Fully Human CD38 Antibodies Efficiently Trigger ADCC of Multiple Myeloma Cell Lines and Primary Tumor Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3377-3377 ◽  
Author(s):  
Matthias Peipp ◽  
Michel de Weers ◽  
Thomas Beyer ◽  
Roland Repp ◽  
Paul Parren ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Cells ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Treatment Modalities ◽  
Potential Candidate ◽  
Isolated Tumor Cells ◽  
Myeloma Cells ◽  
Multiple Myeloma Cell

Abstract Although new treatment modalities have recently been added to the standard regimens for multiple myeloma, the clinical outcome for patients with advanced disease is often limited. Monoclonal antibodies are increasingly used for tumor therapy, and may also represent interesting options for multiple myeloma patients. CD38 is one of the most promising target antigens on malignant plasma cells, which are evaluated in preclinical and early clinical studies as targets for antibody therapy. CD38 is a type II transmembrane protein with ectoenzymatic activity, which is involved in calcium mobilization. Human CD38 is predominantly expressed by bone marrow precursor cells and by terminally differentiated plasma cells. Multiple myeloma cells show moderate to high expression levels - making CD38 a potential candidate as target for immunotherapy. A panel of 42 fully human CD38 antibodies was generated by immunizing human Ig transgenic mice. Immunofluorescence studies with CD38 transfected cells demonstrated antigen-specific, high affinity binding, and cross-blocking experiments revealed four distinct epitope groups. Seven antibodies, representing each of the four groups, were selected for further analyses. ADCC and CDC activity against CD38-positive myeloma cell lines (AMO-1 and JK6), and against freshly-isolated primary multiple myeloma cells was investigated. Human whole blood served as effector source, which was then fractionated into plasma (containing human complement), mononuclear (MNC) or granulocytic (PMN) effector cells. All antibodies mediated concentration-dependent killing of both multiple myeloma cell lines - using human mononuclear cells as effector source. Also complement-dependent killing of freshly isolated myeloma cells was observed. However, none of the antibodies recruited PMN for tumor cell lysis. Importantly, CD38 antibodies also killed freshly isolated tumor cells from a rare patient with a CD38/138- positive plasma cell leukemia, which was chemotherapy- refractory at the time of analysis. Furthermore, CD38 antibodies effectively prevented outgrowth of CD38-positive tumor cells in SCID mouse xenograft models. Antibody 005 was significantly more effective in these assays compared to the remaining panel of CD38 antibodies. In conclusion, CD38 antibodies efficiently mediated killing of multiple myeloma cell lines as well as freshly isolated tumor cells and prevented tumor outgrowth in xenografted SCID mice. Antibody 005 was superior in mediating CDC and ADCC via MNC - particularly at low antibody concentrations.

Expression of functional interleukin-15 receptor and autocrine production of interleukin-15 as mechanisms of tumor propagation in multiple myeloma

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 610-618 ◽  
Author(s):  
Inge Tinhofer ◽  
Ingrid Marschitz ◽  
Traudl Henn ◽  
Alexander Egle ◽  
Richard Greil
Keyword(s):  
Multiple Myeloma ◽  
Cell Survival ◽  
Cell Lines ◽  
Plasma Cells ◽  
Constitutive Expression ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Cytotoxic Treatment ◽  
Interleukin 15 ◽  
Induced Apoptosis

Interleukin-15 (IL-15) induces proliferation and promotes cell survival of human T and B lymphocytes, natural killer cells, and neutrophils. Here we report the constitutive expression of a functional IL-15 receptor (IL-15R) in 6 of 6 myeloma cell lines and in CD38high/CD45low plasma cells belonging to 14 of 14 patients with multiple myeloma. Furthermore, we detected IL-15 transcripts in all 6 myeloma cell lines, and IL-15 protein in 4/6 cell lines and also in the primary plasma cells of 8/14 multiple myeloma patients. Our observations confirm the existence of an autocrine IL-15 loop and point to the potential paracrine stimulation of myeloma cells by IL-15 released from the cellular microenvironment. Blocking autocrine IL-15 in cell lines increased the rate of spontaneous apoptosis, and the degree of this effect was comparable to the pro-apoptotic effect of depleting autocrine IL-6 by antibody targeting. IL-15 was also capable of substituting for autocrine IL-6 in order to promote cell survival and vice versa. In short-term cultures of primary myeloma cells, the addition of IL-15 reduced the percentage of tumor cells spontaneously undergoing apoptosis. Furthermore, IL-15 lowered the responsiveness to Fas-induced apoptosis and to cytotoxic treatment with vincristine and doxorubicin but not with dexamethasone. These data add IL-15 to the list of important factors promoting survival of multiple myeloma cells and demonstrate that it can be produced and be functionally active in an autocrine manner.

