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1.  Elotuzumab enhances natural killer cell activation and myeloma cell killing through interleukin-2 and TNF-α pathways 
Elotuzumab is a humanized monoclonal antibody specific for signaling lymphocytic activation molecule-F7 (SLAMF7, also known as CS1, CD319, or CRACC) that enhances natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) of SLAMF7-expressing myeloma cells. This study explored the mechanisms underlying enhanced myeloma cell killing with elotuzumab as a single agent and in combination with lenalidomide, to support ongoing phase III trials in patients with relapsed/refractory or newly-diagnosed multiple myeloma (MM). An in vitro peripheral blood lymphocyte (PBL)/myeloma cell co-culture model was developed to evaluate the combination of elotuzumab and lenalidomide. Expression of activation markers and adhesion receptors was evaluated by flow cytometry, cytokine expression by Luminex and ELISPOT assays, and cytotoxicity by myeloma cell counts. Elotuzumab activated NK cells and promoted myeloma cell death in PBL/myeloma cell co-cultures. The combination of elotuzumab plus lenalidomide demonstrated superior anti-myeloma activity on established MM xenografts in vivo and in PBL/myeloma cell co-cultures in vitro than either agent alone. The combination enhanced myeloma cell killing by modulating NK cell function that coincided with the upregulation of adhesion and activation markers, including interleukin (IL)-2Rα expression, IL-2 production by CD3+CD56+ lymphocytes, and tumor necrosis factor (TNF)-α production. In co-culture assays, TNF-α directly increased NK cell activation and myeloma cell death with elotuzumab or elotuzumab plus lenalidomide, and neutralizing TNF-α decreased NK cell activation and myeloma cell death with elotuzumab. These results demonstrate that elotuzumab activates NK cells and induces myeloma cell death via NK cell-mediated ADCC, which is further enhanced when combined with lenalidomide.
doi:10.1007/s00262-014-1610-3
PMCID: PMC4282702  PMID: 25287778
Elotuzumab; Interleukin-2; Lenalidomide; Multiple myeloma; Natural killer cell activation; SLAMF7
2.  Human-like mouse models for testing the efficacy and safety of anti-β2-microglobulin monoclonal antibodies to treat myeloma 
Purpose
We recently demonstrated that anti-β2-microglobulin (β2M) mAbs have remarkably strong apoptotic effects on myeloma cells in vitro and in SCID(-hu) mice. However, whether the mAbs will be therapeutic and safe in the treatment of myeloma patients, in whom every tissues express low densities of MHC class I molecules and elevated levels of soluble β2M are present, remains to be determined.
Experimental Design
In this study, human-like myeloma mouse models (HLA-A2-transgenic NOD/SCID mice) were developed, which express mature and functional human MHC class I (HLA-A2 and human β2M) on murine organs and present high levels of circulating human β2M derived from human myeloma cells. Myeloma-bearing mice were treated intraperitoneally with anti-β2M mAbs, and the distribution and effects of the mAbs on normal organs and established tumors were examined.
Results
Our results show that anti-β2M mAbs were effective in suppressing myeloma growth in treated mice. The therapeutic efficacy of the mAbs in these mice are comparable to those observed in myeloma-bearing nontransgenic NOD/SCID mice in which no human MHC class I is expressed on murine organs. Furthermore, although the mAbs can be detected on different organs, no tissue damage or cell apoptosis was observed in the mice.
Conclusion
Based on the antimyeloma efficacy and low toxicity in the mice, our study suggests that anti-β2M mAbs may be safe and the tissue-expressing and soluble β2M may not compromise their therapeutic effects in myeloma patients. This study provides further support for the future application of the mAbs as therapeutic agents for MM.
doi:10.1158/1078-0432.CCR-08-1823
PMCID: PMC2659684  PMID: 19188166
Multiple myeloma; anti-β2 mAbs; mouse models; HLA-A2-transgenic
3.  Dendritic Cells and Malignant Plasma Cells: An Alliance in Multiple Myeloma Tumor Progression? 
The Oncologist  2011;16(7):1040-1048.
Defective function of dendritic cells is examined as a mechanism for myeloma cell escape in multiple myeloma tumor progression.
Learning Objectives
After completing this course, the reader will be able to: Describe defective immunological features that have been identified in dendritic cells in multiple myeloma and explain how immunologic dendritic cell defects could reduce the clinical efficacy of dendritic cell-based vaccines.Outline possible therapeutic strategies based on current knowledge of the bone marrow crosstalk between myeloma cells and immature dendritic cells.
This article is available for continuing medical education credit at CME.TheOncologist.com
The crosstalk of myeloma cells with accessory cells drives the expansion of malignant plasma cell clones and the hyperactivation of osteoclastogenesis that occurs in multiple myeloma (MM). These reciprocal interactions promote defective dendritic cell (DC) function in terms of antigen processing, clearance of tumor cells, and efficacy of the immune response. Thus, myeloma cells exert immune suppression that explains, at least in part, the failure of therapeutic approaches, including DC vaccination. Impairment of DCs depends on high bone marrow levels of cytokines and adhesion molecules that affect both maturation and expression of costimulatory molecules by DCs. Moreover, DCs share with osteoclasts (OCs) a common ontogenetic derivation from the monocyte lineage, and thus may undergo OC-like transdifferentiation both in vitro and in vivo. Immature DCs (iDCs) induce clonogenic growth of malignant plasma cells while displaying OC-like features, including the ability to resorb bone tissue once cultured with myeloma cells. This OC-like transdifferentiation of iDCs is dependent on the activation of both the receptor activator of nuclear factor κB (RANK)–RANK ligand (RANK-L) and CD47–thrombospondin (TSP)-I axes, although interleukin 17–producing T helper-17 clones within the bone microenvironment may also take part in this function. Therefore, iDCs allied with malignant plasma cells contribute to MM osteoclastogenesis, although other molecules released by tumor cells may independently contribute to the bone-resorbing machinery.
doi:10.1634/theoncologist.2010-0327
PMCID: PMC3228131  PMID: 21659611
Multiple myeloma; Dendritic cells; Bone disease; Immune system
4.  Bortezomib Reduces the Tumorigenicity of Multiple Myeloma via Downregulation of Upregulated Targets in Clonogenic Side Population Cells 
PLoS ONE  2013;8(3):e56954.
