Multiple Myeloma Cell Lines U266 Research Articles

Differential Effects of Hsp70 Inhibition within the Tumor and Microenvironment in Multiple Myeloma.

Multiple myeloma (MM) remains fatal despite prolonged survival by recent advances in treatment, including the use of proteasome inhibitors. It has been proposed that proteasome inhibitors target MM by modulating the NF-κB pathway. Alternatively, proteasome inhibitors may target MM cells because the proteasome helps alleviate the unfolded protein response (UPR) that results from the accumulation of aberrant proteins in the endoplasmic reticulum (ER), which in turn may trigger apoptosis. Indeed, the UPR is induced in neoplastic plasma cells, possibly because of increased immunoglobulin (IG) synthesis that exceeds the protein folding capacity of the ER. Consequently, the degree to which inhibition of the proteasome induces apoptosis may be related to the concentration of unfolded light chains within the ER in MM. We therefore examined whether inhibition of a protein-folding mediator, the Hsp70 molecular chaperone, results in synergistic induction of MM cell apoptosis when combined with a proteasome inhibitor. To this end, the effects of MAL3-101, a novel inhibitor of Hsp70 function, both alone and in combination with a proteasome inhibitor (MG132) on three MM cell lines as well as primary patient MM cells were examined. Dose-response and time course studies in MM cell lines U266, RPMI-8226, and NCI-H929 showed increasing apoptosis and inhibition of proliferation after 16 hours of exposure to MAL3-101 (IC50: 0.9 μM) or to MG132 (IC50: 7 μM). Strikingly, when sub-effective concentrations of MG132 and MAL3-101 were combined, a strong, synergistic apoptotic response was observed in the NCI cell line after 16 hours, and synergistic effects were observed in all cell lines after 36 hours of exposure to the two drugs. Next, we studied MM cells and endothelial progenitor cells (EPCs) derived from the bone marrow of five untreated patients. These two cell populations, which were shown to bear clonotypic similarities, also showed sensitivity to dual targeting. However, these effects occurred at drug concentrations different than those found to be most potent in the cell lines. Moreover, semi-quantitative RT-PCR studies indicated that RPMI cells but not U266 cells exhibited a strong UPR induction after 16 hours of exposure to 7 μM MG132. These data correlated with the greater degree of IG secretion observed in RPMI cells compared to U266 cells as assessed by pulse-chase analysis. Taken together, these results suggest that the apoptotic response of MM cells via targeting the UPR and ER stress pathways may be dependent on basal protein production, including IG synthesis. Studies relating the secretion “index” to UPR induction and sensitivity to Hsp70 and proteasome inhibition in primary tumor cells as well as in microvascular cells from MM patients are ongoing.

Fully Retargeted Oncolytic Measles Virus for Multiple Myeloma Therapy.

Multiple Myeloma (MM) is a disseminated plasma cell malignancy with approximately 14,600 new cases diagnosed in the USA annually. Despite recent progress in current therapeutical options the median survival is 3 to 5 years and cure is extremely rare. Therefore the evaluation of new treatment modalities for MM is highly warranted. An attractive approach to treat Myeloma with a minimum of undesired side effects is the use of a tumour antigen specific for MM cells. Wue-1, a monoclonal antibody binds very selectively normal and malignant plasma cells (50 of 51 MM samples, 14 of 15 immunocytoma and 13 of 13 MALT type lymphomas with plasma cell differentiation were Wue-1 positive, normal tissue including hematopoietic cells were negative) and offers the possibility to define MM cells as targets. The tool for selective killing of MM cells recognized by Wue-1 monoclonal antibody is in this study the measles virus vaccine strain Edmonston B in an ablated variant (MV-Wue) which no longer binds the usual measles receptors CD46 and CD150 (SLAM) expressed on almost every human cell type displaying a single-chain antibody (scFv) derived from the monoclonal Wue-1-antibody which has been tethered to the C-terminus of the H protein to restrict and retarget its interaction to malignant plasma cells especially MM cells. In addition, MV-Wue encodes EGFP facilitating the read out of infected cells. To determine if the fully retargeted MV-Wue would be able to infect MM cell lines and primary MM cells selectively an array of infection assays were performed using the MM cell lines U266 as well as primary CD138 positive MM cells expressing the Wue-1 antigen as expected targets and CD138 negative cells and normal B cells as controls negative for Wue-1. In these experiments selective infections of the MM cell line and primary MM cells were observed whereas the control cells were not infected with MV-Wue. In all cell types GFP expression indicating replicative infection correlated with the expression of the Wue-1 antigen determined by FACS. Infection experiments performed in the presence of monoclonal Wue-1 antibody showed a decreased GFP expression of about 78% in CD138 positive MM cells demonstrating specificity of the infection by MV-Wue. These results indicate that the engineered virus can be a safe and potential curative oncolytic agent to face the main problem in Multiple Myeloma which is responsible for frequent relapses, the minimal residual disease (MRD).

Myeloma Cell Proliferation Is Inhibitied by the Activation of Adenosine Monophosphate Activated Protein Kinase (AMPK).

