RPMI-8226 Multiple Myeloma Cells Research Articles

Increased Sclerostin Secretion in Multiple Myeloma Results in Stimulation of Osteoclastogenesis and Inhibition of Osteoblastogenesis

Abstract 1819 Objectives:Osteoblasts (OB) and osteoclasts (OC), are an integral part of the bone microenvironment, and play a crucial role in myeloma growth and survival. Their imbalance results in osteolytic disease and elucidating the mechanisms underlying osteolytic lesions is important not only for the improvement of osteolytic bone disease but also for the treatment of multiple myeloma (MM).The osteocyte-secreted protein sclerostin, encoded by the SOST gene, is a potent inhibitor of osteoblastogenesis. It is regarded as a good target for osteoporosis treatment, but its role in MM remains to be determined. Our objective was to study the role of sclerostin in MM bone disease and determine if sclerostin directed strategies were a reasonable approach in MM. Methods and Results:Sclerostin concentration in patients’ blood plasma and MM cell line supernatant stimulated by IL-6, FGF-2, TNFalpha, BMP7 and TGFbeta was detected by ELISA (ALPCO immunoassays). Increased level of sclerostin was detected in MM patient plasma (n=20, median: 4.73 ng/mL, range: 1.5–19.5 ng/mL). Plasma concentrations were significantly higher (p<0.01) when compared to sclerostin concentration in the plasma of leukemia patients (n=3), gastric cancer patients (n=40) and healthy volunteers (n=4). High sclerostin levels were not associated with extent of bone disease but rather correlated with tumor burden (High B2M, creatinine and LDH, and low Hb) suggesting an autocrine loop for sclerostin production. Because sclerostin is derived from mature OB or orteocytes, we measured levels during OB differentiation but we were unable to detect increased levels. We then measured sclerostin levels in RPMI-8226 MM cell line supernatant either alone or stimulated by cytokines D Systems). Conclusions:These data demonstrate that increased sclerostin levels in MM patients inhibit osteoblastogenesis and stimulate osteoclastogenesis. Taken together, sclerostin may be good target to inhibit myeloma bone disease and help restore normal bone homeostasis. Disclosures:Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.

The Translation Initiation Factor eIF4E Is Overexpressed in Multiple Myeloma and Is Critical for Myeloma Cell Proliferation and Survival

Abstract 1853 Methods:The translation initiation factor eIF4E is central to protein synthesis in general, and overexpression and/or activation of eIF4E is associated with a malignant phenotype by regulating oncogenic protein translation. Several previous publications indicate that aberrant control of protein synthesis contributes to lymphoma genesis but the exact role of protein translation in multiple myeloma (MM) is less clear. Therefore, understanding the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. The goal of this study was to determine the role and regulation of eIF4E, as well as the effects of protein translation controlling drugs in MM. Results:By western blot analysis as well as RT-PCR we found that eIF4E protein and mRNA levels are significantly elevated (up to 20 fold) in MM cell lines (H929, RPMI-8226, MM.1S and OPM2) and primary myeloma cells compared to normal plasma cells. Silencing of eIF4E gene expression in RPMI-8226 MM cells by a stable and inducible shRNA system significantly decreased viability of myeloma cells (by ∼ 43%) but not of HEK 293 suggesting a higher dependency of MM cells to protein translation. Next we evaluated different drugs including pomalidomide, rapamycin, pp242, 4EGI-1 and ribavirin, that are known to inhibit protein synthesis for their effects on protein translation in MM. By m7GTP pull down assays we evaluated the effects of the different drugs on eIF4E expression and activity. Rapamycin blocked the phosphorylation of 4EBP1 and eIF4E release, and subsequently inhibited eIF4G binding. The compound 4EGI-1 decreased the interaction between eIF4E and eIF4G. Pomalidomide decreased eIF4E protein expression. All drugs inhibited MM cell DNA synthesis measured by 3H-Thymidine incorporation. Treatment with pomalidomide (10uM), rapamycin (40nM), pp242 (10uM), 4EGI1 (50uM) or ribavirin (50uM) for 48h significantly decreased (p<0.05) proliferation by 43–62% indicating that drugs controlling protein translation inhibit MM growth. We also found that all drugs decreased expression of eIF4E dependent targets such as cyclin D1 and c-myc. Conclusion:Here we show that eIF4E, a key player in translational control, is highly expressed in MM cells and critical for MM growth and survival. Therefore our study helps to understand the function and regulatory mechanism of eIF4E in MM. Further the evaluation of drugs targeting protein translation provides the basis for the optimization of current MM treatment or to open up new strategies such as targeting protein translation in future MM therapy. Disclosures:Lentzsch:Celgene Corp: Consultancy, Research Funding; Onyx: Consultancy; Genzyme: Consultancy; prIME Oncology: Honoraria; Imedex: Honoraria; Clinical Care Options: Honoraria.

TRAF6 Dominant Negative Peptides Block Intracellular Signaling Resulting in Anti-Myeloma and Anti-Bone Resorptive Effects in Multiple Myeloma