Unique Pattern of Raf-1 Kinase Inhibitory Protein (RKIP) Expression in Multiple Myeloma (MM) Compared to Other Tumor Types: Overexpression of RKIP and a Phosphorylated RKIP Is Common in MM Tumor Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4852-4852
Author(s):  
Stavroula Baritaki ◽  
Sara Huerta-Yepez ◽  
Kam Yeung ◽  
Manuel Penichet ◽  
Haiming Chen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Cell ◽  
Signaling Pathways ◽  
Tumor Cells ◽  
Cell Lines ◽  
Immune Surveillance ◽  
Myeloma Cell ◽  
Inhibitory Protein ◽  
Multiple Myeloma Cell ◽  
Survival Signaling

Abstract Objective and Rationale Raf-1 kinase inhibitory protein (RKIP) is a modulator of cell signaling and survival that functions as an endogenous inhibitor of multiple kinases, including kinases involved in the Raf/MEK/ERK and NF-κB pathways. RKIP has been identified as a metastasis suppressor gene and an immune surveillance cancer gene, since loss of RKIP protein expression has been associated with tumor progression, metastasis and escape from immune surveillance. Further, RKIP expression has been associated with prognostic significance in many cancers. Recently, we have demonstrated that induction of RKIP expression in tumors with low RKIP levels results in increased tumor cell sensitivity to immuno- or chemo-therapy via inhibition of the above pathways. However, multiple myeloma (MM) cells have been shown to express high RKIP levels compared to other tumors and still remain highly resistant to conventional cytotoxic therapies. These findings were unexpected and thus, it was plausible that the high level of RKIP expression was not functionally active. It has been reported that phosphorylation of RKIP at Ser-153 renders the cells inactive (Rosner et al., 2003, J Biol Chem 278:13061–8). Thus, we examined the expression and the phosphorylation status of the RKIP protein in several multiple myeloma cell lines and tissues and compared them with other cell lines with low RKIP expression. Hypothesis We hypothesized that MM tumor cells express high levels of the inactive phoshorylated RKIP protein which antagonizes the active non-phoshorylated RKIP form in the inhibition of the survival signaling pathways. Experimental Designs and Methods Multiple myeloma (IM-9, RPMI 8226, MM1S, U266 cell lines and fresh bone marrow samples from MM patients), PC-3 prostatic carcinoma and Ramos B-NHL cell lines were examined for total and phosphorylated RKIP expression by IHC and Western Blot analyses. The total RKIP protein was significantly elevated in multiple myeloma cell lines compared to the prostate and B-NHL lines. The predominant RKIP form in multiple myeloma tumors was the phosphorylated RKIP protein with high nuclear localization, as assessed by IHC, while the phosphorylated RKIP levels in the non-myeloma tumors were relatively low. It has been reported that the phosphorylation of RKIP is mediated by protein kinase C (Rosner et al., 2003, J Biol Chem 278:13061–8). Additional studies in multiple myeloma cell lines also revealed high expression of the zeta isoform of PKC (PKCζ), known to phosphorylate and inactivate RKIP. Conclusions and Implications The present findings demonstrate that the aberrant RKIP phosphorylation in multiple myeloma tumors may result in the inhibition of the suppressive effect of RKIP on tumor survival signaling pathways. We postulate that the high expression of RKIP may be due to inhibition of proteasome degradation. The present findings also suggest that screening of RKIP levels and RKIP phosphorylation status in MM may be useful as prognostic factors of tumor cell response to anti-tumor therapies.

