Myeloma cells exhibit an increase in proteasome activity and an enhanced response to proteasome inhibition in the bone marrow microenvironment in vivo

Abstract The proteasome inhibitor bortezomib (VELCADE®) represents an important advance in the treatment of multiple myeloma. Despite its very significant beneficial effects, its precise mechanism of action remains unclear. There is currently evidence to suggest that bortezomib has the potential to directly affect both myeloma cells and osteoblasts. However, it is not possible to discern the precise mechanism of action of bortezomib in patients with myeloma. To address this we used a well-characterized murine model of myeloma combined with MALDI mass spectrometry (MALDI MS) to study the effect of bortezomib on bone formation and tumor burden in vivo and protein profiles of myeloma cells. To more closely reflect the clinical setting, we used an established treatment protocol, in which bortezomib treatment was initiated upon development of myeloma. 5TGM1-GFP myeloma cells were inoculated into C57BlKaLwRij mice, resulting in tumor growth within the bone marrow and the development of an osteolytic bone disease. Tumor burden was monitored by measurement of serum IgG2bκ, and a significant increase was detected 14 days following tumor inoculation. Mice were then randomized to receive either bortezomib (0.5mg/kg, 3x week) or vehicle control for the remainder of the experiment. Treatment with bortezomib resulted in a significant reduction in tumor burden, as determined by serum IgG2bκ concentrations and by flow cytometric analysis of GFP-positive cells in the bone marrow. The tibia was analyzed by microCT, and bortezomib was found to significantly increase trabecular bone volume and reduce the number of osteolytic bone lesions. In order to study bone formation, mice were treated with 2 doses of calcein at a 7 day interval. Myeloma-bearing mice were associated with a significant reduction in rates of bone formation, which was prevented by treatment with bortezomib. GFP-positive myeloma cells were isolated from bone marrow of control- and bortezomib-treated mice and purified by fluorescence-activated cell sorting. Protein profiling by MALDI MS identified a number of proteins, both known and unknown, which were regulated by bortezomib treatment in vivo. Free ubiquitin (m/z 8565) was down-regulated, which is a known effect of proteasome inhibition resulting from the inhibition of protein degradation and release of free ubiquitin. Regulated proteins also included the up-regulation of thymosin beta-10 (m/z 4936) following bortezomib treatment. Thymosin beta 10 was undetectable in myeloma cells from control-treated mice. In addition, the calcium binding protein calgranulin A (S100A8) (m/z 10163) was up-regulated in myeloma cells isolated from bortezomib-treated mice. Neither thymosin-beta 10 nor calgranulin A have been previously linked to the activity of the proteasome, and thus may indicate novel molecular mechanisms involved in the anti-myeloma effect of bortezomib. Our data demonstrate that treatment with bortezomib from the time of established myeloma not only reduces myeloma tumor burden but also significantly increases bone formation in vivo. In addition, proteomic analysis of myeloma cells following in vivo treatment with bortezomib reveals a number of regulated proteins, providing novel insights into the molecular mechanism of action of proteasome inhibition in multiple myeloma in vivo.

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Dendritic cells differentiate into osteoclasts in bone marrow microenvironment in vivo

Blood ◽

10.1182/blood-2008-09-180836 ◽

2009 ◽

Vol 113 (1) ◽

pp. 264-265 ◽

Cited By ~ 11

Author(s):

Mawadda Alnaeeli ◽

Yen-Tung A. Teng

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Bone Marrow Microenvironment

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Increasing Wnt signaling in the bone marrow microenvironment inhibits the development of myeloma bone disease and reduces tumor burden in bone in vivo

Blood ◽

10.1182/blood-2007-03-077685 ◽

2008 ◽

Vol 111 (5) ◽

pp. 2833-2842 ◽

Cited By ~ 118

Author(s):

Claire M. Edwards ◽

James R. Edwards ◽

Seint T. Lwin ◽

Javier Esparza ◽

Babatunde O. Oyajobi ◽

...

