PRL-3 Regulates Myeloma Cell Survival, Proliferation, Adhesion and Migration

Abstract Abstract 1321 Introduction: Multiple myeloma (MM) is a neoplastic monoclonal proliferation of bone marrow plasma cells. Despite advances in treatment in recent years, MM is still a fatal disease. Phosphatase of regenerating liver-3 (PRL-3) is a protein expressed in primary MM cells and MM cell lines, but not in normal plasma cells. A recent study showed that siRNA against PRL-3 suppresses MM-cell migration (Fagerli et al, 2008) and another study has identified PRL-3 as a marker gene for a subgroup of patients with MM (Broyl et al, 2010). Methods: The human myeloma cell lines INA-6, ANBL-6, IH-1, OH-2 and RPMI-8226 were used in this study. Apoptosis was measured by Annexin V-FITC binding by flow cytometry, proliferation was measured by methyl-3H-thymidine incorporation, migration against a stromal cell derived factor-1α (SDF-1α) gradient was studied in a Transwell two-chamber assay and adhesion was measured as percentage adhered cells to fibronectin after stimulation with the pro-adhesive cytokines hepatocyte growth factor (HGF), insulin like growth factor-1 (IGF-1) and SDF-1α. RT-PCR and Western blotting were used to measure expression of pro- and anti-apoptotic proteins. Western blotting was used to map intracellular signaling pathways. INA-6 cells stably expressing exogenous PRL-3 were generated using retroviral transduction. The small molecular inhibitor PRL-3 Inhibitor I was used for PRL-3 inhibition. Results: PRL-3 inhibitor I reduced survival of the myeloma cell lines INA-6, ANBL-6, IH-1, OH-2 and RPMI-8226. IC50 was between 15 and 50 μM depending on cell line. Treatment with the inhibitor down-regulated the anti-apoptotic protein Mcl-1 and led to activation of the intrinsic apoptotic pathway. Inhibition of PRL-3 reduced interleukin (IL)-6-induced phosphorylation of STAT-3. Treatment with 10 μM of PRL-3 inhibitor I also significantly reduced migration against a SDF-1α gradient and HGF-, IGF-1- and SDF-1α -mediated adhesion of the cell line INA-6. By retroviral transduction we made PRL-3-overexpressing INA-6 cells. INA-6 cells are dependent on IL-6 to grow, but overexpression of PRL-3 led to a major increase in cell proliferation even in the absence of IL-6 as well as increased survival at suboptimal concentrations of IL-6. Conclusion: PRL-3 is expressed in MM cells but not in normal plasma cells and can represent a cancer-specific target. Overexpression of PRL-3 in the IL-6-dependent human myeloma cell line INA-6 renders the cells less dependent of IL-6 and increases survival and proliferation. On the other hand, the use of an inhibitor against PRL-3 significantly decreases survival of five MM-cell lines and reduces adhesion and migration of the cell line INA-6. IL-6-STAT3 signaling is important in MM cell survival and proliferation and we here show that PRL-3 possibly influences cell survival as a positive regulator of this signaling pathway. This study indicates that PRL-3 could be important in the pathogenesis of MM and a potential target in the treatment of MM. Disclosures: No relevant conflicts of interest to declare.

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CD28 and CD86 Are Necessary for Myeloma Cell Survival.

Blood ◽

10.1182/blood.v120.21.2946.2946 ◽

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.

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A Role for Syntenin-1 in Multiple Myeloma Cell Survival

Blood ◽

10.1182/blood-2018-99-113594 ◽

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.

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

Blood ◽

10.1182/blood.v95.2.610 ◽

2000 ◽

Vol 95 (2) ◽

pp. 610-618 ◽

Cited By ~ 87

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.

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Plasma cells induce apoptosis of pre-B cells by interacting with bone marrow stromal cells

Blood ◽

10.1182/blood.v87.8.3375.bloodjournal8783375 ◽

1996 ◽

Vol 87 (8) ◽

pp. 3375-3383 ◽

Cited By ~ 30

Author(s):

