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.

Identification and Hierarchy of Clonogenic Factors for Human Myeloma Cells: IL-6 for CD45+ Versus IL-6 and Growth Factors for CD45-.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3407-3407
Author(s):  
Martine Amiot ◽  
Regis Bataille ◽  
Madeleine Collette ◽  
Geraldine Descamps ◽  
Catherine Pellat-deceunynck
Keyword(s):  
Growth Factors ◽  
Cell Lines ◽  
Plasma Cells ◽  
Treatment Strategies ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Growth And Survival ◽  
Essential Growth ◽  
Cell Malignancy ◽  
Clonogenic Cells

Abstract Multiple Myeloma (MM) is a fatal plasma-cell malignancy characterized by the accumulation of malignant plasma cells within the bone marrow. IL-6 and IGF-1 are known to be essential growth and survival factors in this malignancy. Beside these well characterized growth factors, other growth factors such as HGF, HB-EGF and FGF have been involved in this malignancy. Even though all of them seem to be involved in myeloma cell proliferation, the hierarchy of each growth factor remains to be established. In the present study, a serum-free cytokine-free and collagen-based assay which does not allow the generation of endogeneous myeloma colonies was used to identify the clonogenic factors of fourteen myeloma cell lines. We selected seven myeloma cell lines expressing CD45 on 100% of cells and seven other cell lines lacking CD45 expression. IL-6 is the only clonogenic factor able to stimulate both CD45+ and CD45- myeloma cell lines, generating myeloma colonies from 10 of 14 myeloma cell lines. For some cell lines (LP-1, L363 and XG-2) the percentage of clonogenic cells reached 40% to 50% indicating that clonogenic cells are CD138+ and do not represent a particular CD138-subpopulation. In contrast, the other growth factors (IGF-1, FGF, HGF and HB-EGF) stimulate only some CD45-myeloma cell lines and at a less extend than IL-6. Among them, IGF-1 is the most potent, generating myeloma colonies from 5 of 8 CD45- myeloma cell lines. We searched for a correlation between myeloma-colony formation and the signaling pathway induced by IGF-1 and FGF in MM cells. It appears that activation of both the PI3Kinase and ERK pathway by IGF-1 and FGF fully correlates with their capacity to stimulate the clonogenicity of CD45-myeloma cell lines. In conclusion, the CD45 phenotype of myeloma cells discriminates their signaling and proliferative response to IL-6 and growth factors. These results give a strong rational to the stratification of prognostic value discriminating CD45+ from CD45- MM. Furthermore, treatment strategies for CD45- patients should combined the disruption of signaling induced by both IL-6 and IGF-1 (or other growth factors) whereas CD45+ patients could be targeted through IL-6 only.

Preclinical activity of the JAK1/2 inhibitor ruxolitinib on malignant plasma cell growth and survival.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e18565-e18565 ◽  
Author(s):  
Renate Burger ◽  
Tim Bugdahn ◽  
Matthias Staudinger ◽  
Matthias Peipp ◽  
Andreas Günther ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Cell Lines ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Thymidine Uptake ◽  
Jak Inhibitor ◽  
Growth And Survival ◽  
Stat3 Phosphorylation

e18565 Background: In multiple myeloma, cytokines in the tumor environment, in particular interleukin-6 (IL-6), support the growth and survival of malignant plasma cells. Binding of IL-6 to its receptor leads to gp130 dimerization and activation of JAKs and STAT3. Ruxolitinib (INC424)is the first small molecule JAK inhibitor approved for the treatment of patients with myelofibrosis. The aim of our study was to evaluate the effects of ruxolitinib on malignant plasma cells. Methods: Cell growth was studied in seven myeloma cell lines including the IL-6 dependent INA-6. Ruxolitinib was tested at 0.0625 µmol/L to 8 µmol/L. Proliferation of plasma cell enriched patient samples was measured by [3]H-thymidine uptake, apoptosis by flow cytometry upon annexin V/7-AAD staining. Levels of STAT3 and ERK1/2 phosphorylation were determined by Westernblot analysis. IC50 concentrations and combination index were calculated with CalcuSyn. IL-6 levels were determined by ELISA. Results: A significant inhibition of plasma cell growth with ruxolitinib was achieved in IL-6 dependent INA-6 cells (IC50 0.22 µmol/L). Complete growth inhibition at 1 µmol/L was seen in the absence and presence of bone marrow stromal cells. Stromal cell viability and IL-6 production were not affected. The number of apoptotic INA-6 cells upon treatment with ruxolitinib at 1 µmol/L increased 3.6- and 7.2-fold (after 48 and 72 hours, respectively), consistent with the reduction of IL-6 induced STAT3 phosphorylation. A similar strong inhibitory activity of ruxolitinib (IC50 0.16 µmol/L) was observed in tumor cells of a patient with plasma cell leukemia proliferating in response to IL-6. In contrast, none of the myeloma cell lines that grow autonomously were sensitive, pointing to the kinase specificity of the drug. Using INA-6 as a model, combinations with other signaling inhibitors revealed additive to synergistic effects with PI3K, mToR and IGF-1R inhibitors. Conclusions: In multiple myeloma, ruxolitinib has a strong cytotoxic activity against malignant plasma cells that require IL-6 for growth and survival. This warrants further clinical testing but also points to the need of identifying molecular markers to predict benefit from JAK inhibitor treatment.

