Serum Of Multiple Myeloma Patients Research Articles

MiRNA in Serum and Bone Marrow Plasma Cells From Multiple Myeloma Patients.

AbstractAbstract 2921 Background:Multiple myeloma (MM) is a clonal B-cell malignancy characterized by the aberrant expansion of clonal plasma cells (PCs) within the bone marrow. Malignant PCs produce intact or partial monoclonal immunoglobulin (M protein) and cause organ damage. More than 20,000 new cases of multiple myeloma (MM) are diagnosed every year in the US with approximately 10,700 deaths occurring. The pathogenesis of MM is still largely unclear, but several reports suggest that interaction of tumor cells with the bone marrow microenvironment and microRNAs (miRNAs) deregulation may play a role in the etiology and progression of MM. miRNAs are small non-coding RNAs capable of regulating protein expression by binding to mRNA, and have been implicated in the development of MM. First identified inside cells, miRNAs can also be detected in body fluids, including serum and plasma, and may be a valid biomarker. Few studies have investigated the agreement between circulating miRNAs and intracellular myeloma PC miRNAs at diagnosis. Methods:Using Nano-String nCounter technology we first performed a screening analysis on serum samples obtained from MM patients and healthy controls. We identified a candidate set of miRNAs differentially expressed in the serum of MM patients. The levels of these miRNA markers were validated by RT-PCR in both serum and bone marrow PCs from the same cohort. Agreement of the quantitative miRNA marker levels between sample types was evaluated using intraclass correlation coefficients (ICC) (both for normalized and log2 measures). Results:Thirty-nine MM patients (21 male, 18 female) with a median age of 72 years (range: 65 – 83) were included in the analysis. Most were ISS stage I or II (59% vs. 41% ISS stage III) and 39% were high risk according to FISH abnormalities – 21% of patients carried del17p, 24% t(4;14) and 5% t(14;16). Medians and ranges for lab markers were as follows: hemoglobin 10.0 g/dl (7.2 to 15.1), beta2-microglobulin 5.18 ug/ml (1.38 – 12.1), creatinine 0.94 mg/dl (0.65 – 2.49), CRP = 1.6 mg/dl (0.02 – 116.0). Nine age-matched healthy controls were also used for the analysis. After the screening analysis, the following miRNAs were differentially expressed between healthy subjects and MM patients in serum samples: miR-92a, miR-451, miR-19b, miR-21, miR-16, miR-25, miR-30a, and miR-126. There was no significant agreement or correlation between serum and myeloma cell samples using either untransformed as well as log2measures (all p>0.40) (Table 1). Conclusion:Our preliminary results suggest a difference between circulating miRNAs in myeloma patients from controls. This indicates that future studies are needed to better define the role of miRNAs in the peripheral blood as a prognostic and even diagnostic biomarker in myeloma. From our preliminary data it also appears that circulating miRNAs are not simply secreted into the peripheral blood by myeloma PCs as it seems that circulating miRNAs do not reflect those of myeloma PCs. Differential expression could be determined by other cells that can release and or modify their miRNA expression in response to MM. Ongoing studies are examining the origin and function of miRNAs in the peripheral blood.Table 1Results of the intraclass correlation coefficients, where the closer to 1, the better the agreement between serum vs PCmiR markerNICC coefficientICC p-valueICC coefficient for log2(marker)ICC p-value for log2(marker)miR-92a370.0440.40−0.0170.54miR-19b36−0.1380.79−0.2450.93miR-2132−0.3280.97−0.4150.99miR-1637−0.2870.96−0.2860.96miR-2530−0.430.99−0.7860.99miR-30a30−0.2650.93−0.4740.997miR-12625−0.0390.58−0.0110.52 Disclosures:No relevant conflicts of interest to declare.

Serum B‐cell maturation antigen is elevated in multiple myeloma and correlates with disease status and survival

Although TNFRSF17 (also designated as B-cell maturation antigen (BCMA)) is expressed on tumour cells in B-cell malignancies, it has not been found in serum. The present study found that BCMA concentrations were higher in the supernatants of cultured bone marrow mononuclear cells from multiple myeloma (MM) patients than in healthy subjects. Serum BCMA levels were measured in samples from MM patients (n = 209), monoclonal gammopathy of undetermined significance (MGUS) individuals (n = 23) and age-matched controls (n = 40). BCMA was detected in the serum of untreated MM patients (n = 50) and levels were higher than in MGUS patients (P = 0·0157) and healthy subjects (P < 0·0001). Serum BCMA levels were higher among patients with progressive disease (n = 80) compared to those with responsive disease (n = 79; P = 0·0038). Among all MM patients, overall survival was shorter among patients whose serum BCMA levels were above the median (P = 0·001). We also demonstrated that sera from mice with human MM xenografts contained human BCMA, and levels correlated with the change in tumour volume in response to melphalan or cyclophosphamide with bortezomib. These results suggest that serum BCMA levels may be a new biomarker for monitoring disease status and overall survival of MM patients.

Effect of matrix metallopeptidase 13 (MMP13) on multiple myeloma (MM) cells, osteoclast (OCL) activity, and bone resorption.

8099 Background: MM cells produce OCL-activating factors that induce excessive bone resorption resulting in lytic lesions. The role of MMPs in invasion/progression of solid tumors is well-known, but its function in MM has not been well elucidated. Our group has shown that MMP13 is highly expressed in primary MM cells and in sera of MM patients. Levels of MMP13 significantly correlate with the extent of bone disease. MMP13 is induced by IL-6 via AP-1 activation in MM cells and enhances fusion of OCL precursors resulting in excessive bone resorption. OCL formation using MNCs of mmp-13-/- mice resulted in a fusion defect, significantly decreased OCL size and activity, which could be reversed by exogenous MMP13 (ASH 2009, IMW 2011). Methods: Methods will be presented in the Results section. Results: RT-PCR and western blotting revealed that IL-6 treatment of MM cells induced MMP13 transcription (30-fold) and secretion (&gt;1000-fold). Protein expression of the AP-1 members c-Jun and c-Fos was induced by IL-6, which correlated with MMP13 upregulation. Our data further indicate that the catalytic activity of MMP13 is not required to enhance OCL formation and bone resorption. To prove this, we generated the MMP13 activity-dead mutation MMP13-E223A construct by site-directed mutagenesis PCR-based cloning. The mutated protein was overexpressed in HEK293 cells and purified from the supernatant to confirm whether loss of catalytic activity blocks MMP13 function. To further investigate the in vivo role of MMP13 in MM bone disease, MMP13 expression was knocked down (KD) in murine 5TGM1-MM cells by pKLO. 1 puro lentiviral infection containing sh-RNA targeting mouse MMP13 sequence. MMP13-KD 5TGM1-MM cells or WT-5TGM1-MM cells were intratibially injected into RAG2-/- mice. Development of lytic bone lesions are monitored by micro-QCT and data will be available at the time of presentation. Conclusions: Our data suggest that MMP13, secreted by MM cells, plays a critical role in the development of lytic lesions. Targeting MMP13 represents a promising approach to treat or to prevent bone disease in MM.

