Molecular Characterization of Human Multiple Myeloma Cell Lines by Genome-Wide Profiling.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1793-1793
Author(s):  
Carolina Elosua ◽  
Purificacion Catalina ◽  
Cristina Palma ◽  
Brian A Walker ◽  
Nicholas J Dickens ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Uniparental Disomy ◽  
Myeloma Cell ◽  
Molecular Pathogenesis ◽  
Gene Localization ◽  
Multiple Myeloma Cell

Abstract Abstract 1793 Poster Board I-819 Multiple Myeloma (MM) is a malignancy depicted by clonal expansion of plasma cells in the bone marrow. There are two broad genetic subtypes of multiple myeloma as defined as hyperdiploid multiple myeloma (H-MM), characterized by trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19, and 21, and nonhyperdiploid multiple myeloma (NH-MM) associated with primary translocations involving the immunoglobulin heavy chain (IgH). These two subtypes of multiple myeloma have two different molecular pathogenesis and characteristic changes of each have been already observed. In our study, we previously described the patterns of genetic lesions and molecular pathogenesis of 12 HMCLs with Affymetrix 500K Single Nucleotide Polymorphism-based mapping arrays, now we re-depict those patterns using the Illumina 1M array set and compare the two studies. These techniques allow the examination and identification of copy number changes, bi-allelic deletions and the identification of loss of heterozygosity (LOH) due to loss and uniparental disomy (UPD), as well as gene localization and identification. The 12 HMCLs utilized are characterized for their structural alterations and not by hyperdiploidy. In addition, so as to fulfill the selection criteria, a minimum of 3 cell lines must present the alterations cited below. Previously described gains were observed in 1q, 7q, 8, 11q, 18, 19, and 20q; but also found at 4q. The bi-allelic deletions were ascertained on 3p. Similarly, we identified the regions of mono-allelic deletions on 1, 2q, 6q, 8q, 9p, 11q, 12, 13q, 14q, 17p, and 20p. In addition, described regions of bi-allelic deletions were detected on 1p, 6q, 8p, 13q, 16q, and 22q, and furthermore located on 2q, 3, 4q, 9, 10q, 12p, and 20p. Finally, the UPD obtained were traced on 1q, 4q, 8q, 10q, and 22q. The use of the new platform has allowed us to re-identify, significantly increase in information above the original set and finely delimit the regions previously described. Taken together, the dysregulated genes from the myeloma genome indicate that the crucial pathways in myeloma include NF-kB, apoptosis, cell-cycle and critical intracellular signaling pathways including the JAK/STAT, Wnt signaling, RAS/RAF/MAPK and PI3k/AKT. Disclosures No relevant conflicts of interest to declare.

Molecular Characterization of Human Multiple Myeloma Cell Lines by Genome-Wide Profiling Identifies Kinase Pathway Alterations.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1694-1694
Author(s):  
Carolina Elosua ◽  
Purificacion Catalina ◽  
Brian A Walker ◽  
Nicholas J Dickens ◽  
Athanasia Avramidou ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Myeloma Cell ◽  
Molecular Pathogenesis ◽  
Gene Localization ◽  
Cycle Progression ◽  
Multiple Myeloma Cell ◽  
Human Multiple Myeloma