Side population (SP) cells in cancers, including multiple myeloma, exhibit tumor-initiating characteristics. In the present study, we isolated SP cells from human myeloma cell lines and primary tumors to detect potential therapeutic targets specifically expressed in SP cells. We found that SP cells from myeloma cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11) express CD138 and that non-SP cells include a CD138-negative population. Serial transplantation of SP and non-SP cells into NOD/Shi-scid IL-2γnul mice revealed that clonogenic myeloma SP cells are highly tumorigenic and possess a capacity for self-renewal. Gene expression analysis showed that SP cells from five MM cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11, JJN3) express genes involved in the cell cycle and mitosis (e.g., CCNB1, CDC25C, CDC2, BIRC5, CENPE, SKA1, AURKB, KIFs, TOP2A, ASPM), polycomb (e.g., EZH2, EPC1) and ubiquitin-proteasome (e.g., UBE2D3, UBE3C, PSMA5) more strongly than do non-SP cells. Moreover, CCNB1, AURKB, EZH2 and PSMA5 were also upregulated in the SPs from eight primary myeloma samples. On that basis, we used an aurora kinase inhibitor (VX-680) and a proteasome inhibitor (bortezomib) with RPMI 8226 and AMO1 cells to determine whether these agents could be used to selectively target the myeloma SP. We found that both these drugs reduced the SP fraction, though bortezomib did so more effectively than VX-680 due to its ability to reduce levels of both phospho-histone H3 (p-hist. H3) and EZH2; VX-680 reduced only p-hist. H3. This is the first report to show that certain oncogenes are specifically expressed in the myeloma SP, and that bortezomib effectively downregulates expression of their products. Our approach may be useful for screening new agents with which to target a cell population possessing strong tumor initiating potential in multiple myeloma.
doi:10.1371/journal.pone.0056954
PMCID: PMC3587640  PMID: 23469177
5.  Roles of Idiotype-Specific T Cells in Myeloma Cell Growth and Survival: Th1 and CTL Cells Are Tumoricidal While Th2 Cells Promote Tumor Growth 
Cancer research  2008;68(20):8456-8464.
Idiotype protein (Id) secreted by myeloma cells is a tumor-specific antigen. Id-based immunotherapy has been explored in patients with myeloma, and results are disappointing. Although previous studies have demonstrated that Id-specific CTLs are able to lyse myeloma cells, it is unclear whether other types of Id-specific T cells, such as type-1 T-helper (Th1) and type-2 T helper (Th2) cells, are also able to suppress or kill myeloma cells. Using a 5T murine myeloma model, we generated T-cell clones of different subsets and examined their function in the context of myeloma cells. Id-specific CTLs specifically lysed myeloma cells via MHC class I, perforin and Fas ligand, and Th1 but not Th2 cells lysed the myeloma cells by Fas ligand-Fas interaction. CTL and Th1 cells also suppressed the growth and function of myeloma cells whereas Th2 cells promoted the proliferation of and enhanced secretion of Id protein and cytokines by myeloma cells. CTL and Th1 but not Th2 cells were able to eradicate established myeloma in vivo after adoptive transfer. These results demonstrate that Id-specific CTL and Th1 are promising effector cells while Th2 provide no protection and may even promote tumor progression in vivo.
doi:10.1158/0008-5472.CAN-08-2213
PMCID: PMC2575640  PMID: 18922919
Multiple myeloma; idiotype; T-cell subsets; immunotherapy; murine model
6.  High expression of BCL3 in human myeloma cells is associated with increased proliferation and inferior prognosis 
European Journal of Haematology  2009;82(5):354-363.
Background:
BCL3 is a putative oncogene encoding for a protein belonging to the inhibitory κB-family. We experienced that this putative oncogene was a common target gene for growth-promoting cytokines in myeloma cell lines.
Methods:
Gene expression of BCL3 was studied in 351 newly diagnosed myeloma patients, 12 patients with smouldering myeloma, 44 patients with monoclonal gammopathy of undetermined significance and 22 healthy individuals. Smaller material of samples was included for mRNA detection by RT-PCR, protein detection by Western blot and immunohistochemistry, and for cytogenetic studies. A total of eight different myeloma cell lines were studied.
Results:
Bcl-3 was induced in myeloma cell lines by interleukin (IL)-6, IL-21, IL-15, tumor necrosis factor-α and IGF-1, and its upregulation was associated with increased proliferation of the cells. In a population of 351 patients, expression levels of BCL3 above 75th percentile were associated with shorter 5-yr survival. When this patient population was divided into subgroups based on molecular classification, BCL3 was significantly increased in a poor risk subgroup characterized by overexpression of cell cycle and proliferation related genes. Intracellular localization of Bcl-3 was dependent on type of stimulus given to the cell.
Conclusion:
BCL3 is a common target gene for several growth-promoting cytokines in myeloma cells and high expression of BCL3 at the time of diagnosis is associated with poor prognosis of patients with multiple myeloma (MM). These data may indicate a potential oncogenic role for Bcl-3 in MM.
doi:10.1111/j.1600-0609.2009.01225.x
PMCID: PMC2704939  PMID: 19191868
multiple myeloma; Bcl-3; nuclear factor-κB
7.  Role of Bruton's tyrosine kinase (BTK) in growth and metastasis of INA6 myeloma cells 
Blood Cancer Journal  2014;4(8):e234-.