In multiple myeloma (MM), a network of cytokines in the bone marrow microenvironment promotes myeloma cell proliferation. Consequent inhibition of intracellular signalling in the myeloma cells seems to be a promising strategy to encounter disease progression.The multiple myeloma cell lines U266, OPM-2, RPMI-8226 and NCI-H929 were incubated with the AMPK activators AICAr and D942. Basal and cytokine stimulated proliferation rates of myeloma cells were measured by the WST-1 assay. Alterations of the cell cycle were determined by flow cytometry after staining with propidium iodide. Intracellular signalling was shown by western blotting.The AMPK activators 5-aminoimidazole-4-carboxamide (AICAr) and D942 induced inhibition of proliferation in multiple myeloma cell lines. AICAr also induced a S-phase cell cycle arrest in all four tested cell lines and led to phosphorylation and herewith activation of AMPK. Furthermore, the inhibition of a nucleoside transporter by nitrobenzyl-thio-9-β-D-ribofuranosylpurine (NBTI), inhibition of the adenosine kinase by iodotubericidine and inhibition of AMPK by AMPKI Compound C reversed AICAr effects, indicating that the cellular effects of AICAr were mediated by AMPK. Activation of AMPK inhibited basal extracellular-signal regulated kinase (ERK), mTOR and P70S6 kinase (P70S6K) signalling and blocked cytokine induced increase of proliferation, which again was due to inhibition of ERK and P70S6K signalling. Troglitazone, a representative of a group of anti-diabetic drugs, similarly inhibited myeloma cell proliferation, activated AMPK and decreased ERK and P70S6K signalling.We demonstrate for the first time that myeloma cell proliferation is controlled by AMPK activity. Consequently, targeting this pathway by inhibitors like glitazones provides a novel strategy in myeloma therapy.

Oncolytic Virotherapy for Multiple Myeloma.

Oncolytic virotherapy for cancer employs viruses which specifically lyse tumour cells, leaving normal cells intact. Double-deleted vaccinia virus (vvDD) is tumour-specific due to mutations that restrict viral replication to dividing cells. This is the first use of vvDD to treat a hematologic malignancy. vvDD expressing enhanced green fluorescent protein (EGFP) was used to infect multiple myeloma (MM) cell lines. We used flow cytometry (FACS) analysis to quantify EGFP positive cells in parallel to colorimetric (MTT) and microscopic cell viability assays. FACS results indicated vvDD efficiently (27–73%) infected the MM cell lines RPMI8226, MY5, H929, U266 and LP1 by 72 hours post infection. MTT assays revealed a 7–8 fold decrease in cell viability of infected cells compared to controls. MM cell lines were grown as subcutaneous xenografts (My5) or systemically (RPMI8226) in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, and tumour size/paralysis was monitored after systemic delivery of vvDD. My5 tumours responded to ip vvDD, resulting in a significant delay of tumour growth compared to controls, while 80% of mice treated with vvDD 3 days after iv RPMI8226 tumour injection were cured. Analysis of mouse tissues for viral biodistribution and pathogenicity showed that the ovary was the second highest viral containing tissue though it was 4 logs lower than tumour. Ovaries showed some evidence of follicular necrosis in treated tissues. All other tissues were unaffected. Primary human MM (CD138+) cells obtained from bone marrow aspiration were readily (50–60%) infected with vvDD while normal mononuclear cells from the same samples were not infected. At the same time primary human CD138+ cells were infected with vvDD at an MOI of 1 or mock infected and then plated in matrigel. vvDD treated cells showed significantly reduced colony formation compared to mock-infected controls. These results suggest that vvDD is a safe and effective treatment for multiple myeloma and should be considered for phase I testing in these patients.

PK11195, a Ligand of the Peripheral Benzodiazepine Receptor, Inhibits Myeloma Cell Growth In Vitro and Chemosensitizes Myeloma Cells In Vivo in SCID-hu Models of Human Multiple Myeloma.

The peripheral benzodiazepine receptor (mPBR) appears to be a potential target to induce apoptosis in tumor cells. The expression of this receptor has been linked to a poor prognosis in cancer patients. PK11195 may represent a new, well-tolerated potent chemosensitizing agent that affects multiple resistance mechanisms within malignant cells. We have evaluated whether PK11195 inhibits multiple myeloma (MM) cell growth in vitro; and, furthermore, whether this drug can chemosensitize a melphalan resistant human MM tumor, LAGλ-1 (Campbell et al, International Journal of Oncology 2006), to arsenic trioxide (ATO) and melphalan using an in vivo SCID-hu model. The MM cell lines RPMI8226 and U266 were treated with varying concentrations of PK11195 (1 – 100 mM). After incubating with PK11195 for 24 hours, cell growth was measured by MTT assay. Those cells treated with PK11195 showed decreased proliferation at concentrations as low as 1 mM compared to the untreated cells. Next, we investigated the chemosensitizing effects of PK11195 using an in vivo model of human MM. To accomplish this, each immunodeficient (SCID) mouse was implanted with a 2.0 – 4.0 mm3 LAGλ-1 tumor fragment into the left superficial gluteal muscle. The tumors were allowed to grow for 14 days at which time human IgG levels were detectable in the mouse serum or when tumors became palpable (21 days) and mice were blindly assigned into treatment groups. PK11195 (10, 50 and 100 mg/kg) was administered via oral gavage once weekly when combined with melphalan and once daily five times per week when combined with ATO. Melphalan (3 mg/kg) was administered once weekly via intraperitoneal (i.p.) injection. ATO (1.25 mg/kg) was administered once daily five times per week via i.p. injection. Mice receiving the combination of PK11195 and melphalan (3 mg/kg) showed marked inhibition of tumor growth (PK11195 10 mg/kg, P = 0.03; PK11195 50 mg/kg, P = 0.02; PK11195 200 mg/kg, P < 0.01) compared to mice receiving no therapy. Animals treated with melphalan, as a single agent, did show minimal tumor growth inhibition and reduced paraprotein levels whereas mice treated with single agent PK11195 showed tumor growth similar to the control mice. Mice receiving the combination of PK11195 and low dose ATO (1.25 mg/kg) also showed inhibition of tumor growth (PK11195 200 mg/kg, P < 0.01) whereas treatment with either single agent PK11195 or ATO demonstrated growth similar to the control groups. Treatment with the highest dose of PK11195 (200 mg/kg) was not associated with any observed toxicity suggesting that high doses can be safely administered and are well tolerated. In this study, we showed PK11195 inhibits MM cell growth in vitro at very low concentrations and can chemosensitize drug resistant tumor cells in vivo at doses that have no observable toxicity. We are further evaluating PK11195 as a single agent and in combination therapy both in vitro and in vivo..