Autoantibodies against the signal peptide domain of MUC1 in patients with multiple myeloma: Implications for disease diagnosis and prognosis

Naturally generated autoantibodies to tumor-associated antigens such as MUC1 can assist in cancer diagnosis and prognosis. While previous studies have concentrated on the tandem repeat array domain of MUC1, here we focused on MUC1's signal peptide domain. We used ELISA assays with MUC1-specific epitopes and antibodies to quantify soluble MUC1 antigen and anti-MUC1 autoantibodies against the tandem repeat array and signal peptide domains in 15 naïve donors and 27 multiple myeloma cancer patients. We showed a significant increase in up to 24-fold (P<0.004) only in the levels of anti-MUC1 signal peptide autoantibodies in the sera of multiple myeloma patients vs. naïve donors. This increase stemmed chiefly from the preferred immunogenicity of the signal peptide. Moreover, a significant positive correlation (R(2)=0.5361, P<0.048, Pearson correlation) was shown between the levels of soluble MUC1 and anti-MUC1 signal peptide autoantibodies in multiple myeloma patients with progressive disease while under therapy. This is an initial report on the existence of autoantibodies to a signal peptide domain in general and to the MUC1 signal peptide domain in particular in cancer patients. The autoantibodies had MUC1 rather than signal peptide specificity. The specific nature of the antigen leading to generation of these autoantibodies is still unclear because it is unlikely that the target antigen is a major histo-compatibility complex-peptide complex and we could not trace soluble MUC1 signal peptide fragments in naïve donors and multiple myeloma patients. Further validation of these findings may improve diagnostic and prognostic capabilities for MUC1-positive multiple myeloma patients and potentially, patients with other MUC1-positive cancers, as well.

Evaluation of protein quantification using standard peptides containing single conservative amino acid replacements

Structural analogs are evaluated as peptide internal standards for protein quantification with liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM); specifically, single conservative amino acid replacements (SCAR) are performed to create tagged standards that differ by the addition or subtraction of a single methylene group in one amino acid side chain. Because the performance of stable isotope-labeled standards (SIS) has been shown to be superior to structural analogs, differences in both development and quantitative performance between assays based on SIS and SCAR peptides are explored. To establish an assay using the structural analogs, analysis of endogenous, SCAR and SIS peptides was performed to examine their ion signal, fragmentation patterns and response in LC-MRM. Performance of SCAR and SIS peptides was compared for quantification of epidermal growth factor receptor from lung cancer cell lysates and immunoglobulin M in the serum of multiple myeloma patients.

Abstract 802: Cytokine profiles in bone marrow supernatant and peripheral blood of patients with multiple myeloma and Waldenström's macroglobulinemia

Abstract Cytokines serve vital roles in the regulation of immune responses. In patients diagnosed with hematologic malignancies, cytokines may play a role in the tumor-host microenvironment affecting tumor survival and progression. Previous studies have quantified the variation in cytokine concentrations between malignant and normal states in patients’ serum. However the research is sparser in relation to measurement of cytokine concentrations in the bone marrow microenvironment. Multiple myeloma (MM) and Waldenström's macroglobulinemia (WM) are two such malignancies that arise in the B-lymphoid lineage and proliferate within the bone marrow (BM) microenvironment. In this study, an ultra-sensitive IL-6 and a human TH1/TH2 multiplex ELISA system were utilized to determine protein concentrations of the following cytokines: IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 p70, IL-13, IFN-gamma, and TNF-alpha, in the BM supernatant and peripheral blood (PB) of 20 patients with WM, 16 patients with MM, and 7 controls. The BM supernatant concentration of TNF-alpha was significantly increased in MM compared to controls (10.650 pg/ml vs. 4.980 pg/ml, p=0.0158). IL-1beta was significantly decreased in the bone marrow supernatant of MM (0.038 pg/ml vs. 2.352 pg/ml, p=0.0469) and WM (0.046 pg/ml vs. 2.352 pg/ml, p=0.0435). IL-8 was also significantly decreased in the BM supernatant of patients with MM (11.520 pg/ml vs. 43.590 pg/ml, p=0.0158) and WM (7.348 pg/ml vs. 43.590 pg/ml, p=0.0036). Differences in BM supernatant concentrations of IL-6 were not significant in MM, WM, and normal controls. Analysis of PB showed IL-2, IL-5, IL-6, IL-8, IL-10, and TNF-alpha were significantly increased in both MM and WM versus controls, and IFN-gamma was significantly increased in WM. Hierarchical clustering analysis of the cytokine profiles suggested two distinct subgroups existed within both MM and WM. TNF-alpha was the only cytokine in our study that was increased in both the bone marrow and serum of MM patients. The discordant results obtained in the BM supernatant vs. PB for IL-6 and IL-8 in both MM and WM indicate that cytokine profiles vary somewhat between the PB and BM compartments. The BM supernatant and PB cytokine profiles in patients with MM and WM highlight the important role cytokines may play in the microenvironment and biology of MM and WM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 802. doi:10.1158/1538-7445.AM2011-802

Lack of Binding of Monoclonal Protein Produced by Multiple Myeloma (MM) Cells to FcγRIIb Reduces Immunoreceptor Tyrosine-Based Inhibitory Motif (ITIM) Signaling and Provides the Rationale for a Novel Therapeutic Approach to MM