Bruton's tyrosine kinase (BTK) and the chemokine receptor CXCR4 are linked in various hematologic malignancies. The aim of the study was to understand the role of BTK in myeloma cell growth and metastasis using the stably BTK knockdown luciferase-expressing INA6 myeloma line. BTK knockdown had reduced adhesion to stroma and migration of myeloma cells toward stromal cell-derived factor-1. BTK knockdown had no effect on short-term in vitro growth of myeloma cells, although clonogenicity was inhibited and myeloma cell growth was promoted in coculture with osteoclasts. In severe combined immunodeficient-rab mice with contralaterally implanted pieces of bones, BTK knockdown in myeloma cells promoted their proliferation and growth in the primary bone but suppressed metastasis to the contralateral bone. BTK knockdown myeloma cells had altered the expression of genes associated with adhesion and proliferation and increased mammalian target of rapamycin signaling. In 176 paired clinical samples, BTK and CXCR4 expression was lower in myeloma cells purified from a focal lesion than from a random site. BTK expression in random-site samples was correlated with proportions of myeloma cells expressing cell surface CXCR4. Our findings highlight intratumoral heterogeneity of myeloma cells in the bone marrow microenvironment and suggest that BTK is involved in determining proliferative, quiescent or metastatic phenotypes of myeloma cells.
doi:10.1038/bcj.2014.54
PMCID: PMC4219470  PMID: 25083818
8.  Bone marrow stromal cells create a permissive microenvironment for myeloma development: a new stromal role for Wnt inhibitor Dkk1 
Cancer research  2012;72(9):2183-2189.
The rapid progression of multiple myeloma is dependent upon cellular interactions within the bone marrow microenvironment. In vitro studies suggest that bone marrow stromal cells (BMSCs) can promote myeloma growth and survival and osteolytic bone disease. However, it is not possible to recreate all cellular aspects of the bone marrow microenvironment in an in vitro system, and the contributions of BMSCs to myeloma pathogenesis in an intact, immune competent, in vivo system are unknown. To investigate this, we utilized a murine myeloma model that replicates many features of the human disease. Co-inoculation of myeloma cells and a BMSC line isolated from myeloma-permissive mice in otherwise non-permissive mice resulted in myeloma development, associated with tumor growth within bone marrow and osteolytic bone disease. In contrast, inoculation of myeloma cells alone did not result in myeloma. BMSCs inoculated alone induced osteoblast suppression, associated with an increase in serum concentrations of the Wnt signaling inhibitor, Dkk1. Dkk1 was highly expressed in BMSCs and in myeloma-permissive bone marrow. Knockdown of Dkk1 expression in BMSCs decreased their ability to promote myeloma and the associated bone disease in mice. Collectively, our results demonstrate novel roles of BMSCs and BMSC-derived Dkk1 in the pathogenesis of multiple myeloma in vivo.
doi:10.1158/0008-5472.CAN-11-2067
PMCID: PMC3775476  PMID: 22374979
9.  Enhancement of clonogenicity of human multiple myeloma by dendritic cells 
The Journal of experimental medicine  2006;203(8):1859-1865.
Infiltration by dendritic cells (DCs) is a common feature of most human tumors. Prior studies evaluating the interaction of DCs with tumors have focused largely on their immunologic properties (for review see Banchereau, J., and R.M. Steinman. 1998. Nature. 392:245–252). In this study, we show that the clonogenicity of several human tumor cell lines and primary tumor cells from myeloma patients is enhanced by their interactions with DCs. Myeloma cells cultured in the presence of DCs have an altered phenotype with an increased proportion of cells lacking terminal plasma cell differentiation marker CD138. DC–tumor interaction also leads to the up-regulation of B cell lymphoma 6 expression in myeloma cells. Effects of DCs on myeloma cells are inhibited by blockade of the receptor activator of NF-kB (RANK)–RANK ligand and B cell–activating factor–APRIL (a proliferation-inducing ligand)-mediated interactions. Together, these data suggest that tumor–DC interactions may directly impact the biology of human tumors, particularly multiple myeloma, and may be a target for therapeutic intervention.
doi:10.1084/jem.20052136
PMCID: PMC2034506  PMID: 16880256
10.  Enhancement of clonogenicity of human multiple myeloma by dendritic cells 
The Journal of Experimental Medicine  2006;203(8):1859-1865.
Infiltration by dendritic cells (DCs) is a common feature of most human tumors. Prior studies evaluating the interaction of DCs with tumors have focused largely on their immunologic properties (for review see Banchereau, J., and R.M. Steinman. 1998. Nature. 392:245–252). In this study, we show that the clonogenicity of several human tumor cell lines and primary tumor cells from myeloma patients is enhanced by their interactions with DCs. Myeloma cells cultured in the presence of DCs have an altered phenotype with an increased proportion of cells lacking terminal plasma cell differentiation marker CD138. DC–tumor interaction also leads to the up-regulation of B cell lymphoma 6 expression in myeloma cells. Effects of DCs on myeloma cells are inhibited by blockade of the receptor activator of NF-kB (RANK)–RANK ligand and B cell–activating factor–APRIL (a proliferation-inducing ligand)-mediated interactions. Together, these data suggest that tumor–DC interactions may directly impact the biology of human tumors, particularly multiple myeloma, and may be a target for therapeutic intervention.
doi:10.1084/jem.20052136
PMCID: PMC2034506  PMID: 16880256
11.  Identification of a new HLA-A2-restricted T-cell epitope within HM1.24 as immunotherapy target for multiple myeloma 
Experimental Hematology  2006;34(4):486-496.
Objective
Aim of this study was the identification of HLA-A2-restricted T-cell epitopes within the HM1.24-antigen as target for multiple myeloma (MM) directed specific peptide-based immunotherapy.