Downregulation of PU.1 Is Required for Subtype of Multiple Myeloma.

There are several recent reports that certain mutations in critical transcription factors could be associated with leukemogenesis; however, it remains unclear whether such mutations could be linked to the pathogenesis of multiple myeloma (MM). In the present study, we attempted to determine whether mutations in transcription factors could be found in the patients with MM, utilizing MM cell lines and freshly isolated MM cells, purified with CD138 antibody and magnetic beads. When we looked at the expression of Blimp-1, XBP-1, NF-kB (p65 and p50), and PU.1 using semi-quantitative RT-PCR, all except PU.1 were found expressed in all MM cell lines and freshly isolated MM cells examined. All the PCR products were sequenced, but no significant mutations were identified. As examined with RT-PCR, PU.1 was found completely downregulated in 6 of 8 MM cell lines, while only faint to no expression of PU.1 was seen in 12 of 41 freshly isolated MM cells. As PU.1 message was sequenced, no mutations were found in 29 PU.1-positive MM cells or 2 MM cell lines that had relatively high PU.1 expression. Considering that PU.1 is essential for myeloid and lymphoid differentiation and that mice with null mutation in the PU.1 gene have no granulocytes, monocytes/macrophages or B lymphocytes, together with a recent report by others that AML developed in mice with PU.1 downregulation by 20%, we asked whether PU.1 downregulation might be associated with the oncogenesis or maintenance of MM. To examine the effects of PU.1 expression on MM cells, we generated tet-off conditional PU.1 expression model of PU.1-negative U266 MM cell line. Surprisingly the induction of PU.1 expression resulted in cell cycle arrest and apoptosis of those cells. Cell cycle analysis using flow cytometry revealed that PU.1-expressing U266 cells were in the G1 arrest. May-Giemsa staining revealed that such cells were swollen, enlarged, and vacuolous. These changes were completely reverted when the expression of PU.1 was silenced by tetracycline addition. There was no change in expression levels of cell cycle-related genes (p27, cyclin D1, cyclin E) and apoptosis-related genes (Bcl-2, Bcl-XL, and A1), while caspase3 cleavage was observed by 72 hr following PU.1 induction. We are currently examining the mechanism(s) of PU.1 downregulation in MM cells and a downstream cascade of PU.1 expression during cell death.

Stimulation with K562 Cells Transfected with 4-1BBL and IL-15 Expands and Activates Natural Killer (NK) Cells with Specific Cytotoxicity for Multiple Myeloma (MM).

Introduction: Although stem cell transplant (PBSCT) supported high dose chemotherapy can induce remission in MM patients with high risk features (e.g. high CKS1-B expression and abnormal cytogenetics) there is a high rate of relapse. Immunologic therapies can augment high dose chemotherapy by eradicating chemo-resistant disease. We have previously established that KIR-ligand mismatched NK cells from haplo-identical donors are cytotoxic to primary MM cells. In this study we investigate whether co-incubation of NK cells with irradiated K562 cells transfected with 41BBL and membrane-bound IL15 (K562-41BBL-mIL15) could expand NK cells and augment cytotoxicity to primary MM cells.Methods: Peripheral blood mononuclear cells (PBMC) of normal donors were co-cultured with irradiated K562-41BBL -mIL15 at a 1.5:1 ratio with varying concentrations of IL2 and re-stimulated on day 7. The cultures were then analyzed for proliferation, expansion, immunophenotype and cytotoxicity.Results: By testing a range of IL2 concentrations (10–300 IU/ml) we first established that 100IU of IL2/ml induced optimal proliferation in H3 thymidine uptake assays and this concentration was used in all subsequent experiments. After two weeks PBMC co-cultured with K562-41BBL-mIL15 were enriched for NK cells (93%) and contained few T cells (0.8%) compared to PBMC cultured with IL2 alone (NK cells 11.9% and T cells 58.7% respectively). In absolute numbers there was as 60-fold (±0.6) expansion of NK cells in the K562-41BBL -IL15 cultures compared to only a 1.5-fold expansion of NK cells cultured with IL2 alone. NK cells were analyzed by flow cytometry for expression of KIR2DL1, KIR2DL2/3, and KIR3DL1 pre- and post-incubation with K562-41BBL-mIL15 and we observed no change in NK immunophenotype. 51Cr release assays of expanded NK cells showed specific killing of K562 cells (84±4%), the MM cell line U266 (88±3%), and primary MM cells (58±9%), without killing of non-myeloma patient cells (PHA-blasts) or autologous cells at a ratio of 7 NK effectors to 1 target cell. Expanded NK cells achieved much higher specific lysis and at lower E:T ratios of primary MM cells when compared side-by-side with non-expanded NK cells.Conclusion: K562-41BBL-mIL15 transfectants stimulated vigorous expansion of NK cells without expanding T lymphocytes. Analysis of the KIR repertoire showed that there was no change in NK cell subpopulations after expansion. The expanded NK cells were highly active and killed patient MM cells better than non-expanded NK cells without significant lysis of normal cells. Expanded/activated NK cells may prove especially helpful in treating patients with relapsed/refractory MM who have a high tumor burden.

DKK-1 Is a Widely Expressed, Potent Tumor-Associated Antigen in Multiple Myeloma Recognized by Cytotoxic T Lymphocytes.