AbstractAbstract 455Functional B cell and plasma cell immune responses are dependent upon an exquisitely controlled process integrating signals from activating and inhibitory receptors present on the surface of these cells. These activating and inhibitory signaling pathways regulate both the quality and quantity of immunoglobulin (Ig) production. These signaling motifs, termed immunoreceptor tyrosine-based activation motif (ITAM) and ITIM provide the basis for two opposed signaling modules that duel for control of plasma cellular activation within the immune system. The inhibitory Fc receptor FcγRIIb is expressed on plasma cells and controls their persistence in the bone marrow and their ability to produce most serum Ig. Activation of FcγRIIb leads to the phosphorylation of ITIM and recruitment of SH2-containing protein tyrosine phosphatase-1 (SHP-1) SHP-2 and the SH2-containing inositol 5-phosphatase (SHIP) in plasma cells. FcγRIIb-mediated SHIP phosphorylation results in enhanced degradation of PtdIns (3,4,5)P, which is required for B-cell antigen receptor (BCR)-induced recruitment and activation of downstream ITAM signaling molecules. The inhibitory IgG Fc receptor FcγRIIB was the first discovered and remains the best studied example of an ITIM-containing receptor. In this study, we first investigated the IgG-binding ability of 18 MM patients and 10 normal donors to FcγRIIb using flow cytometric analysis. Each serum sample was incubated with MHC1 cells that only express FcγRIIb but do not express FcγRI and FcγRIIa. After washing three times with 1 × PBS, anti-human IgG antibody conjugated with FITC was added to the cells for another 30 minutes. The results showed MM patients' serum IgG have much lower FcγRIIb-binding ability than normal human IgG (P<0.05). Immunohistochemical staining also demonstrated MM patients' sera showed obviously less binding to the FcγRIIb on MHC1 cells compared to serum samples from normal human donors. We further analyzed the FcγRIIb-SHIP signaling pathway in normal B-cells following exposure to MM and normal human sera. Fresh human B-cells were isolated using anti-CD20 antibodies with magnetic bead selection and the cells were washed in an acidic solution (pH 4) for 1 minute to remove any bound antigen from the cell surface. Raji B-cells were also acid-treated. First, these cells were exposed to MM patients' or normal human sera for 5, 15, 30, or 60 minutes to determine the maximum time point of SHIP phosphorylation. Using a standard Western blot protocol, phosphorylated SHIP and total SHIP protein expression were visualized using an enhanced chemiluminescence detection system. The maximum time point of SHIP phosphorylation was 15 minutes. The results showed that both normal human B-cells and Raji B-cells showed markedly lower SHIP phosphorylation/total SHIP following exposure to MM patient serum compared to normal human serum which is consistent with the lack of binding of human Ig to FcγRIIb. Our findings suggest that the monoclonal protein produced by MM patients has very low FcγRIIb-binding ability and is incapable of signaling through inhibitory ITIM pathway. Most importantly, FcγRIIb is expressed on plasma cells and controls their persistence in the bone marrow in addition to Ig production. Cross-linking of FcγRIIb induces apoptosis of plasma cells (Xiang Z et al Nat Immunol 8: 419-29, 2007). Thus, it is possible that this effect prevents the induction of apoptosis in MM cells. Previous studies have reported that normal Ig is capable of inducing B-cell apoptosis. Thus, it is possible that exposure of MM cells to normal immunoglobulin may be capable of inducing apoptosis of these cells and reducing their production of M-protein. We are currently evaluating the ITIM signal transduction pathway, M-protein production and apoptosis in MM and normal B-cells following exposure to normal and MM M-protein IgG. Disclosures:No relevant conflicts of interest to declare.

Serotonin Dysregulation Correlates with Both Bone and Active Disease In Multiple Myeloma

AbstractAbstract 1920The biologic mechanisms involved in the pathogenesis of multiple myeloma (MM)-induced osteolytic bone disease are poorly understood. Physiological interactions between the serotoninergic and skeletal systems have been implicated by clinical observations. Brainstem-derived serotonin positively regulates bone mass following binding to 5-HT2C receptors on ventromedial hypothalamic neurons. This is opposed by platelet-derived serotonin that induces bone lysis and osteoclast activation. Moreover, the serotonin transporter SLCA6A4 is universally present among the malignant B cell clones.We examined serotonin dysregulation in two sample types from MM patients: peripheral blood and bone marrow. In the blood we measured by ELISA the ratio of serotonin in serum compared to platelets in patients with MM (n=10), MGUS (n=10) or healthy controls (n=5). We found higher levels of serotonin in platelets for the MM patients compared to the MGUS & healthy controls (p=0.017). Concomitantly there was less serotonin in the serum of MM patients compared to compared to the MGUS & healthy (p=0.002). This implies an imbalance in the compartmentalization of serotonin associated with presence of MM bone disease.Our multiplexed protein kinase signal pathway mapping technology, reverse phase protein microarrays (RPMA), was applied to bone marrow samples for quantifying post-translational modifications (e.g. phosphorylation, cleavage, acetylation) and/or total cell signaling kinase levels.Using reverse-phase protein microarray (RPMA) we retrospectively measured bone remodeling signal pathway perturbations in 15 bone marrow core biopsies from patients diagnosed with MM, at different clinical stages, and correlated this with the presence of MM-related bone disease (documented by scan or MRI). Bone marrow core biopsies exhibited significant elevation of cellular Serotonin, RANK, MMP-11, TNFα, TNF-R1, and Ezrin Tyr353 in MM with active bone disease (n=9) compared to patients without bone disease (n=6) (respectively p=0.031, p=0.038, p=0.0082, p=0.0221, p=0.01, p=0.028).To further evaluate the serotonin dysregulation in bone marrow cells, we measured serotonin bound to plasma cells (CD138+) compared to CD138- cells in bone marrow aspirates (n=21). Bone marrow aspirate were collected from patients undergoing standard of care hematological work up for multiple myeloma at any stage or treatment course. Patients with symptomatic myeloma (defined as presence of at least one of CRAB symptom) had lower serotonin levels in the CD138- bone microenvironment cells compared to non-symptomatic patients (p=0.0235). Plasma cells (CD138+) exhibited larger amounts of serotonin compared CD138- bone microenvironment cells (p=0.016).Taken together, our data show a dysregulation of serotonin in MM suggesting an altered distribution of serotonin in blood and bone marrow. This provides potential insights into diagnosis, prognosis, and/or treatment. Disclosures:No relevant conflicts of interest to declare.

