Interactions between Myeloma Cells and Osteoclasts Are Controlled by Unique Histological Structures Involving Vascular Compartments.

Abstract Although myeloma development and enhanced bone resorption are intimately related, the mechanism responsible of this relation is not known. In vitro studies have stressed the critical role of direct cell contacts between myeloma cells and osteoclast. In vivo, however, little is known about the organization of the cells present at osteolytic lesions, because of the complexity of cell-cell interactions in the bone marrow of myeloma patients. Therefore, we conducted an immunohistochemical study with multiple stainings allowing the simultaneous identification of different cell types at resorption sites of bone marrow biopsies of myeloma patients. The biopsies showed that in average 1% of the bone surface was lined by mature multinucleated TRAP+ osteoclasts, but that only 6% of these osteoclasts showed direct contacts with myeloma cells. The biopsies showed also TRAP+ mononucleated pre-osteoclasts in the bone marrow compartment, and 40% of these pre-osteoclasts showed direct contacts with myeloma cells. Bone marrow pre-osteoclasts show thus much more frequent contacts with myeloma cells, compared with mature osteoclasts lining bone surfaces. These respective values remained unchanged, whether the myeloma cells were identified through CD138 or through light chain expression (counts in biopsies from 13 patients). Importantly, we found that 80% of the osteoclasts lining the bone surfaces, were separated from the bone marrow compartment by a specialized cell wall (seen in all biopsies of the 15 patients, that were analyzed). This wall consists of a single layer of (sometimes very) flattened cells lining the bone marrow, and expressing NCAM, propeptide of type III collagen, and osteocalcin, but not CD34. When performing 3D reconstructions by using serial sections, this wall appeared as a continuous roof covering the bone surfaces undergoing remodeling, and connected to the bone lining cells at its periphery. Furthermore, CD34 staining revealed that capillaries are abundant at the bone marrow side of this cell wall, and that some show an opening into the wall. These capillaries may thus allow communication between the bone marrow and the bone surfaces undergoing remodeling, and render the bone remodeling compartments vascular-like. In addition, the TRAP+ preosteoclast detected in the bone marrow space, were positioned along the capillaries leading to the vascular/remodeling compartments. In conclusion, this study shows that in vivo, interactions between myeloma cells and osteoclasts are mediated only rarely through direct cell contacts, and identifies for the first time unique cell arrangements that are likely to play a role in these interactions: a specialized cell wall separates the bone marrow from the vascular/remodeling compartments in most resorption sites (80% of the osteoclasts), and the cells of this wall are thus in a privileged situation to control myeloma-osteoclast interactions; capillaries connect the marrow cavity with the vascular/remodeling compartment, thereby allowing guidance of pre-osteoclasts from the bone marrow to the resorption sites; the generation of these pre-osteoclasts may be stimulated by the high incidence of their direct contacts with myeloma cells in the bone marrow.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

High levels of soluble syndecan-1 in myeloma-derived bone marrow: modulation of hepatocyte growth factor activity

Blood ◽

10.1182/blood.v96.9.3139.h8003139_3139_3146 ◽

2000 ◽

Vol 96 (9) ◽

pp. 3139-3146 ◽

Cited By ~ 1

Author(s):

Carina Seidel ◽

Magne Børset ◽

Øyvind Hjertner ◽

Dianjun Cao ◽

Niels Abildgaard ◽

...

Keyword(s):