Abstract 2919Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in regulating the NF-kB and JNK signal transduction pathways; and, thus, is likely to promote tumor cell proliferation and osteoclast formation. We have previously reported inhibition of cell proliferation and increase of apoptosis in multiple myeloma (MM) cells through regulation of these intracellular pathways through silencing of TRAF6 C-domain mRNA. To determine TRAF6 protein expression in fresh MM tumor cells, we performed an immunofluorescence assay (IFA). The results showed that expression of this factor in tumor cells from bone marrow (BM) from MM patients with progressive disease is higher than in cells from patients with monoclonal gammopathies without disease progression or normal controls. We further examined the effects of TRAF6 negative dominant peptides on intracellular signaling pathways. Briefly, cells from the RPMI8226 or MM1s MM cell lines or primary MM BM samples were treated with or without TRAF6 inhibition peptide for 24 hours and then stimulated with either IGF1 (30ng/ml) or IL1 β (20ng/ml) for 30 minutes. The cells were lysed and Western blot analysis performed to determine protein phosphorylation and RT-PCR for gene expression. TRAF6 has been found to be an E3 ligase for Akt ubiquitination. We found that IGF1 increased the phosphorylation of AKT and treatment with TRAF6 inhibition peptide markedly decreased its phosphorylation compared to treatment with a control peptide in RPMI8226 and primary MM tumor cells. Downstream of AKT, C-Raf phosphorylation was also significantly reduced with treatment with TRAF6 inhibition peptide. Notably, cyclin D gene expression in MM tumor cells treated with TRAF6 inhibition peptide was reduced as determined with an RT-PCR. In contrast, the gene expression of mTOR was increased in RPMI8226 cells treated with TRAF6 inhibition peptide whereas there was no change in its expression in MM1s and primary MM tumor cells. It is quite possible that the increase in mTOR expression in RPMI8226 cells may act as a negative feedback which results from blockage of the ubiquitination of TRAF6. We further examined the effect of the TRAF6 inhibition peptide on NF-kB and JNK signaling as determined through evaluation of JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. Phospho-NF-kB protein was reduced and phosphorylation of JNKK was clearly decreased with exposure to the TRAF6 inhibition peptide. We examined c-Jun, a component of the transcription factor complex AP-1, which binds and activates transcription at TRE/AP-1 elements. Total endogenous c-Jun is reduced following exposure of RPMI8226 cells to the TRAF6 inhibition peptide. Consistent with our past findings, TRAF6 inhibition peptide significantly inhibited osteoclast formation from CD14+ induced by RANKL and M-CSF with in a concentration dependent fashion whereas control peptides showed no effects on osteoclast formation. In addition, inhibition of the TRAF6 signaling blocked not only myeloma cell proliferation induced by AKT and NF-kB activation but also osteoclast cell formation mediated through transcription at TRE/AP-1 elements. The study has been extended to our SCID-hu murine model of human myeloma. Disclosures:No relevant conflicts of interest to declare.

Monitoring a Nuclear Factor-κB Signature of Drug Resistance in Multiple Myeloma

The emergence of acquired drug resistance results from multiple compensatory mechanisms acting to prevent cell death. Simultaneous monitoring of proteins involved in drug resistance is a major challenge for both elucidation of the underlying biology and development of candidate biomarkers for assessment of personalized cancer therapy. Here, we have utilized an integrated analytical platform based on SDS-PAGE protein fractionation prior to liquid chromatography coupled to multiple reaction monitoring mass spectrometry, a versatile and powerful tool for targeted quantification of proteins in complex matrices, to evaluate a well-characterized model system of melphalan resistance in multiple myeloma (MM). Quantitative assays were developed to measure protein expression related to signaling events and biological processes relevant to melphalan resistance in multiple myeloma, specifically: nuclear factor-κB subunits, members of the Bcl-2 family of apoptosis-regulating proteins, and Fanconi Anemia DNA repair components. SDS-PAGE protein fractionation prior to liquid chromatography coupled to multiple reaction monitoring methods were developed for quantification of these selected target proteins in amounts of material compatible with direct translation to clinical specimens (i.e. less than 50,000 cells). As proof of principle, both relative and absolute quantification were performed on cell line models of MM to compare protein expression before and after drug treatment in naïve cells and in drug resistant cells; these liquid chromatography-multiple reaction monitoring results are compared with existing literature and Western blots. The initial stage of a systems biology platform for examining drug resistance in MM has been implemented in cell line models and has been translated to MM cells isolated from a patient. The ultimate application of this platform could assist in clinical decision-making for individualized patient treatment. Although these specific assays have been developed to monitor MM, these techniques are expected to have broad applicability in cancer and other types of disease.

Triptolide-induced apoptosis by inactivating nuclear factor-kappa B apoptotic pathway in multiple myeloma in vitro

The effect of triptolide on proliferation and apoptosis of human multiple myeloma RPMI-8226 cells in vitro, as well as the roles of nuclear factor-kappa B (NF-κB) and IκBα was investigated. The effect of tritptolide on the growth of RPMI-8226 cells was studied by MTT assay. Apoptosis was detected by Hoechest 33258 staining and Annexin V/PI double staining assay. The expression of NF-κB and IκBα was observed by Western blot and confocal microscopy. The results showed that triptolide inactivated NF-κB apoptotic pathway in human multiple myeloma RPMI-8226 cells. Triptolide at nM range induced proliferation inhibition in a dose- and time-dependent manner and apoptosis in a dose-dependent fashion in RPMI-8226 cells. Besides, we observed the inhibition of NF-κB /p65 in the nuclear fraction was correlated with the increase in the protein expression of IκBα in the cytosol. These results suggested that triptolide might exhibit its strong anti-tumor effects via inactivation of NF-κB/p65 and IκBα.

Inhibition of IGF-1 Signalling Enhances the Apoptotic Effect of AS602868, an IKK2 Inhibitor, in Multiple Myeloma Cell Lines

Multiple myeloma (MM) is a B cell neoplasm characterized by bone marrow infiltration with malignant plasma cells. IGF-1 signalling has been explored as a therapeutic target in this disease. We analyzed the effect of the IKK2 inhibitor AS602868, in combination with a monoclonal antibody targeting IGF-1 receptor (anti-IGF-1R) in human MM cell lines. We found that anti-IGF-1R potentiated the apoptotic effect of AS602868 in LP1 and RPMI8226 MM cell lines which express high levels of IGF-1R. Anti-IGF-1R enhanced the inhibitory effect of AS602868 on NF-κB pathway signalling and potentiated the disruption of mitochondrial membrane potential caused by AS602868. These results support the role of IGF-1 signalling in MM and suggest that inhibition of this pathway could sensitize MM cells to NF-κB inhibitors.

