Surface Of Multiple Myeloma Cells Research Articles

Podia in Multiple Myeloma (MM) Cells Promote Adhesion with Bone Marrow (BM) Fibroblastic Stromal Cells

Abstract 626The physical contact between MM cells and the BM microenvironment can lead to cell adhesion mediated drug resistance. Although some components of adhesive structures in MM cells have been identified very little is known about their exact organisation and dynamics. We have observed formation of proteoglycan CD138 and F-actin containing membrane extensions in MM cells in in vitro cultures of BM aspirates as well as in sections of BM trephines of MM patients. MM membrane extensions elongated on the surface of BM stromal cells or interconnected MM cells forming seemingly cellular networks. Morphologically, MM membrane extensions appear similar to other structures in haematopoietic cells such as nanotubes formed by T-cells involved in cell communication and podia in CD34+ cells and leukemia cell lines whose function remains unknown. Dexamethasone (a drug commonly used clinically against MM) increased the percentage of MM cells displaying membrane extensions and the length of these structures. This correlated with enhanced expression of surface CXCR4, increased adhesion of MM cells on fibroblastic stromal cells and protection of MM cells against Dexamethasone-mediated apoptosis. Treatment with Bortezomib at clinically achievable doses decreased CXCR4 levels in MM cells and did not induce podia formation/extension and correlated with inhibition of MM cell adhesion and induction of MM cell apoptosis in a dose dependent manner. Blocking CXCR4 signalling with Plerixafor (AMD3100) inhibited the rate of formation and length of MM membrane extensions and correlated with sensitisation of MM cells to Dexamethasone. Similar inhibitory results were obtained using Dexamethasone in combination with the c-Abl/Src kinase inhibitor Dasatinib. Neither Dasatinib nor Plerixafor at doses achievable in patients directly induced apoptosis in MM or BM stromal cells. We conclude that membrane extensions/podia formed by MM cells are involved in the interaction with BM stromal cells, require CXCR4 and Src and/or c-Abl activity and could be involved in resistance to treatment with Dexamethasone. Disclosures:No relevant conflicts of interest to declare.

Systemic Therapy of Disseminated Myeloma in Passively Immunized Mice Using Measles Virus-infected Cell Carriers

Multiple myeloma (MM) is bone marrow plasma cell malignancy. A clinical trial utilizing intravenous administration of oncolytic measles virus (MV) encoding the human sodium-iodide symporter (MV-NIS) is ongoing in myeloma patients. However, intravenously administered MV-NIS is rapidly neutralized by antiviral antibodies. Because myeloma cell lines retain bone marrow tropism, they may be ideal as carriers for delivery of MV-NIS to myeloma deposits. A disseminated human myeloma (KAS 6/1) model was established. Biodistribution of MM1, a myeloma cell line, was determined after intravenous infusion. MM1 cells were found in the spine, femurs, and mandibles of tumor-bearing mice. Lethally irradiated MM1 cells remained susceptible to measles infection and transferred MV to KAS 6/1 cells in the presence of measles immune sera. Mice-bearing disseminated myeloma and passively immunized with measles immune serum were given MV-NIS or lethally irradiated MV-NIS-infected MM1 carriers. The antitumor activity of MV-NIS was evident only in measles naive mice and not in passively immunized mice. In contrast, survivals of both measles naive and immune mice were extended using MV-NIS-infected MM1 cell carriers. Hence, we demonstrate for the first time that systemically administered cells can serve as MV carriers and prolonged survival of mice with pre-existing antimeasles antibodies.