Idiotype proteins have been used for immunotherapy in multiple myeloma (MM); and the results thus far have been disappointing. Therefore, the identification and use of novel and more potent tumor-associated antigens are urgently needed to improve the efficacy of the treatment. Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by the tumor cells of most myeloma patients and may be responsible for suppressed osteoblast formation. Based on these data and the fact that DKK1 is not expressed in normal tissues except placenta, prostate, and mesenchymal stem cells, we hypothesized that DKK1 is a novel tumor-associated antigen in MM. In this study we examined the capacity of DKK1 to trigger the induction of antimyeloma cytotoxic T lymphocyte (CTL) responses. Using DKK-1 peptide-pulsed dendritic cells (DCs), we successfully generated DKK-1 peptide (DKK120-29 and DKK166-74V)-specific CTL lines and clones from HLA-A2+ MM patients and healthy individuals. In our experiments, mature DCs obtained from cultures of blood monocytes were pulsed with HLA-A2-restricted DKK1 peptides. Autologous T cells were then stimulated weekly with these DCs, and cytotoxicity was examined against DKK1 peptide-pulsed T2 cells, myeloma cell lines, and primary myeloma cells isolated from patients. After 4 cycles of in vitro stimulation and subsequent T cell cloning and expansion, specific CD8+ CTL lines and clones were obtained. These CTLs not only had cytolytic activity against DKK1 peptide-pulsed T2 cells, but also significantly lysed HLA-A2+ MM cell line U266 and primary tumor cells in vitro. No killing was observed against HLA-A2+ normal lymphocytes including B cells or HLA-A2− myeloma cell lines or primary myeloma cells from patients. The CTL response could be inhibited by anti-HLA-A2 antibody indicating that the response was indeed restricted by HLA-A2 molecules. IFN-γ, but not IL-4, was secreted by the T cells indicating that the effector cells were type-1 CD8+ T cells. Further functional studies are underway. Thus, these results demonstrate that DKK1-specific CTLs are able to lyse myeloma tumor cells including primary myeloma cells from patients, and identify DKK1 as a potentially important antigenic target for antimyeloma immunotherapeutic strategies

Clonal Restriction in Endothelial Progenitor Cells in Patients with Multiple Myeloma.

In multiple myeloma (MM), neoplastic plasma cells and bone marrow microvascular cells have paracrine cross-regulatory interactions, and display abnormal features suggesting that these neoangiogenic cells are myeloma-related. Tumor neovascularization is closely linked to neoplastic growth. We have recently shown that circulating endothelial progenitor cells (EPCs) reflect disease severity as assessed by M protein and serum β2-microglobulin levels in MM. These observations linking circulating EPCs to disease activity in MM prompted us to study the genetic characteristics of EPCs using X-chromosome inactivation (XCI) analysis to determine their clonal restriction profile. In addition, we examined EPCs for the presence of genetic markers common to neoplastic MM cells by examining immunoglobulin heavy chain (IgH) rearrangement of germline DNA. Isolation and outgrowth of EPCs was done as previously described. Clonality within EPCs was evaluated by quantitation of XCI patterns in female patients by a human androgen receptor gene (AR) assay (HUMARA). DNA was prepared from EPCs, hair root, or PBMCs and digested by a methylase-sensitive restriction enzyme. The highly polymorphic X-linked human androgen receptor gene was amplified by PCR. In polyclonal populations, both alleles are seen as two distinct bands, while clonal populations yield a single band. Amplification of genomic DNA extracted from PBMCs showed that XCI status was informative in 7 of the 16 MM patients and in 7 of the 15 healthy controls. Next, EPCs and hair root cells from informative patients were studied for XCI patterns to determine clonality. DNA obtained from confluent EPCs outgrown from 7 of the 10 informative patients displayed extremely skewed XCI patterns (90% inactivation of one allele), while hair follicle cells in the same patients displayed a random pattern. These results suggest that EPCs share characteristics of clonal plasma cells in MM, and display clonal restriction that is lineage-specific. To further explore the genetic similarity between EPCs and MM cells, EPCs were assessed for clonotypic immunoglobulin heavy chain (IgH) VDJ sequences by PCR amplification of genomic DNA. In these experiments, DNA was extracted from bone marrow EPCs and used in a series of 7 PCR amplifications containing a specific VH forward primer and a consensus JH reverse primer. In EPCs from 2 patients, including one with skewed XCI, we found a single PCR product identical to the IgH VDJ rearrangement found in bone marrow containing 60% and 65 % plasma cells, respectively, suggesting that an identical IgH VDJ rearrangement was present in EPCs and plasma cells in the same patient. Controls for this experiment included HUVEC genomic DNA, which did not yield PCR products even when spiked with a positive control DNA (up to 50 %) extracted from the MM cell line U266, and PBMC genomic DNA from controls which yielded multiple bands produced from several of the VH primers. Our results show that EPCs in a population of MM patients display clonal restriction and genetic similarity to plasma cells. Thus, it is possible that neoplastic MM cells and EPCs are linked not only within a functional context by their paracrine cross-regulatory effects within bone marrow microenvironment, but also by their genetic ancestory. These conclusions await confirmation from ongoing molecular genetic comparisons of the two cell types.

Arsenic Trioxide Targets the Interleukin-6 Cascade in Multiple Myeloma by Promoting Lysosomal Degradation of the Interleukin-6 Receptor Complex.