Serum/Glucocorticoid-Regulated Kinase 1 (SGK1) Is a Prominent Target Gene of the Transcriptional Response to Cytokines In Multiple Myeloma and Supports the Growth of Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1915-1915
Author(s):  
Unn-Merete Fagerli ◽  
Thorsten Stühmer ◽  
Toril Holien ◽  
Randi Utne Holt ◽  
Ove Bruland ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factors ◽  
Cell Lines ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Transcriptional Response ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Growth And Survival ◽  
Stat3 Phosphorylation

Abstract Abstract 1915 Multiple myeloma is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We hypothesized that the intracellular signals evoked by cytokines converge and regulate transcription of a set of genes that are common targets for several growth factors and therefore constitute pivotal mediators of the tumor-promoting effects of autocrine or paracrine stimuli. To identify such targets, we determined the changes in gene expression induced by IL-6, TNFalpha, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase SGK1, which is a down-stream effector of PI3-kinase and highly homologous to AKT. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the JAK/STAT pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, shRNA-mediated knock-down of STAT3 reduced basal and induced SGK1 levels, demonstrating the involvement of the JAK/STAT3 signaling pathway in SGK1 induction. Furthermore, down-regulation of SGK1 by shRNAs resulted in decreased proliferation and viability of myeloma cell lines. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their growth and survival and represents an attractive candidate for further evaluation as a therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Metabolomic, Proteomic and Genomic Profiling Identifies Biomarakers of Sensitivity to Glutaminase Inhibitor CB-839 in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1802-1802 ◽  
Author(s):  
Andrew L MacKinnon ◽  
Mark Bennett ◽  
Matt Gross ◽  
Julie Janes ◽  
Weiqin Li ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Tca Cycle ◽  
Myeloma Cell ◽  
Akt Inhibitor ◽  
Employment Equity ◽  
Myeloma Cells ◽  
Multiple Myeloma Cell ◽  
Equity Ownership ◽  
Resistant Cells

Abstract Introduction Glutaminase is a mitochondrial enzyme that converts glutamine to glutamate to support several metabolic processes including amino acid and nucleotide synthesis, maintenance of cellular redox homeostasis, and the replacement of TCA cycle intermediates. Selective glutaminase inhibitors BPTES and CB-839 have anti-proliferative activity in several pre-clinical cancer models including breast, pancreatic, lung, renal, brain, leukemia, and lymphoma. Across a panel of twenty-nine multiple myeloma cell lines, we found that glutaminase inhibition with CB-839 caused tumor cell death or growth inhibition in only a subset of cell lines. To identify biomarkers that predict sensitivity to CB-839 in multiple myeloma cells, we profiled cellular metabolites, mRNA transcripts, and signaling pathways in eight multiple myeloma cell (four CB-839-sensitive and four CB-839-resistant). Results Proteomic analysis showed that CB-839 treatment suppressed the activity of the amino-acid sensing kinase mTORC1 in CB-839-sensitive cells, leading to down regulation of protein synthesis and expression of metabolic genes. Analysis of steady-state levels of intra-cellular metabolites revealed that CB-839-sensitive cells had more profound decreases in nucleotide levels and less pronounced increases in essential amino acids upon CB-839 treatment compared to CB-839-resistant cells. This suggests that the metabolic response to glutaminase inhibition is fundamentally different in sensitive versus resistant multiple myeloma cell lines. Consistent with the in vitro data, in a xenograft model with the CB-839-sensitive cell line RPMI8226, CB-839 treatment produced a 71% reduction in tumor growth that was associated with reduced levels of intratumoral nucleotides and no changes in the levels of essential amino acids. We next explored protein biomarkers that predict resistance to CB-839 and found that pyruvate carboxylase (PC) expression strongly correlated with resistance. siRNA-mediated knockdown of PC reduced TCA cycle activity and sensitized cells to CB-839 treatment, suggesting that PC can rescue cells from glutaminase inhibition by supporting anapleurotic utilization of glucose. This hypothesis was further substantiated by the observation that treatment of CB-839-resistant cells with the AKT inhibitor MK2206 led to a decrease in glucose utilization, and when combined with CB-839, produced a significant decrease in TCA cycle activity and a profound synergistic anti-proliferative response. Conclusion Multiple myeloma cells show varying anti-proliferative responses to glutaminase inhibition by CB-839. CB-839 treatment inhibits mTORC1 pathway signaling and causes decreases in nucleotides in sensitive multiple myeloma cells. Multiple myeloma cells that are resistant to glutaminase inhibition have high expression of PC, which may allow these cells to utilize glucose instead of glutamine to resupply TCA cycle intermediates. Knockdown of PC or treatment with an AKT inhibitor causes cells to utilize less glucose and sensitizes resistant cells to glutaminase inhibition with CB-839. CB-839 is currently being evaluated in Phase 1 clinical trials for the treatment of various solid and hematological cancers including multiple myeloma. We are exploring the utility of PC and mTORC1 pathway signaling biomarkers to identify multiple myeloma patients that may respond to CB-839 treatment. Disclosures MacKinnon: Calithera Biosciences: Employment, Equity Ownership. Bennett:Calithera Biosciences: Employment, Equity Ownership. Gross:Calithera Biosciences: Employment, Equity Ownership. Janes:Calithera Biosciences: Employment, Equity Ownership. Li:Calithera Biosciences: Employment, Equity Ownership. Rodriquez:Calithera Biosciences: Employment, Equity Ownership. Wang:Calithera Biosciences: Employment, Equity Ownership. Zhang:Calithera Biosciences: Employment, Equity Ownership. Parlati:Calithera Biosciences: Employment, Equity Ownership.