Mechanism of Action of a Novel c-Met Inhibitor MK2461 In Multiple Myeloma

AbstractAbstract 4078 Background:Cytokine stimulated signaling pathways contribute to multiple myeloma (MM) disease progression and in acquired resistance to current treatment options making MM an incurable malignancy. It is very well documented that HGF is a cytokine that is secreted by bone marrow stromal cells which has an autocrine and paracrine role in the disease progression in a myeloma setting. HGF binds to its receptor tyrosine kinase on MM cells, MET and this binding at the extracellular domain results in activation of MET which interacts with several of its target proteins resulting in increased survival, increased proliferation, cell cycle progression, motility, migration and invasion. In normal bone marrows, co-expression of MET and its ligand HGF is a rarity whereas co-expression is a common feature of MM. Elevated levels of HGF have been observed in serum and bone marrows of MM patients with a negative correlation to disease progression. In addition, increased HGF levels cause abnormal and reduced bone formation in patients. HGF gene levels in MM samples have been observed to be significantly up regulated in myeloma cells when compared to normal cells. Recently, studies have identified that HGF facilitates the MM cells to adhere to fibronectin, a bone marrow matrix protein, thereby positively impacting MM cell invasion and proliferation. Overall, HGF and its receptor mediated pathway influences tumor progression in myeloma by targeting several different aspects of the disease biology and hence is a very attractive and potentially very important target for improving treatment regimens in a myeloma setting. Methods:MK2461 was synthesized by Merck Inc. (Whitehouse Station, NJ, USA). Stock solutions were made using DMSO and working stock solutions were made using RPMI 1640 media containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. MTT assay was performed to study drug induced cytotoxicity and thymidine uptake was used as a measure to study differences in proliferation. Flow cytometry using Annexin V-FITC and propidium iodide (PI) was used to measure drug induced apoptosis in cell lines and patient cells. In order to study the mechanism of action of the drug, immunoblotting studies were performed on lysates made from cell lines incubated with the drug for various durations. Results:MK2461 treatment led to dose and time dependent cytotoxicity in a few myeloma cell lines (OPM2, DOX40, RPMI8226 and LR5) but not in others (MM1S, MM1R, H929 and U266). The IC50 values for the sensitive lines varied from 1μ M (OPM2) to 10μ M (DOX40, RPMI8226 and LR5). However, MK2461 significantly inhibited the proliferation of MM cells at sub IC50 concentrations in all cell lines tested except MM1S. This inhibition of proliferation was observed when cells were co-cultured with stromal cells or cytokines, namely VEGF, IL6 or HGF. Culturing MM cells with increasing doses of HGF was still unable to protect them from drug induced inhibition of proliferation. MK2461 was able to induce time dependent increase in apoptosis (as measured by annexin/PI), decrease in proliferation (as measured by BrdU assay) and induction of cell cycle arrest in the drug sensitive cell lines. This effect was not observed in MM1S cells. Exploring the mechanism of action of the drug indicated that MK2461 treatment led to down regulation of pc-Met, pGab1, pAkt and pErk in both the drug sensitive (OPM2) and drug resistant (MM1S) cell lines. However, proteins down stream of Akt in the PI3K/Akt pathway, namely pGSK3β, p70S6K, Bcl2, cyclin E and cyclin D3 were down regulated only in OPM2 cells. On the contrary, we observed up-regulation of these proteins in the drug resistant cell line offering a possible explanation for the drug resistant phenotype. We have also examined combinations of MK2461 with inhibitors of PI3K/Akt pathway. Conclusion:These studies demonstrate significant in-vitro activity of MK2461 in MM. Our results suggest the presence of two populations one very sensitive to MK2461 and one insensitive. Differential effects on the signaling pathways provide important clues to the mechanisms of action of c-met inhibitors in myeloma. The results form the basis for clinical evaluation of MK2461 in MM. Disclosures:Kumar:Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

Antibody‐mediated sialidase activity in blood serum of patients with multiple myeloma

Cell surface sialylation is known to be tightly connected with tumorigenicity, invasiveness, metastatic potential, clearance of aged cells, while the sialylation of IgG molecules determines their anti-inflammatory properties. Four sialidases - hydrolytic enzymes responsible for cleavage of sialic residues - were described in different cellular compartments. However, sialidases activity in body fluids, and specifically in blood serum, remains poorly studied. Here, we characterize first known IgG antibodies possessing sialidase-like activity in blood serum of multiple myeloma (MM) patients. Ig fractions were precipitated with ammonium sulfate (50% of saturation) from blood serum of 12 healthy donors and 14 MM patients, and screened for the presence of sialidase activity by using 4-MUNA (2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid) as substrate. High level of sialidase activity was detected in the MM patients, but not in healthy donors. Subsequent antibody purification by protein-G affinity chromatography and HPLC size exclusion chromatography at acidic conditions demonstrated that sialidase activity was attributable to IgG molecules. Sialidase activity was also specific for (Fab)(2) fragment of IgG and blocked by sialidase inhibitor DANA. Sialidase activity of IgG molecule was also confirmed by in gel assay for cleavage of sialidase substrate. Kinetic parameters of the catalysis reaction were described by Michaelis-Menten equation with K(m) = 44.4-108 µM and k(cat) = 2.7-23.1 min(-1). The action of IgG possessing sialidase-like activity towards human red blood cells resulted in a subsequent increase in their agglutination by the peanut agglutinin, that confirms their desialylation by the studied IgG. This is the first demonstration of the intrinsic sialidase activity of IgG isolated from blood serum of MM patients.

Junctional Adhesion Molecule-A/ F11 Receptor (JAM-A/ F11R) Expression in Multiple Myeloma (MM): a Candidate Biomarker of Aggressive Disease.

Abstract 2830Poster Board II-806 Background:Multiple myeloma (MM) is an incurable disease of clonal plasma cells that accumulate in the bone marrow (BM), causing monoclonal IG production, bone marrow failure, osteolytic lesions and kidney disease. Although initially treatable, MM ultimately becomes refractory to treatment, and is invariably fatal, when tumor cells that harbor genetic mutations expand without regulation. Therefore novel treatment targets need to be identified. A key mechanism in MM pathogenesis is regulation of tumor growth by the bone marrow (BM) microenvironment, particularly by bone marrow neo-vascularization and adhesion of tumor cells to the marrow stroma. Aberrantly expressed genes that regulate angiogenesis by MM cells enhance MM progression and constitute targets in its treatment.JAM-A/F11R is an endothelial cell (EC) adhesion molecule of the immunoglobulin superfamily which is a multifunctional cell membrane protein that mediates intracellular signaling events that alter EC migration and paracellular permeability. For example, in breast cancer, attenuation of JAM-A increases tumor invasion and metastasis through a decrease in tumor adhesion (Ulas Naik Cell Adh Migr. 2008 Oct;2(4):249-51.). In this study we explored the JAM-A/F11R expression in MM tumor cells and in patients to determine the potential role of this molecule in the pathogenesis and progression of MM. Methods:The MM cell lines examined were RPMI-8266, U266, and NCI-H929. Human umbilical vein endothelial cells (HUVECs) served as controls. Informed consent was obtained from patients and control subjects. Primary BM tumor cells were enriched to > 95% CD138+ cells by positive selection using anti-CD138 MACS MicroBeads. The CD138-negative fraction was used for outgrowth of confluent EPCs (> 98% vWF/CD133/KDR+). JAM-A mRNA expression was assessed using an microarray gene expression profile, JAM-A probe based real-time PCR, and JAM-A levels in each sample were measure using a standard curve and normalized to GADPH. JAM-A protein levels in MM cell lines and primary tumor cells were measured by flow cytometry and immunofluorescence. For serum studies, peripheral blood was obtained from 25 newly diagnosed MM patients and 8 healthy, age- and sex-matched controls, and JAM-A levels were measured using an ELISA. Statistical analysis was performed using Student's t-test, two-tailed, with P ' .05 considered significant. Results:JAM-A mRNA levels were significantly increased in MM cell lines RPMI-8266, U266, and NCI-H929 compared to HUVECs (U266, P = 3×10-5; RPM1-8266, P = 1×10-6; NC1-H929, P= 5×10-4). The JAM-A mRNA levels were significantly greater in RPMI-8226; P < .04 compared to TNFα-activated HUVECs for 24 hours which is a proangiogenic switch for HUVEC gene expression. The elevated mRNA expression of the JAM-A in MM cell lines was confirmed by immunofluorescence and flow cytometry which showed the presence of both membrane and cytoplasmic JAM-A protein. Microarray analysis of gene expression profiles from 20 patients' corresponding tumor cells and microenvironmental EPCs showed that JAM-A had a higher level of expression in tumor cells versus MM EPC by 12.62 fold, (P=.0000642). Furthermore, JAM-A had a higher level of expression in MM EPC versus normal control EPC by 2.41 fold, (P=.00113) reflecting a complex regulatory role of F11 signaling in MM, similar to breast cancer (Naik, U. et al 2008). JAM-A was also found to be 12.6 fold greater in tumor cells compared to EPCS (P=.0000642). In addition, circulating levels of soluble JAM-A were found to be significantly greater in the serum of MM patients compared to controls (P < .005), with an average 2-fold increase. Serum levels of JAM-A in MM patients also decreased 71% with treatment n=5, P<.05. Conclusion:We show for the first time that JAM-A expression is highly elevated in MM tumor cells and its levels respond to treatment. In addition, MM patients have higher circulating JAM-A levels compared to healthy individuals and circulating JAM-A levels were reduced following treatment, suggesting that JAM-A may serve as a novel biomarker in MM. Current studies in the lab are aimed at correlating these levels with clinical parameters to determine whether JAM-A levels reflect disease severity and response to treatment. Results of these analyses, as well as results of ongoing experiments using JAM-A siRNA and antibody-inhibition approaches to target JAM-A in myeloma tumor and ECs will be presented. Disclosures:No relevant conflicts of interest to declare.