Effect of cobalt‐60 (γ radiation) on multidrug‐resistant multiple myeloma cell lines

Emergence of resistance to chemotherapy and radiotherapy is a major obstacle for the successful treatment of MM (multiple myeloma). Prednisone, vincristine and melphalan are commonly used chemotherapeutic agents for the treatment of MM. In the current study, we examined the presence of possible cross-resistance between these drugs and gamma (γ) radiation. Prednisone, vincristine and melphalan resistant RPMI-8226 and U-266 MM cells were generated by stepwise increasing concentrations of the drugs. The sensitive and resistant cells were exposed to 200- and 800 cGy γ radiation, and proliferation was examined by XTT {2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide} assay. The results showed that Prednisone- and melphalan-resistant RPMI-8226 cells were also cross-resistant to 200 and 800 cGy γ radiation application, while vincristine-resistant cells did not show resistance. On the other hand, Prednisone-, vincristine- and melphalan-resistant U-266 cells showed cross-resistance to 200- and 800 cGy γ radiation application. These results demonstrated that MM cells resistant to anticancer agents respond to radiation in different levels. These findings may be important in the clinical applications of radiation therapy in the treatment of vincristine resistant MM.

Erufosine, an alkylphosphocholine, with differential toxicity to human cancer cells and bone marrow cells

To investigate the activity and myeloprotective properties of erufosine, a novel alkylphosphocholine (APC), on human malignant cells and normal bone marrow cells. Human or mouse bone marrow cells were exposed to erufosine, miltefosine, perifosine, or edelfosine in CFU-GM assays. Human MDA-MB-231 breast carcinoma, Panc-1 pancreatic carcinoma, and RPMI8226 multiple myeloma cells were exposed to erufosine in colony formation assays. Colony formation of Panc-1 tumor cells and mouse bone marrow cells ex vivo were quantified following intravenous administration of erufosine to tumor-bearing mice. Western blotting methods were applied to human U87 glioblastoma cells exposed to erufosine to investigate Akt inhibition. Erufosine was less toxic to human and mouse bone marrow cells than perifosine, miltefosine, and edelfosine and was equally toxic to human and mouse CFU-GM. The human cancer cells MDA-MB-231 breast, Panc-1 pancreatic, and RPMI8226 MM cells were more sensitive to erufosine in a colony formation assay than were human bone marrow cells generating an approximately tenfold differential in IC(90) values. Erufosine injected intravenously significantly reduced Panc-1 tumor cell colony formation ex vivo but not mouse bone marrow CFU-GM. Erufosine inhibited Akt phosphorylation in human U87 glioblastoma cells. Erufosine offers potential as a novel therapeutic for cancer with a reduced toxicity profile to bone marrow cells compared with other agents in this class. Human cancer cells were more sensitive to erufosine than human or mouse bone marrow cells indicating a favorable therapeutic window for erufosine.

Isopentenyl Pyrophosphate (IPP), a Metabolite produced in Myeloma Cells Induces the Chemotaxis of γδT Cells

AbstractAbstract 2766γδT cells, which control the innate immune system, are classified into three subtypes on the basis of Vγ chain. Of these subtypes, Vγ2Vδ9 T cells display anti-tumor immunity. We have demonstrated that nitrogen-containing bisphosphonate (N-BP) treatment expands Vγ2Vδ9 T cells ex vivo and that these expanded cells can kill tumor cells in a major histocompatibility complex-unrestricted manner (Sato, Int J Cancer, 2005; Uchida, Biochem Biophys Res Commun, 2007; Sato, Cancer Immunol Immunother, 2008.). N-BP inhibits farnesyl pyrophosphate synthase in the mevalonate pathway, resulting in the accumulation of isopentenyl pyrophosphate (IPP), which is a stimulatory antigen for Vγ2Vδ9T cells. In the present study, we investigated the chemotactic factors for Vγ2Vδ9T cells by using a micro total analysis system-based microfluidic cellular analysis device (Kanai, Sens Actuators A, 2004; Munaka, Analyst, 2007.). This microchip possesses a minute-volume (240 nL) chamber integrated with a micro-sample injector that permits the injection of a small amount (several nL) of a solute (Figure 1). Because of the minute size of this chamber, a concentration gradient can be maintained free from the influence of fluid convection and stirring, and the solute can consequently spread in a diffusion-dependent manner. Therefore, administration of a humoral factor via the sample injector mimics its release from the cell surface. We first investigated whether the supernatant of RPMI8226 multiple myeloma (MM) cells treated with zoledronic acid (ZOL) induced chemotaxis of γδT cells. We treated RPMI8226 MM cells with ZOL (1 mM) overnight and collected the supernatant. Human γδT cells were obtained by the culture of peripheral blood mononuclear cells as previously reported (Uchida, Biochem Biophys Res Commun, 2007.), and these cells were cultured in the microchip. After the injection of supernatant, γδT cells migration was observed under a microscope and continuous time-lapse recording was performed for 30 min. γδT cells migrated toward the injector, indicating that the supernatant of ZOL-treated RPMI8226 cells includes a chemoattractant factor for γδT cells. We next applied soluble MICA (sMICA), sICAM-1, sVCAM-1, and IPP and examined the migration of γδT cells. Among them, sMICA and IPP were chemoattractive for γδT cells, and the velocity of γδT cell migration was increased by the injection of IPP compared to the solvent control (Figure 2). These observations indicate that IPP, a metabolite of the mevalonate pathway in MM cells, or sMICA is a chemotactic factor for γδT cells when the target MM ells are treated with ZOL. [Display omitted] [Display omitted] Disclosures:Munaka:Shimadzu Corporation: Employment. Kanai:Shimadzu Corporation: Employment. Abe:Shimadzu Corporation: Employment.