Inhibition of NF-κB and Akt pathways by an antibody-avidin fusion protein sensitizes malignant B-cells to cisplatin-induced apoptosis

Multiple myeloma (MM) is an incurable disease of malignant plasma cells. Recent therapeutic advancements have resulted in improved response rates, however, there is no improvement in overall survival, therefore, new therapeutics are needed. Since the transferrin receptor is upregulated on the surface of MM cells, we previously developed an antibody fusion protein consisting of an IgG3 specific for the human transferrin receptor 1 (TfR1, CD71) genetically fused to avidin at its carboxy-terminus (ch128.1Av). We have previously shown that ch128.1Av exhibits intrinsic cytotoxicity against certain malignant B-cells by disrupting the cycling of the TfR and decreasing TfR cell surface expression resulting in lethal iron starvation. In addition, ch128.1Av can sensitize malignant cells to apoptosis induced by gambogic acid, a herbal drug used in Chinese medicine. In this study, we hypothesized that ch128.1Av may also sensitize drug-resistant malignant B-cells to chemotherapeutic agents by inhibiting key survival pathways. In this study we show that ch128.1Av sensitizes malignant B-cells to apoptosis induced by cisplatin (CDDP). The sensitization by ch128.1Av resulted in the inhibition of the constitutively activated Akt and NF-kappaB survival/antiapoptotic pathways and downstream decreased expression of antiapoptotic gene products such as BclxL and survivin. The direct role of the inhibition of the Akt and NF-kappaB pathways by ch128.1Av in CDDP-mediated cytotoxicity was demonstrated by the use of specific chemical inhibitors and siRNA which mimicked the effects of ch128.1Av. Overall, this study provides evidence of the therapeutic potential of ch128.1Av as a chemo-sensitizing agent in drug-resistant tumor cells.

FcRL5 as a Target of Antibody-Drug Conjugates for the Treatment of Multiple Myeloma.

Abstract 3836Poster Board III-772Antibody-drug conjugates (ADCs), potent cytotoxic drugs linked to antibodies via specialized chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Target selection is a key component to the success of an ADC. In addition to expression on the tumor, the target should have limited expression on normal tissues. The most prevalent and best-studied surface antigens on multiple myeloma (MM) cells, CD138, CD38, CD72, and CD56, all have relatively broad expression patterns that may lead to target-dependent toxicities with ADCs. We have previously shown that FcRL5/FcRH5/IRTA2 is expressed only on B-cells and plasma cells. Here we show that FcRL5 is expressed on the surface of MM cells from 85% of patients and thus could be a target for antibody and ADC treatments of MM. As experience in humans with anti-CD20 antibodies suggests that depletion of B-cells does not present a major safety issue, FcRL5 appears to have an excellent expression pattern as an ADC target for MM. Internalization of target antigens is known to increase the efficacy of ADCs and we found that FcRL5 is internalized upon antibody binding, an ideal trait. We made two different ADCs consisting of anti-FcRL5 antibodies 1) conjugated through cysteines to monomethylauristatin E (MMAE) by a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vcPAB) linker that is designed to be cleaved by cathepsins (Anti-FcRL5-MC-vcPAB-MMAE); and 2) conjugated via lysines to the maytansinoid DM4 through a disulified linker designed to be cleaved by reducing conditions (anti-FcRL5-SPDB-DM4). These ADCs were tested for cell killing in vitro and in xenograft studies using OPM2 cells stably expressing FcRL5. While unconjugated anti-FcRH5 and control ADCs were not effective, anti-FcRL5-MC-vcPAB-MMAE and anti-FcRL5-SPDB-DM4 were effective at killing this MM cell line both in vitro and in vivo. Furthermore, the anti-FcRL5-SPDB-DM4 conjugate was effective in a SCID-rabbit bone model of MM using the LD cell line. These data suggest that FcRL5 ADCs could be an effective treatment for MM. Disclosures:Polson:Genentech, Inc.: Employment, Equity Ownership. Zheng:Genentech, Inc.: Employment, Equity Ownership. Elkins:Genentech, Inc.: Employment, Equity Ownership. Lau:Genentech, Inc.: Employment, Equity Ownership. Go:Genentech, Inc.: Employment, Equity Ownership. Scales:Genentech, Inc.: Employment, Equity Ownership. Yu:Genentech, Inc.: Employment, Equity Ownership. Chesi:Genentech, Inc.: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Merck: Research Funding. Bergsagel:Genentech, Inc.: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Merck: Research Funding. Ebens:Genentech, Inc.: Employment, Equity Ownership, Patents & Royalties.