Bone Marrow ◽

Growth Factor ◽

Hepatocyte Growth Factor ◽

Heparan Sulfate ◽

Cell Line ◽

Osteosarcoma Cell ◽

Heparin Binding ◽

Myeloma Cells ◽

Hepatocyte Growth

Syndecan-1 is a heparan sulfate proteoglycan expressed on the surface of, and actively shed by, myeloma cells. Hepatocyte growth factor (HGF) is a cytokine produced by myeloma cells. Previous studies have demonstrated elevated levels of syndecan-1 and HGF in the serum of patients with myeloma, both of negative prognostic value for the disease. Here we show that the median concentrations of syndecan-1 (900 ng/mL) and HGF (6 ng/mL) in the marrow compartment of patients with myeloma are highly elevated compared with healthy controls and controls with other diseases. We show that syndecan-1 isolated from the marrow of patients with myeloma seems to exist in an intact form, with glucosaminoglycan chains. Because HGF is a heparan-sulfate binding cytokine, we examined whether it interacted with soluble syndecan-1. In supernatants from myeloma cells in culture as well as in pleural effusions from patients with myeloma, HGF existed in a complex with soluble syndecan-1. Washing myeloma cells with purified soluble syndecan-1 could effectively displace HGF from the cell surface, suggesting that soluble syndecan-1 can act as a carrier for HGF in vivo. Finally, using a sensitive HGF bioassay (interleukin-11 production from the osteosarcoma cell line Saos-2) and intact syndecan-1 isolated from the U-266 myeloma cell line, we found that the presence of high concentrations of syndecan-1 (more than 3 μg/mL) inhibited the HGF effect, whereas lower concentrations potentiated it. HGF is only one of several heparin-binding cytokines associated with myeloma. These data indicate that soluble syndecan-1 may participate in the pathology of myeloma by modulating cytokine activity within the bone marrow.

Download Full-text

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

American Journal of Hematology ◽

10.1002/ajh.21374 ◽

2009 ◽

Vol 84 (5) ◽

pp. 268-272 ◽

Cited By ~ 31

Author(s):

Claire M. Edwards ◽

Seint T. Lwin ◽

Jessica A. Fowler ◽

Babatunde O. Oyajobi ◽

Junling Zhuang ◽

...

Keyword(s):

Bone Marrow ◽

Proteasome Inhibition ◽

Bone Marrow Microenvironment ◽

Proteasome Activity ◽

Myeloma Cells

Download Full-text

Exosomal miR-135b shed from hypoxic multiple myeloma cells enhances angiogenesis by targeting factor-inhibiting HIF-1

Blood ◽

10.1182/blood-2014-05-576116 ◽

2014 ◽

Vol 124 (25) ◽

pp. 3748-3757 ◽

Cited By ~ 287

Author(s):

Tomohiro Umezu ◽

Hiroko Tadokoro ◽

Kenko Azuma ◽

Seiichiro Yoshizawa ◽

Kazuma Ohyashiki ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Signaling Pathway ◽

Cell Lines ◽

Tube Formation ◽

Myeloma Cells ◽

Endothelial Tube Formation ◽

Key Points ◽

Resistant Cells

Key Points We established hypoxia-resistant cells that can mimic in vivo conditions of hypoxic bone marrow. Exosomal miR-135b derived from these cell lines enhanced endothelial tube formation under hypoxia via the HIF-FIH signaling pathway.

Download Full-text

Mcl-1 Is Overexpressed in Multiple Myeloma and Correlates with Disease Severity.

Blood ◽

10.1182/blood.v104.11.1419.1419 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1419-1419

Author(s):

Soraya Wuilleme-Toumi ◽

Nelly Robillard ◽

Patricia Gomez-Bougie ◽

Philippe Moreau ◽

Steven Le Gouill ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Disease Severity ◽

Cell Lines ◽

Plasma Cells ◽

Myeloma Cell ◽

Lymphocytic Leukemia ◽

Myeloma Cells

Abstract Multiple Myeloma (MM) is a fatal malignancy of B-cell origin characterized by the accumulation of plasma cells within the bone marrow. The expression of the pro-survival members of the Bcl-2 family has been shown to be a key process in the survival of myeloma cells. More particularly, Mcl-1 expression turned out to be critical for their survival. Indeed, knockdown of Mcl-1 by antisenses induces apoptosis in myeloma cells. Finally, Mcl-1 was found to be the only anti-apoptotic Bcl-2 family member which level of expression was modified by cytokine treatment of myeloma cells. For these reasons, we have evaluated the expression of Mcl-1 in vivo in normal, reactive and malignant plasma cells (PC) i.e., myeloma cells from 55 patients with MM and 20 human myeloma cell lines using flow cytometry. We show that Mcl-1 is overexpressed in MM in comparison with normal bone marrow PC. Forty-seven percent of patients with MM at diagnosis (p=.017) and 80% at relapse (p=.014 for comparison with diagnosis) overexpress Mcl-1. Of note, only myeloma cell lines but not reactive plasmocytoses have abnormal Mcl-1 expression, although both plasmocyte expansion entities share similar high proliferation rates (>20%). Of interest, Bcl-2 as opposed to Mcl-1, does not discriminate malignant from normal PC. This shows that the overexpression of Mcl-1 is clearly related to malignancy rather than to proliferation. It will be important to know whether the overexpression of Mcl-1 is related to an abnormal response to cytokines like Interleukin-6 or to mutations of the promoter of the Mcl-1 gene as already described in B chronic lymphocytic leukemia. Finally, level of Mcl-1 expression is related to disease severity, the highest values being correlated with the shortest event-free survival (p=.01). In conclusion, Mcl-1 which has been shown to be essential for the survival of human myeloma cells in vitro is overexpressed in vivo in MM and correlates with disease severity. Mcl-1 represents a major therapeutical target in MM.