scholarly journals Blocking the Don't Eat Me Signal (CD47-SIRPα Axis) to Improve Antibody-Based Immunotherapy of Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2684-2684
Author(s):  
Katja Klausz ◽  
Carina Lynn Gehlert ◽  
Ammelie Svea Boje ◽  
Marta Lustig ◽  
Steffen Krohn ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Tumor Cells ◽  
Cell Lines ◽  
Myeloid Cells ◽  
Myeloma Cell ◽  
Cell Phenotype ◽  
Blocking Antibody ◽  
Myeloma Cells

Abstract The addition of monoclonal antibodies daratumumab, elotuzumab and isatuximab to the treatment of patients with multiple myeloma significantly improved the outcome and prolonged survival. Unfortunately, although many patients benefit, depth and duration of response are a problem. In order to improve efficacy of antibody-based immunotherapy, we aimed to combine CD38-directed antibodies daratumumab and isatuximab as well as SLAMF7-targeting elotuzumab with a CD47 blocking antibody to enhance phagocytosis of myeloma cells. Antibody-dependent cellular phagocytosis (ADCP) of malignant plasma cells is described to be one important mode of action of daratumumab, isatuximab and elotuzumab, respectively. Of note, CD47 is highly expressed on myeloma cells and allows evading immune recognition by myeloid cells, i.e. monocytes, macrophages and neutrophils. Binding of CD47 to SIRPα expressed on myeloid cells provides a strong 'don't eat me' signal and diminishes phagocytosis of tumor cells. Blocking the CD47-SIRPα axis, by a monoclonal antibody against CD47 or a SIRPα-Fc fusion protein can restore recognition of tumor cells by macrophages and enhance phagocytosis. In patients with Non-Hodgkin's lymphoma the combination of CD20 antibody rituximab with CD47 antibody magrolimab was clinically successful (Advani et al., NEJM 379:1711, 2018). To test the applicability of blocking the CD47-SIRPα axis and improve ADCP of myeloma cells by CD38-targeting or SLAMF7-directed myeloma antibodies, we generated a CD47 IgG2σ antibody carrying an engineered Fc domain not binding to Fcγ receptors (FcγR). This CD47 antibody was subsequently used in phagocytosis experiments in combination with antibodies daratumumab, isatuximab as well as elotuzumab and various myeloma cell lines. The cell lines AMO-1, JK-6L, L363, RPMI-8226, and U266 express different levels of CD47, CD38 and SLAMF7 as determined by quantitative flow cytometry. M0 macrophages expressing FcγRs were generated from healthy donor PBMC monocytes by cultivation with M-CSF for 10-14 days prior use in 6 hour real-time live cell imaging phagocytosis experiments with pHrodo-labeled myeloma cells - turning red only when engulfed by macrophages. Macrophages and myeloma cells were used at an effector-to-target cell ratio of 1:1. Importantly, ADCP of myeloma cells induced by all three monoclonal antibodies, daratumumab, isatuximab or elotuzumab, can be enhanced by the addition of the CD47 blocking antibody. However, improvement in phagocytosis strongly differs between myeloma cell lines although all have high CD47 level on their cell surface. In responsive myeloma cell lines, ADCP mediated by CD38 antibodies daratumumab or isatuximab was found more efficient than that by SLAMF7 antibody elotuzumab. This may be related to the significantly higher CD38 than SLAMF7 expression at the myeloma cell surface. Our findings demonstrate that ADCP of approved IgG antibodies targeting CD38 or SLAMF7 can be enhanced by blocking the CD47-SIRPα axis and this may depend on the particular malignant plasma cell phenotype. The inhibition of this myeloid 'don't eat me' signal with a CD47 blocking antibody may open a new avenue for powerful myeloma immunotherapy. Since combination treatments with proteasome inhibitors and IMiDs are commonly used, these interactions also require attention. Initial data indicate that pre-treatment of myeloma cells with proteasome inhibitor carfilzomib did not negatively impact improvement of ADCP by blocking the CD47-SIRPα axis in responsive cell lines. Taken together, particularly CD38-targeting antibodies may have a significant potential to further improve immunotherapy in multiple myeloma patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Role for Syntenin-1 in Multiple Myeloma Cell Survival