A Novel SIRT1 Activator SIRT1720 Triggers In Vitro and In Vivo Cytotoxicity In Multiple Myeloma Via ATM-Dependent Mechanism

AbstractAbstract 3007 Background and Rationale:SIRT1 belongs to the silent information regulator 2 (Sir2) family of proteins and functions as NAD+-dependent deacetylase. Previous studies showed that resveratrol, a polyphenolic SIRT1 activator, inhibits tumorigenesis in various solid tumor and hematologic malignancies, including human multiple myeloma (MM) cells. This notion led to the discovery and development of more potent and selective pharmacological activators of SIRT1 as potential anti-cancer therapeutics. In this context, a recent medicinal chemistry research using high-throughput screening, and mass spectrometry identified SRT1720, a small molecule activator of SIRT1 that is structurally distinct from resveratrol. Here, we examined the anti-tumor activity of SRT1720 in MM cells using in vitro and in vivo model systems. Methods and Model:We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12BM, H929, and INA-6 (an IL-6 dependent) human MM cell lines, as well as purified tumor cells from patients with MM relapsing after prior therapies including lenalidomide or bortezomib. Cell viability, proliferation, and apoptosis assays were performed using trypan blue, MTT, thymidine incorporation, and Annexin V staining. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Results:We first confirmed the functional specificity of SRT1720 against SIRT1 using different experimental strategies. First, we utilized Fluor de Lys Deacetylase Assay to measure whether SRT1720 affects the SIRT1 deacetylase enzymatic activity. Treatment of MM.1R and RPMI-8226 MM cells with SRT1720 markedly increased the deacetylating activity; conversely, pre-treatment of cells with nicotinamide (NAM) - an inhibitor of SIRT1 – significantly blocked SRT1720-triggered deacetylating activity. Second, immunoblot analysis using antibodies specific against acetylated p53 (a known substrate of SIRT1) showed a marked decrease in acetylated state of p53 in SRT1720-treated MM cells. These findings in MM cells confirm SIRT1 as a selective target of SRT1720. We next examined the efficacyof SRT1720 in MM cells. Treatment of MM cell lines and primary patient cells for 24h significantly decreased their viability (IC50 range 3–7 uM) (P < 0.005; n=3) without markedly affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index for SRT1720. SRT1720-triggered apoptosis was confirmed in MM.1R and RPMI-8226 cells, evidenced by a marked increase in Annexin V+ and PI- cell population (P < 0.001, n=3). Importantly, SRT1720 induced apoptosis in MM cells even in the presence of bone marrow stromal cells. Mechanistic studies showed that SRT1720-triggered apoptosis in MM cells is associated with 1) activation of caspase-8, caspase-9, caspase-3, and PARP; 2) activation of pATM, CHK2, endoplasmic reticulum stress molecules pEIF2, and BIP; as well as an increase in reactive oxygen species (ROS); 3) inhibition of MM cell growth and survival pathway via NF-kB; and 4) inhibition of VEGF-induced migration of MM cells and associated angiogenesis. Importantly, blockade of pATM using a biochemical inhibitor KU-5593 significantly attenuated SRT1720-induced MM cell death (P value < 0.002; n=2). These data suggest that SRT1720-induced MM cell apoptosis is predominantly mediated by an ATM-dependent apoptotic pathway. We next examined the in vivo efficacy of SRT1720 using a human plasmacytoma xenograft mouse model. Treatment of tumor-bearing mice with SRT1720 (200 mg/kg, 5 days a week for three weeks), but not vehicle alone, significantly (P < 0.008) inhibits MM tumor growth in these mice. Finally, the combination of SRT1720 with bortezomib or dexamethasone triggered synergistic anti-MM activity. Conclusions:These preclinical studies provide the rationale for novel therapeutics targeting SIRT1 to improve patient outcome in MM. Disclosures:Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Lenalidomide Enhances Multiple Myeloma Cytotoxicity Induced by a Novel Fc Domain-Engineered Anti-HM1.24 Monoclonal Antibody with Augmented NK Cell Degranulation