Keyword(s):

Bone Marrow ◽

Tumor Growth ◽

Wnt Signaling ◽

Bone Disease ◽

Critical Role ◽

Tumor Burden ◽

Bone Marrow Microenvironment ◽

Myeloma Bone Disease ◽

Osteolytic Bone Disease

There is increasing evidence to suggest that the Wnt signaling pathway plays a critical role in the pathogenesis of myeloma bone disease. In the present study, we determined whether increasing Wnt signaling within the bone marrow microenvironment in myeloma counteracts development of osteolytic bone disease. C57BL/KaLwRij mice were inoculated intravenously with murine 5TGM1 myeloma cells, resulting in tumor growth in bone and development of myeloma bone disease. Lithium chloride (LiCl) treatment activated Wnt signaling in osteoblasts, inhibited myeloma bone disease, and decreased tumor burden in bone, but increased tumor growth when 5TGM1 cells were inoculated subcutaneously. Abrogation of β-catenin activity and disruption of Wnt signaling in 5TGM1 cells by stable overexpression of a dominant-negative TCF4 prevented the LiCl-induced increase in subcutaneous growth but had no effect on LiCl-induced reduction in tumor burden within bone or on osteolysis in myeloma-bearing mice. Together, these data highlight the importance of the local microenvironment in the effect of Wnt signaling on the development of myeloma bone disease and demonstrate that, despite a direct effect to increase tumor growth at extraosseous sites, increasing Wnt signaling in the bone marrow microenvironment can prevent the development of myeloma bone disease and inhibit myeloma growth within bone in vivo.

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CD4+ T-cell killing of multiple myeloma cells is mediated by resident bone marrow macrophages

Blood Advances ◽

10.1182/bloodadvances.2020001434 ◽

2020 ◽

Vol 4 (12) ◽

pp. 2595-2605 ◽

Cited By ~ 1

Author(s):

Ole Audun W. Haabeth ◽

Kjartan Hennig ◽

Marte Fauskanger ◽

Geir Åge Løset ◽

Bjarne Bogen ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Antigen Presenting Cells ◽

Bone Marrow Microenvironment ◽

Cd4 T Cell ◽

Myeloma Cells ◽

Antigen Presenting

Abstract CD4+ T cells may induce potent antitumor immune responses through interaction with antigen-presenting cells within the tumor microenvironment. Using a murine model of multiple myeloma, we demonstrated that adoptive transfer of idiotype-specific CD4+ T cells may elicit curative responses against established multifocal myeloma in bone marrow. This finding indicates that the myeloma bone marrow niche contains antigen-presenting cells that may be rendered tumoricidal. Given the complexity of the bone marrow microenvironment, the mechanistic basis of such immunotherapeutic responses is not known. Through a functional characterization of antitumor CD4+ T-cell responses within the bone marrow microenvironment, we found that killing of myeloma cells is orchestrated by a population of bone marrow–resident CD11b+F4/80+MHC-IIHigh macrophages that have taken up and present secreted myeloma protein. The present results demonstrate the potential of resident macrophages as powerful mediators of tumor killing within the bone marrow and provide a basis for novel therapeutic strategies against multiple myeloma and other malignancies that affect the bone marrow.

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In Vivo Mobilization of Multiple Myeloma Cells Out of the Bone Marrow Using the CXCR4 Inhibitor AMD3100 and Bortezomib: Implications for Sensitization of Myeloma Cells to Apoptosis.

Blood ◽

10.1182/blood.v110.11.2501.2501 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2501-2501 ◽

Cited By ~ 1

Author(s):

Judith M. Runnels ◽

Abdelkareem Azab ◽

Costas Pitsillides ◽

Anne-Sophie Moreau ◽

Feda Azab ◽

...