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.

Amphiregulin Is Produced by Myeloma Cells and Is Involved in Tumor-Environment Interactions in Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4306-4306
Author(s):  
Karène Mahtouk ◽  
Dirk Hose ◽  
Thierry Reme ◽  
John De Vos ◽  
Michel Jourdan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Growth Factors ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Erbb Receptors ◽  
Heparin Binding ◽  
Myeloma Cells ◽  
Heparin Binding Growth Factors ◽  
Egf Family

Abstract Multiple myeloma (MM) is characterized by the accumulation of clonal malignant plasma cells in the bone marrow. One of the hallmarks of plasma cells is the expression of the heparan-sulfate proteoglycan syndecan-1. In epithelial cells, syndecan-1 plays a major role as a coreceptor for heparin-binding growth factors and chemokines. This stresses that heparin-binding growth factors may play a major role in the biology of MM cells. Recently we have demonstrated that heparin-binding EGF-like growth factor (HB-EGF), one of the ten members of the Epidermal Growth Factor (EGF) family, is produced by the tumor microenvironment and is able to trigger myeloma cell growth. As amphiregulin (AREG) is another member of the EGF family that also binds heparan-sulphate chains, we investigated its role in MM. We looked for AREG expression on a panel of 7 normal plasmablastic cells (PPCs), 7 normal bone marrow plasma cells (BMPCs), purified MM cells from 65 patients and 20 myeloma cell lines (HMCLs), with Affymetrix U133A+B microarrays. We showed that primary MM cells overexpress AREG compared to normal BMPCs and PPCs. We then investigated the expression of the ErbB receptors with real-time RT-PCR. Myeloma cells variably expressed the 4 ErbB receptors. Normal BMPCs also expressed ErbB1 and ErbB2 unlike PPCs that did not express any ErbB receptors. We demonstrated that the high AREG expression by primary myeloma cells may have a dual effect. On the one hand, AREG stimulated IL-6 production and growth of bone-marrow stromal cells that highly express the AREG ErbB1 receptor. On the other hand, AREG could promote HMCL proliferation, suggesting that a functional autocrine loop involving AREG and ErbB receptors is involved in MM cell growth. Finally, we looked for the effect of ErbB inhibitors on MM cells of 14 patients cultured for 6 days together with their bone marrow environment. A pan-ErbB inhibitor (PD-169540, Pfizer) and an ErbB1-inhibitor (IRESSA, Astrazeneca) induced strong MM cell apoptosis in respectively 71% of patients (10 of 14) and 29% of patients (4 of 14). Of major interest, when PD169540 or IRESSA were combined with dexamethasone, they induced a dramatic myeloma cell death (respectively 92% and 69% inhibition of MM cell survival), while non-myeloma cells were unaffected. Thus ErbB activation is critical to trigger MM-cell survival in short-term culture. In conclusion, our findings provide evidence for a major role of AREG and HB-EGF in the biology of multiple myeloma and identify ErbB receptors as putative therapeutic targets. These data emphasize the interest of clinical evaluation of specific-ErbB-inhibitors in patients with MM, either used alone or in combination with dexamethasone.

CD28 and CD86 Are Necessary for Myeloma Cell Survival.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2946-2946
Author(s):  
Catherine M Gavile ◽  
Jayakumar R Nair ◽  
Kelvin P Lee ◽  
Sagar Lonial ◽  
Lawrence H. Boise
Keyword(s):  
T Cells ◽  
Growth Factor ◽  
Cell Survival ◽  
Cell Line ◽  
Cell Lines ◽  
Plasma Cells ◽  
Surface Expression ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Growth And Survival