Methods
The HM1.24 sequence was scanned for immunogenic peptides using the HLA binding prediction softwares “SYFPEITHI” and “BIMAS”. Peripheral blood mononuclear cells (PBMC) from HLA-A2+ normal donors (ND) were stimulated with autologous HM1.24-peptide loaded dendritic cells (DC), and expanded in-vitro. Activation of T-cells was assessed by ELISpot and cytotoxicity by 51Cr-release-assays. T2-cells pulsed with irrelevant peptide, the HM1.24−/HLA-A2+ breast-carcinoma cell-line MCF-7 and the HM1.24+/HLA-A2− myeloma cell-line RPMI-8226 were used as negative controls. Expression of the HM1.24-gene (BST2) was assessed using purified plasma cells and Affymetrix-U133A+B-microarrays.
Results
Of the 8 nona-peptides with the highest probability of binding to HLA-A2, the HM1.24-aa22-30-peptide (LLLGIGILV) showed the highest activation of T-cells. The antigen-recognition by the HM1.24-aa22-30-specific CD8+ T-cells was HLA-A2 restricted (ELISpot with HLA-A2-blocking antibodies: median, 15; range, 14–18 spots/well; isotype-control-antibodies: median, 47; range, 44–48). HM1.24-aa22-30-specific CD8+ T-cells lysed HLA-A2+ myeloma-derived cell-lines (51Cr-release-assay: XG-1 vs. MCF-7, 91% vs. 0%; U266 vs. MCF-7, 38% vs. 4.2%; IM-9 vs. RPMI-8226, 22% vs. 0%). The HM1.24-gene was expressed at comparable levels by plasma cells from 65 MM-patients, 7 patients with monoclonal gammopathy of undetermined significance (MGUS), and 7 ND.
Conclusion
HM1.24-aa22-30 is a new HLA-A2-restricted T-cell epitope processed and presented by MHC-I-complexes. Specific CD8+ T-cells can lyse MM cell-lines. We conclude that HM1.24-aa22-30 is a suitable candidate target for a specific peptide-based immunotherapy of MM.
doi:10.1016/j.exphem.2006.01.008
PMCID: PMC1913933  PMID: 16569595
Antigens, CD; Cell Line, Tumor; Dendritic Cells; immunology; Epitopes, T-Lymphocyte; immunology; therapeutic use; HLA-A2 Antigen; immunology; Humans; Immunotherapy; methods; Isoantigens; immunology; therapeutic use; Lymphocyte Activation; immunology; Membrane Glycoproteins; immunology; therapeutic use; Multiple Myeloma; immunology; therapy; Neoplasm Proteins; immunology; therapeutic use; Oligopeptides; immunology; therapeutic use; Plasma Cells; immunology; T-Lymphocytes, Cytotoxic; immunology
12.  Lenalidomide enhances anti-myeloma cellular immunity 
Lenalidomide is an effective therapeutic agent for multiple myeloma that exhibits immunomodulatory properties including the activation of T and NK cells. The use of lenalidomide to reverse tumor-mediated immune suppression and amplify myeloma-specific immunity is currently being explored. In the present study, we examined the effect of lenalidomide on T-cell activation and its ability to amplify responses to a dendritic cell-based myeloma vaccine. We demonstrate that exposure to lenalidomide in the context of T-cell expansion with direct ligation of CD3/ CD28 complex results in polarization toward a Th1 phenotype characterized by increased IFN-γ, but not IL-10 expression. In vitro exposure to lenalidomide resulted in decreased levels of regulatory T cells and a decrease in T-cell expression of the inhibitory marker, PD-1. Lenalidomide also enhanced T-cell proliferative responses to allogeneic DCs. Most significantly, lenalidomide treatment potentiated responses to the dendritic cell/myeloma fusion vaccine, which were characterized by increased production of inflammatory cytokines and increased cytotoxic lymphocyte-mediated lysis of autologous myeloma targets. These findings indicate that lenalidomide enhances the immunologic milieu in patients with myeloma by promoting T-cell proliferation and suppressing inhibitory factors, and thereby augmenting responses to a myeloma-specific tumor vaccine.
doi:10.1007/s00262-012-1308-3
PMCID: PMC4098790  PMID: 22733396
Lenalidomide; Multiple myeloma; Dendritic cell vaccine; PD-1
13.  Clonogenic Multiple Myeloma Progenitors, Stem Cell Properties, and Drug Resistance 
Cancer research  2008;68(1):190-197.
Many agents are active in multiple myeloma, but the majority of patients relapse. This clinical pattern suggests most cancer cells are eliminated, but cells with the clonogenic potential to mediate tumor regrowth are relatively chemoresistant. Our previous data suggested that CD138+ multiple myeloma plasma cells cannot undergo long-term proliferation but rather arise from clonogenic CD138neg B cells. We compared the relative sensitivity of these distinct cell types to clinical antimyeloma agents and found that dexamethasone, lenadilomide, bortezomib, and 4-hydroxycyclophosphamide inhibited CD138+ multiple myeloma plasma cells but had little effect on CD138neg precursors in vitro. We further characterized clonogenic multiple myeloma cells and stained cell lines using the Hoechst side population and Aldefluor assays. Each assay identified CD138neg cells suggesting that they possess high drug efflux capacity and intracellular drug detoxification activity. We also found that multiple myeloma cells expressing the memory B-cell markers CD20 and CD27 could give rise to clonogenic multiple myeloma growth in vitro and engraft immunodeficient nonobese diabetes/severe combined immunodeficient mice during both primary and secondary transplantation. Furthermore, both the side population and Aldefluor assays were capable of identifying circulating clonotypic memory B-cell populations within the peripheral blood of multiple myeloma patients. Our results suggest that circulating clonotypic B-cell populations represent multiple myeloma stem cells, and the relative drug resistance of these cells is mediated by processes that protect normal stem cells from toxic injury.
doi:10.1158/0008-5472.CAN-07-3096
PMCID: PMC2603142  PMID: 18172311
14.  Immune escape from NY-ESO-1-specific T cell therapy via loss of heterozygosity in the MHC 
Gene therapy  2014;21(3):337-342.