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Therapeutic Agents ◽

In Vivo Studies ◽

Thymidine Uptake ◽

Myeloma Cells ◽

Fourfold Increase ◽

Bortezomib Treatment ◽

In Vivo Flow Cytometry

Abstract Background: MM is characterized by widespread involvement of the bone marrow (BM) as the result of successful homing, engraftment and growth of myeloma cells. The BM provides protection and resistance of MM to therapeutic agents. Therefore, disruption of the interaction of MM cells with their microenvironment should lead to enhanced sensitivity to therapeutic agents. We hypothesized that disrupting CXCR4/SDF-1 axis will induce mobilization of MM cells from the BM into the circulation. Methods: MM.1S cells were co-cultured with bone marrow stromal cells (BMSCs) in the presence of AMD3100 (50uM, Sigma), bortezomib (0–2.5nM, Millennium) or combination of both; and cell proliferation was measured using [3H]-thymidine uptake. We then tested the in vivo AMD3100-induced mobilization of MM cells after they homed to the BM. MM1.S cells that had been fluorescently labeled with DiD (Invitrogen) were injected into mice through their tail veins. Beginning 24 hours later and for three subsequent days, the mice were treated with 5mg/kg AMD3100 sq daily, injected with fluorescently labeled c-kit antibody for HSC detection and immediately monitored for presence of circulating MM1-S cells or HSCs, using in vivo flow cytometry. To test whether AMD3100 induces mobilization of MM cells in established tumors, a GFP+ and luciferase+ osteotropic MM.1S cell line (Luc+GFP+MM.1S) was developed. This mouse model was used for continuous, real-time quantitation of MM cells mobilization. Bioluminescence imaging was used to determine tumor growth in vivo. Mice were treated with AMD3100 (5mg/kg, daily), bortezomib (1mg/kg biweekly) or the combination. Results: There was a significant increase in proliferation of MM cells in co-culture with BMSCs compared to MM cells alone. Moreover, AMD3100 alone did not inhibit proliferation; however, it significantly enhanced the cytotoxic effect of bortezomib in the presence of stromal cells. In vivo studies revealed that, unlike HSC, no appreciable mobilization of MM1S cells occurred after the first AMD3100 injection; however, the second AMD3100 treatment induced a fourfold increase in circulating MM cell numbers above background, while no further remarkable increase in circulating HSC was observed. The effect of AMD on an established tumor model showed that by the end of the first week of AMD3100 or bortezomib treatment, a 2-fold increase of circulating MM cells was observed compared to control mice, a trend which continued for three weeks. Moreover, compared to AMD3100 or bortezomib treatment alone, a further increase of circulating MM cells was observed in the peripheral blood of mice treated with combination of AMD3100 and bortezomib following the second bortezomib treatment. In the AMD3100 only treated mice, the counts of circulating MM cell continued to increase and tumors continued to progress, while in the AMD3100-bortezomib-treated mice, the circulating MM cell count decreased and the tumors regressed. Conclusion: These data support the hypothesis that disruption of the CXCR4/SDF-1 axis mobilizes MM cells, and that AMD3100 can be used to enhance the effects of therapeutic agents such as bortezomib.

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Developing In Vivo Model for Studying Mechanisms of Osteoblast Suppression in Multiple Myeloma.

Blood ◽

10.1182/blood.v110.11.4731.4731 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4731-4731

Author(s):

Chang-Sook Hong ◽

Alisa Huston ◽

Flavia Esteve ◽

Judy Anderson ◽

Ken Patrene ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Osteoblast Differentiation ◽