Introduction. Treatment of multiple myeloma (MM), a B-cell neoplasm characterized by clonal expansion of plasma cells in the bone marrow, remains unsuccessful in a significant proportion of patients, so that innovative strategies are needed. Arsenic trioxide (As2O3) has shown notable efficacy against MM in vitro and in clinical studies. Multiple cellular pathways in MM are targeted by As2O3. As cellular growth of MM cells is interleukin-6 (IL-6) dependent, we investigated if As2O3 also targeted the IL-6 cascade. Materials and methods. The IL-6-dependent MM cell line U266 was used as an in vitro model. Cell growth was measured by MTT assay, and apoptosis by flow cytometry. Protein phosphorylation was studied by Western blotting with specific antibodies. Expression of IL-6 receptor (IL-6R) was investigated by Western blotting and flow cytometry. Gene expression was detected by quantitative polymerase chain reaction (Q-PCR). Results. As2O3 showed a time and dose related inhibition of U266 cellular proliferation by induction of apoptosis. At clinically achievable concentrations (2 – 4 μmol/L), As2O3-induced apoptosis was associated with inhibition of constitutive tyrosine phosphorylation of JAK2 and STAT3, in a time and dose-dependent manner. Furthermore, pre-treatment of U266 cells with As2O3 prevented rescue of phosphorylation of JAK2 and STAT3 by exogenous IL-6, implying that the IL-6 cascade was targeted. Using Western blot analysis, we showed that As2O3 induced a time and dose-dependent down-regulation of both components of the IL-6R complex: IL-6R alpha subunit (IL-6Rα) and gp130 signal transducer. These results were confirmed by flow cytometry, showing that As2O3 treatment led to a down-regulation of surface expression of the IL-6Rα. Interestingly, Q-PCR did not reveal any change in the mRNA levels of the two genes with As2O3 treatment, suggesting that As2O3 downregulated IL-6R complex via a post-transcriptional mechanism. It is known that under physiological conditions, the IL-6R complex is internalized upon ligand binding and is targeted to lysosomes for degradation. Treatment of the U266 cell line with the lysosome inhibitor ammonium chloride totally abrogated As2O3-induced degradation of IL-6Rα and gp130. These results suggested that As2O3 might promote lysosomal degradation of IL-6Rα and gp130 by inducing a ligand-independent internalization of the receptor complex. Conclusion. Our results demonstrated that As2O3 suppressed IL-6-induced JAK/STAT3 signaling pathway in MM cells and this might be, at least partly, mediated by promoting ligand-independent internalization and lysosomal degradation of the IL-6R complex. These results have significant implications on the use of As2O3 in the treatment of patients with MM and other malignancies that are IL-6 dependent.

Type I MAGE Proteins: Novel Markers of Proliferation in Multiple Myeloma Progenitor Cells.

CT7 (MAGE-C1) and MAGE-A3, two members of the type I MAGE family of Cancer-Testis antigens, are commonly expressed at both the mRNA and protein levels in primary tumor specimens from multiple myeloma patients. In previous analyses, tumors with higher percentages of type I MAGE-expressing cells had a positive correlation with abnormally elevated plasma cell proliferation. These data support the hypothesis that type I MAGE proteins are molecular markers of proliferating myeloma progenitor cells (the so-called “myeloma stem cell”) and may play a role in the pathobiology of this disease. To test this hypothesis, we examined the expression of type I MAGE in proliferating myeloma cells by flow cytometry. Human multiple myeloma cell lines U266, RPMI-8226, and KMS-11 were co-cultured for 12 or 24 hours with the nucleoside analog bromodeoxyuridine (BrdU), then fixed, permeabilized, and stained with CT7-33, a monoclonal antibody (mAb) to CT7, or M3H67 (to MAGE-A3), followed by a phycoerythrin (PE)-conjugated secondary mAb. The cells were then treated with DNAse and stained with a fluoroisothiocyanate (FITC)-conjugated mAb against BrdU. Proliferating cells that incorporated BrdU into their DNA exhibited high FITC fluorescence. For mAb M3H67, dual color analysis of this population showed that greater than 99% demonstrated a significant shift in PE fluorescence in all three of these cell lines as measured by Mean Fluorescence Index (MFI= geometric mean fluorescence [specific primary antibody]/mean fluorescence [no primary antibody], table 1). For CT7-33 mAb, greater than 85% demonstrated a shift in two of three lines (U266 and KMS-11), but not in RPMI-8226. For all three of these cell lines, dual color analysis of the BrdU-low population demonstrated less than 65% staining with either type I MAGE mAb. Interestingly, RT-PCR with CT7-specific primers of total RNA from RPMI-8226 revealed a product of lower molecular weight than expected, suggesting that a gene deletion occurred in this cell line possibly resulting in a stop codon, decreased translation, or decreased protein stability. This PCR product is being sequenced to determine the nature of the deletion. These results demonstrate that type I MAGE proteins are expressed in proliferating myeloma cells and are molecular markers of this population. These data suggest that novel therapeutics such as vaccines that target type I MAGE may preferentially eliminate the cycling myeloma cells, resulting in long-term cures.Table 1Type I MAGE expression in proliferating (BrdU+) myeloma cellsCell lineMFI CT7-33MFI M3H67U26665.862.5KMS-119.948.4RPMI-82263.112.3

The Histone Deacetylase Inhibitor Sodium Valproate Has In Vitro Anti-Neoplastic Activity Against Primary Multiple Myeloma Cells and Cell Lines Both Alone and in Combination with Velcade.