Adoptive immunotherapy of tumors with T cells specific for the cancer-testis antigen NY-ESO-1 has shown great promise in preclinical models and in early stage clinical trials. Tumor persistence or recurrence after NY-ESO-1-specific therapy occurs, however, and the mechanisms of recurrence remain poorly defined. In a murine xenograft model of NY-ESO-1+ multiple myeloma, we observed tumor recurrence after adoptive transfer of CD8+ T cells genetically redirected to the prototypic NY-ESO-1157-165 peptide presented by HLA-A*02:01. Analysis of the myeloma cells that had escaped from T cell control revealed intact expression of NY-ESO-1 and B2M, but selective, complete loss of HLA-A*02:01 expression from the cell surface. Loss of heterozygosity in the Major Histocompatibility Complex (MHC) involving the HLA-A locus was identified in the tumor cells, and further analysis revealed selective loss of the allele encoding HLA-A*02:01. Although loss of heterozygosity involving the MHC has not been described in myeloma patients with persistent or recurrent disease after immune therapies such as allogeneic hematopoietic cell transplantation (HCT), it has been described in patients with acute myelogenous leukemia who relapsed after allogeneic HCT. These results suggest that MHC loss should be evaluated in patients with myeloma and other cancers who relapse after adoptive NY-ESO-1-specific T cell therapy.
doi:10.1038/gt.2013.87
PMCID: PMC4040020  PMID: 24451117
NY-ESO-1; adoptive immunotherapy; immune escape
15.  Enhanced interferon-γ secretion and antitumor activity of T-lymphocytes activated by dendritic cells loaded with glycoengineered myeloma antigens 
Chinese medical journal  2007;120(19):1678-1684.
Background
Immunotherapy is emerging as a promising cure for cancer. However, a severe problem in this area is the immune tolerance to tumor cells and tumor-associated antigens, as evidenced by the ability of cancer to escape immune surveillance. To overcome this problem this work examined the potential of improving the antigenicity of myeloma by metabolic engineering of its cell surface carbohydrate antigens (i.e., glycoengineering) and presentation of the modified tumor antigens by dendritic cells (DCs) to generate cytotoxic T-lymphocytes (CTLs).
Methods
CD138+ myeloma cells were isolated from 11 multipe myeloma (MM) patients by the immunomagnetic bead method. The MM cells were treated with N-propionyl-D-mannosamine (ManNPr), a synthetic analog of N-acetyl-D-mannosamine (ManNAc), the natural biosynthetic precursor of N-acetyl sialic acid (NeuNAc), to express unnatural N-propionylated sialoglycans. The glycoengineered cells were then induced to apoptosis, and the apoptotic products were added to cultured functional DCs that could present the unnatural carbohydrate antigens to autologous T-lymphocytes.
Results
It was found that the resultant DCs could activate CD4+ and CD8+ T-lymphocytes, resulting in increased expression of T cell surface markers, including CD8CD28 and CD4CD29. Moreover, upon stimulation by glycoengineered MM cells, these DC-activated T-lymphocytes could release significantly higher levels of IFN-γ (P<0.05). Lactate dehydrogenase (LDH) assays further showed that the stimulated T-lymphocytes were cytotoxic to glycoengineered MM cells.
Conclusions
This work demonstrated that glycoengineered myeloma cells were highly antigenic and the CTLs induced by the DCs loaded with the unnatural myeloma antigens were specifically cytotoxic to the glycoengineered myeloma. This may provide a new strategy for overcoming the problem of immune tolerance for the development of effective immunotherapies for MM.
PMCID: PMC3178877  PMID: 17935669
multiple myeloma; dendritic cells; T-lymphocytes; immunotherapy
16.  Heparanase enhances local and systemic osteolysis in multiple myeloma by upregulating the expression and secretion of RANKL 
Cancer research  2010;70(21):8329-8338.
Excessive bone destruction is a major cause of morbidity in myeloma patients. However, the biologic mechanisms involved in the pathogenesis of myeloma-induced bone disease are not fully understood. Heparanase, an enzyme that cleaves the heparan sulfate chains of proteoglycans, is upregulated in a variety of human tumors, including multiple myeloma. We previously demonstrated that heparanase promotes robust myeloma tumor growth and supports spontaneous metastasis of tumor cells to bone. In the present study, we demonstrate, for the first time, that the expression of heparanase by myeloma tumor cells remarkably enhances bone destruction locally within the tumor microenvironment. In addition, enhanced heparanase expression in the primary tumor also stimulated systemic osteoclastogenesis and osteolysis, thus mimicking the systemic osteoporosis often seen in myeloma patients. These effects occur, at least in part, as the result of a significant elevation in the expression and secretion of RANKL by heparanase-expressing myeloma cells. Moreover, analysis of bone marrow biopsies from myeloma patients reveals a positive correlation between the level of expression of heparanase and RANKL. Together these discoveries reveal a novel and key role for heparanase in promoting tumor osteolysis and demonstrate that RANKL is central to the mechanism of heparanase-mediated osteolysis in myeloma.
doi:10.1158/0008-5472.CAN-10-2179
PMCID: PMC2970667  PMID: 20978204
Heparanase; osteolysis; multiple myeloma; RANKL; tumor microenvironment
17.  MHC Class II Transactivator (CIITA) Expression is up Regulated in Multiple Myeloma Cells by IFN-γ 
Molecular immunology  2007;44(11):2923-2932.