APRIL and TACI interact with syndecan‐1 on the surface of multiple myeloma cells to form an essential survival loop

BLyS and APRIL share two receptors - transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA) - and BLyS binds to a third receptor, BAFF-R. We previously reported that TACI gene expression is a good indicator of a BLyS-binding receptor in human multiple myeloma cell lines (HMCLs), unlike BCMA, which is expressed by all HMCLs or BAFF-R which is typically not expressed by late-stage B cells. We hypothesised a link between APRIL and TACI through syndecan-1, similar to the situation reported for FGF and FGFR. We observed very strong binding of APRIL, but not BLyS, at the surface of all syndecan-1(+) HMCLs and primary multiple myeloma cells (MMC). All syndecan-1(+) HMCLs and MMC could also bind TACI-Fc, but not BCMA-Fc or BAFF-R-Fc molecules. Binding of APRIL or TACI-Fc was abrogated by heparin or cell pretreatment with heparitinase, which cleaves heparan sulfate chains. The growth factor activity of APRIL on MMC was also inhibited by heparin. Our data identify syndecan-1 as a co-receptor for APRIL and TACI at the cell surface of MMC, promoting the activation of an APRIL/TACI pathway that induces survival and proliferation in MMC.

Expression of genes encoding for proteins involved in heparan sulphate and chondroitin sulphate chain synthesis and modification in normal and malignant plasma cells

Syndecan-1 is a proteoglycan that concentrates heparin-binding factors on the surface of multiple myeloma cells, and probably plays a major role in multiple myeloma biology. As heparan sulphate and chondroitin sulphate are the bioactive components of syndecan-1, we analysed the signature of genes encoding 100 proteins involved in synthesis of these chains, i.e. from precursor uptake to post-translational modifications, using Affymetrix microarrays. The expression of enzymes required for heparan sulphate and chondroitin sulphate biosynthesis was shown to increase in parallel with syndecan-1 expression, throughout the differentiation of memory B cells into plasmablasts and normal bone marrow plasma cells. Sixteen genes were significantly different between normal and malignant plasma cells, nine of these genes -EXT2, CHSY3, CSGALNACT1, HS3ST2, HS2ST1, CHST11, CSGALNACT2, HPSE, SULF2 - encode proteins involved in glycosaminoglycan chain synthesis or modifications. Kaplan-Meier analysis was performed in two independent series of patients: B4GALT7, CSGALNACT1, HS2ST1 were associated with a good prognosis whereas EXT1 was linked to a bad prognosis. This study provides an overall picture of the major genes encoding for proteins involved in heparan sulphate and chondroitin sulphate synthesis and modifications that can be implicated in normal and malignant plasma cells.

Heparanase influences expression and shedding of syndecan-1, and its expression by the bone marrow environment is a bad prognostic factor in multiple myeloma

AbstractThe heparan sulfate (HS) proteoglycan, syndecan-1, plays a major role in multiple myeloma (MM) by concentrating heparin-binding growth factors on the surface of MM cells (MMCs). Using Affymetrix microarrays and real-time reverse transcriptase–polymerase chain reaction (RT-PCR), we show that the gene encoding heparanase (HPSE), an enzyme that cleaves HS chains, is expressed by 11 of 19 myeloma cell lines (HMCLs). In HSPEpos HMCLs, syndecan-1 gene expression and production of soluble syndecan-1, unlike expression of membrane syndecan-1, were significantly increased. Knockdown of HPSE by siRNA resulted in a decrease of syndecan-1 gene expression and soluble syndecan-1 production without affecting membrane syndecan-1 expression. Thus, HPSE influences expression and shedding of syndecan-1. Contrary to HMCLs, HPSE is expressed in only 4 of 39 primary MMC samples, whereas it is expressed in 36 of 39 bone marrow (BM) microenvironment samples. In the latter, HPSE is expressed at a median level in polymorphonuclear cells and T cells; it is highly expressed in monocytes and osteoclasts. Affymetrix data were validated at the protein level, both on HMCLs and patient samples. We report for the first time that a gene's expression mainly in the BM environment (ie, HSPE) is associated with a shorter event-free survival of patients with newly diagnosed myeloma treated with high-dose chemotherapy and stem cell transplantation. Our study suggests that clinical inhibitors of HPSE could be beneficial for patients with MM.