Pleiotrophin Is Highly Produced by Solid Tumors and Transdifferentiates Human Monocytes Into Endothelial Cells.

Abstract 3050Poster Board II-1026We have previously shown that tumor cells from multiple myeloma (MM) patients express pleiotrophin (PTN). This protein is elevated in the serum of MM patients and we have shown that, in combination with M-CSF, it induces the transdifferentiation of monocytes into endothelial cells both in vitro and in vivo (Chen et al. Blood 2009). In this study, we determined the level of PTN expression in a variety of solid tumor types including breast, ovarian, prostate, and pancreatic cancers, and examined whether PTN produced by tumor cells from these different types of solid tumors could also induce transdifferentiation of human monocytes into endothelial cells both in vitro and in vivo. Our results showed that breast, ovarian, prostate, and pancreatic cancers all produce high levels of PTN as determined by RT-PCR, Western blot analysis, immunohistochemical staining, and measurement of the level of this growth factor secreted into the medium derived from cultured tumor cells whereas normal peripheral blood mononuclear cells (PBMCs) showed no expression of this protein. Next, monocytes derived from CD14-selected PBMCs or the THP-1 cell line were exposed to breast, ovarian, prostate, and pancreatic cancer cells on collagen I-coated Transwell plates with M-CSF. Following culture of these cells for 5-14 days, both fresh CD14+ cells and THP-1 cells changed their morphology into endothelial-like cells and expressed the endothelial genes and proteins Flk-1 and Tie-2 as determined with RT-PCR and Western blot analysis, respectively. These endothelial cell-inducing effects on monocytes were blocked with anti-PTN antibodies. Next, we determined whether human monocytes could be incorporated into blood vessels and express endothelial cell markers in vivo within solid tumors that express PTN. Human breast cancer cells (MDA-MB-231) alone, THP-1 monocytes transduced with the green fluorescent protein (GFP) gene, or the combination of both cell types were injected subcutaneously into severe combined immunodeficient (SCID) mice. Mice were sacrificed 8 weeks later and the tumor tissue was fixed and cut into frozen sections. Breast cancer cells or GFP+ THP-1 monocytes alone did not demonstrate the presence of GFP-marked cells within tumor blood vessels. When breast cancer cells and GFP+ THP-1 cells were injected together, GFP-marked cells were found within tumor blood vessels; and, moreover, double staining of serial sections of the breast cancer with anti-Tie-2 and CD31 antibodies showed a similar distribution pattern of staining as the blood vessel cells showing the presence of GFP. We also examined endothelial gene expression in these samples using RT-PCR. The results showed that the THP-1 monocytes alone or breast cancer cells alone did not express endothelial genes whereas THP-1 monocytes mixed with breast cancer cells showed endothelial gene (FLK-1, Tie-2) expression. These data show that solid tumors through expression of PTN support new blood vessel formation by the transdifferentiation of monocytes into endothelial cells and provide a new potential target for inhibiting early blood vessel formation within tumors. DisclosuresNo relevant conflicts of interest to declare.

CCL3 Impairs Osteoblast Function Via Downregulation of Osteocalcin.

Abstract 739A common feature of bone disease in cancer is hyperactivity of osteoclasts (OC). However, osteoblast (OB) inhibition is critical to the development of osteolytic lesions. Tumor-OC interactions have been extensively studied, the mechanism of OB inhibition however still remains elusive. Several chemokines are upregulated in the multiple myeloma (MM)/bone microenvironment. CCL3 (MIP-1αa), in particular, mediates MM cell migration and stimulates OC differentiation, directly, by stimulating precursor cell fusion, and indirectly, by inducing OB secretion of RANKL. Here, we investigate whether CCL3 interferes with OB differentiation and activity. In vitro osteoblastogenesis consists of differentiation of alkaline phosphatase (ALP) positive cells, followed by secretion and mineralization of the extracellular matrix. We observed that ALP-positive cells express both CCL3 receptors, CCR1 and CCR5, late in differentiation (40% and 32%, respectively, isotype control 9%). No significant CCL3 secretion was detected in OB culture supernatant, suggesting that paracrine CCL3 may primarily affect matrix mineralization. Indeed, exogenous CCL3 (25 to 100 ng/ml) did not modify OB number, instead it decreased calcium deposition (10% decrease at 25ng/ml, 33% decrease at 50 and 100 ng/ml, p<0.05). We next assessed the expression levels of proteins critical to matrix formation and mineralization including osteopontin, bone-sialoprotein and osteocalcin. Osteopontin expression was not affected by CCL3, while both bone-sialoprotein and osteocalcin were downregulated. Importantly, either continuous exposure to CCL3 or 24h stimulation of mature OB impaired RNA expression of osteocalcin (30% to 80%) and bone-sialoprotein (26% to 60%) but not ALP, confirming that CCL3 interferes with OB function rather than formation. We also observed a correlation between osteocalcin expression by IHC on BM biopsies and CCL3 levels in BM serum of MM patients. These data suggest that MM-derived CCL3 interferes with bone mineralization by inhibiting osteocalcin expression. Using neutralizing antibodies against CCL3 we restored both osteocalcin and bone sialoprotein RNA expression levels in the presence of CCL3. Importantly, pretreatment with a small molecule CCR1 inhibitor, MLN3897 (Millennium Pharmaceuticals) completely abrogated the inhibition on osteocalcin and partially reversed bone-sialoprotein expression, suggesting CCR1 as the main mediator of CCL3 effects. We further verified these results in an in-vivo setting of MM bone disease, using the SCID-hu model. CB17 SCID mice bearing a human fetal bone implant were engrafted with CCL3-expressing INA6 MM cells and treated orally with MLN3897 for a total of 49 doses. After 4 weeks of treatment the bones were harvested and stained for TRAP activity, hematoxylin-eosin and osteocalcin. The number of OC/400x field was significantly reduced in the treated group (2.7 vs 1.9, p<0.05), thus confirming in vivo the anti-osteoclastogenic effect of CCR1 inhibition. Moreover, in the presence of INA6 MM cells osteocalcin levels were downregulated compared to non-injected bones and treatment with the CCR1 inhibitor partially restored osteocalcin expression. These results suggest a new role for the CCL3/CCR1 pathway in the development of osteolytic lesions in MM, as inhibitor of OB function other than OC growth factor. Targeting this pathway represents a promising strategy for the treatment of bone disease. Disclosures: Veiby:Millennium Pharmaceuticals: Employment. Anderson:Millennium: Research Funding. Raje:Astrazeneca, Novartis, Celgene: Research Funding.

