. To determine if we could further enhance the effects of XRK3F2 on bone formation and tumor burden, we evaluated if bortezomib, an anti-MM drug with potential bone anabolic effects, enhanced XRK3F2-induced MM cell cytotoxicity. Human MM cell lines were treated with combinations of XRK3F2 and bortezomib at concentrations below the IC50 of each drug for each cell line. H929, MM1.S, U266, ANBL6, and RPMI8226 cells were treated for 48 hours with XRK3F2, bortezomib (1nM), or the combination, and analyzed by MTT assay for viability. All cell lines demonstrated a highly significant decrease in viability in response to treatment with the XRK3F2-bortezomib combination as compared with either alone (p < 0.01). These results suggest that XRK3F2 or its derivatives, in combination with bortezomib or other proteasome inhibitors, should have a profound therapeutic effect on MM tumor burden and bone disease. Disclosures Silbermann: Amgen: Consultancy; Celgene: Research Funding. Roodman:Eli Lilly and Co.: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees.

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