Abstract Multiple Myeloma (MM) is a malignancy depicted by clonal expansion of plasma cells in the bone marrow. There are two broad genetic subtypes of multiple myeloma as defined as hyperdiploid multiple myeloma (H-MM), characterized by trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19, and 21, and nonhyperdiploid multiple myeloma (NH-MM) associated with primary translocations involving the immunoglobulin heavy chain (IgH). These two subtypes of multiple myeloma have two different molecular pathogenesis given that characteristic changes of each have been already observed. In order to contribute to the understanding of this malignancy and to unveil the different molecular pathogenesis, our interest is focused on Human Multiple Myeloma Cell lines (HMCLs), as a model, and a broad but specific group of enzymatic proteins: the Kinases. Kinase hyperactivity or lack of it often results in disregulation of cellular pathways involved in proliferation and survival. In our study, we describe the patterns of genetic lesions and molecular pathogenesis of 11 HMCLs with Single Nucleotide Polymorphism (SNP)-based mapping arrays from Affymetrix Human Mapping 500K array set. This technique allows the examination and identification of copy number changes, bi-allelic deletions and the identification of loss of heterozygosity (LOH) due to loss and uniparental disomy, as well as gene localization and identification. The 11 HMCLs utilized are characterized for their structural alterations and not by hyperdiploidy. In addition, so as to fulfill the selection criteria, a minimum of 3 cell lines must present the alterations cited below. The most frequently identified alterations were located as follows: Previously described gains were observed in 1q, 7q, 8, 11q, 18, 19, and 20q; but also found at 4q. The bi-allelic deletions were ascertained on 3p. Similarly, we identified the regions of hemizygotic deletions on 1, 2q, 6q, 8q, 9p, 11q, 12, 13q, 14q, 17p, and 20p. In addition, described regions of homozygotic deletions were detected on 1p, 6q, 8p, 13q, 16q, and 22q, and furthermore located on 2q, 3, 4q, 9, 10q, 12p, and 20p. Finally, the uniparental disomies (UPDs) obtained were traced on 1q, 4q, 8q, 10q, and 22q. These identified alterations are affecting a series of enzymatic genes belonging to targeted pathways. Within the chromosomes 1, 10, 11, 14, and 16 we have localized kinases that are part of the PI3K/AKT pathway, which affect to a number of intracellular and extracellular myeloma growth cytokines. In the chromosomes 1, 6, 12, and 19 we identified a series of Cyclin-Dependent Kinases that are critical regulators of cell cycle progression and RNA transcription, since they regulate and control the cyclins, cell cycle regulatory proteins, which can provoke dysregulation and abnormally accelerated cell cycle progression. And finally on chromosomes 1, 2, 14, 21, and 22 we observed certain Aurora and related kinases, as another family of the cell cycle regulators and often aberrantly activated in human tumor cells, they facilitate transit from G2 through cytokinesis. These mutated kinases may be potential targets for therapeutics. Our data demonstrates the genomic complexity of multiple myeloma enhancing our understanding of the molecular pathogenesis of the disease and the importance of the HMCLs as a model.

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.

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.

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.

BI 836909, a Novel Bispecific T Cell Engager for the Treatment of Multiple Myeloma Induces Highly Specific and Efficacious Lysis of Multiple Myeloma Cells in Vitro and Shows Anti-Tumor Activity in Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2999-2999 ◽  
Author(s):  
Susanne Hipp ◽  
Petra Deegen ◽  
Joachim Wahl ◽  
Diann Blanset ◽  
Oliver Thomas ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Xenograft Model ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell ◽  
Anti Tumor Activity ◽  
Dose Dependent