CS1 promotes multiple myeloma cell adhesion, clonogenic growth, and tumorigenicity via c-maf–mediated interactions with bone marrow stromal cells

AbstractCS1 is highly expressed on tumor cells from the majority of multiple myeloma (MM) patients regardless of cytogenetic abnormalities or response to current treatments. Furthermore, CS1 is detected in MM patient sera and correlates with active disease. However, its contribution to MM pathophysiology is undefined. We here show that CS1 knockdown using lentiviral short-interfering RNA decreased phosphorylation of ERK1/2, AKT, and STAT3, suggesting that CS1 induces central growth and survival signaling pathways in MM cells. Serum deprivation markedly blocked survival at earlier time points in CS1 knockdown compared with control MM cells, associated with earlier activation of caspases, poly(ADP-ribose) polymerase, and proapoptotic proteins BNIP3 and BIK. CS1 knockdown further delayed development of MM tumor and prolonged survival in mice. Conversely, CS1 overexpression promoted myeloma cell growth and survival by significantly increasing myeloma adhesion to bone marrow stromal cells (BMSCs) and enhancing myeloma colony formation in semisolid culture. Moreover, CS1 increased c-maf–targeted cyclin D2-dependent proliferation, -integrin β7/αE-mediated myeloma adhesion to BMSCs, and -vascular endothelial growth factor-induced bone marrow angiogenesis in vivo. These studies provide direct evidence of the role of CS1 in myeloma pathogenesis, define molecular mechanisms regulating its effects, and further support novel therapies targeting CS1 in MM.

Elotuzumab (HuLuc63) Activates CD56dim Natural Killer Cells and Monocytes Resulting in the Release of IP-10 and MCP-1

Elotuzumab (formerly known as HuLuc63) is a humanized monoclonal antibody targeting CS1 (CD319, SLAMF7), a protein which is highly and uniformly expressed on myeloma cells. CS1 is also expressed at lower levels on selected lymphocyte subsets in humans, including natural killer (NK) cells and a subset of CD8+ T cells. Elotuzumab mediates killing of multiple myeloma cell lines (OPM-2, L363) via antibody-dependent cellular cytotoxicity in vitro and has significant anti-tumor activity in mouse xenograft models. Elotuzumab is currently being investigated in Phase I safety studies in relapsed multiple myeloma (MM). To further characterize the effects of elotuzumab on different immune cell subsets, we evaluated its effects in human whole blood cultures in vitro. In resting human whole blood, >95% of the CD56dim (CD3−CD56+CD16++) NK population expressed CS1. In contrast, only 50–75% of CD56bright NK cells expressed CS1. The expression level of CS1, as measured by mean fluorescence intensity, was also lower in CD56bright than in CD56dim NK cells. In 24-hr whole blood cultures, elotuzumab selectively activated CD56dim NK cells, as evidenced by up-regulation of CD69, CD11b, CD54, and down-modulation of CD16 expression, while having little or no effect on CD56bright NK cells. Monocytes in the same whole blood culture were also activated as determined by up-regulation of CS1, HLA-DR, and CD54. Monocyte activation was significantly correlated with CD56dim NK cell activation. In contrast, elotuzumab induced little to no activation of either CD4+ or CD8+ T cell subsets. In whole blood cultures, elotuzumab induced low levels of IFN-γ and only negligible levels of TNF-α. However, elotuzumab induced robust levels of the chemokines IP-10 and MCP-1 which correlated significantly with the level of CD56dim NK cell activation. The induction of chemokines was both dose and Fc-dependent, as F(ab′)2 fragments of elotuzumab failed to induce either NK cell activation or release of chemokines. Interestingly, when elotuzumab was added to purified NK cell cultures, no increase in the levels of IP-10 and MCP-1 was observed. Addition of neutralizating anti-IFN-g mAb to elotuzumab-treated whole blood cultures had little effect on NK cell activation but partially reduced monocyte activation and IP-10 release. Finally, addition of anti-CD18 mAb abrogated the elotuzumab-mediated activation of NK cells and monocytes suggesting cell-cell contact plays an important role in elotuzumab-induced activation of CD56dim NK cells and monocytes. In conclusion, the result from this study demonstrated that elotuzumab may activate NK cells via CS1 and CD16 signaling, resulting in the release of low levels of IFN-g and subsequently monocyte-dependant chemokine production including IP-10 and MCP-1. When dosed in a phase I clinical trial, elotuzumab induced a transient elevation of the chemokines IP-10 and MCP-1 in the serum of MM patients, indicating that the whole blood culture system enables the identification of appropriate pharmacodynamic markers.

CS1 Promotes Multiple Myeloma Cell Adhesion, Clonogenic Growth, and Tumorigenicity Via C-Maf-Mediated Interactions with Bone Marrow Stromal Cells (BMSCs)