The MHC Class II transactivator (CIITA) acts in the cell nucleus as the master regulator of MHC class II (MHC II) gene expression. It is important to study CIITA regulation in multiple myeloma since MHC expression is central to ability of myeloma cells to present antigen and to the ability of the immune system to recognize and destroy this malignancy. Regulation of CIITA by IFN-γ in B lymphocytes occurs through the CIITA type IV promoter (pIV), one of the four potential promoters (pI-pIV) of this gene. To investigate regulation of CIITA by IFN-γ in multiple myeloma cells, first the ability of these cells to respond to IFN-γ was examined. RTPCR analyses show that IFN-γR1, the IFN-γ-binding chain of the IFN-γ receptor, is expressed in myeloma cells and IRF-1 expression increases in response to IFN-γ treatment. Western blotting demonstrates that STAT1 is activated by phosphorylation in response to IFN-γ. RT-PCR and functional promoter analyses show that IFN-γ up regulates the activity of CIITA pIV, as does ectopic expression of IRF-1 or IRF-2. In vivo protein/DNA binding studies demonstrate protein binding at the GAS, E box and IRF-E sites. In vitro studies confirm the binding of IRF-1 and IRF-2 to CIITA pIV. Although multiple myeloma cells express PRDI-BF1/Blimp-1, a factor that represses both the CIITA type III and IV promoters, they retain the capability to up regulate CIITA pIV and MHC II expression in response to IFN-γ treatment. These findings are the first to demonstrate that although PRDI-BF1/Blimp-1 diminishes the constitutive ability of these cells to present antigen by limiting CIITA and MHC II expression, it is possible to enhance this expression through the use of cytokines, like IFN-γ.
doi:10.1016/j.molimm.2007.01.009
PMCID: PMC1892219  PMID: 17300840
myeloma; CIITA; IFN-γ, IFN-γR1; STAT1; IRF-1; IRF-2; PRDI-BF1; Blimp-1
18.  Integrative analysis of differential miRNA and functional study of miR-21 by seed-targeting inhibition in multiple myeloma cells in response to berberine 
BMC Systems Biology  2014;8:82.
Background
Berberine is a natural alkaloid derived from a traditional Chinese herbal medicine. It is known to modulate microRNA (miRNA) levels, although the mechanism for this action is unknown. Here, we previously demonstrate that the expression of 87 miRNAs is differentially affected by berberine in multiple myeloma cells. Among 49 miRNAs that are down-regulated, nine act as oncomirs, including miR-21. Integrative analysis showed that 28 of the down-regulated miRNAs participate in tumor protein p53 (TP53) signaling and other cancer pathways. miR-21 is involved in all these pathways, and is one of the most important oncomirs to be affected by berberine in multiple myeloma cells.
Results
We confirmed that berberine down-regulated miRNA-21 expression and significantly up-regulated the expression of programmed cell death 4 (PDCD4), a predicted miR-21 target. Luciferase reporter assays confirmed that PDCD4 was directly regulated by miR-21. Bioinformatic analysis revealed that the miR-21 promoter can be targeted by signal transducer and activator of transcription 3 (STAT3). Down-regulation of interleukin 6 (IL6) by berberine might lead to inhibition of miR-21 transcription through STAT3 down-regulation in multiple myeloma. Furthermore, both berberine and seed-targeting anti-miR-21 oligonucleotide induced apoptosis, G2-phase cell cycle arrest and colony inhibition in multiple myeloma cell lines. Depletion of PDCD4 by short interfering RNA could rescue berberine-induced cytotoxicity in multiple myeloma cells.
Conclusions
Our results suggest that berberine suppresses multiple myeloma cell growth, at least in part, by down-regulating miR-21 levels possibly through IL6/STAT3. This led to increased PDCD4 expression, which is likely to result in suppression of the p53 signaling pathway. These findings may also provide new mechanistic insight into the anti-cancer effects of certain compounds in traditional Chinese herbal medicines.
doi:10.1186/1752-0509-8-82
PMCID: PMC4096730  PMID: 25000828
Berberine; miRNA-21; Multiple myeloma; Programmed cell death 4; Bioinformatic; Tumor protein p53; Cell cycle; Apoptosis
19.  An inhibitor of the EGF receptor family blocks myeloma cell growth factor activity of HB-EGF and potentiates dexamethasone or anti-IL-6 antibody-induced apoptosis 
Blood  2003;103(5):1829-1837.
We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells, we studied the role of HB-EGF on myeloma cell growth.
The HB-EGF gene was expressed by bone marrow mononuclear cells of 8/8 patients with myeloma, particularly by monocytes and stromal cells, but not by purified primary myeloma cells. 6/9 myeloma cell lines and 9/9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low IL-6 concentration, HB-EGF stimulated the proliferation of the six ErbB1+ or ErbB4+ cell lines, through the PI-3K/AKT pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients’ myeloma cells cultured with their tumor environment. It also increased patients’ myeloma cell apoptosis induced by an anti-IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between MM cells and stromal cells. It was not toxic for non-myeloma cells present in patients’ bone marrow cultures or for the growth of hematopoietic progenitors. Altogether, these data identify ErbB receptors as putative therapeutic targets in multiple myeloma.
doi:10.1182/blood-2003-05-1510
PMCID: PMC2386161  PMID: 14576062
1-Phosphatidylinositol 3-Kinase; metabolism; Antigens; CD14; biosynthesis; Antineoplastic Agents; Hormonal; pharmacology; Apoptosis; Blotting; Western; Bone Marrow Cells; cytology; Cell Adhesion; Cell Differentiation; Cell Division; Cell Line; Tumor; Cell Separation; Cells; Cultured; Dexamethasone; pharmacology; Drug Synergism; Electrophoresis; Polyacrylamide Gel; Enzyme Inhibitors; pharmacology; Epidermal Growth Factor; metabolism; physiology; Hematopoietic Stem Cells; metabolism; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-6; biosynthesis; immunology; metabolism; Leukocytes; Mononuclear; metabolism; Monocytes; metabolism; Multiple Myeloma; metabolism; Receptor; Epidermal Growth Factor; antagonists & inhibitors; biosynthesis; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity
20.  Stemness of B cell progenitors in multiple myeloma bone marrow 
Purpose
In myeloma, B cells and plasma cells show a clonal relationship. Clonotypic B cells may represent a tumor-initiating compartment or cancer stem cell responsible for minimal residual disease in myeloma.