Heparanase Influences Expression and Shedding of Syndecan-1, and Its Expression by the Bone Marrow Environment Is a Bad Prognostic Factor in Multiple Myeloma.

The heparan sulfate (HS) proteoglycan (PG) syndecan-1 plays a major role in multiple myeloma (MM), in part by concentrating HS-binding growth factors on the surface of MM cells (MMC). The function of HSPG is regulated by extracellular enzymes that modulate the structure of HS-chains. One such enzyme is heparanase (HPSE), an endo-glucuronidase that cleaves HS chains at only a few sites, resulting in fragments of 10–20 sugar units long that are biologically active.Using Affymetrix microarrays and real-time RT-PCR, we showed that the gene encoding HPSE was expressed by 11/19 myeloma cell lines (HMCLs). In HSPE-positive HMCLs, syndecan-1 mRNA expression and production of soluble syndecan-1, unlike expression of membrane syndecan-1, were significantly increased. Downregulation of HPSE by HPSE-siRNA resulted in a decrease of syndecan-1 mRNA expression and soluble syndecan-1 production, without affecting membrane syndecan-1. Contrary to HMCLs, HPSE was expressed in only 4/39 primary MMC samples at a low level, whereas it was highly expressed in 36/39 whole bone marrow (WBM) microenvironment samples. In the latter, HPSE was expressed at a median level in polynuclear cells and T cells, at high level in monocytes and osteoclasts, and it was not expressed in BM stromal cells. In order to investigate the connection of clinical parameters with HPSE gene expression, 30/39 myeloma patients treated with high dose chemotherapy and autologous stem cell transplantation where classified into two groups: 15 patients with the lowest or the highest HPSE expression in the WBM. The HPSEhigh group had an increased percentage of patients with elevated C-reactive protein and β2-microglobulin. According to the international staging system (ISS), the HPSEhigh group included a significant higher frequency of patients with stage III MM and a lower frequency of patients with stage I MM. HPSEhigh patients had a shorter event free survival (EFS) (p=.017) and overall survival (OAS) (p=.023) than HPSElow patients. Classifying these 30 patients into two groups according to HPSE expression in MMC did not yield to any EFS or OAS significant differences between the two groups.Altogether, those data provide evidence that HPSE not only modulate syndecan-1 activity through the cleavage of HS chains but also regulates syndecan-1 mRNA expression and shedding. We report for the first time that the expression of a gene in the BM, i.e. HSPE, is an indicator of poor prognostic for MM patients. Therefore, HPSE inhibitors like PI-88, which is currently in clinical trial, will be of interest for the treatment of patients with MM.

Anti-Myeloma Effects Induced by Myeloma Idiotype Pulsed Dendritic Cells In Vitro.