Cell membrane protein CS1 is highly expressed by tumor cells from the majority of multiple myeloma (MM) patients (>95%) regardless of cytogenetic abnormalities and response to current treatments. Furthermore, CS1 is detected in MM patient sera and correlates with active MM. However, its role in MM pathophysiology is undefined. In the present study, we first generated CS1 null OPM2 MM cells using lentiviral CS1 short interfering RNA. Specific CS1 knockdown was confirmed by depletion of CS1 mRNA and membrane protein, whereas CS1 was expressed in parental OPM2 and OPM2 cells infected with control lentiviral vector (cntOPM2). Immunoblotting of phopho-site of multiple kinase screen analysis showed decreased phosphorylation of ERK1/2, AKT, and STAT3 in CS1null OPM2 cells vs. cntOPM2 cells. Serum deprivation markedly blocked survival at earlier time points in CS1null OPM2 cells vs. cntOPM2 cells. Earlier apoptosis in CS1null OPM2 cells correlated with earlier activation of caspases, PARP cleavage, and increased proapoptotic proteins BNIP3, BIK. CS1 knockdown further delayed development of OPM2 tumor and prolonged survival in mice. CS1null OPM2 cells failed to grow tumors in the majority of mice (n=8) at 5 weeks after cell inoculation, whereas cntOPM2 cells formed tumors within 1.5 weeks in all animals (n=8). Interestingly, CS1 was expressed in tumors that developed late in mice injected with CS1null OPM2 cells. Concomitantly, we overexpressed CS1 in CS1-low expressing U266 cells by transfecting an expressing plasmid pflagCS1 or control vector. Enforced CS1 expression enhanced U266 cell growth and survival. In contrast to the majority of U266 cells (>95%) that grow in suspension in standard tissue culture flasks, all U266CS1 cells exhibited adherent growth and homotypic adhesion. Importantly, overexpressed CS1 increased adhesion of U266 and MM1S cells to BMSCs. Furthermore, U266CS1 cells formed more and larger colonies in methylcellulose than U266 cells. Interestingly, tumors that developed in mice injected with U266 cells expressed significantly higher levels of CS1 than injected U266 cells; moreover, exercised tumors grew in an adherent manner in vitro. Overlapping differentially expressed genes in U266CS1 vs. U266 and CS1null OPM2 vs. cntOPM2 was next analyzed by gene expression profiling. Importantly, c-maf pathway was significantly upregulated in U266CS1 vs. U266 cells and downregulated in CS1null OPM2 vs. cntOPM2 cells, as evidenced by differentially expressed c-maf and its target genes, i.e., cyclin D2, integrin αE/β7 at both mRNA and protein levels. Myeloma cell adhesion-induced VEGF secretion by BMSCs was greater with U266CS1 than U266 cells. Finally, immunoblotting showed upregulation of c-maf and cyclin D2 in U266 tumors overexpressing CS1. These studies provide direct evidence of the role of CS1 in myeloma pathogenesis, define molecular mechanisms regulating its effects, and further support novel therapies targeting CS1 in MM.

Canonical and Non Canonical Activation of Hedgehog Pathway in Multiple Myeloma

To identify genes or Tumor-Associated Antigens (TAA) potentially involved in the transition from Monoclonal Gammopathy of Undetermined Significance (MGUS) to Multiple Myeloma (MM) and in the MM pathogenesis, we used SEREX and screened serum of MGUS and MM patients with a MM cDNA library. We identified 10 novel antigens and focused our attention on Oral-facial-digital syndrome 1 gene (Ofd1) due to a specific antibody response in MGUS (20.6%) as well as post-transplant MM patients (27.2%). Ofd1 is an embryonal antigen with an important role in the organogenesis and fetal survival; however, in the adult life its expression decreases significantly and is restricted to few tissues. Additionally it is a basal body/centrosome-related protein and a component of Tip60-HAT complex which is involved in cilia formation and functional regulation of Hedgehog (Hh)- and Wnt- pathways in mouse models. We first studied the expression of Ofd1 by western blotting analysis and observed that 11 MM cell lines and 4 primary MM patient cells express high levels compared with PBMCs. To define its role in MM pathogenesis, we specifically knocked-down Ofd1 using 100nM and 200nM of specific siRNA in MM1s cell line. Time-dependent but dose-independent inhibition of Ofd1 does not affect viability and proliferation of MM1s at 48h, as assayed by MTT and thymidine-uptake, respectively. Since mutations in proteins of cilia may block the Hh-signal transduction downstream to Smoothened (Smo) but upstream to Gli transcription factors, we next evaluated the effects of Ofd1 inhibition on the expression of Gli1 and Patched1 (Ptch1), hallmarks of Hh-activity. Western blotting analysis showed that specific knock-down of Ofd1 leads to down-regulation of Gli1 or Ptch1, and β-catenin. Therefore Ofd1 has a role in the functional regulation of Hh- and Wnt-pathway, and its overexpression might lead to constitutive activation of these tumorigenic pathways. These data strongly indicate the existence of an alternative and not canonical activation of Hh-pathway in MM. We also investigated if the canonical way is active in MM. To demonstrate that Hh-pathway is important in CD138+ MM patient cells, we used double staining with antibody anti-CD138 and anti-Sonic (Shh) to show that MM cells co-express Shh, the most common Hh-ligand, suggesting that MM cells might have autocrine and/or paracrine Hh-activity. We confirmed the expression of Gli1 and Ptch1, by western blotting, in 10 on 11 MM cell lines tested and using antibody anti-Gli1 we demonstrated, by immunofluorescence, predominant nuclear localization of Gli1 suggesting constitutive Hh-pathway activation. Treatment with 5μM of a specific Smo-inhibitor for 12–24 and 36 hours, induces down-regulation of Gli1 and Ptch1 in 50% MM cell lines. While down-regulation of Ptch1, a Gli-target gene, is an early event starting at 12 hours, down-regulation of Gli1 starts later at 36 hours. Therefore Gli1 is not the most important transducer of Hh-signaling in MM. In 50% MM cell lines, however, we didn't observe any down-regulation of Gli1 and Ptch1 after treatment with Smo-inhibitor; in these MM cell lines, the predominant nuclear localization of Gli1 is completely abrogated by treatment for 24 hours with 10μM of forskolin, a compound inhibiting the nuclear translocation of Gli1 via PKA. Taken together our data show canonical as well as alternative not canonical activation of Hh-pathway associated with overexpression of ciliary protein Ofd1. Further delineation of the role of primary cilia in regulating cellular response to Hh-signaling in MM will both define their role in disease pathogenesis and support novel therapies targeting Hh-signaling to improve patient outcome in MM.

Anti-CS1 humanized monoclonal antibody HuLuc63 inhibits myeloma cell adhesion and induces antibody-dependent cellular cytotoxicity in the bone marrow milieu

AbstractCurrently, no approved monoclonal antibody (mAb) therapies exist for human multiple myeloma (MM). Here we characterized cell surface CS1 as a novel MM antigen and further investigated the potential therapeutic utility of HuLuc63, a humanized anti-CS1 mAb, for treating human MM. CS1 mRNA and protein was highly expressed in CD138-purified primary tumor cells from the majority of MM patients (more than 97%) with low levels of circulating CS1 detectable in MM patient sera, but not in healthy donors. CS1 was expressed at adhesion-promoting uropod membranes of polarized MM cells, and short interfering RNA (siRNA) targeted to CS1 inhibited MM cell adhesion to bone marrow stromal cells (BMSCs). HuLuc63 inhibited MM cell binding to BMSCs and induced antibody-dependent cellular cytotoxicity (ADCC) against MM cells in dose-dependent and CS1-specific manners. HuLuc63 triggered autologous ADCC against primary MM cells resistant to conventional or novel therapies, including bortezomib and HSP90 inhibitor; and pretreatment with conventional or novel anti-MM drugs markedly enhanced HuLuc63-induced MM cell lysis. Administration of HuLuc63 significantly induces tumor regression in multiple xenograft models of human MM. These results thus define the functional significance of CS1 in MM and provide the preclinical rationale for testing HuLuc63 in clinical trials, either alone or in combination.