There is growing evidence that histone deacetylase inhibitors (HDAC-I) may have benefit in the treatment of multiple myeloma (MM). We assessed the in vitro responses of the MM cell lines H929, JJN3, KMS11 and U266 to the HDAC-Is trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA) and sodium valproate (SV). All three HDAC-Is induced dose dependent cell killing and the ranked sensitivity of the lines was remarkably constant with U266 cells being most resistant to all three inhibitors. Real time RT-PCR based analyses of the HDAC expression profiles, identified that the greater resistence of U266 cells was associated with 5–10 fold increased expression of a single HDAC from the sirtuin family of deacetylases (SIRT5). Each HDAC-I displayed different potencies against the cell lines; TSA killed in the range 0.1–1μM, SAHA in the range 1–10μM and SV at the higher doses of 1–10mM. Despite being less potent, the doses of SV are commensurate with those that can be achieved in vivo. Furthermore, we have identified synergy/cooperation between suboptimal SV concentrations and the proteasome inhibitor Velcade (Bortezomib). MTT assays using JJN3 cells, demonstrated that 1mM SV had little effect reducing survival to 89% whereas 0.6 nM velcade reduced cell survival to 42±5.5%. Killing by velcade was not significantly enhanced by the additional presence of 1μM dexamethasone (Dex)(36±3.4%). However, the combination of SV, Dex and Velcade resulted in near total cell killing (4.7±2.6% survival). Collectively, these observations suggested that SV may have value in MM therapy. However, we wished to also investigate the potency of SV against primary MM cells. We have developed an in vitro culture system in which primary MM plasma cells survive for up to 30 days. In this culture system we have observed efficient and selective killing of MM cells by 1mM SV that has been comparable or indeed greater than that observed in response to 1μM SAHA. Thus it appears that primary MM cells are more SV sensitive than MM cell lines. Together our findings indicate that SV may have value in MM therapy and in particular when combined with proteasome inhibitors. Our data also indicate that SIRT5 overexpression may be a resistance factor against such therapies.

Blocking Pleiotrophin Activity Inhibits Multiple Myeloma (MM) Cell Growth In Vitro and in a Severe Combined Immunodeficient (SCID)-hu Murine Model of Human MM.

Pleiotrophin (PTN) is a heparin-binding growth factor that binds CD138 and stimulates angiogenesis, tumor growth and metastasis in some solid tumors. Recently, we have shown that this factor is highly produced by multiple myeloma (MM) cell lines including RPMI8226 and U266 and fresh malignant plasma cells, and is secreted into the culture medium following short-term culture of bone marrow from MM patients. We investigated the effects of PTN on MM growth in vitro and in vivo using a SCID-hu murine MM model. We determined the anti-proliferative effects of suppressing PTN by cloning a whole PTN sense or anti-sense cDNA construct containing the green fluorescent protein (GFP) gene into the MM cell lines RPMI8226 and U266. Cells transduced with sense PTN showed markedly increased proliferation compared to cells transduced with vector alone whereas the anti-sense-containing MM cells showed reduced cell numbers. In addition, we treated RPMI8226 and U266 cells with a polyclonal anti-PTN antibody and evaluated its effect on MM growth. These cells were cultured for 48 hours in the presence of the anti-PTN antibody at a concentration of 100 micrograms/ml or a control antibody, and effects on cell growth assessed with an MTT assay. Marked anti-MM effects were observed with the anti-PTN antibody compared to the control antibody in both cell lines [RPMI8226 (p < 0.01) and U266 (p < 0.001)]. In order to further define the importance of PTN in the growth of MM in a more clinically relevant in vivo setting, we determined whether this polyclonal anti-PTN antibody could suppress tumor growth and human paraprotein secretion using our SCID-hu murine model of human myeloma LAGλ-1. LAGλ-1 has been previously shown by our group to produce large amounts of PTN as measured in mouse serum by ELISA and by RT- PCR analysis on freshly isolated LAGλ-1 tumor cells. Thirty SCID mice (n = 5 mice/group) were implanted with a 0.4 – 0.6 cm3 LAGλ-1 tumor fragment into the left hind limb muscle. Fourteen days following implantation, mice were randomized into treatment groups, and received treatment intraperitoneally (IP) with anti-PTN antibody at doses of 0.1, 0.3, 1.0, 3.0 or 10 mg/kg or vehicle alone twice weekly. Mice receiving anti-PTN antibody at the highest doses (3.0 and 10 mg/kg) showed marked inhibition of tumor growth [3.0 mg/kg (p < 0.03), 10 mg/kg (p < 0.008)] as well as decreases in levels of human paraprotein [3.0 mg/kg (p < 0.004), 10 mg/kg (p < 0.003)]. Notably, immunohistochemical staining with an anti-CD138 antibody showed a marked reduction in cells with CD138 positivity in the LAGλ-1 tumors from animals treated with anti-PTN antibody compared to mice treated with vehicle alone. These in vitro and in vivo results demonstrate that PTN may be a potential new target for the treatment of MM. The effects of this therapy on angiogenesis and cell signaling are currently under investigation.

Apoptosis of multiple myeloma cells induced by agonist monoclonal antibody against human CD28

CD28 is expressed abnormally on human multiple myeloma (MM) cells but the significance had not been identified until now. In this paper, we are suggesting that abnormal expression of CD28 might be a marker of tumour progression. We therefore took the approach of generating a hybridoma cell line capable of secreting agonist monoclonal antibody directed against human CD28 (agonist anti-CD28 mAb) and then determined the expression of CD28 molecules on the MM cell lines U266 and XG1. The biological effects of agonist anti-CD28 mAb on cell growth and proliferation of U266 and XG1 cell lines were then analysed. Our results showed that the expression of CD28 on U266 and XG1 was significantly higher than that of PBTC or Jurkat cells. We found that by adding the agonist anti-CD28 mAb to cultures of U266 and XG1 cells their rate of growth and proliferation was obviously inhibited. Further morphological and molecular analyses found that U266 and XG1 incubated with agonist anti-CD28 mAb showed signs of nuclear condensation, chromatin marginal changes, cells membrane breaking, and cytoplasmic shrinkage. Vacuoles and apoptotic bodies were also observed using a transmission electron microscope and the development of typical DNA laddering patterns were found by the use of electrophoresis assays, suggesting that U266 and XG1 cells were undergoing apoptosis induced by agonist anti-CD28 mAb in vitro.

Depletion of the Intracellular Akt Kinase, and Its Downstream Signaling, Mediates Synergy between the Proteasome Inhibitor MG-132 and the Heat Shock Protein 90 Inhibitor 17-AAG in the Multiple Myeloma Cell Line U266.