Treated Group ◽

Bone Lesions ◽

Neoplastic Disease ◽

In Vivo Models ◽

Myeloma Cells ◽

Ganciclovir Treatment

Abstract Multiple myeloma (MM) is an incurable neoplastic disease characterized by an accumulation of plasma cells in bone marrow. Osteolytic bone lesions are the major source of morbidity in MM patients and are associated with bone pain and fractures and hypercalcemia. The bone lesions result from increased osteoclastic bone destruction in areas adjacent to the myeloma cells. New bone formation that normally happens at sites of previous bone resorption still occurs in early stages of the disease but is absent in advanced MM. Although the molecular basis for the increased osteoclastic activity has been intensely investigated, the basis for the decreased osteoblast activity is just beginning to be understood. Recently, inhibitors of WNT signaling pathway, Dickkorpf1 (DKK1) and secreted Frizzle-Related Protein-2 (sFRP2) have been identified as factors involved in osteoblast suppression in MM. In addition, IL-3 and IL-7 are increased in plasma of MM patients and suppress osteoblastogenesis in cell culture models. However, the role of those factors in the osteoblastic activity in MM patients is unclear. Studies in patients are confounded by cytotoxic therapy as well as bisphosphonates, which are standard therapy for MM patients. Therefore, preclinical in vivo models are required to delineate the mechanisms responsible for the profound osteoblast suppression in MM. We have developed a mouse model of myeloma bone disease in which genetically modified myeloma cells can be selectively ablated without the confounding effects of cytotoxic therapies and allows us to tract the growth of MM cells. The 5TGM1 cell line which is the most common version of murine MM, was stably transfected with the thymidine kinase (TK) gene from herpes simplex virus, which permits eradication of myeloma cells with ganciclovir, as well as GFP and luciferase genes to detect the presence of MM cells. One ug/ml ganciclovir treatment in culture results in 100% death of the transfected 5TGM1 cells in 4 days. Importantly, ganciclovir treatment of primary marrow cell cultures had no effect on growth and differentiation of osteoblast and hematopoietic progentitors. Co-culturing of primary marrow cells with 5TGM1 expressing TK has no bystander effect on osteoblast differentiation with ganciclovir treatment. Subcutaneously implanted 5TGM1 cells into SCID mice were eradicated by intraperitoneal injection of 20mg/kg ganciclovir/d for 2 weeks. The dose of ganciclovir did not affect osteoblast differentiation of primary marrow culture from the mice treated with ganciclovir. Then we injected the 5TGM1 cells into tibia of SCID nude mice (n=4 per group). After measuring the increase of serum IgG2b level, half of the mice were treated with ganciclovir for 2 weeks and the other with saline. Our preliminary data show that osteogenic cultures of bone marrow from the ganciclovir treated mice had significantly higher alkaline phosphatase activity than cultures derived from the saline treated group (p=0.03). In addition, the ganciclovir treated mice had tendency of higher trabecular bone volume than the saline-treated group (p=0.08). These results demonstrate that this model should be useful for studying mechanisms of osteoblast suppression in MM.

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Inhibition of the Proteasome in Bone Marrow-Derived CD138+ Tumor Cells Following Carfilzomib Administration in Relapsed or Refractory Myeloma Patients.

Blood ◽

10.1182/blood.v114.22.1845.1845 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1845-1845

Author(s):