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1008-1008
Author(s):  
Tyler Moser-Katz ◽  
Catherine M. Gavile ◽  
Benjamin G Barwick ◽  
Sagar Lonial ◽  
Lawrence H. Boise
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Death ◽  
Cell Survival ◽  
Cell Lines ◽  
Plasma Cells ◽  
Surface Expression ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Rpmi 8226

Abstract Multiple myeloma is the second most common hematological malignancy in the U.S. with an estimated 30,700 new diagnoses in 2018. It is a clonal disease of plasma cells that, despite recent therapeutic advances, remains incurable. Myeloma cells retain numerous characteristics of normal plasma cells including reliance on survival signals in the bone marrow for long term viability. However, malignant transformation of plasma cells imparts the ability to proliferate, causing harmful bone lesions in patients, and in advanced stages independence of the bone-marrow microenvironment. Therefore, we are investigating the molecular mechanisms of myeloma cell survival that allow them to become extramedullary. We identified syntenin-1 (SDCBP) as a protein involved in myeloma cell survival and a potential therapeutic target. Syntenin-1 is an adapter protein that has been shown to regulate surface expression of several transmembrane proteins by binding with membrane phospholipids and mediating vesicular trafficking of proteins throughout the cell. Syntenin-1 regulates the surface expression of CD138, a plasma/myeloma cell marker. Syntenin-1 has been shown to regulate apoptosis in numerous cancer cell lines including breast cancer, glioma, and pancreatic cancer but its role in multiple myeloma survival has not been studied. To determine if syntenin-1 expression has an effect on myeloma cell survival, we utilized the CoMMpass dataset (IA12), a longitudinal study of myeloma patients that includes transcriptomic analysis throughout treatment. We found that patients with the highest expression of syntenin-1 mRNA (top quartile) had significantly worse overall survival, progression-free survival, and a shorter response duration than those in the bottom quartile of expression. To determine if syntenin-1 has a role in myeloma cell survival, we used short hairpin RNA to knock down syntenin-1 (shsyn) in RPMI 8226 and MM1.s myeloma cell lines. We then determined the amount of cell death using Annexin-V staining flow cytometry four days following lentiviral infection. We found increased cell death in syntenin-1-silenced cells compared to our empty vector control in both RPMI 8226 (control=42.17%, shsyn=71.53%, p=0.04) and MM1.s cell lines (control=8.57%, shsyn=29.9%, p=0.04) suggesting that syntenin-1 is important for myeloma cell survival. Syntenin-1 contains two PDZ domains that allow it to bind to receptor proteins via their corresponding PDZ-binding motifs. We therefore wanted to look at correlation of syntenin-1 expression with CD138 and CD86, two PDZ-binding domain containing proteins expressed on the surface of myeloma cells. Using the CoMMpass dataset, we found patients with high expression of syntenin-1 had a median expression of CD86 that was twice as high as the total population (P<0.0001) while syntenin-1-low patients expressed CD86 at levels that were half as much as the population (P<0.0001). In contrast, there was no clear relationship between syntenin-1 and CD138 mRNA expression. Indeed if one takes into account all patients, there is a positive correlation between CD86 and syntenin-1 expression (r=0.228, P<0.0001) while there is a negative correlation between CD138 and syntenin-1 (r=-0.1923, P<0.0001). The correlation with CD86 but not CD138 suggests a previously undescribed role for syntenin-1 in myeloma cells. Our lab has previously shown that expression of CD86 is necessary for myeloma cell survival, and signals via its cytoplasmic domain to confer drug resistance. Silencing syntenin-1 results in a decrease in CD86 surface expression. However, there is no change in CD86 transcript or total cellular CD86 protein levels in our shsyn treated cells. Moreover, knockdown of CD86 resulted in increased protein expression and transcript levels of syntenin-1. Taken together, these data suggest that syntenin-1 may regulate CD86 expression on the cell surface. Our data supports a novel role for syntenin-1 in myeloma cell viability and as a potential regulator of CD86 surface expression. The role of syntenin-1 has not previously been explored in multiple myeloma and determining its molecular function is warranted as it may be an attractive target for therapeutic treatment of the disease. Disclosures Lonial: Amgen: Research Funding. Boise:AstraZeneca: Honoraria; Abbvie: Consultancy.