Experimental Design
We report a study of 58 patients with myeloma at time of diagnosis or relapse. B cells in bone marrow were evaluated by multicolor flow cytometry and sorting. Clonality was determined by light chain and/or immunoglobulin chain gene rearrangement PCR. We also determined aldehyde dehydrogenase activity and colony formation growth. Drug sensitivity was tested with conventional and novel agents.
Results
Marrow CD19+ cells express a light chain identical to plasma cells and are therefore termed light chain restricted (LCR). The LCR B cell mass is small in both newly diagnosed and relapsed patients (≤1%). Few marrow LCR B cells (~10%) are CD19+/CD34+, with the rest being more differentiated CD19+/CD34− B cells. Marrow LCR CD19+ B cells exhibit enhanced aldehyde dehydrogenase activity versus healthy controls. Both CD19+/CD34+ and CD19+/CD34− cells showed colony formation activity, with colony growth efficiency optimized when stroma-conditioned medium was used. B cell progenitors showed resistance to melphalan, lenalidomide, and bortezomib. Panobinostat, a histone deacetylase inhibitor, induced apoptosis of LCR B cells and CD138+ cells. LCR B cells are CD117, survivin, and Notch positive.
Conclusions
We propose that antigen-independent B cell differentiation stages are involved in disease origination and progression in myeloma. Further investigations of myeloma putative stem cell progenitors may lead to novel treatments to eradicate the potential reservoir of minimal residual disease.
doi:10.1158/1078-0432.CCR-12-0531
PMCID: PMC3500436  PMID: 22988056
multiple myeloma; B cell progenitors; bone marrow; stemness
21.  Human Placenta-Derived Adherent Cells Prevent Bone loss, Stimulate Bone formation, and Suppress Growth of Multiple Myeloma in Bone 
Stem cells (Dayton, Ohio)  2011;29(2):10.1002/stem.572.
Human placenta has emerged as a valuable source of transplantable cells of mesenchymal and hematopoietic origin for multiple cytotherapeutic purposes, including enhanced engraftment of hematopoietic stem cells, modulation of inflammation, bone repair, and cancer. Placenta-derived adherent cells (PDACs) are mesenchymal-like stem cells isolated from postpartum human placenta. Multiple myeloma is closely associated with induction of bone disease and large lytic lesions, which are often not repaired and are usually the sites of relapses. We evaluated the antimyeloma therapeutic potential, in vivo survival, and trafficking of PDACs in the severe combined immunodeficiency (SCID)–rab model of medullary myeloma-associated bone loss. Intrabone injection of PDACs into non-myelomatous and myelomatous implanted bone in SCID-rab mice promoted bone formation by stimulating endogenous osteoblastogenesis, and most PDACs disappeared from bone within 4 weeks. PDACs inhibitory effects on myeloma bone disease and tumor growth were dose-dependent and comparable with those of fetal human mesenchymal stem cells (MSCs). Intrabone, but not subcutaneous, engraftment of PDACs inhibited bone disease and tumor growth in SCID-rab mice. Intratumor injection of PDACs had no effect on subcutaneous growth of myeloma cells. A small number of intravenously injected PDACs trafficked into myelomatous bone. Myeloma cell growth rate in vitro was lower in coculture with PDACs than with MSCs from human fetal bone or myeloma patients. PDACs also promoted apoptosis in osteoclast precursors and inhibited their differentiation. This study suggests that altering the bone marrow microenvironment with PDAC cytotherapy attenuates growth of myeloma and that PDAC cytotherapy is a promising therapeutic approach for myeloma osteolysis.
doi:10.1002/stem.572
PMCID: PMC3175303  PMID: 21732484
Myeloma; Bone disease; Cytotherapy; Osteoblasts; Placenta; Mesenchymal stem cells
22.  Regulation of Bcl-2-family proteins in myeloma cells by three myeloma survival factors: interleukin-6, interferon-alpha and insulin-like growth factor 1 
Cell Death and Differentiation  2000;7(12):1244-1252.
Summary
As survival regulation is a key process in multiple myeloma biology, we have studied the Bcl-2 family proteins that can be regulated by 3 myeloma cell survival factors: interleukin-6 (IL-6), interferon-α (IFN-α) and insulin-like growth factor 1 (IGF-1). Eleven myeloma cell lines, whose survival and proliferation are dependent on addition of IL-6, variably expressed 10 anti-apoptotic or pro-apoptotic proteins of the Bcl-2-family. When myeloma cells from 4 cell lines were IL-6 starved and activated with IL-6 or IFN-α, we observed that only Mcl-1 expression was up-regulated with myeloma cell survival induction. Nor was obvious regulation of these 10 pro-apoptotic or anti-apoptotic proteins found with IGF-1, another potent myeloma cell survival factor. Our results indicate that the myeloma cell survival activity of IL-6 linked to Bcl-xL regulation cannot be generalized and emphasize that Mcl-1 is the main target of IL-6 and IFN-α stimulation. However, other changes in the activity of the Bcl-2 protein family or other apoptosis regulators must be identified to elucidate the IGF-1 action mechanism.