Abstract BI 836909 is a Bispecific T cell Engager (BiTE®), designed to redirect the body's endogenous T cells towards cells expressing B cell maturation antigen (BCMA) on the cell surface. BCMA is a highly plasma cell specific antigen and shows homogeneous expression on the cell surface of multiple myeloma, plasma cell leukemia and plasmacytoma cells. In normal tissues, BCMA expression is restricted to plasma cells, while other normal tissues do not express BCMA. This highly selective expression pattern makes BCMA an ideal target for T cell redirecting therapy. The pharmacological effect of BI 836909 depends on its simultaneous binding to both the CD3 epsilon subunit of the T cell receptor complex on T cells as well as to BCMA on multiple myeloma cells, resulting in the lysis of the BCMA-expressing cells. In vitro, unstimulated peripheral blood mononuclear cells (PBMCs) were co-cultured with several multiple myeloma cell lines and increasing concentrations of BI 836909, and tumor cell lysis, T cell activation, and induction of cytokine release were assessed. BI 836909 induced dose-dependent redirected lysis of human multiple myeloma cell lines with EC90 values ranging from 16 to 810 pg/mL. Viability of BCMA-negative cells was not affected, demonstrating the specificity of BI 836909 for BCMA. The expression of the activation markers CD69 and CD25 on T cells and the release of cytokines by T cells were target-dependent and occurred only in the presence of BCMA-positive cells. In vivo anti-tumor activity of BI 836909 was assessed in NOD/SCID mice reconstituted with human T cells and bearing subcutaneous or orthotopic xenografts derived from human multiple myeloma cell lines. In the subcutaneous NCI-H929 xenograft model, animals were treated with BI 836909 by daily intravenous or subcutaneous bolus injections. Statistically significant dose-dependent anti-tumor activity was observed at doses of 50 µg/kg/day and higher. The efficacy of BI 836909 was comparable after intravenous and subcutaneous administration, when the difference in bioavailability of the different routes was considered. In an orthotopic L-363 xenograft model, treatment with BI 836909 resulted in a statistically significant prolonged survival at doses of 5 µg/kg/day and higher. BI 836909 shows comparable cross-reactive binding to both BCMA and CD3 epsilon of human and macaque origin at picomolar and low nanomolar affinities respectively, thus allowing the assessment of pharmacodynamics, pharmacokinetics, and safety in non-human primates. In toxicity studies, cynomolgus monkeys were administered doses of up to 135 µg/kg/day of BI 836909 via continuous intravenous infusion, and up to 405 µg/kg/day via daily subcutaneous injection for up to 28 days. A dose- dependent decrease in plasma cells was observed in the bone marrow of treated animals compared to the vehicle control group, consistent with BCMA expression on cynomolgus monkey plasma cells, this demonstrated the pharmacological activity of BI 836909. These pre-clinical data demonstrate that BI 836909 is a highly potent, efficacious and BCMA-selective T cell redirecting agent and support clinical testing of BI 836909 in multiple myeloma patients. Disclosures Hipp: Boehringer Ingelheim RCV GmbH & Co KG, Dr. Boehringer-Gasse 5-11, 1121 Vienna, Austria: Employment. Deegen:Amgen Research (Munich) GmbH, Staffelseestrasse 2, 81477 Munich, Germany: Employment. Wahl:Amgen Research (Munich) GmbH, Staffelseestrasse 2, 81477 Munich, Germany: Employment. Blanset:Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, CT 06877, USA: Employment. Thomas:Amgen Research (Munich) GmbH, Staffelseestrasse 2, 81477 Munich, Germany: Employment. Rattel:Amgen Research (Munich) GmbH, Staffelseestrasse 2, 81477 Munich, Germany: Employment. Adam:Boehringer Ingelheim RCV GmbH & Co KG, Dr. Boehringer-Gasse 5-11, 1121 Vienna, Austria: Employment. Friedrich:Amgen Research (Munich) GmbH: Employment.

scholarly journals The Mucolipin TRPML2 Channel Enhances the Sensitivity of Multiple Myeloma Cell Lines to Ibrutinib and/or Bortezomib Treatment

Biomolecules ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 107
Author(s):  
Giorgio Santoni ◽  
Consuelo Amantini ◽  
Federica Maggi ◽  
Oliviero Marinelli ◽  
Matteo Santoni ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Plasma Cells ◽  
Biological Effects ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell ◽  
Bortezomib Treatment

Multiple myeloma (MM) is a haematological B cell malignancy characterised by clonal proliferation of plasma cells and their accumulation in the bone marrow. The aim of the present study is the evaluation of biological effects of Ibrutinib in human MM cell lines alone or in combination with different doses of Bortezomib. In addition, the relationship between the expression of TRPML2 channels and chemosensitivity of different MM cell lines to Ibrutinib administered alone or in combination with Bortezomib has been evaluated. By RT-PCR and Western blot analysis, we found that the Ibrutinib-resistant U266 cells showed lower TRPML2 expression, whereas higher TRPML2 mRNA and protein levels were evidenced in RPMI cells. Moreover, TRPML2 gene silencing in RPMI cells markedly reverted the effects induced by Ibrutinib alone or in combination with Bortezomib suggesting that the sensitivity to Ibrutinib is TRPML2 mediated. In conclusion, this study suggests that the expression of TRPML2 in MM cells increases the sensitivity to Ibrutinib treatment, suggesting for a potential stratification of Ibrutinib sensitivity of MM patients on the basis of the TRPML2 expression. Furthermore, studies in vitro and in vivo should still be necessary to completely address the molecular mechanisms and the potential role of TRPML2 channels in therapy and prognosis of MM patients.

scholarly journals Expression of the bcl-2 gene in human multiple myeloma cell lines and normal plasma cells

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 495-502 ◽  
Author(s):  
M Pettersson ◽  
H Jernberg-Wiklund ◽  
LG Larsson ◽  
C Sundstrom ◽  
I Givol ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
B Cell Differentiation ◽  
Multiple Myeloma Cell ◽  
Normal Plasma ◽  
Human Multiple Myeloma