Mcl-1 Is Overexpressed in Multiple Myeloma and Correlates with Disease Severity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1419-1419
Author(s):  
Soraya Wuilleme-Toumi ◽  
Nelly Robillard ◽  
Patricia Gomez-Bougie ◽  
Philippe Moreau ◽  
Steven Le Gouill ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Disease Severity ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Lymphocytic Leukemia ◽  
Myeloma Cells

Abstract Multiple Myeloma (MM) is a fatal malignancy of B-cell origin characterized by the accumulation of plasma cells within the bone marrow. The expression of the pro-survival members of the Bcl-2 family has been shown to be a key process in the survival of myeloma cells. More particularly, Mcl-1 expression turned out to be critical for their survival. Indeed, knockdown of Mcl-1 by antisenses induces apoptosis in myeloma cells. Finally, Mcl-1 was found to be the only anti-apoptotic Bcl-2 family member which level of expression was modified by cytokine treatment of myeloma cells. For these reasons, we have evaluated the expression of Mcl-1 in vivo in normal, reactive and malignant plasma cells (PC) i.e., myeloma cells from 55 patients with MM and 20 human myeloma cell lines using flow cytometry. We show that Mcl-1 is overexpressed in MM in comparison with normal bone marrow PC. Forty-seven percent of patients with MM at diagnosis (p=.017) and 80% at relapse (p=.014 for comparison with diagnosis) overexpress Mcl-1. Of note, only myeloma cell lines but not reactive plasmocytoses have abnormal Mcl-1 expression, although both plasmocyte expansion entities share similar high proliferation rates (&gt;20%). Of interest, Bcl-2 as opposed to Mcl-1, does not discriminate malignant from normal PC. This shows that the overexpression of Mcl-1 is clearly related to malignancy rather than to proliferation. It will be important to know whether the overexpression of Mcl-1 is related to an abnormal response to cytokines like Interleukin-6 or to mutations of the promoter of the Mcl-1 gene as already described in B chronic lymphocytic leukemia. Finally, level of Mcl-1 expression is related to disease severity, the highest values being correlated with the shortest event-free survival (p=.01). In conclusion, Mcl-1 which has been shown to be essential for the survival of human myeloma cells in vitro is overexpressed in vivo in MM and correlates with disease severity. Mcl-1 represents a major therapeutical target in MM.