As the most potent antigen-presenting cells, Dendritic cells (DCs), capable of inducing immune responses from naive T cells, are operative tools for tumor immunotherapy. Derived DCs are extremely effective in capturing and presentation of antigens to T cells and play a key role in the induction of cytotoxic T lymphocytes (CTLs). In vitro culture system containing the combination of GM-CSF, IL-4 and TNF-α cytokine can affect CD14 + progenitor cells from mononuclear cells (MNCs) of peripheral blood (PB) developing into functional DCs, which have enough quantities for application in vitro researches and clinical practices. Multiple myeloma cells(MM)are able to secrete a great quantity of immunoglobulin (Ig) expressing idiotypic antigen called idiotype (Id) in its mutational hotspot. This kind of idiotypic structure regions also expressing on the surface of MM cells are high specific autologous tumor associated antigen (TAA). The combination use of DCs and tumor specific antigen can improve the immunogenicity of MM cells and stimulate specific anti-tumor immunological response effectively, so by using this new kind of DC tumor vaccine, following high dose chemical therapy, the tiny residual pathological changes might be cleared totally in the future.To investigate the specific antitumor immune response induced by Id-pulsed dendritic cells(DCs) in vitro. DCs were generated from peripheral blood monocytes of the multiple myeloma(MM) patients using GM-CSF, IL-4, and TNF-α. pulsed with idiotype protein at the immature stage, DCs could activate T cells to become tumor specific cytotoxic T lymphocytes (CTLs). The morphologic characteristics of those cells were observed with light and electron microscopes. The phenotypic figures were analyzed with FACS analysis. Methy-thiazoly-Tetrazolium (MTT) assay was employed to evaluate the effect of proliferation of autologous T cells and the inhibition rate of CTL on MM cells. DCs precursors in peripheral blood could be induced to typical mature DCs in medium containing GM-CSF, IL-4 and TNF-α. Mature DCs with Id could operatively increase proliferation of the autologous T cells and active naive T cells to become tumor specialized CTLs. Any doses of CTLs had significant inhibition or killing ability on autologous MM cells. These results suggest in suitable cytokine environment, the precursors in peripheral blood of MM patients could be induced to functional DCs, and vaccination with Id-pulsed DCs could induce active antitumor immune response. Multiple cycles of immunization using DC as APC in vitro can be beneficial in generating antigen- specific T cells from normal PBMC, and Id an auto-specific tumor antigen, can be got with ammonium sulfate four-step precipitated method, By digestion of pepsin and affinity chromatography so as to stimulate MM specific immunological responce, and Id-pulsed mature DCs from MM patients can stimulate not only the proliferation of autologous T cells, but also the specific CTL immune response against autologous MM cells. In addition, in vitro immunization may provide an alternative approach to in vivo immunization of MM. We believe that DCs vaccine can bring the breakthrough of therapy to MM in the near future.

Development of Novel CD138 Antigen-Specific Peptide Capable of Eliciting Myeloma-Specific Cytotoxic T Lymphocytes Response.

Multiple myeloma (MM) is characterized by the accumulation of mature plasma cells in the bone marrow. Despite progress in treatment, the disease still remains incurable, therefore novel therapeutic approaches are required to achieve better treatment. Development of peptide-based immunotherapies against specific tumor-associated antigens offers an attractive approach that may lead to tumor-targeted cellular therapy in MM patients. CD138 (Syndecan-1) is an unique target expressed on the surface of MM cells and normal plasma cells with very limited other distribution or expression. CD138 has been reported to play an important role in myeloma cell survival and, infact, is used to purify MM cells in laboratory experiments. The purpose of this study was to develop immunogenic peptides derived from CD138 antigen for the induction of antigen-specific cytotoxic T lymphocytes (CTLs) against MM. We have identified an immunogeneic HLA-A2-specific CD138 peptide that is capable of inducing MM specific-CTLs. To induce the specific CTLs, HLA-A2-positive normal human T lymphocytes were stimulated with autologous dendritic cells or T2 cells pulsed with the CD138 peptide. The generated CTLs showed a distinct phenotype consisting of 67% of CD69+/CD45RO+ and 1% of CD45RA+/CCR7+ T cells, characteristic of effector memory cell population. A significant (p < 0.05) increase in T cell proliferation and IFN-g secretion were observed by the CTLs following restimulation with HLA-A2+/CD138+ MM cells. The CTLs displayed HLA-A2-restricted CD138-specific cytotoxic activity against MM cell, demonstrated as 76% and 86% cytotoxicity at Effector:Target ratios of 20:1 or 60:1, respectively. The MM cell-specific cytotoxicity was confirmed in “cold” target inhibition assays using HLA-A2.1+/CD138+ MM cells as “cold” inhibitor, demonstrated 25% and 39% reduction in cytotoxicity when incubated with cold inhibitors at E:T ratios of 20:1 or 60:1, respectively. A distinct CD138 peptide-specific tetramer+/CD8+ population was also confirmed. Taken together, these data demonstrate the efficacy of the immunogenic CD138 peptide in inducing a cellular immune response in MM for future clinical application.

Anti-Myeloma Activity of the Maytansinoid Immunoconjugate of Internalizing Human Monoclonal Antibody Specific for HM1.24/BST2 (CD317).