Abstract Abstract 2946 Multiple myeloma (MM) is a hematologic malignancy characterized by the aberrant proliferation of plasma cells. Myeloma cells retain most of the physiological characteristics of their normal counterpart – the long-lived plasma cell. Myeloma cells secrete immunoglobulin and reside in the bone marrow, where they rely heavily on interactions with the stroma for survival signals. While recent advances in therapeutics have led to an increase in median survival post-diagnosis, the disease remains incurable. Understanding the pathways which mediate growth and survival of these cells will help in identifying new targets that can potentially further improve patient outcomes. CD28 is a receptor better known for its role in T-cell signaling through interaction with its ligands, CD80 or CD86. Interaction between CD28 on T-cells and CD80/86 on antigen-presenting cells leads to survival and proliferation of T-cells. Recent work has shown that the CD80/86-CD28 pathway also plays an important role in normal plasma cell generation and survival. Interestingly, high expression of CD28 and CD86 are poor prognostic markers for myeloma patients. Previous work has shown that CD28 activation provides survival signals for myeloma cells in growth-factor deficient conditions. It has also been shown that CD28 on the myeloma cell interacts with CD80/86 on the dendritic cell, which induces secretion of IL-6 (by the DC), an important myeloma growth factor. However, it is not known if CD28 or CD86 play a role in steady state growth and survival of myeloma cells. In order to determine the role of each of these 2 molecules in myeloma physiology, we knocked-down either CD28 or CD86 on the myeloma cell via lentivirus-mediated shRNAs. We found that knockdown of CD86 leads to apoptosis in 3 myeloma cell lines (RPMI8226, MM1.s, and KMS18). Four days after infection with the lentivirus containing shCD86, 45.7±4.9 and 60.3±4.6 percent control apoptosis was observed in RPMI8226 and MM1.s respectively, while less death was observed in KMS18 (17.6±1.6). CD28-knockdown resulted in apoptosis as well (24.9±4.3 for RPMI8226, 26.8±4.1 for MM1s, 21.8±3.8 for KMS18, percent control apoptosis). Consistent with these findings, we were unable to establish a myeloma cell line with stable knockdown of either CD28 or CD86. Additionally, RPMI8226 cells stably transfected to over-express either Bcl-2, Bcl-xL, or Mcl-1 are protected from cell death induced by CD86 or CD28 silencing. These data suggest that CD28 and CD86 are essential to prevent apoptosis of myeloma cells in vitro. To confirm these findings we determined the effects of CTLA4-Ig on myeloma survival. CTLA4-Ig inhibits CD86-CD28 signaling by binding to CD86, blocking its interaction with CD28. We found that treatment of RPMI8226 and MM1.s cells with CTLA4-Ig caused apoptosis in the myeloma cells after 2 days (23.9±3.9 for RPMI8226 and 20.4±6.2 for MM1.s, percent control apoptosis). Thus like normal plasma cells, CD28 and CD86 are required for the survival of myeloma cells. To determine why silencing of CD86 has a more potent effect than CD28 silencing on myeloma cell survival in 2 out of 3 cell lines, we investigated the effects of silencing on cell surface expression of each of these proteins. CD28 and CD86 mRNA and protein levels were silenced to similar levels by their cognate hairpins. However, in MM.1s and RPMI8226 we found that silencing of CD28 resulted in an increase in CD86 surface expression. This increase was also observed at the mRNA level and in the cells over-expressing Bcl-2 family members, indicating that this is not simply due to the selection of the highest expressing cells. These data suggest a feedback loop exists to regulate CD28-CD86 signaling in myeloma cells. Surprisingly, in the KMS18 cell line, we observe the converse effect, where silencing of CD86 resulted in upregulation of CD28. This provides a likely explanation for why these cells are less susceptible to CD86 silencing than the other two lines. Interestingly, blocking CD86 with CTLA4-Ig treatment also resulted in a modest upregulation in CD28 surface expression of MM.1s and RPMI8226, which suggests that silencing CD86 and binding of CD86 with a soluble receptor are not equivalent, and that multiple signaling feedback pathways exist to regulate the expression of this receptor-ligand pair that is necessary for myeloma cell survival. 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.

scholarly journals De Novo Nethyltransferases, DNMT3a and DNMT3b Are Underexpressed in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4818-4818 ◽  
Author(s):  
Pavla Latalova ◽  
Jiri Minarik ◽  
Katerina Smesny Trtkova
Keyword(s):  
Dna Methylation ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Plasma Cells ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Dna Methyltransferases ◽  
Rt Pcr ◽  
Cpg Dinucleotides