Suzanne Trudel ◽

Susan Lee ◽

Christopher J. Kirk ◽

Nashat Gabrail ◽

Sagar Lonial ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Tumor Cells ◽

Whole Blood ◽

Research Funding ◽

Proteasome Inhibition ◽

Proteasome Activity ◽

Advisory Committees ◽

Post Dose ◽

Blood Samples

Abstract Abstract 1845 Poster Board I-871 Background: Proteasome inhibition is an effective strategy for the treatment of multiple myeloma. In patients, proteasome inhibition has primarily been measured in peripheral blood samples (whole blood or mononuclear cells). However, it is unknown whether myeloma cells in the bone marrow (BM) are equally sensitive to proteasome inhibitors such as bortezomib (BTZ) and carfilzomib (CFZ). Aim: To measure proteasome inhibition in purified tumor cells from BM samples taken from patients enrolled in two ongoing Phase 2 trials of single agent CFZ in relapsed or refractory myeloma: PX-171-003 (003) and PX-171-004 (004). Methods: CFZ was administered as an IV bolus of 20 mg/m2 on Days 1, 2, 8, 9, 15 and 16 of a 28-day cycle on both trials. Bone marrow samples, from an optional sub-study of both trials, were taken during screening and Day 2 (post-treatment) and sorted into CD138+ and CD138− cells. Proteasome activity was measured by an enzymatic assay using a fluorogenic substrate (LLVY-AMC) for the chymotrypsin-like (CT-L) activity and an active site ELISA (ProCISE) to quantitate levels of the CT-L subunits of the constitutive proteasome (Beta5) and immunoproteasome (LMP7) and the immunoproteasome subunit MECL1. Results: Whole blood samples from patients treated with CFZ showed inhibition of CT-L activity of ∼80+, similar to values obtained in Phase 1 studies. A total of 10 CD138+ screening samples, 6 from 004 and 4 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed for proteasome levels and activity. In addition, 15 CD138−screening samples, 7 from 004 and 8 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed. When compared to the average base-line activity, CFZ treatment resulted in 88% CT-L inhibition in CD-138+tumor cells from 004 patients (P = 0.0212 by unpaired t-test) and 59% CT-L inhibition in CD-138+ tumor cells from 003 patients (P = 0.25). Baseline CT-L activity in CD138+ tumor cells was 3-fold higher in 004 than 003, which includes a more heavily pre-treated patient population with greater prior exposure to BTZ. Higher specific enzymatic activity was due to increased levels of both constitutive and immunoproteasomes in tumor cells, where immunoproteasomes account for >75% of total cellular proteasomes. No differences between trials were seen in baseline CT-L activity from non-tumor (CD138−) cells. Inhibition in CD138− cells was 84% (P = 0.0380 and 42% (P = 0.38) in 004 and 003, respectively. Using ProCISE, we measured inhibition of LMP7 (66%), beta5 (48%) and MECL1 (64%) in CD138+ tumor cells from 004 patients. Three patients from 004 and one from 003 had both a screening and post-dose tumor cell samples available for analysis. Inhibition of CT-L activity was >80% in two of the 3 patients on 004; the third patient showed no proteasome inhibition by ProCISE and was unavailable for analysis by CT-L. CT-L activity in the CD138+ tumor cells in the 003 patient was not inhibited, however, inhibition was seen in non-tumor cells. Conclusions: CFZ inhibits the proteasome activity of myeloma cells in the bone marrow of relapsed and refractory myeloma patients. The levels of inhibition were similar to those measured in whole blood samples, supporting the use of the blood-based assay as a surrogate marker for proteasome inhibition in tumor cells. CFZ treatment resulted in inhibition of both CT-L subunits as well as additional subunits of the immunoproteasome in tumor cells. Reduced baseline activity in the more heavily pretreated 003 patients may reflect reduced tumor-dependency on the proteasome and may be related to prior treatment with BTZ in these patients. More samples are needed in order to make correlations between levels of proteasome inhibition in bone marrow tumor cells and prior therapies or response. These observations support further evaluation of proteasome activity and the effects of this promising new agent in primary tumors cells from myeloma patients. Disclosures: Trudel: Celgene: Honoraria, Speakers Bureau; Ortho Biotech: Honoraria. Lee:Proteolix, Inc.: Employment. Kirk:Proteolix, Inc.: Employment. Lonial:Celgene: Consultancy; Millennium: Consultancy, Research Funding; BMS: Consultancy; Novartis: Consultancy; Gloucester: Research Funding. Wang:Proteolix, Inc.: Research Funding. Kukreti:Celgene: Honoraria. Stewart:Genzyme, Celgene, Millenium, Proteolix: Honoraria; Takeda, Millenium: Research Funding; Takeda-Millenium, Celgene, Novartis, Amgen: Consultancy. Jagannath:Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. McDonagh:Proteolix: Research Funding. Zonder:Celgene: Speakers Bureau; Pfizer: Consultancy; Seattle Genetics, Inc.: Research Funding; Amgen: Consultancy; Millennium: Research Funding. Bennett:Proteolix: Employment.