HM1.24/BST-2 (CD317) is a type II transmembrane protein that was originally identified as a cell surface antigen overexpressed on multiple myeloma (MM) cells. Our previous studies have demonstrated that HM1.24 has a potential as an attractive target molecule for antibody-based or cellular-mediated immunotherapy of MM. Subsequent studies have revealed that rat homologue of HM1.24 at the cell surface can be internalized and localized to the intracellular trans-Golgi network, suggesting that HM1.24 may play an important role in trafficking and signaling. To evaluate the potential effect of HM1.24-targeting therapy in MM, we generated internalizing fully human monoclonal antibodies specific for human HM1.24 by using KM mice. Among these monoclonal antibodies, b-76-8 had the highest affinity to HM1.24 and induced ADCC activity and complement-dependent cytotoxicity in the presence of immune effectors. Importantly, b-76-8 internalized rapidly after cell surface binding and approximately 80% of b-76-8 was delivered to the intracellular location within 30 min as determined by immunofluorescence staining of b-76-8-treated RPMI 8226 cells. We next developed the immunoconjugate of b-76-8 with the analog of the cytotoxic drug maytansine, DM1 [N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine] and further evaluated its potential impact on MM cells. The ratio of DM1 molecules linked per antibody molecule was 1.8–3.0. Several MM cell lines as well as primary MM cells were cultured with either b-76-8, DM1, b-76-8-DM1, or control IgG-DM1, and cell viability was determined by a WST-8 assay. Treatment with b-76-8-DM1 (2–10 nM, based on the concentration of DM1) induced 50–70% of cell death in RPMI 8226, KMS12-BM, and IM-9 cells. In contrast, the relevant amount of b-76-8, DM1 (10 nM), or control IgG-DM1 (10 nM) did not inhibit growth of these MM cells. Of interest, T-cell lines such as MOLT-4 and CEM also expressed HM1.24; however, these cells showed minimal cytotoxicity (<10%) to b-76-8-DM1 (10 nM) despite having a similar sensitivity to DM1. On the other hand, primary MM cells from 5 patients were relatively resistant to DM1, but maximal cytotoxicity (10–40%) was achieved at the concentration of 250 nM by b-76-8-DM1 but not by control IgG-DM1. This dose of b-76-8-DM1 did not mediate significant cytotoxicity against normal bone marrow mononuclear cells. We finally examined the anti-tumor activity of b-76-8-DM1 in an xenograft model of human MM. SCID mice were inoculated intravenously with IM-9 cells, and groups of 8 mice were treated with control IgG, b-76-8, b-76-8 mixed with free DM1, or b-76-8-DM1 on day 5–8. Treatment with b-76-8 (160 μg/dose) alone or b-76-8 together with DM1 (1.9 nmol/dose) significantly prolonged survival of these mice (p<0.01). In contrast, administration of b-76-8-DM1 (160 μg/dose) resulted in more significant tumor regression including complete remission in 3 of 8 mice (p<0.003). These results suggest that HM1.24 might be involved in trafficking between the cell surface and intracellular sites of MM cells, and that the immunoconjugate targeting HM1.24 provides a novel therapeutic approach in patients with MM.

CD40 Ligand Stimulation of Multiple Myeloma Cells Results in Upregulation of Surface Expressed Heat Shock Proteins and Increased Antigenicity.