Low Levels of Circulating CS1, a Newly Identified Multiple Myeloma (MM) Antigen for a Novel Humanized HuLuc63 Monoclonal Antibody, Is Detected in MM Patient Sera and Correlates with Active Disease.

A new humanized anti-CS1 mAb HuLuc63 significantly induces antibody-dependent cellular cytotoxicity (ADCC) against autologous CS1-expressing patient MM cells. Here we examined whether soluble CS1 is detectable in MM patient sera and affects HuLuc63-induced cytotoxicity against MM cells. Using a sandwich ELISA, CS1 was detected in 44% (23/52) of sera of 52 MM patients (1.2 to 35.3 ng/ml). Immunoprecipitation of sera from CS1 ELISA-positive and -negative MM patients using anti-CS1 mAbs (HuLuc63 and ChLuc90) followed by immunoblotting with HuLuc63 and 1G9 recognizing different epitopes of CS1, further confirmed two forms of CS1 only in CS1-positive MM patient sera. Detection of CS1 is associated with MM (p < 0.0001), since it is detected in MM patient sera, but not in MGUS (n=15) or in healthy donors (n=100). In additional sera of 199 MM patients with newly diagnosed MM, 90% (181/199) of MM patients have detectable CS1 (1 to >80 ng/ml). Median CS1 levels for International Stage System (ISS) I (n=100), II (n=53), and III (n=46) patients are 5.87, 9.37, and 8.37 ng/ml, respectively. The correlation between ISS and CS1 is moderate (spearman correlation coefficient = 0.197, p=0.005). Patients with ISS II/III had significantly higher CS1 levels compared with those with ISS I (median 9.0 vs. 5.9 ng/ml, p=0.006), suggesting a correlation of serum CS1 with active MM. Since patients with ISS II and III require therapy, while those with ISS I do not, these results suggest that circulating CS1 may indicate need for therapy and further support clinical investigation of anti-CS1 therapy using HuLuc63 in MM. Importantly, HuLuc63 has demonstrated significant dose-dependant activity, leading to reduced or eliminated human myeloma tumors (MM1S, OPM2, and L363) in xenograft mice models. Anti-MM activity of HuLuc63 in mice could be observed at sustained serum levels of >2 mg/ml, which is well above the levels of circulating CS1 protein observed in MM patients. Thus, it is proposed that serum CS1 will be an unlikely antibody sink in patients treated with optimal doses of HuLuc63. Since NK cells also express CS1, albeit at lower levels than MM cells, we next determined the effects of HuLuc63 on NK cell function. Pretreatment of NK cells with HuLuc63 (0.1 mg/ml) for 3 days did not alter NK-mediated ADCC against CS1-expressing MM cells (MM1S, H929, INA-6) via HuLuc63. Moreover, recombinant CS1-Fc at physiological serum levels (<200 ng/ml) did not significantly inhibit HuLuc63-induced ADCC against MM cells. Inhibition of HuLuc63-induced MM cell lysis by CS1-Fc was observed at higher concentrations (>200 ng/ml), confirming specific HuLuc63-CS1 binding. Together, these studies strongly support continued clinical investigation of HuLuc63 in MM.

Increased Expression of Pro-Angiogenic and Decreased Expression of Osteogenic Genes by Multipotential Stromal Cells after Stimulation with Myeloma-Derived Microvesicles.

Despite recent advances in the treatment protocols, Multiple Myeloma (MM) is still an incurable disease with median patients' survival of five years. This dismal prognosis is mostly due to resistance of MM to conventional therapies. Recently, attention has been directed toward studying the role of bone marrow microenvironment in MM progression in order to find new ways to inhibit tumor growth by blocking interaction between tumor cells and their microenvironment. Microvesicles (MV) are small membrane fragments released by eukaryotic cells during cell membrane turnover or cell activation. Our group presented the evidence that PMV can facilitate haematopoietic stem cells engraftment in myeloablated recipients (Janowska-Wieczorek et al Blood 2001) and also that PMV could stimulate normal and malignant haematopoietic cells growth and survival (Baj-Krzywozeka et al Exp. Hematology 2001). Recently, it has been shown that PMV may transfer several platelet-derived adhesion molecules to the lung (Janowska-Wieczorek et al IJC 2005) or breast cancer cells (Janowska-Wieczorek et al Transfusion 2006) and thus increase their metastatic potential. In this study we assessed the influence of myeloma-derived microvesicles (MMV) on multipotential stromal cells (MSC). We also compared the gene expression profile of MSC from ten healthy donors (hMSC) and ten MM patients (mMSC). MMV were isolated from MM cell lines supernatants. Expression level of mRNA for genes involved in angiogenesis, invasion and MSC proliferation and osteoblastic differentiation were evaluated using qRT-PCR. hMSC were exposed to 20 ug/ml of MMV for 8 and 24 hours and changes in gene expression were quantified. We noticed increased level of IL8 expression: 4.5 fold after 8 hours and 2 fold after 24 hours stimulation. Upregulation of MMP9 expression was seen at 8 and 24 hours. HGF expression was decreased by approximately 2 folds at both 8 and 24 hours. Interestingly, 8-hour exposure of MSC to MMV resulted in downregulation of RUNX2, collagen1 and osteocalcin mRNA by 1.5, 3 and 2 folds, respectively. After 24 hours, level of downregulation of RUNX2 and collagen1 remained constant and level of osteocalcin decreased further to 3.5 folds. Western blot analysis revealed expression of Dickkopf 1 (Dkk1) protein by MMV. Dkk1 is a well know osteogenic inhibitor and it has been shown to be present in serum of MM patients (Tian E et at N Engl J Med 2003). We analyzed gene expression in mMSC in comparison to hMSC and observed increased level of IL8 (14 folds), VEGF and MMP9 (3 folds), and decreased level of HGF (2 folds). Difference in osteogenic genes expression in mMSC were also observed. RUNX2, collagen1 and osteocalcin were downegulated by 6, 11 and 5 folds respectively. Our study showed for the first time that MMV can induce expression of angiogenic and invasion promoting genes and inhibit expression of osteoblastic genes in MSC. This suggests presence of pro-tumorigenic activators and osteogenic inhibitors in MMV (including Dkk1). Moreover, MSC isolated from MM patients have skewed expression of several pro-tumorigenic and osteogenic genes in comparison to healthy controls. This data might suggest that MMV produced by MM cell in vivo could negatively influence bone marrow environment leading to bone lesions and pathological angiogenesis.