Download Full-text

Selective Inhibition of CDK4 and CDK6 Primes Chemoresistant Myeloma Cell for Cytotoxic Killing through Induction of Cell Cycle-Coupled Mitochondrial Dysfunction and Apoptosis

Blood ◽

10.1182/blood.v112.11.3678.3678 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3678-3678 ◽

Cited By ~ 1

Author(s):

Xiangao Huang ◽

Maurizio Di Liberto ◽

Tracey Louie ◽

David S Jayabalan ◽

Scott Ely ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Bone Marrow ◽

Mitochondrial Dysfunction ◽

Low Dose ◽

Selective Inhibition ◽

Cytotoxic Agents ◽

G1 Arrest ◽

Myeloma Cells

Abstract Dysregulation of cyclin-dependent kinase (CDK)4 or CDK6 activity by gain of function or loss of inhibition is one of the most frequent aberrations in cancer. This includes multiple myeloma (MM), where overexpression of CDK4 (CDK6) precedes unrestrained proliferation of CD138+ bone marrow myeloma cells in vivo, in particular during aggressive tumor growth and relapse. In complex with the D-type cyclin, CDK4 and CDK6 promote cell cycle entry and progression through G1 by inactivating the retinoblastoma protein Rb and antagonizing the INK4 family of CDK inhibitors, suggesting that inhibition of CDK4/6 is a promising approach for cell cycle control in MM. We have now developed a novel approach to both halt cell proliferation and enhance cytotoxic killing of MM cells by selective inhibition of CDK4/6 in combination with cytotoxic agents. We show that knocking down CDK4 and CDK6 expression by shRNA interference or inhibiting CDK4/6 activity with PD 0332991, the only known CDK4/6-specific small molecule inhibitor, leads to sustained G1 arrest and induction of synchronous cell cycle progression upon removal of PD 0332991. Induction of sustained early G1 arrest is not accompanied by apoptosis. However, it primes MM cells for synergistic killing by low dose cytotoxic agents of diverse modes of action, which is further augmented during synchronous S phase entry. Most importantly, induction of sustained G1 arrest with PD 0332991 primes freshly isolated chemoresistant CD138+ bone marrow myeloma cells for killing by low dose proteasome inhibitors in the presence of bone marrow stromal cells. Synergistic killing by PD 0332991 combined with low dose bortezomib (2–6 nM) in early G1 (referred to as PD-B) is mediated by increased neutralization of Mcl-1 and Bcl-2 in the absence of Noxa, as PD-B augments bortezomib activation of Bim and Mcl-1 transcription while silencing Noxa in early G1. This leads to aggregation of Bak, but not Bax, on the mitochondria, mitochondrial membrane depolarization, preferential release of Smac/DIABLO, but not cytochrome c, from mitochondria, reduction of c-IAP and caspase-9 activation. Apoptosis is further amplified through activation of caspase-8 without inducing TRAIL, FASL and TNF-α, the major ligands that trigger the extrinsic apoptosis pathway. Cytotoxic killing by PD-B is recapitulated in synergistic tumor suppression in animal models. Collectively, our ex vivo and in vivo data demonstrate that PD-B induces synergistic killing of MM cells through cell cycle-coupled regulation of Bcl-2 family genes and induction of mitochondrial dysfunction. As PD 0332991 is orally bio-available, potent and low in toxicity, our approaches have formed the basis for an ongoing, first-inclass Phase I/II clinical trial to selectively target CDK4/6 with PD 0332991 in combination with bortezomib and dexamethasone in multiple myeloma. Selective targeting CDK4 and CDK6 in combination with cytotoxic killing, therefore, provides a new and promising mechanism-based therapeutic strategy for multiple myeloma and potentially other cancers.