Effective immune-based therapies against the plasma cell malignancy, multiple myeloma (MM), are currently lacking. Identification of novel antigens (Ag) on the surface of MM cells to use as cellular targets for the destruction of cancer cells by the body's immune system has been of great interest. We and others have demonstrated that CD40 stimulation of MM cells results in marked upregulation of membrane bound proteins such as Ku86. Using CD40 triggered MM cells as immunogens and hybridoma technology; we generated a monoclonal antibody (mAb), 6D11, that recognizes a CD40 induced cell membrane Ag on MM cells. This Ag is detectable on the surface of MM cells using indirect immunofluorescence flow cytometric analysis. Moreover, in Western immunoblotting assays, 6D11 mAb reacts with a 94 kDa protein, which is strongly associated with a 78 kDa protein. Using high performance liquid chromatography and protein microsequencing, we confirm that these proteins are the heat shock proteins (HSP), glucose-regulated peptide 94 (GRP94) and GRP78, respectively. These data were confirmed using co-immunprecipitation experiments. Furthermore, we demonstrate through indirect immunofluorescence flow cytometric analysis and quantitative real time reverse transcription polymerase chain reaction (RQ-PCR) that CD40 ligand (CD40L) stimulation of MM cells results in rapid upregulation of both GRP94 and GRP78. Since HSPs have been shown to play a role in both Ag presentation, as well as the intracellular transport of cellular Ags, it is tempting to speculate that cell membrane expression of tumor-specific peptides could also be induced via CD40 triggering. Accordingly, CD40 induced cell membrane HSP expression resulted in increased antigenicity as determined by increased co-stimulatory molecule expression on Ag presenting cells (APC) and by increased immunoreactivity in mixed lymphocyte reactions (MLR). This suggests that CD40 induced HSP expression may indeed result in increased recognition of MM cancer by the immune system. Our study therefore supports the development of CD40-based targeted cell therapies against MM.

Plasma Levels of SDF-1 and Expression of SDF-1 Receptor on Multiply Myeloma Cells of Human Multiply Myeloma.

Multiple myeloma(MM) is a monoclonal expansion of malignant cells with a plasmablast-plasma cell morphology that is almost exclusively localized to the bone marrow, except at the final stages of disease, when they proliferate in the extramedullary area. The mechanisms of the selective homing of MM cells to the bone marrow compartment are poorly understood. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 contribute to stem cell homing and play a role in trafficking of leukemic cells. In this study we have investigated expression and biological behavior of SDF-1/CXCR4 in MM-derived cell lines and primary MM cells. FACS and RT-PCR analysis was used to study the expression of CXCR4 and ICAM-1(CD54) on the surface of MM cells from 4 IL-6 dependant cell lines (XG1,XG2,XG6 and XG7) and 25 freshly isolated tumor samples from patients with diagnosed MM. Mononuclear cells were purified by positive selection of magnetical and FACS sorting. Chemotaxis assay through transwell bore polycaronate and ELISA assay were employed to monitor the SDF-1, IL-6, and sICAM-1 levels. We found that[circ1]Fresh MM cells and MM cell lines expressed various levels of functional CXCR4 ranging from 23.1% to 77.7%,which was correlated with the in vitro migration ability of MM cells[(23.2±1.08)%, P<0.01]; [circ2]SDF-1 levels in the bone marrow(BM) of MM patients were significantly higher than the those of healthy persons (3489.23±651.63)pg/ml, (2818.57±597.79)pg/ml, P<0.05; but plasma levels of SDF-1 in peripheral blood of MM patients were lower than those of healthy persons[(1973±133)pg/ml, (2334.857±574.92), P=0.062]; [circ3]Plasma levels of PCL(4097.14±680.71) were significantly higher than those of healthy persons, P<0.01. The results firstly demonstrated abnormal expression of SDF-1 and its receptor CXCR4 on Human MM cells, which is closely correlated with the migration of MM cells. Furthermore, we discovered that SDF-1 could up-regulate the expression of ICAM-1 on MM cells; the plasma level of soluble ICAM-1 was correlated with the expression of CXCR4 on MM cells. These findings suggested that SDF-1/CXCR4 axis play a key role on the trafficking of MM cells via mediating the effect of adhesion molecules. Moreover, we observed higher plasma levels of IL-6 in PB of 60% MM patients compared with those of healthy individuals. Finally, the levels of IL-6 were closely correlated with SDF-1 levels (γ=0.8, P<0.01), These data indicated that in the IL-6-dependent myeloma cell lines or fresh myeloma samples and myeloma cell growth triggered by SDF-1 maybe due to up-regulation of autocrine and paracrine IL-6 by myeloma cells and stromal cells in BM. The results suggested that the expression of CXCR4 have an essential role in the proliferation and migration of myeloma cells in patients with multiple myeloma.In conclusion, MM cells expressed various levels of functional CXCR4, which were correlated with the migration ability of MM cells in vitro; SDF-1/CXCR4 axis plays a key role in the trafficking of MM cells via mediating the effect of adhesion molecules; The plasma levels of IL-6 closely correlated with SDF-1 plasma levels, myeloma cell growth triggered by SDF-1 may be due to up-regulation of autocrine and paracrine IL-6 by myeloma cells and stromal cells in BM. All these suggested that the expression of CXCR4 play an essential role in the proliferation and migration of myeloma cells in patients with multiple myeloma.