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

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5045-5045
Author(s):  
Philipp Baumann ◽  
Sonja Mandl-Weber ◽  
Bertold Emmerich ◽  
Christian Straka ◽  
Daniel Franke ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Adenosine Kinase ◽  
Intracellular Signalling ◽  
Phase Cell ◽  
Myeloma Cells ◽  
Multiple Myeloma Cell

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

Survival Signals, Growth Cytokines, Immunosuppressive Factors and Their Cellular Perpetrators in the Myeloma Tumor Microenvironment.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1664-1664
Author(s):  
Jayakumar R Nair ◽  
Louise M Carlson ◽  
Noreen Ersing ◽  
Asher Alban Chanan-Khan ◽  
Kelvin P. Lee
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Microenvironment ◽  
Cell Lines ◽  
Plasma Cells ◽  
Immune Escape ◽  
Human Monocyte ◽  
Fold Increase ◽  
Myeloma Cell ◽  
Myeloma Cells

Abstract Multiple myeloma (MM) is an incurable neoplasia of terminally differentiated plasma cells in the bone marrow. Essential interactions of MM cells with host bone marrow stromal cells (BMSC) induce growth factors essential for MM progression and pathogenesis, as well as induce an immunosuppressive environment that inhibits endogenous and therapeutically-induced immune responses against the MM cells. However, despite their importance, little is known about the identity of these BMSC cells or the molecular basis of their interaction with myeloma cells. A potential MM surface protein that could be involved in these interactions is CD28, based on its known pro-survival role in T cells. Clinical studies have shown that expression of CD28 in multiple myeloma highly correlates (p=0.006) with myeloma disease progression. Moreover, CD28+ MM cells invariably express the CD28 ligand CD86. A survival role for MM-CD28 might involve interactions with cellular partners that express the B7 (CD80/CD86) ligands. Potential candidates would include CD86+ myeloma cells themselves or B7+ dendritic cells (DC) that are known to be closely associated with myeloma cells in the patient bone marrow. When myeloma-myeloma interactions were disrupted by using the high affinity CD80/CD86 blocker CTLA4Ig (Abatacept®), increased sensitivity to arsenic trioxide (ATO) and melphalan (MEL) was observed in all the three MM cell lines U266, RPMI8226 and MM1S. For U266 viability was 93% in media alone, 84% with CTLA4Ig (100 μg/ml) alone, 86% with 2 μM ATO alone and was significantly reduced to 36% with CTLA4Ig + ATO. Similar drops in viability were observed with 25 μM MEL in combination with CTLA4Ig (33% as opposed to 71–74 % with CTLA4Ig or MEL alone). Our data suggests that this does not involve the downregulation of anti-apoptotic proteins Bcl-2, Bcl-xL or Mcl-1, commonly associated with drug resistance in myeloma. In the second part of the study, we demonstrate that myeloma cell lines or primary CD138+ myeloma cells can enhance via direct contact the ability of human monocyte derived immature DC to produce the immunosuppressive tryptophan depleting enzyme indoleamine 2,3 dioxygenase (IDO, as estimated by kynurenine (Kyn) (a tryptophan catabolite) levels in the supernatant) and also the pro-plasma cell survival cytokine IL-6. In co-cultures of IFNg treated immature DCs with either MM cell lines or with primary CD138+ myeloma cells from patient BM aspirates, the activity of IDO was enhanced ~ 2–8 fold (81 mM kyn with U266 and 20–43mM with primary cells) over that observed in control IFNg-treated DCs (9.7 mM Kyn). Western analysis also demonstrated increased IDO expression relative to IFNg activated DC controls. Blocking MM-CD28 with (Fab)2 fragments of anti-hCD28 mAb 9.3 downregulated IDO activity (9.3 mM) close to that of control, demonstrating the involvement of MM-CD28 in these interactions. We also demonstrated a significant up-regulation of the pro-myeloma survival cytokine IL-6 when immature DCs were co-cultured with CD28+ MM1S (90–300 pg/ml), a 4–9 fold increase over that of DC only control (25 – 35 pg/ml). This was further enhanced when immature DCs cultured with IL-10 (+ GM-CSF + IL-4) was used in co-cultures with MM-1S (800 – 1300 pg/ml), or with primary CD138+ myeloma cells from patient bone marrow aspirates (128–1142 pg/ml). In conclusion, our data demonstrates that blocking myeloma-CD28 - myeloma-CD86 “autocrine” interaction can enhance drug cytotoxicity, while interactions with DCs produce the essential growth cytokines IL-6 and immunosuppressive enzyme IDO with potential implications in MM survival and immune escape. Use of clinically approved agents (e.g. Abatacept®) to block myeloma-CD28 binding to its B7 ligands (increase chemotherapeutic efficacy), 1-MT to inhibit IDO and targeting DCs in the microenvironment to disrupt the tumor microenvironment could be viable therapeutic strategies for the future treatment of multiple myeloma.