Abstract The bcl-2 gene, encoding a mitochondrial membrane protein suggested to play an important role in cell survival, is translocated into the Ig loci in about 80% of human follicular lymphomas, which results in a high level of expression. This report shows that bcl-2 was expressed in eight of eight human multiple myeloma cell lines and in normal lymph node and bone marrow plasma cells. In the majority of the myeloma lines, the level of expression was comparable with that observed in Karpas 422, a follicular lymphoma cell line carrying a 14;18 translocation of the bcl-2 gene. DNA rearrangements of the bcl-2 locus were evident in only one of the myeloma cell lines, U-266–1970. In this cell line, which exhibited the highest bcl-2 expression, a fourfold increased copy number of the bcl-2 gene was estimated by Southern analysis. This amplification was lost in cells of later passages (U-266– 1984), suggesting that bcl-2 might possibly have played a role in the tumor development in vivo. Our results are in contrast to previous observations in murine plasmacytoma, in which bcl-2 was shown to be silent. The results also contradict the published observation that bcl- 2 is not expressed at terminal stages of B-cell differentiation. It is at present unclear whether the high expression of bcl-2 in human myeloma is the result of a deregulated expression associated with the malignant phenotype or a mere reflection of the bcl-2 expression typical of normal plasma cells.

Heterogeneous Bcl-2 Family Expression In Waldenström Macroglobulinemia Determines Response To Inducers Of Intrinsic Apoptosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4287-4287
Author(s):  
Brian T Gaudette ◽  
Kasyapa S. Chitta ◽  
Stephanie Poulain ◽  
Kelvin P Lee ◽  
Asher Chanan-Khan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Intrinsic Apoptosis ◽  
Myeloma Cell Line ◽  
Multiple Myeloma Cell ◽  
Activating Mutation

Abstract Waldenström Macroglobulinemia (WM) is a disorder of lymphoplasmacytoid cells that inhabit lymph nodes and the bone marrow. WM cells are characterized by secretion of monoclonal pentameric IgM. These cells are CD19+, CD20+, CD22+, CD38+, CD138+/- and phenotypically resemble IgM plasmablasts or plasma cells. In addition, 91% of WM cases carry an activating mutation of MyD88 (L265P). Mature resting B cells can be driven to differentiate to IgM secreting plasmablasts and plasma cells with similar phenotypes using the TLR4 ligand lipopolysaccharide (LPS). We have demonstrated that LPS (+ cytokine)-differentiated cells become Bcl-xL dependent during this process, rendering them sensitive to the Bcl-xL/Bcl-2 inhibitor ABT-737. For this reason, we hypothesized that activation of MyD88 in WM cells could drive Bcl-xL dependence in a similar manner conferring ABT-737 sensitivity. We treated three WM cell lines, BCWM.1, MWCL-1 and RPCI-WM1 which all harbor the MyD88 (L265P) mutation with ABT-737. We found varying levels of resistance to ABT-737 with an IC50 > 2 μM for all three lines as compared with the ABT-737 sensitive multiple myeloma cell line MM.1s which has an IC50 of 0.4 μM. The RPCI-WM1 cell line was the most insensitive to ABT-737-induced apoptosis with no apoptosis above baseline up to 1.6 μM of drug. Since the WM cell lines were not sensitive to direct inhibition of intrinsic survival regulators, we then examined the sensitivity of these cell lines to other activators of the intrinsic apoptosis pathway. Two of the three cell lines were moderately sensitive to bortezomib with IC50 ≈ 5 nM as compared with the sensitive multiple myeloma cell line MM.1s with an IC50 of 2 nM. The RPCI-WM1 cell line was insensitive to bortezomib with no apoptosis above baseline up to 20 nM bortezomib. Similarly, we found that two of the cell lines were moderately sensitive to arsenic trioxide with an IC50 ≈ 6 μM as compared with the multiple myeloma cell line MM1.s (IC50 ≈ 4 μM). The RPCI-WM1 cell line was insensitive to ATO as well with an IC50 > 20 μM. Given the lack of sensitivity of the three WM cell lines we tested to Bcl-xL/Bcl-2 inhibition with ABT-737 treatment, and that RPCI-WM1 appears insensitive to multiple inducers of intrinsic apoptosis, we examined the expression levels of Bcl-2 family members in these cells. Both BCWM.1 cells and MWCL-1 cells expressed Bim mRNA at very low levels with MWCL-1 expressing no detectable Bim at the protein level. Surprisingly, more moderate levels of Bim were detected in RPCI-WM1 cells. These findings were confirmed at the mRNA level by qRT-PCR. Bcl-xL and Mcl-1 were detectable in all three lines at moderate levels while Bcl-2 which was only expressed at significant levels in MWCL-1 cells and undetectable in BCWM.1 cells. We examined the expression levels of the Bax and Bak in these cells and remarkably there was no detectable Bax and very small amounts of Bak protein in RPCI-WM1 cells. Consistent with a defect in gene expression, Bax mRNA was also low in RPCI-WM1. This was not due to copy number variation, as determined by array-CGH in both the initial patient isolate and the established cell line. Additionally, no loss of Bax, Bak or Bim (Bcl2l11) was observed in SNP array analysis of 46 patients with WM. Interestingly, Bak mRNA levels in RPCI-WM1 were similar to the other WM lines, suggesting a defect in translation or post-translational regulation is responsible for the low protein expression. These results lead us to conclude that these WM cell lines are not sensitive to Bcl-xL/ Bcl-2 inhibition despite activation of MyD88. We have further shown that there are multiple and distinct differences in Bcl-2 family protein expression that lead to this insensitivity. While low levels of Bim combined with expression of Mcl-1 confer resistance to ABT-737 in MWCL-1 and BCWM.1, the lack of Bax and Bak confers resistance to intrinsic apoptotic stimuli in general in RPCI-WM1. Moreover, the loss of Bax and Bak protein expression occur through distinct mechanisms. These WM cell lines demonstrate that sensitivity to agents that kill through the intrinsic apoptotic pathway may vary within a disease that is characterized by a single activating mutation and suggests that additional heterogeneous events regulate the expression of Bcl-2 family proteins in WM. Disclosures: Leleu: CELGENE: Honoraria; JANSSEN: Honoraria. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy. Boise:Onyx Pharmaceuticals: Consultancy.