The clinical potential of proteasome inhibition

The development of the proteasome inhibitor bortezomib is a prime example of successful bench-to-bedside research. NF-κB is implicated in the pathogenesis of multiple myeloma (MM), and bortezomib blocks NF-κB activation and its sequelae. Additional bortezomib mechanisms include interactions at the MM cell surface and mitochondrial level and in downstream apoptotic signaling. In the pivotal bortezomib study, the response rate was 35%; time to progression (TTP), 7 months; and median duration of response, 14.3 months. Responses were associated with improvements in quality of life, increases in laboratory parameters, and decreased transfusions. Bortezomib is thus indicated in the US for patients with relapsed, progressive MM and recommended for approval in Europe. The confirmatory phase III trial has been stopped early due to a significant improvement in TTP with bortezomib. The success of bortezomib in MM demonstrates the utility of targeting the tumour:host interaction and BM milieu, in addition to the tumour itself.

The clinical potential of proteasome inhibition

The development of the proteasome inhibitor bortezomib is a prime example of successful bench-to-bedside research. NF-κB is implicated in the pathogenesis of multiple myeloma (MM), and bortezomib blocks NF-κB activation and its sequelae. Additional bortezomib mechanisms include interactions at the MM cell surface and mitochondrial level and in downstream apoptotic signaling. In the pivotal bortezomib study, the response rate was 35%; time to progression (TTP), 7 months; and median duration of response, 14.3 months. Responses were associated with improvements in quality of life, increases in laboratory parameters, and decreased transfusions. Bortezomib is thus indicated in the US for patients with relapsed, progressive MM and recommended for approval in Europe. The confirmatory phase III trial has been stopped early due to a significant improvement in TTP with bortezomib. The success of bortezomib in MM demonstrates the utility of targeting the tumour:host interaction and BM milieu, in addition to the tumour itself.

Translocation of Ku86/Ku70 to the multiple myeloma cell membrane: functional implications.

Since the central hallmarks of human multiple myeloma (MM) are abnormalities in immunoglobulin (Ig) gene rearrangement, IgH class switching, and DNA damage repair, and since Ku86 and Ku70 proteins are central to these processes, aberrant Ku function may play a role in MM pathogenesis. Our prior studies demonstrated a 69-kDa Ku86 variant in freshly isolated patient MM cells that confers sensitivity to DNA damage. We also showed that Ku86 on the cell surface of CD40-activated MM cells mediates homotypic tumor cell adhesion, as well as heterotypic adhesion to bone marrow stromal cells. We here define the mechanism and functional significance of CD40-induced Ku translocation from the cytoplasm to the cell membrane in MM cells vs normal B cells. We examined Ku86 and Ku70 translocation following CD40 activation in human MM cells vs normal tonsillar B lymphocytes. We then identified the functional sequelae of membrane Ku86 and Ku70 expression on CD40-activated human MM cells. CD40 activation induces translocation of both Ku86 and Ku70 to the cell surface of MM cells, but not normal tonsillar B cells. Moreover, CD40 activation triggers Ku association with CD40 only in CD40-activated MM cells. Finally, CD40-activated MM cells adhere to fibronectin and are protected against apoptosis triggered by irradiation or doxorubicin; conversely, antibodies to Ku both inhibit tumor cell binding and restore sensitivity to these agents. These results demonstrate functional significance of Ku translocation to the cell membrane of CD40-activated human MM cells. Therefore, targeting Ku86 and Ku70, with blocking peptides for example, might serve as a novel treatment strategy in human MM.