While proteasome inhibitors are effective therapy for multiple myeloma (MM), their efficacy could be improved by synergistic targeting of apoptosis. The intracellular serine/threonine kinase Akt has been demonstrated to have a central role in MM cell growth, survival, and drug resistance. Akt is activated by extracellular cytokines such as IGF-1 and IL-6, and contributes to MM resistance by ameliorating the apoptotic effects of proteasome inhibition. Akt requires chaperone proteins for proper stability and function, including the 90 kD molecule Heat Shock Protein 90 (HSP-90). HSP-90 function is abrogated by geldanamycin and its derivative, 17-allylamino-17-demethoxygeldanamycin (17-AAG).In the U266 MM cell line, the IC50 of the proteasome inhibitor MG-132 and 17-AAG was 100 nM and 800 nM, respectively. Following exposure of U266 MM cells to either drug alone, or the combination at a fixed-ratio of their IC50s (1:8), apoptosis was determined by Annexin V staining and FACScan analysis. Synergy analysis was performed using Calcusyn (Biosoft, Cambridge, UK). We found that the combination index (CI) was synergistic (CI<1) throughout the dose range, with a CI = 0.449 ± 0.298 at the combination IC50 (highly significant). For example, the apoptotic effect of 50 nM MG-132 and 400 nM 17-AAG was 6 ±2 % and 23 ±3 %, respectively, whereas the 50:400 nM combination produced apoptosis in 68 ± 2 % of the cells.To analyze effects on Akt and its substrates, we incubated U266 MM cells with MG-132 (50 nM), 17-AAG (400 nM), or the combination. We harvested lysates after zero, two, six, and 24 hours incubation, and Western blot analysis was performed. Co-incubation with MG-132 and 17-AAG, but not either alone, depleted Akt by 24 hours post-therapy. Co-treatment also produced significantly greater upregulation of HSP-90 and HSP-70 than 17-AAG alone, thus demonstrating greater functional inhibition of Akt. The combination also demonstrated the greatest abrogation of Akt-mediated effects on mitochondrial apoptosis: Co-treatment produced the greatest expression of BAD, decreased BCL-XL expression, reduced phosphorylation of GSK-3, and produced the greatest activation of caspase 3.Monotherapy with 17-AAG upregulated HSP-90 and HSP-70, reduced BCL-XL expression, and activated caspase 9. MG-132 monotherapy produced none of these effects. These findings demonstrate that synergy between proteasome inhibitors and 17-AAG is mediated by Akt depletion and abrogation of Akt signaling, predominantly by MG-132 augmentation of 17-AAG-mediated decay of Akt. Down-regulation of Akt-mediated resistance allows dual-apoptotic signaling and synergistic effect of combination therapy. These findings demonstrate a mechanistic rationale for utilizing heat shock protein inhibitors in combination with proteasome inhibitors as therapy for MM.

Targeting IKK Inhibits Multiple Myeloma (MM) Cell Growth in the Bone Marrow Microenvironment.

NF-κB is a transcriptional factor promoting tumor cell growth and inhibition of apoptosis via regulating expression of proteins modulating cell cycle and anti-apoptosis. NF-κB is constitutively associated with an inhibitor (IκB), which is phosphorylated by IκB kinase (IKK) upon cell stimulation (ie, TNFα) and subsequently degraded by ubiquitin-proteasome pathway, thereby allowing NF-κB to translocate to nucleus. We have previously shown that an IKK inhibitor PS-1145 partially (20–50%) inhibits MM cell proliferation; however, it inhibits both IL-6 secretion from BMSCs triggered by MM cell adhesion and proliferation of MM cells adherent to BMSCs. Targeting IKKβ is therefore a possible therapeutic option for inhibition of MM cell growth in the bone marrow microenvironment by downregulating NF-κB activity.In this study, we further delineated the biologic significance of IKK inhibition in MM cells using IKKβ specific inhibitor MLN120B (Millennium Pharmaceuticals, Cambridge, MA). MLN120B induced 60–90% growth inhibition in cells from the MM cell lines RPMI8226, RPMI/Dox40, RPMI/LR5, U266, and INA-6; on the other hand, it induced only 25–30% inhibition in MM.1S and MM.1R cells, assessed by 72 h tritiated-thymidine uptake. Importantly, neither IL-6 nor IGF-1 overcomes the growth inhibitory effect of MLN120B in both MM.1S and U266 cells. Interestingly, MLN120B significantly augmented TNFα-induced cytotoxicity in MM.1S cells. We next examined whether MLN120B could enhance the cytotoxicity of other agents. MLN120B augmented growth inhibition triggered by doxorubicin and melphalan in RPMI8226 and IL-6 dependent INA-6 cell line. We therefore studied growth inhibitory effect of MLN120B in the presence of bone marrow stromal cells (BMSCs). MLN120B inhibited 70–80% of constitutive IL-6 secretion from BMSCs, without toxicity. Importantly, MLN120B almost completely blocked stimulation of MM.1S, U266 and INA-4 cell growth and IL-6 secretion from BMSCs induced by binding of tumor cells to BMSCs. Finally, MLN120B overcame the protective effect of BMSCs, cell adhesion mediated drug resistance, against dexamethasone in MM.1S cells. Taken together, our data demonstrate that an IKKβ inhibitor induces cytotoxicity in MM cells in the BM milieu, providing the framework for clinical trials of these novel agents to improve patient outcome in MM.

Inhibition of Multiple Myeloma Cell Adhesion to Fibronectin by Ephrin Ligation.