scholarly journals Expression of the bcl-2 gene in human multiple myeloma cell lines and normal plasma cells

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 495-502 ◽  
Author(s):  
M Pettersson ◽  
H Jernberg-Wiklund ◽  
LG Larsson ◽  
C Sundstrom ◽  
I Givol ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
B Cell Differentiation ◽  
Multiple Myeloma Cell ◽  
Normal Plasma ◽  
Human Multiple Myeloma

The bcl-2 gene, encoding a mitochondrial membrane protein suggested to play an important role in cell survival, is translocated into the Ig loci in about 80% of human follicular lymphomas, which results in a high level of expression. This report shows that bcl-2 was expressed in eight of eight human multiple myeloma cell lines and in normal lymph node and bone marrow plasma cells. In the majority of the myeloma lines, the level of expression was comparable with that observed in Karpas 422, a follicular lymphoma cell line carrying a 14;18 translocation of the bcl-2 gene. DNA rearrangements of the bcl-2 locus were evident in only one of the myeloma cell lines, U-266–1970. In this cell line, which exhibited the highest bcl-2 expression, a fourfold increased copy number of the bcl-2 gene was estimated by Southern analysis. This amplification was lost in cells of later passages (U-266– 1984), suggesting that bcl-2 might possibly have played a role in the tumor development in vivo. Our results are in contrast to previous observations in murine plasmacytoma, in which bcl-2 was shown to be silent. The results also contradict the published observation that bcl- 2 is not expressed at terminal stages of B-cell differentiation. It is at present unclear whether the high expression of bcl-2 in human myeloma is the result of a deregulated expression associated with the malignant phenotype or a mere reflection of the bcl-2 expression typical of normal plasma cells.

Bone sialoprotein mRNA and protein expression in human multiple myeloma cell lines and patients

2000 ◽  
Vol 111 (4) ◽  
pp. 1118-1121 ◽  
Author(s):  
A. Bellahcene ◽  
I. Van Riet ◽  
C. de Greef ◽  
N. Antoine ◽  
M. F. Young ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Expression ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Bone Sialoprotein ◽  
Multiple Myeloma Cell ◽  
Human Multiple Myeloma ◽  
Mrna And Protein Expression
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