AbstractAbstract 4064HM1.24, an immunological target highly expressed on majority of multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). Recently, XmAb5592, a novel Fc-domain engineered humanized anti-HM1.24 mAb with specific Fc-domain modification, was shown to induce >10-fold antibody-dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis against MM cells, when compared with the humanized normal anti-HM1.24 IgG1 (XmAb5627) from which it is derived (ASH Abstract#609, 2009). Here we investigated whether XmAb5592, when combined with other anti-MM drugs, further enhanced ADCC against MM cell lines and primary patient MM cells using Calcein-AM release ADCC assays and flow cytometric analysis for cell membrane CD107a to specifically quantitate NK cell (CD56+CD3-) activation. Addition of lenalidomide (10 μ M) in standard ADCC assays increased XmAb5592 (0.1 μ g/ml)-induced cell lysis against MM1S, MM1R, and RPMI8226 cells in the presence of peripheral blood mononuclear cells (PBMCs) from normal donors (n=2). Specifically, XmAb5592 (0.01, and 0.1 μ g/ml) combined with lenalidomide (2.5, 5, 10 μ M), in the presence or absence of IL-2 (100 units/ml), synergistically induced NK-mediated RPMI8226 MM cell lysis, as evidenced by combination index (CI) < 1 (0.08-0.89). Pre-incubation of PBMCs with additional IL-2 (100 units/ml) enhanced even stronger XmAb5592-induced cytotoxicity against MM cells than pretreatment with lenalidomide alone. Lenalidomide also enhanced PBMC effectors to kill primary patient MM cells. Cell surface CD107a, as a functional marker for NK cell activation dependent on granzyme B secretion, was further determined following target myeloma cell incubation, with or without NK cells and in the presence of mAbs. NK cells were activated by as low as 0.001 μ g/ml of XmAb5592 only in the presence of MM1S tumor cells. In contrast, neither XmAb6166, an Fc-domain knockout of XmAb5592 without NK binding, nor XmAb4614, a similar Fc-engineered mAb targeting respiratory syncytial virus (RSV) antigen, induced any cell surface CD107a on NK cells incubated with MM cells. Specific XmAb5592-induced CD107a-dependent NK cell degranulation further correlated with IFNγ secretion (r=0.7, p=0.03). XmAb5592 induced > 10-fold more potent NK degranulation than XmAb5627, which significantly correlated with MM cell lysis by ADCC, even in the presence of bone marrow stromal cells (BMSCs). Moreover, no significant HM1.24 was expressed on NK cells stimulated with or without IFN-α, suggesting minimal NK toxicity. XmAb5592 more potently (>10-fold) than XmAb5627 induces homotypic aggregation and adhesion of NK cells, which was further enhanced by lenalidomide. Finally, minimal HM1.24 expression was confirmed on different PBMC subsets including CD15+ (PMN), CD19+ (B), CD14+ (MC), and CD3+ (T) cells. These results indicate that lenalidomide further potentiates XmAb5592-induced myeloma cell killing via NK-mediated ADCC, providing a rationale to combine both novel drugs to improve patient outcome in MM. Disclosures:Muchhal:Xencor Inc: Employment. Desjarlais:Xencor Inc: Employment. Richardson:Gentium: Membership on an entity's Board of Directors or advisory committees, Research Funding. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Multiple Myeloma (MM) Highly Expresses CGEN-928 and the Anti-CGEN-928 TM21 Antibody Markedly Inhibits the Growth of MM Cells and Enhances the Anti-Myeloma Effects of Dexamethasone, Melphalan and Bortezomib

AbstractAbstract 4067Although patients with multiple myeloma (MM) initially respond to current treatment modalities, it remains an incurable disease. Many new therapeutic options have become available during the past several years but nearly all patients develop resistance to currently available therapeutic options. In addition, there is no tumor marker that is uniformly expressed in all MM cells although CD138 is considered to be present on the surface of tumor cells in most cases of MM but generally is only present in a subset of the patients' tumor population and may be absent in the most resistant part of the tumor clone. In order to address these unmet clinical needs, we queried Compugen's MED Platform, an expression database which covers over 40,000 microarray experiments, for genes overexpressed in B cell-derived malignancies including MM and that exhibit low expression levels in normal cells and tissues. One of the most prominent candidates was CGEN-928, which was validated as over-expressed in MM at the mRNA level using an independent panel of both hematological malignancies and normal tissues. In this study, we first investigated whether the previously unidentified membrane antigen CGEN-928 is expressed in cells from human MM cell lines, human MM xenografts and fresh bone marrow (BM) aspirates derived from MM patients using flow cytometric analysis and immunohistochemical staining with the anti-CGEN-928 TM21 polyclonal antibody (Compugen Ltd, Tel Aviv, Israel). Using this antibody, we found that CGEN-928 was highly expressed in cells from the MM1s, U266 and RPMI8226 MM cell lines. Next, we examined CGEN-928 antigen expression in fresh tumor cells from BM aspirates from 17 MM patients and also showed high expression of CGEN-928. Notably, expression of this antigen was not only found on CD138+ MM cells but also on MM tumor cells lacking CD138 expression. We also examined the expression of CGEN-928 using our human MM xenograft models LAGκ-1A (bortezomib-sensitive), LAGκ-1B (bortezomib-resistant) and LAGλ-1 (melphalan-resistant). The bortezomib-sensitive MM tumor LAGκ-1A expresses CD138 whereas the bortezomib-resistant version LAGκ-1B developed from the same patient after the patient developed bortezomib reisistance does not express CD138. Cells from all three tumor types showed high levels of reactivity with the TM21 antibody. Similar to the fresh MM BM samples, CGEN-928 expression was not only found on CD138+ MM cells but also on CD138- tumor cells derived from these human MM xenografts. Because this molecule is highly expressed on MM cells, we hypothesized that the anti-CGEN-928 antibody may show anti-MM effects and enhance the anti-MM effects of other anti-MM drugs. To evaluate this, we examined the effect of the TM21 antibody alone and in combination with dexamethasone, melphalan and bortezomib in vitro using cell proliferation MTT assays. Anti-TM21 polyclonal antibody (100 mg/ml) decreased MM tumor cell proliferation and increased apoptosis in cells from the MM1s, RPMI8226 and U266 cell lines. Next, we determined the effects of combining the anti-CGEN-928 antibody with bortezomib, melphalan or dexamethasone on MM1s cells. Cell proliferation assays demonstrated marked enhanced anti-proliferative effects when CGEN-928 antibody at concentrations of 5, 10, 50 and 100 mg/ml was combined with bortezomib, melphalan or dexamethasone. Further investigations are defining the mechanisms and signal transduction pathways that produce the anti-MM effects of CGEN-928. These preliminary studies suggest that the CGEN-928 antigen is highly expressed in MM and treatment with an anti-CGEN-928 polyclonal antibody produces anti-MM effects alone and in combination with other anti-MM agents; and thus, this antigen may be a target for the treatment of multiple myeloma. Currently, a monoclonal anti-CGEN-928 antibody is in development that will be used by our group to evaluate its anti-MM effects both in vitro and in vivo using our SCID-hu models of human MM. Disclosures:Levy:Compugen Ltd.: Employment. Dassa:Compugen Ltd.: Employment. Cojocaru:Compugen Ltd.: Employment. Berenson:Compugen Ltd.: Research Funding. Levine:Compugen Ltd.: Employment.