Abstract Background and aims: Presently, there is growing evidence that along with the important role of genetic abnormalities, epigenetic aberrations are relevant factors in multiple myeloma (MM). As was recently found, genome-wide analysis of DNA methylation reveals epigenetic alterations in plasma cells from patients with MM and individuals with monoclonal gammopathy of undetermined significance (MGUS). MGUS is characterized by predominant hypomethylation. Transformation into MM is accompanied by progressive hypermethylation with maximum methylation seen in relapsed disease. DNA methyltransferases (DNMTs) catalyze DNA methylation through transfer of methyl group to cytosine of the CpG dinucleotides, resulting in 5-methylcytostine. DNMT1 maintains patterns of methylated cytosine residues in human genome. DNMT3A and DNMT3B are de novo DNA methyltransferases, whose role is to maintain new methylation pattern that forms due to formation of the cancer. Methods: 30 bone-marrow aspirates from individuals with MGUS or MM patients before the treatment initiation were used. The cDNA was synthesized using 100 ng of total RNA in a 20 µl reaction volume (Roche, Diagnostics, Basel, Switzerland). Quantification of DNMT1, DNMT3a and DNMT3b levels by TaqMan® probes (Life Technologies, Grand Island, NY) with Xceed qPCR Master Mix (IAB, BioTech-Europe, Czech Republic) was performed. For normalization, the GAPDH was used. Results: Although MM is characterized by widespread alterations in DNA methylation, we observed that DNMT3a and DNMT3b de novo methyltransferases were underexpressed in both, MGUS individuals and MM patients when compared to DNMT1 expression level (Figure 1). The transcribed genes have increased levels of 5-hydroxymethylcytosine, then the DNMTs activities might compensate for active hydroxymethylation - demethylation. Conclusions: Our results confirm that the expression of de novo DNA methyltransferases is deregulated in MM cell lines. The presented analysis is first of its kind that was performed on human myeloma cell lines, especially with the focus on the residual expression of Dnmt3a. With support of the grant NT14393. Figure 1. Quantitative RT-PCR for DNMT1, DNMT3a and DNMT3b in MGUS individuals and MM patients. Figure 1. Quantitative RT-PCR for DNMT1, DNMT3a and DNMT3b in MGUS individuals and MM patients. Disclosures No relevant conflicts of interest to declare.

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.

Binding of TACI and APRIL to Syndecan-1 Confer on Them a Major Role in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3522-3522
Author(s):  
Jerome Moreaux ◽  
Anne-Catherine Sprynski ◽  
Karene Mahtouk ◽  
Philippe Moine ◽  
Nicolas Robert ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factors ◽  
Heparan Sulfate ◽  
Plasma Cells ◽  
Specific Binding ◽  
Myeloma Cell ◽  
Matrix Proteins ◽  
Myeloma Cells ◽  
Cellular Processes ◽  
Binding Partners

Abstract B-cell activating factor (BAFF) and A Proliferation-Inducing Ligand (APRIL) are produced by the bone marrow microenvironment of patients with multiple myeloma (MM) and are growth factors of multiple myeloma cells (MMC). BAFF and APRIL share two receptors - TACI and BCMA - and BAFF binds to a third receptor, BAFF-R. We previously reported that TACI expression is a good indicator of a BAFF-binding receptor in human myeloma cell lines (HMCL), unlike BCMA that is expressed on all HMCL. BAFF-R is lacking. More recently, Ingold et al (J Exp Med; 2005) and Hendriks at al (Cell Death Differ; 2005) identified proteoglycans as the APRIL-specific binding partners. Bischof et al (Blood; 2006) demonstrated that TACI binds also heparan sulfate (HS) chains, in particular to syndecan-1, syndecan-2 and syndecan-4. Syndecan-1 is expressed by plasma cells and epithelial cells and is involved in several cellular processes relying on interactions with extra-cellular matrix proteins, growth factors, chemokines and adhesion molecules. We found a very large binding of APRIL (mean fluorescence activity ≥ 500), unlike BAFF, at the surface of all syndecan-1+ HMCL. These syndecan-1+ HMCL also highly bound TACI-Fc molecules, unlike BCMA-Fc or BAFF-R-Fc molecules. This large binding of APRIL or TACI-Fc molecules was abrogated by heparitinase pretreatment of MMC, removing the HS chains or by preincubation of APRIL and TACI-Fc with heparin. These data were extended to patients primary myeloma cells. In agreement with this large binding to MMC, APRIL was 15 to 40 fold more efficient than BAFF to stimulate the growth of HMCL. The APRIL growth factor activity could be inhibited using heparin, unlike that of BAFF. Our data establish that syndecan-1, by concentrating large levels of APRIL and TACI at MMC surface, can promote APRIL/TACI signaling that induces survival and proliferation of MMC. Heparan sulfate chain inhibitors could be helpful to synergize with BAFF/APRIL inhibitors in MM disease.

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.

Sign in / Sign up

Export Citation Format

Share Document