Triggering of Toll-Like Receptor-4 In Human Multiple Myeloma Cells Promotes Proliferation and Alters Cell Responses to Immune and Chemotherapy Drug Attack

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1905-1905
Author(s):  
Zhen Cai ◽  
Hanying Bao ◽  
Peilin Lu ◽  
Lijuan Wang ◽  
Donghua He ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Response To Chemotherapy ◽  
Tlr4 Ligand ◽  
Induced Apoptosis

Abstract Abstract 1905 Multiple myeloma (MM) is a fatal plasma cell malignancy mainly localized in the bone marrow. The clonal expansion of tumor cells is associated with the disappearance of normal plasma cells and with a marked depression in the production of normal immunoglobulin (Ig). This makes MM patients highly vulnerable to bacterial, fungal and viral infections and recurrent infections remain to be a major cause of death in MM patients. It has been shown that most primary myeloma cells and cell lines express multiple Toll-like receptors (TLRs). Among them, TLR4 is most frequently expressed. To investigate TLR-initiated responses in MM cells including proliferation, anti-apoptosis and immune escape, we first screened four commonly used human myeloma cell line (HMCL) for the expression of major TLRs by RT-PCR. Surprisingly, all the HMCL expressed multiple TLRs. We also examined primary myeloma cells from 4 patients with MM and our results showed that TLR4 was expressed by all the tumor cells. We incubated myeloma cells with LPS, the natural ligand for TLR4, and found that cell proliferation increased significantly. Targeting TLRs on malignant B cells can induce resistance to chemotherapeutic agents but can also be exploited for combined therapeutic approaches. As mechanisms involved in the resistance to apoptosis play a major role in MM escape to therapies, we sought to determine the capacity of TLR4 ligand to promote the survival of HMCL cells. Myeloma cells were pretreated for four hours with LPS before being induced apoptosis by adriamycin. Results showed that LPS pretreatment partially protected the cells from adriamycin-induced apoptosis. The TLR signaling pathway activates several signaling elements, including NF-kB and ERK/JNK/p38 MAPKs, which regulate many immunologically relevant proteins. Time-dependent MAPK phosphorylation was measured to assess the activation of these kinases upon treatment with LPS in cell lines. ERK1/2, p38, and JNK phosphorylation and NF-kB were significantly up-regulated following LPS treatment. Moreover, our findings demonstrated that LPS-induced cell proliferation was dependent on JNK, ERK and p38 signaling. IL-18, a recently described member of the IL-1 cytokine superfamily, is now recognized as an important regulator of innate and acquired immune responses. In this study, we found that LPS induced IL-18 secretion and activated MAPK and NF-kB signaling simultaneously. Therefore, our results suggest that activation of the MAPK signaling and secretion of IL-18 are interconnected. Tumors evade immune surveillance by multiple mechanisms, including the production of factors such as TGF-β and VEGF, which inhibit and impair tumor-specific T cell immunity. Our study also showed that T cell proliferation induced by allostimulatory cells decreased when the HMCL were pre-treated with LPS. Moreover, immunoregulatory molecules on HMCL, such as B7-H1, B7-H2 and CD40, were upregulated after treatment with LPS, suggesting that TLR4 ligand LPS facilitates tumor cell evasion of the immune system. Our results show that TLRs are functional on myeloma tumor cells, and the ligands to these TLRs have a functional role in affecting myeloma cell proliferation, survival, and response to chemotherapy and immune attacks. Disclosures: No relevant conflicts of interest to declare.

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