Anti-Myeloma Activity of Enzymatically Activated Melphalan Prodrug J1

AbstractAbstract 1838 Background and Rationale:The alkylating drug melphalan is routinely used in clinical protocols for the treatment of multiple myeloma (MM). Importantly, clinical trials in MM have effectively utilized combination of melphalan with proteasome inhibitor bortezomib and prednisolone (VMP regimen) to reduce toxicity, overcome drug resistance and enhance cytotoxicity. These findings highlight the utility of conventional alkylating agent, and importantly, provide impetus to develop conventional agents based prodrugs with a potent cytotoxic activity. In this context, pharmacological screening of alkylating oligopeptides led to the identification of a novel melphalan-containing prodrug J1 (L-melphalanyl-p-L-fluoro phenylalanine ethyl ester) - a new molecular entity with a more distinct activity profile than melphalan (Gullbo J, et al., Anticancer Drugs 2003,14:617–24; Gullbo J, et al., Invest New Drugs 2004, 22:411–20; Wickstrom M, et al., Mol Cancer Ther 2007, 6:2409–17). J1 is rapidly incorporated into the tumor cells cytoplasm, followed by intracellular hydrolysis in part mediated by aminopeptidase N (APN), resulting in a 10-fold greater release of free intracellular melphalan than exposure to melphalan at the same molar concentration (Gullbo J, et al., J Drug Target 2003,11:355–63; Wickstrom et al., Biochem Pharmacol 2010, 79(9):1281-90). In vitro studies showed a greater cytotoxic potency of J1 versus melphalan against different human solid cancers; however, its effect in MM is undefined. In the present study, we examined the anti-tumor activity of J1 in MM cells using both in vitro and in vivo model systems. Methods and Models:We utilized MM.1S, MM.1R, RPMI-8226, melphalan-resistant derivative of RPMI-8226 (LR-5), KMS12BM, and INA-6 (an IL-6 dependent) human MM cell lines, as well as purified tumor cells from patients with MM relapsing after prior therapies including lenalidomide or bortezomib. Cell viability-, proliferation-, and apoptosis assays were performed using Trypan blue, MTT, thymidine incorporation, and Annexin V/Propidium iodide staining. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymologic assays. Statistical significance of data was determined using Student t test. Results:As aminopeptidase N (APN) has been shown to play a key role in conversion of J1 into melphalan in solid tumors, we first examined both expression and enzymatic activity of APN in MM cells. Immunoblot analysis showed a high expression of APN in various MM cell lines. Similarly, colorimetric analysis of APN enzymatic activity using the APN substrate L-alanine-4-nitro-anilide demonstrated elevated APN activity in MM cells. Moreover, pre-treatment of MM cells with APN inhibitor Bestatin showed a moderate, but significant blockade of J1-induced cytotoxicity in MM cells (P < 0.05; n=3). We next examined the effects of J1 in MM cells. Treatment of MM cell lines and primary patient cells for 24h significantly decreased their viability (IC50 range 0.5 – 1.0 uM; P < 0.001; n=3) without markedly affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index forJ1. Of note, the IC50 range of melphalan for MM cell lines is 10–40 uM. J1-triggered apoptosis was confirmed in MM.1R and RPMI-8226 cells, evidenced by marked increase in Annexin V+ and PI-cell population (P < 0.001, n=3). Importantly, J1induced apoptosis in MM cells even in the presence of MM bone marrow stromal cells. Mechanistic studies showed that J1-triggered apoptosis in MM cells is associated with 1) activation of caspase-7, caspase-8, caspase-9, caspase-3, and PARP; 2) induction of phospho-c-Jun and phospho-JNK, p53, and p21; 3) release of mitochondrial apoptogenic protein cytochrome-c; 4) inhibition of VEGF-induced migration of MM cells and angiogenesis; and 5) induction of DNA damage response, evidenced by increase in phospho-histone H2AX. Pre-treatment of MM cells with pan-caspase inhibitor Z-VAD-fmk attenuated J1-triggered MM cell apoptosis (P value < 0.001; n=3). Finally, treatment of tumor-bearing mice with J1 (3 mg/kg, twice a week for 2 weeks), but not vehicle alone, significantly (P < 0.008) inhibits MM tumor growth in these mice. Conclusions:Our study provides the rationale for clinical protocols evaluating J1, either alone or in combination, to improve patient outcome in MM. Disclosures:Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Spira:Oncopeptide AB: Employment, Equity Ownership. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Artesunate inhibiting angiogenesis induced by human myeloma RPMI8226 cells