Multiple myeloma (MM) cell adhesion to fibronectin (FN), mediated via VLA-4 and VLA-5, has been shown to induce resistance to several chemotherapeutic drugs. Disruption of MM cell adhesion to FN and other marrow microenvironment elements might therefore enhance the effects of therapy. We now present the first evidence that Eph-ephrin signaling may be exploited to inhibit MM cell binding to fibronectin. Ephs are transmembrane tyrosine kinases and ephrins are their cell-surface ligands. There are two classes of Ephs and ephrins, A and B. Both Ephs and ephrins can transduce repulsive signals that cause interacting cells to lose contact with each other and with extracellular matrix. We are not aware of any previous systematic study of Eph and ephrin expression or function in MM cells. We have found MM cell lines H929, U266, and RPMI 8226 express members of the A classes of both Ephs and ephrins, but not the B classes. First, we demonstrated ligation with commercially available anti-ephrin A3 antibody was followed by ephrin capping and shedding from the cell surface. We next explored whether ephrin ligation affects MM cell adhesiveness in culture. Whereas H929, U266, and RPMI 8226 cells adhered rapidly to fibronectin-coated plastic surfaces, all three cell lines failed completely to adhere to a mixed coating of FN and rabbit anti-ephrin A3 antibody for a period of 2 hrs. This effect was not seen with FN + normal rabbit Ig. This suggests binding of ephrin A3 (or another cross-reacting A-class ephrin) by solid-state antibody triggers intracellular signals that interfere with initial steps of integrin-mediated adhesion. After 2 hr, spontaneous partial recovery of adhesion occurred, reaching a plateau of approximately 30% of control values by 24 hr. We postulate this recovery occurs via clipping of the extracellular ephrin domain by transmembrane metalloproteases, since recovery of FN adhesion was partially prevented by the metalloprotease inhibitor GM6001 (25 uM). Also consistent with this theory, we found in a separate experiment that GM6001 reduced the shedding of cross-linked A-class ephrins from MM cell lines. In summary, we have demonstrated that manipulation of EPH-ephrin signaling can impair MM-cell adhesion to FN, and that this effect is enhanced by simultaneous inhibition of metalloprotease activity. We are currently studying the effect of A-class ephrin ligation on adhesion-mediated drug resistance in MM cell lines. We also intend to evaluate EPH-ephrin expression in marrow specimens from patients with MM.

Pleitrophin Is Highly Expressed by Myeloma Cells, Elevated in the Serum of Myeloma Patients, and Is a New Autocrine Growth Factor for This B Cell Malignancy.

Pleitrophin (PTN) was originally described as a development regulatory cytokine. This protein regulates the growth of neuroectodermal and mesodermal cell lineages during early embryogenesis but becomes down-regulated during late phase of embryogenesis and shows a very restricted expression pattern in adult neural system. This protein has been recently shown to both induce angiogenesis and bind to syndecan. Thus, we determined whether this protein may be found in tumor cells from myeloma patients and its potential to influence their growth. First, we measured serum levels of PTN in myeloma (n=115) and age-matched controls (n=50) using an ELISA-based technique. PTN levels in the serum of myeloma patients were markedly elevated compared to the normal control group (P<0.02). The serum concentration of PTN in MM patients averaged 28 pg (range, 8 to 110 pg). In contrast, the control serum levels averaged only 12 pg (range, 0 to 20 pg. Interestingly, the PTN concentration in two patients with plasma leukemia was much higher than other multiple myeloma patients. Next, we analyzed the expression of PTN using RT-PCR on RNA from myeloma cell lines (RPMI8226, U266), and multiple myeloma patients' and normal control bone marrow aspirates. Results showed that the PTN mRNA was strongly expressed in multiple myeloma cell lines, myeloma bone marrow samples but not in the normal control bone marrow specimens.In order to determine whether PTN stimulates multiple myeloma growth, we further cloned a whole PTN sense or anti-sense sequencing DNA into the multiple myeloma cell lines RPMI8226 and U266. Cells transduced with sense PTN showed markedly increased proliferation compared to cells transduced with antisense or vector alone. The role of PTN in hematological malignancies has not been previously defined. Due to the restricted expression pattern of PTN in adults, PTN is suggested as a potential new target for treatment of multiple myeloma. Further investigations are defining the prognostic value of PTN serum levels and the mechanism by which this cytokine drives myeloma growth.

Prompt tumor formation and maintenance of constitutive NF-kappaB activity of multiple myeloma cells in NOD/SCID/gammacnull mice.

Clinically and biologically relevant animal models are indispensable to evaluate both the pathophysiology and strategies for diagnosis and treatment of multiple myeloma (MM). We examined the tumorigenicity of MM cell lines KMM-1 and U-266 in an in vivo cell proliferation model using NOD/SCID/gammacnull (NOG) mice. Two cell lines were inoculated either subcutaneously (s.c.) in the post-auricular region or intravenously (i.v.) in the tail of NOG mice. The KMM-1 cell line produced a progressively growing large tumor with infiltration of the cells expressing human lambda-chain in various organs of all NOG mice, while the U-266 cell line failed to do so. Tumor cells grown in NOG mice maintained the original histomorphology, as well as expression patterns of tumor markers human lambda Ig light chain and VEGF. Tumor progression in mice also correlated with elevation of serum human soluble IL-6R and gp130. Tumor cells sustained a strong NF-kappaB activity in vivo and induced NF-kappaB components were indistinguishable from those in cells cultured in vitro. The rapid and efficient engraftment of the MM cell line in NOG mice suggests that this is a very useful animal model which could provide a novel system in which to clarify the mechanism of growth of cancer cells, as well as to develop new therapeutic regimens against MM.

Phenylarsonic Acid Compounds with Broad‐Spectrum and Potent Cytotoxic Activity Against Human Cancer Cells.

AbstractFor Abstract see ChemInform Abstract in Full Text.