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Dual Targeting of -TORC1 and -TORC2 as a New Strategy In the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood.v116.21.133.133 ◽

2010 ◽

Vol 116 (21) ◽

pp. 133-133 ◽

Cited By ~ 2

Author(s):

Patricia Maiso ◽

AbdelKareem Azab ◽

Yang Liu ◽

Yong Zhang ◽

Feda Azab ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Cell Lines ◽

Mechanism Of Action ◽

Plasma Cells ◽

Bone Marrow Microenvironment ◽

Advisory Committees

Abstract Abstract 133 Introduction: Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment such as cytokines and growth factors, nutrients and stresses to regulate multiple cellular processes, including translation, autophagy, metabolism, growth, motility and survival. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 and TORC2. Activation of TORC1 leads to the phosphorylation of p70S6 kinase and 4E-BP1, while activation of TORC2 regulates phosphorylation of Akt and other AGC kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin analogues such as RAD001 and CCI-779 have been tested in clinical trials in MM. Their efficacy as single agents is modest, but when used in combination, they show higher responses. However, total inhibition of Akt and 4E-BP1 signaling requires inactivation of both complexes TORC1 and TORC2. Consequently, there is a need for novel inhibitors that can target mTOR in both signaling complexes. In this study we have evaluated the role of TORC1 and TORC2 in MM and the activity and mechanism of action of INK128, a novel, potent, selective and orally active small molecule TORC1/2 kinase inhibitor. Methods: Nine different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: To examine activation of the mTOR pathway in MM, we performed kinase activity assays and protein analyses of mTOR complexes and its downstream targets in nine MM cell lines. We found mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all cell lines tested independently of the status of Deptor, PTEN, and PI3K. All cell lines expressed either Raptor, Rictor or both; excepting H929 and U266LR7 which were negative for both of them. Moreover, primary plasma cells from several MM patients highly expressed pS6R while normal cells were negative for this protein. We found that INK128 and rapamycin effectively suppressed phosphorylation of p6SR, but only INK128 was able to decrease phosphorylation of 4E-BP1. We observed that INK128 fully suppressed cell viability in a dose and time dependent manner, but rapamycin reached a plateau in efficacy at ± 60%. The IC50 of INK128 was in the range of 7.5–30 nM in the eight cell lines tested. Similar results were observed in freshly isolated plasma cells from MM patients. Besides the induction of apoptosis and cell cycle arrest, INK128 was more potent than rapamycin to induce autophagy, and only INK128 was able to induce PARP and Caspases 3, 8 and 9 cleavage. In the bone marrow microenvironment context, INK128 inhibited the proliferation of MM cells and decreased the p4E-BP1 induction. Importantly, treatment with rapamycin under such conditions did not affect cell proliferation. INK128 also showed a significantly greater effect inhibiting cell adhesion to fibronectin OPM2 MM1S, BMSCs and HUVECs compared to rapamycin. These results were confirmed in vivo. Oral daily treatment of NK128 (1.0 mg/kg) decreased tumor growth and improved survival of mice implanted with MM1S. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

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Induction of CXCL1 in Osteoblasts by Myeloma Cells Promotes Migration of Osteoclast Precursors

Blood ◽

10.1182/blood.v120.21.441.441 ◽

2012 ◽

Vol 120 (21) ◽

pp. 441-441

Author(s):

Martin F. Kaiser ◽

Ulrike Heider ◽

Maren Mieth ◽

Jozef Zustin ◽

Andrea Kuehnl ◽

...

Keyword(s):