Multiple myeloma (MM) remains an incurable plasma cell disorder to date; therefore, new biologically target-based therapies are in urgent demand. Our previous studies showed that the antimalarial artesunate (ART) possessed anti-myeloma effect by inhibiting proliferation and inducing apoptosis of myeloma cells. The present study evaluated the effect of ART on human myeloma cell-induced angiogenesis and elucidated its mechanism. The human umbilical vein endothelial cells (HUVECs) migration test, aortic sprouting in fibrin gel in vitro and chicken chorioallantoic membrane (CAM) neovascularization in vivo model were used to examine the effect of ART on angiogenesis induced by human myeloma cells. The results showed that ART could inhibit HUVECs migration, even at a lower concentration (3μmol/l, P<0.01, compared with the result of control group), and suppress efficiently the angiogenic ability of myeloma RPMI8226 cells in a dose-dependent pattern (3-12μmol/l, P<0.05). The levels of VEGF and Ang-1 in the conditioned medium (CM) were quantified by enzyme-linked immunosorbent assay (ELISA). The results confirmed that 3μmol/l ART could significantly decrease VEGF and Ang-1 secretion by RPMI8226 cells (P<0.05), which correlated well with the reduction of angiogenesis induced by myeloma RPMI8226 cells. The present study also showed that ART downregulated the expression of VEGF and Ang-1 in RPMI8226 cells and reduced the activation of extracellular signal-regulated kinase 1 (ERK1) as well. Therefore, ART can block ERK1/2 activation, downregulate VEGF and Ang-1 expression and inhibit angiogenesis induced by human multiple myeloma RPMI8226 cells. Combined with our previous published data, results from the present study indicate that ART possesses potential anti-myeloma effect.

Overexpression of PAX5 induces apoptosis in multiple myeloma cells

PAX5 is an essential transcription factor for the commitment of lymphoid progenitors to the B-lymphocyte lineage. PAX5 suppression results in retrodifferentiation of B lymphocytes to an uncommitted progenitor cell stage, whereas PAX5 suppression in mature B lymphocytes leads to further development into plasma cells. Here, we have analyzed the fate of plasma cell lines following PAX5 reexpression. Human B cell lines were infected with Ad5/F35 adenoviruses encoding either EYFP or PAX5. Expression analysis of specific plasma cell transcription factors (IRF4, Blimp-1 and XBP-1) suggests that PAX5 reexpression does not induce retrodifferentiation of plasma cells into B lymphocytes. Interestingly, the viability of RPMI-8226 and U266 multiple myeloma cell lines markedly declined at 4-7 days post-transduction, whereas other plasma cell lines maintained their viability. Apoptosis analysis through Annexin V measurement also revealed a higher level of apoptosis in PAX5-expressing myeloma cell lines. Finally, Western blot analysis of pro- and anti-apoptotic proteins revealed that the anti-apoptotic protein MCL-1 was down-modulated in PAX5-transduced multiple myeloma cell lines. In conclusion, our results show that the expression of PAX5 in plasma cell lines induces apoptosis exclusively in multiple myelomas. This might represent a potential therapeutic avenue in the treatment of multiple myeloma.

BSc2118 is a novel proteasome inhibitor with activity against multiple myeloma

The ubiquitin-proteasome system emerged as a new therapeutic target in cancer treatment. The purpose of this study was to elucidate the effects of the novel proteasome inhibitor BSc2118 on t(4;14) positive and negative multiple myeloma (MM) cells and normal peripheral blood mononuclear cells (PBMNC). Human MM cell lines OPM-2, RPMI-8226, and U266 and primary MM cells from bone marrow aspirates were exposed to BSc2118. Cytotoxicity levels were evaluated using the MTT-test. BSc2118-induced apoptosis was analyzed by annexin-V assay. Further methods used included proteasomal activity determination, cell cycle analysis, western blot, and transcription factor assays. In OPM-2, RPMI-8226, U266 cell lines and primary MM cells, BSc2118 caused dose-dependent growth inhibitory effects. After 48 h, dose-dependent apoptosis occurred both in cell lines and primary myeloma cells irrespective of t(4;14). A significant G2-M cell cycle arrest occurred after 24 h. Furthermore, we observed a marked inhibition of intracellular proteasome activity, an increase in intracellular p21 levels, and an inhibition of NF-kappaB activation. The toxicity against PBMNC remained low, suggesting a broad therapeutic range of this agent. Taken together, BSc2118 shows significant antimyeloma activity and may be considered as a promising agent in cancer drug development.

Triptolide inhibits cell growth and induces G0- G1 arrest by regulating P21wap1/cip1 and P27 kip1 in human multiple myeloma RPMI-8226 cells

ObjectiveTo investigate the effects of triptolide(TPL) on cell growth, cell cycle and the expressions of p21wap1/cip1 and p27kip1.MethodsMTT assay was used to determine the cell viability after triptolide treatment in human multiple myeloma RPMI-8226 cells. The effect on cell cycle distribution was determined by flow cytometry. Semi-quantitative reverse transcription-PCR was used to examine the mRNA expressions of p21wap1/cip1 and p27kip1. The protein expressions of p21 wap1/cip1 and p27kip1 were determined by Western blot.ResultsTriptolide of varying concentrations induced cell viability inhibition in dose- and time-related fashion and caused G0-G1 phase arrest of cell cycle progression in RPMI-8226 cells. These effects accompanied with up-modulation of the expressions of p21 wap1/cip1 and p27kip1.ConclusionThese results suggest that triptolide inhibit cell proliferation and cell cycle progression via up-regulating p21wap1/cip1 and p27kip1 and triptolide may exert its anti-cancer activity through this pathway.