Download Full-text

Dual Src/Abl Kinase Targeted Inhibition in Myeloma Microenvironment Promotes Myeloma Cell Apoptosis Both in Vitro and In Vivo.

Blood ◽

10.1182/blood.v114.22.2813.2813 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2813-2813

Author(s):

Karthik Ramasamy ◽

Lee Macpherson ◽

Ghulam J Mufti ◽

Stephen Schey ◽

Yolanda Calle

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Stromal Cells ◽

Plasma Cells ◽

Myeloma Cell ◽

Myeloma Cells ◽

Abl Kinase ◽

Osteoclast Function

Abstract Abstract 2813 Poster Board II-789 Osteoclast, in addition to eroding the bone resulting in lytic lesions, enhances plasma cell proliferation and survival via direct cell to cell contact. Src family protein tyrosine kinases (SFKs) and c-Abl kinase play important role downstream of integrin adhesion receptors, and regulate the cytoskeletal organisation, cell motility and gene expression in response to cell adhesion. We hypothesised targeting SFKs and Abl kinase with the small molecule tyrosine kinase inhibitor Dasatinib has potential to reduce adhesion of plasma cells to ECM proteins in the bone marrow and modify the microenvironment by inhibiting osteoclast function, specifically bone resorption. As a result, myeloma cells could be sensitised to drugs with cytotoxic properties such as dexamethasone. Osteoclasts were generated from primary bone marrow mononuclear cells of myeloma and MGUS patients (n=10). Using Immunofluorescence, we found that Dasatinib 100nM but not dexamethasone inhibited osteoclastogenesis and disrupted the actin cytoskeletal organisation with actin clusters formed in the periphery of the cell. There was absence of actin ring formation at sealing zones which is essential for bone resorption. This effect consistently led to impaired osteoclast function, evidenced by fewer resorption pits formed on rabbit dentine slices on toluidine blue staining. Experiments were repeated ≥ 3 times. In plasma cells, the combination of dexamethasone and Dasatinib synergistically (Calcusyn software) inhibited cell proliferation at clinically relevant concentrations and induced apoptosis of human and murine myeloma cell lines alone and in cocultures with human stromal cells ( p<.001). Dasatinib alone at 200 nM concentration does not inhibit plasma cell proliferation with maximal serum concentration achieved in Phase I CML trials being 180nM. Additionally, Dasatinib and Dexamethasone in combination inhibited secretion of IL-6 but not MIL -1 alpha in stromal cell cocultures. Dasatinib but not dexamethasone significantly inhibited adhesion of myeloma cell lines on Fibronectin despite integrin activation with Magnesium EGTA. This effect was mediated through down regulation of both Src and Abl phosphorylation. Both Dasatinib and Dexamethasone inhibited adhesion of PC on stromal cells and osteoclasts. Taken together, our in vitro results suggest that Dasatinib and dexamethasone could be an effective therapeutic combination with Dasatinib impairing adhesion of plasma cells to the bone marrow microenvironment as well as osteoclast function and resultant bone disease thereby sensitising myeloma cells to the cytotoxic effect of dexamethasone. We have also established that the combination of Dasatinib 75mg/kg and dexamethasone 1mg/kg is not toxic to C57BL/KaLwRij mice. The anti-myeloma efficacy of these drugs alone and in combination is being currently studied. The combination of Dasatinib 100 mg OD days 1-28 and Dexamethasone 20mg OD on Day 1-4, 15-18 has resulted in a partial response (EBMT criteria) in 2 multiply relapsed and steroid refractory myeloma patients without significant toxicity. Serum calcium levels fell commensurate with disease response and we are currently performing experiments to analyse the effect of the drug combination on osteoclast function in vivo. These findings warrant exploring this drug combination in steroid resistant myeloma and patients with extensive skeletal disease prospectively in a phase I/II trial. Disclosures: Off Label Use: Dasatinib is not licensed for Myeloma.

Download Full-text