Bone Marrow ◽

Healthy Volunteers ◽

Bone Disease ◽

Bone Marrow Microenvironment ◽

Bone Microenvironment ◽

Bone Marrow Biopsies ◽

Myeloma Cells ◽

Myeloma Bone Disease ◽

Immunomagnetic Sorting

Abstract Abstract 441 Introduction Multiple myeloma (MM) causes a dysbalance in the bone microenvironment between bone building osteoblasts and bone resorbing osteoclasts (OCs), with an increase in OC recruitment, differentiation and activation, leading to myeloma bone disease (MBD). Presence of MBD has a major impact on the quality of life of MM patients and novel treatment approaches for MBD are urgently needed. Several factors have been identified that play a role in this process, e.g. receptor activator of NF-kB ligand (RANKL). However, the pathomechanism of increased osteoclast recruitment and activation is not completely understood. Here, we investigated the role of the chemokine CXCL1 and its receptor CXCR2 in the bone microenvironment in MM. Material and Methods Serum samples from 52 patients with newly diagnosed MM and from 22 healthy volunteers were assayed using a CXCL1 ELISA. Primary human mesenchymal stem cells (hMSCs) were cultured from bone marrow aspirates and primary human differentiated osteoblasts (hOBs) were cultured from trabecular bone fragments, both from healthy volunteers. Osteoclast precursors (pre-OCs) were generated by immunomagnetic sorting of CD14-positive cells from the peripheral blood of healthy volunteers. Human myeloma cell lines (HMCLs) U-266, RPMI-8226 and LP-1 and primary bone marrow myeloma cells (pMMCs) selected using CD138 immunomagnetic sorting were used for the experiments. Co-cultures of HMCLs and pMMCs with hMSCs or hOBs were performed using 0.45 μm transwell inserts, allowing for the exchange of soluble mediators. Migration assays were performed using 8 μm transwell inserts and human recombinant CXCL1. Immunohistochemistry was performed on paraffin-embedded bone marrow biopsies from MM patients using an anti-CXCR2 monoclonal antibody. All experimental procedures involving patient material were approved by the local ethics committee and conducted after informed consent was obtained. Results CXCL1 serum levels were found to be significantly higher in MM patients than in healthy individuals (193.4 pg/mL vs. 137 pg/mL, respectively, p<0.05), indicating a role for CXCL1 in MM pathophysiology. We went on to investigate the role of CXCL1 in MBD and performed co-cultures of HMCLs and pMMCs with hMSCs or hOBs. Baseline CXCL1 expression was absent in HMCLs and low or absent in hMSCs or hOBs at baseline. RNA expression as well as protein excretion by hMSCs and hOBs were induced after co-culture with myeloma cells. For example, pMMCs from different individuals led to a mean 154-fold upregulation of CXCL1 mRNA levels in hMSCs and to a mean upregulation of CXCL1 protein in cell culture supernatants from <31.5 pg/mL at baseline to 2140 pg/mL after co-cultures. In order to investigate the potential function of elevated CXCL1 levels in the bone marrow microenvironment, the expression of CXCR2, the receptor for CXCL1, was analyzed. Pre-OCs as well as a majority of pMMCs expressed CXCR2 mRNA. CXCR2 protein expression in pMMCs was verified using immunohistochemistry on MM bone marrow biopsies. Human recombinant CXCL1 significantly increased pre-OC cell migration in a dose-dependent manner. For example, 50 ng/mL or 100 ng/mL of CXCL1 increased mean pre-OC migration along a CXCL1 gradient 2.5-fold and 5.6-fold over baseline, respectively. In addition, mean pMMC migration was increased 3.8-fold compared to baseline along a 100 ng/mL gradient of recombinant CXCL1. The osteoclastogenic capacity of the migrated pre-OCs was confirmed by TRAP expression after stimulation with RANKL and M-CSF. Conclusion We describe here a novel role for the chemokine CXCL1 in myeloma bone disease. We demonstrate that CXCL1 is induced in hMSCs and hOBs by co-culture with MM cells. CXCL1 leads to chemoattraction of both pre-OCs and pMMCs. These effects could lead to co-localization of OCs and MM cells in the bone marrow microenvironment and contribute to the tumor-promoting interaction between these cell types. Our data indicate the CXCL1-CXCR2 axis as a therapeutic target in myeloma bone disease. Disclosures: No relevant conflicts of interest to declare.

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Silencing The Sialyltransferase Gene ST3GAL6 Inhibits Adhesion and Migration Of Myeloma Cells In Vitro and Reduces The Homing and Proliferation Of Tumor Cells In Vivo

Blood ◽

10.1182/blood.v122.21.275.275 ◽

2013 ◽

Vol 122 (21) ◽

pp. 275-275

Author(s):

Siobhan Glavey ◽

Salomon Manier ◽

Antonio Sacco ◽

Michaela R Reagan ◽

Yuji Mishima ◽

...