Abstract 1569: Antitumor activity of SR16388 against human multiple myeloma in vitro and in vivo

Abstract Multiple myeloma (MM) is the cancer of plasma cells that derive from a clonal disorder of terminally differentiated B cells in the bone marrow. MM is generally thought to be refractory against chemotherapeutics and is incurable. Development of novel agents for the treatment of this disease is urgently needed. Previous studies have indicated that MM cells express estrogen receptors (ERs), and anti-estrogens such as ICI 182,780 and Tamoxifen have demonstrated inhibitory effects on proliferation of MM cell lines as well as on MM cells isolated from patients. MM cells also over-express proangiogenic factors such as VEGF to induce angiogenesis and support their growth and survival. At SRI International, we have developed a steroidal anti-angiogenic molecule (SR16388) that potently inhibits angiogenesis in lung and prostate cancers, and it also binds to ER-α and ER-ß with high affinities in the nanomolar range. In this study, we examined the in vitro and in vivo effects of SR16388 on growth of MM cells. In RPMI-8226 multiple myeloma cells, SR16388 inhibited cell proliferation with an IC50 value of 0.9 µM. Cell cycle analysis revealed that SR16388 arrested the RPMI-8226 cells in G2 phase. In an in vivo murine xenograft model, SR16388 significantly inhibited the growth of RPMI-8226 tumors at the dose levels of 50 and 100 mg/kg with P &amp;lt; 0.05 and P &amp;lt; 0.02 respectively in a dose dependent manner. We are currently examining the molecular effects of SR16388 that contribute to its anti-proliferative and anti-angiogenic effects on MM and other human tumor types. Based on our in vitro and in vivo data, we believe that SR16388 is a promising new therapeutic molecule for the treatment of MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1569.

Abstract A27: Chronic exposure of malignant hematological cell lines to bortezomib induces de novo hot spot mutations in the PSMB5 gene

Abstract The proteasome inhibitor Bortezomib (BTZ, Velcade®) specifically inhibits the catalytic beta 5 subunit of the proteasome. The introduction of BTZ has shown promising results in the treatment of Multiple Myeloma (MM), Non-Hodgkin lymphoma and leukemia. Despite these encouraging results, clinical trials in MM also revealed that a significant proportion of patients acquired resistance to BTZ mono-therapy. We have developed a BTZ-resistant cell line model by chronic exposure to stepwise increasing concentrations of BTZ, including the AML THP-1 cell-line (Oerlemans &amp; Franke et al, Blood 2008), the T-ALL CCRF-CEM-C7 cell-line and the MM RPMI-8226 cell line (Franke et al, Blood 2009 vol 114(22), abstr 940). Cells were initially selected for growth at 7 nM BTZ to acquire low levels of BTZ resistance (2–3 fold higher IC50 concentrations) and subsequently challenged to concentrations of BTZ up to 500 nM to provoke higher resistance levels. Abilities to resist a BTZ concentration of 100 nM could be achieved relatively fast in the CEM cell line (within 16 weeks), intermediate in the THP-1 cell line (within 22 weeks) and relatively slowly in the 8226 cell line (within 60 weeks). Subsequent sequencing of the PSMB5 gene, encoding the beta 5 proteasome subunit, revealed a series of mutations in individual BTZ-resistant subclones, all resulting in amino-acid changes residing within the highly conserved BTZ binding pocket. The relatively fast induction of mutations provoked the question whether there might already exist a subclone within the cell line that harbours the mutation. To distinguish between the outgrowth of a pre-existing resistant subclone and the occurrence of de novo mutations, we generated new BTZ resistant CEM and THP-1 cells (CEM-BR2 and THP-1-BR2). Interestingly, the new CEM-BR2 cells had a different nucleotide change (G322A resulting in a Ala49Thr substitution) from the original BTZ resistant CEM-BR1 (C323T and G332T resulting in Ala49Val and Cys52Phe amino acid substitutions, respectively). Of note, this new CEM-BR2 mutation represented the same mutation as seen in the original BTZ resistant THP-1-BR1. In addition, the new THP-1-BR2 cells also showed a different mutation (A309G) in the PSMB5 gene, introducing a Met45Val amino-acid substitution) compared to the original BTZ resistant THP-1 cells that showed a Met45Iso substitution. These results indicate that the mutations are acquired during BTZ exposure and are mainly induced in specific hot-spots (dominantly Ala49) within the PSMB5 gene. To explore whether mutation-induced resistance could be bypassed, a new proteasome inhibitor, 5-amino-8-hydroxyquinole (5AHQ), acting as a non-competitive inhibitor of the non-catalytic alpha-7 subunit of the proteasome (Li et al. ASH 2008) was studied in our model system. Strikingly, all BTZ-resistant selectants retained full sensitivity towards 5AHQ (IC50: 4–7 μM, measured in a 4-day MTT cytotoxicity assay) as compared to parental cells. To determine whether mutation induction also occurs in patients treated with BTZ, screening for their presence in clinical samples of BTZ refractory patients is warranted. The notion that 5AHQ can overcome BTZ resistance related to single and multiple mutations in the PSMB5 gene, supports further research in this drug and its analogues. This study is supported by VUmc - Stichting Translational Research (STR) and The Netherlands Organization for Health Research and Development (ZonMw). Citation Information: Clin Cancer Res 2010;16(7 Suppl):A27

CD99 signal regulates the proliferation of multiple myeloma cell lines, RPMI8226. (131.10)

Abstract CD99 is a 32-kDa cell surface protein that is variously expressed on hematopoietic cells and hematopoietic-derived cancer cells. CD99 is highly expressed in some multiple myeloma cells including RPMI8226. In this study, we show that engagement of CD99 by agonistic anti-CD99 monoclonal antibody (mAb), YG32, inhibited proliferation of RPMI8226 multiple myeloma cells. However, YG32 mAb did not induce the cell death of RPMI 8226 cells. We carried out the microarray analysis to investigate the expression of genes involved in growth retardation by CD99 engagement. In microarray data, RPMI8226 cells engaged by anti-CD99 mAb had reduced expression of cyclin D1 and D2. Expression of CDKN2A gene, which codes cell cycle inhibitors p16INK4A and p19ARF, were induced by anti-CD99 mAb. These results suggest that CD99 signal regulates the proliferation of multiple myeloma cells by modulating proliferative and anti-prolifearative genes. The study was supported by National Research Foundation (NRF) grant of the Korean government A (R13-2008-023-01003)