doi:10.1038/sj.cdd.4400758
PMCID: PMC2423422  PMID: 11175262
Cell Survival; drug effects; physiology; DNA-Binding Proteins; drug effects; metabolism; Gene Expression Regulation; Neoplastic; drug effects; physiology; Humans; Insulin-Like Growth Factor I; metabolism; pharmacology; Interferon-alpha; metabolism; pharmacology; Interleukin-6; metabolism; pharmacology; Multiple Myeloma; genetics; metabolism; physiopathology; Neoplasm Proteins; drug effects; metabolism; Plasma Cells; drug effects; metabolism; pathology; Proto-Oncogene Proteins c-bcl-2; drug effects; metabolism; STAT3 Transcription Factor; Trans-Activators; drug effects; metabolism; Tumor Cells; Cultured; cytology; drug effects; metabolism; myeloma; IL-6; IFN; IGF-1; apoptosis
23.  Identification of MAGE-C1 (CT-7) epitopes for T-cell therapy of multiple myeloma 
Multiple myeloma is incurable with standard therapies but is susceptible to a T-cell-mediated graft versus myeloma effect after allogeneic stem cell transplantation. We sought to identify myeloma-specific antigens that might be used for T-cell immunotherapy of myeloma. MAGE-C1 (CT-7) is a cancer-testis antigen that is expressed by tumor cells in >70% of myeloma patients and elicits a humoral response in up to 93% of patients with CT-7+ myeloma. No CD8+ T-cell epitopes have been described for CT-7, so we used a combination of reverse immunology and immunization of HLA-A2 transgenic mice with a novel cell-based vaccine to identify three immunogenic epitopes of CT-7 that are recognized by human CD8+ T-cells. CT-7-specific T-cells recognizing two of these peptides are able to recognize myeloma cells as well as CT-7 gene-transduced tumor cells, demonstrating that these epitopes are naturally processed and presented by tumor cells. This is the first report of the identification of immunogenic CD8+ T-cell epitopes of MAGE-C1 (CT-7), which is the most commonly expressed cancer-testis antigen found in myeloma, and these epitopes may be promising candidate targets for vaccination or T-cell therapy of myeloma or other CT-7+ malignancies.
doi:10.1007/s00262-011-1009-3
PMCID: PMC3183483  PMID: 21461886
MAGE-C1; CT-7; Multiple myeloma; Cancer immunology; Immunotherapy
24.  Multiple myeloma/hypercalcemia 
Arthritis Research & Therapy  2007;9(Suppl 1):S4.
Multiple myeloma, a cancer of plasma cells, is associated with excessive tumor-induced, osteoclast-mediated bone destruction. Hypercalcemia remains the most frequent metabolic complication of myeloma in patients, and excessive osteolysis plays a major contributory role in its pathogenesis. The clinical presentation of hypercalcemia in patients varies depending on the level of ionized calcium; it can be life threatening, as in the case of hypercalcemic crisis, requiring immediate medical treatment to prevent death. During the past few years there have been exciting developments in our understanding of the pathogenesis of myeloma bone disease; in particular, key mediators of the osteoclastic bone resorption in myeloma have been identified, including receptor activator of nuclear factor-κB ligand (RANKL) and macrophage inflammatory protein-1α. There is also increasing evidence that Dickkopf 1, which has been shown to be over-expressed in myeloma patients, is also a potent stimulator of osteoclast formation and activity. Importantly, the available data suggest that RANKL is the final common mediator of osteoclastic bone resorption, irrespective of the upstream initiator molecule. This brief review presents an overview of the roles played by these mediators in inducing osteolysis in myeloma bone disease, and it discusses targeting RANKL as a potential new treatment strategy in myeloma bone disease and myeloma-associated hypercalcemia.
doi:10.1186/ar2168
PMCID: PMC1924519  PMID: 17634143
25.  Protein Kinase CK2 Inhibition Down Modulates the NF-κB and STAT3 Survival Pathways, Enhances the Cellular Proteotoxic Stress and Synergistically Boosts the Cytotoxic Effect of Bortezomib on Multiple Myeloma and Mantle Cell Lymphoma Cells 
PLoS ONE  2013;8(9):e75280.
CK2 is a pivotal pro-survival protein kinase in multiple myeloma that may likely impinge on bortezomib-regulated cellular pathways. In the present study, we investigated CK2 expression in multiple myeloma and mantle cell lymphoma, two bortezomib-responsive B cell tumors, as well as its involvement in bortezomib-induced cytotoxicity and signaling cascades potentially mediating bortezomib resistance. In both tumors, CK2 expression correlated with that of its activated targets NF-κB and STAT3 transcription factors. Bortezomib-induced proliferation arrest and apoptosis were significantly amplified by the simultaneous inhibition of CK2 with two inhibitors (CX-4945 and K27) in multiple myeloma and mantle cell lymphoma cell lines, in a model of multiple myeloma bone marrow microenvironment and in cells isolated from patients. CK2 inhibition empowered bortezomib-triggered mitochondrial-dependent cell death. Phosphorylation of NF-κB p65 on Ser529 (a CK2 target site) and rise of the levels of the endoplasmic reticulum stress kinase/endoribonuclease Ire1α were markedly reduced upon CK2 inhibition, as were STAT3 phospho Ser727 levels. On the contrary, CK2 inhibition increased phospho Ser51 eIF2α levels and enhanced the bortezomib-dependent accumulation of poly-ubiquitylated proteins and of the proteotoxic stress-associated chaperone Hsp70. Our data suggest that CK2 over expression in multiple myeloma and mantle cell lymphoma cells might sustain survival signaling cascades and can antagonize bortezomib-induced apoptosis at different levels. CK2 inhibitors could be useful in bortezomib-based combination therapies.
doi:10.1371/journal.pone.0075280
PMCID: PMC3785505  PMID: 24086494

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