Keyword(s):

Bone Marrow ◽

Cell Lines ◽

Research Funding ◽

Advisory Board ◽

Myeloma Cells ◽

Sialyl Lewis ◽

And Migration ◽

Rpmi 8226

Abstract Background Glycosylation is a stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process, especially increased sialylation, have been associated with malignant transformation and metastasis. The adhesion and trafficking of multiple myeloma (MM) cells is strongly influenced by glycosylation and multiple myeloma cells express a variety of adhesion molecules, including selectin ligands and integrins, which are typically dependent on glycosylation for their function. We have previously reported that the sialyltransferase ST3GAL6 is up-regulated in plasma cells from MM patients and that increased expression is associated with inferior overall survival (OS) in MM gene expression profiling (GEP) datasets. The functional significance of increased sialylation of MM cells has not previously been reported. Methods MM cell lines MM1s and RPMI-8226 were confirmed to have high expression levels of ST3GAL6 at the gene and protein level compared to healthy controls. Knockdown of ST3GAL6 was confirmed in MM cell lines RPMI-8226 and MM1s using lentiviral shRNAs targeting different regions in the ST3GAL6 mRNA. Specific ST3GAL6 knockdown was confirmed by reduced ST3GAL6 mRNA and protein expression in comparison to a scrambled control. In a calcein-AM fluorescence based adhesion assay we next evaluated the effects of ST3GAL6 knockdown on MM-cell adhesion to bone marrow stromal cells (BMSC’s) and fibronectin coated plates. Migration to 30nM SDF1-α was assessed using transwell plates comparing ST3GAL6 knockdown cells to scrambled controls. The commercially available sialyltransferase inhibitor 3Fax-Neu5Ac was used to pre-treat MM cells in vitro prior to assessment of apoptosis by flow cytometry. shST3GAL6 MM1s cells positive for green fluorescent protein and luciferin (GFP-Luc+) were injected into tail veins of SCID-Bg mice (5x106 cells, n=5/group) and mice were followed weekly using bioluminescent imaging (BLI) for tumor development. Bone marrow homing of tumor cells was assessed using in vivoconfocal imaging of the skull vasculature (n=3/group). Results Knockdown of ST3GAL6 in MM cell lines resulted in a 50% reduction in cell surface staining with the monoclonal antibody HECA-452. This indicated reduced expression of cutaneous lymphocyte associated antigen (CLA), a carbohydrate domain shared by sialyl Lewis X (sLex) and sialyl Lewis a (sLea) antigens, confirming suppression of ST3GAL6 activity. There was a significant reduction in the ability of knockdown cells to adhere to BMSC’s and fibronectin in-vitro compared to scrambled controls (P=0.016, 0.032 respectively). Migration ability of these cells in response to SDF1-α was also reduced (P=0.01). In vivo in a xenograft SCID-Bg mouse model shST3GAL6 cells demonstrated a reduced tumor burden as assessed by weekly BLI (P=0.017 at week 4). A consolidated map of the skull bone marrow niche in mice injected with shST3GAL6 MM1s GFP-Luc+ cells revealed a reduced homing ability of these cells in comparison to mice injected with scrambled control cells. Treatment of the MM cell lines MM1s and RPMI-8226 with a sialyltransferase inhibitor 3Fax-Neu5Ac resulted in almost complete elimination of cell surface sLex and/or sLea expression as determined by HECA-452 staining. Following pre-treatment with 3Fax-Neu5Ac, MM1S cells grown in co-culture with BMSC’s cells showed increased sensitivity to Bortezomib compared to cells treated with bortezomib alone. Conclusions shRNA knockdown of ST3GAL6 in MM cells significantly inhibits adhesion and migration in vitro with reduced homing and proliferation potential in vivo. In conjunction with the results of enzymatic inhibition this indicates that sialylation may play an important role in the malignant behavior of MM cells. Studies are ongoing to address the potential role of altered glycosylation in MM. Disclosures: Ghobrial: Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

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