T Tsujimoto ◽

IA Lisukov ◽

N Huang ◽

MS Mahmoud ◽

MM Kawano

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Survival ◽

Cell Lines ◽

Stromal Cells ◽

Plasma Cells ◽

Myeloma Cell ◽

Myeloma Cells ◽

B Cell Survival

By using two-color phenotypic analysis with fluorescein isothiocyanate- anti-CD38 and phycoerythrin-anti-CD19 antibodies, we found that pre-B cells (CD38+CD19+) signifcantly decreased depending on the number of plasma cells (CD38++CD19+) in the bone marrow (BM) in the cases with BM plasmacytosis, such as myelomas and even polyclonal gammopathy. To clarify how plasma cells suppress survival of pre-B cells, we examined the effect of plasma cells on the survival of pre-B cells with or without BM-derived stromal cells in vitro. Pre-B cells alone rapidly entered apoptosis, but interleukin-7 (IL-7), a BM stromal cell line (KM- 102), or culture supernatants of KM-102 cells could support pre-B cell survival. On the other hand, inhibitory factors such as transforming growth factor-beta1 (TGF-beta1) and macrophage inflammatory protein- 1beta (MIP-1beta) could suppress survival of pre-B cells even in the presence of IL-7. Plasma cells alone could not suppress survival of pre- B cells in the presence of IL-7, but coculture of plasma cells with KM- 102 cells or primary BM stromal cells induced apoptosis of pre-B cells. Supernatants of coculture with KM-102 and myeloma cell lines (KMS-5) also could suppress survival of pre-B cells. Furthermore, we examined the expression of IL-7, TGF-beta1, and MIP-1beta mRNA in KM-102 cells and primary stromal cells cocultured with myeloma cell lines (KMS-5). In these cells, IL-7 mRNA was downregulated, but the expression of TGF- beta1 and MIP-1beta mRNA was augmented. Therefore, these results suggest that BM-derived stromal cells attached to plasma (myeloma) cells were modulated to secrete lesser levels of supporting factor (IL- 7) and higher levels of inhibitory factors (TGF-beta1 and MIP-1beta) for pre-B cell survival, which could explain why the increased number of plasma (myeloma) cells induced suppression of pre-B cells in the BM. This phenomenon may represent a feedback loop between pre-B cells and plasma cells via BM stromal cells in the BM.

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C/EBPβ Is a Critical Factor for Proliferation and Apoptosis in MM Cells by Controlling Transcription Factors Like IRF-4, PAX5 and Blimp1.

Blood ◽

10.1182/blood.v110.11.1506.1506 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1506-1506

Author(s):

Rekha Pal ◽

Martin Janz ◽

Deborah Galson ◽

Suzanne Lentzsch

Keyword(s):

Transcription Factors ◽

Western Blot ◽

Cell Lines ◽

Plasma Cells ◽

Annexin V ◽

Critical Factor ◽

Myeloma Cell ◽

Down Regulation ◽

Protein Levels ◽

Rpmi 8226

Abstract The development and maturation of plasma cells is dictated by multiple interacting transcription factors (TFs). C/EBPb (NF-IL6) is a TF regulated by IL-6 and has profound effects on the regulation of growth, survival and differentiation of B-cells. Mice deficient in C/EBPb show impaired generation of B lymphocytes suggesting that C/EBPb plays an important role in B lymphopoiesis. In this study we delineated the effect of C/EBPb on transcription factors critical for myeloma cell proliferation by over-expressing and inhibiting C/EBPb in myeloma cells. Multiple myeloma (MM) cell lines MM.1S, RPMI-8226 and H929 were transiently transfected with GFP, C/EBPb (pcNF-IL6), and truncated C/EBPb with a deletion of the internal spII-spII fragment [pcmNF-IL6(Dspl)] by using Bio-Rad Gene Pulser Xcell, followed by G418 selection. A pool of transfected cells was selected and subjected to thymidine incorporation, flow cytometry and western blot analysis. We found that transfection of a truncated form of C/EBPb induced a down-regulation of C/EBPb in MM cell lines (MM.1S, RPMI-8226 and H929) as measured by western blot. Down-regulation of C/EBPβ significantly inhibited proliferation and induced apoptosis of MM cell lines analyzed by annexin V-FITC/PI staining. This was accompanied by a complete down-regulation of the anti-apoptotic protein BCL-2. Further, inhibition of C/EBPb completely decreased IRF-4 expression. In contrast, over-expression of C/EBPb increased protein levels of IRF-4 suggesting that IRF-4 is under control of C/EBPb. IRF-4, which was over-expressed in all our tested MM cells lines, is an essential TF for the generation of plasma cells by regulating TFs like Blimp-1 and PAX-5, which are critical for plasma cell differentiation. Our studies showed that down-regulation of IRF-4 resulted in a complete abrogation of Blimp-1 and PAX-5 suggesting that the expression of these factors is C/EBPb/IRF-4 dependent. In conclusion, our data indicate that C/EBPb is an important key regulator for survival and growth of MM cells. We show for the first time that C/EBPb is a critical regulator upstream of IRF-4. Down-regulation of the C/EBPb and consequently IRF-4 results in complete disruption of the network of TFs necessary for MM growth and survival. Targeting C/EBPb may provide a novel therapeutic approach in the treatment of MM.

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Biological and Clinical Significance of CD81 Expression by Clonal Plasma Cells in High-Risk Smoldering and Symptomatic Multiple Myeloma (MM) Patients,

Blood ◽

10.1182/blood.v118.21.3936.3936 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3936-3936 ◽

Cited By ~ 1

Author(s):

Bruno Paiva ◽

Norma C. Gutierrez ◽

Xi Chen ◽

María-Belén Vidriales ◽

María-Angeles Montalbán ◽

...

Keyword(s):

Cell Proliferation ◽

High Risk ◽

Cell Line ◽

Cell Lines ◽

Therapeutic Target ◽

Prognostic Value ◽

Plasma Cells ◽

Multiparameter Flow Cytometry ◽

Transplant Candidates ◽

Rpmi 8226

Abstract Abstract 3936 The presence of CD19 in myelomatous plasma cells (MM-PC) correlates with adverse prognosis in MM. Although CD19 expression is up-regulated by CD81, this marker has been poorly investigated and its prognostic value in myeloma remains unknown. Herein, we assessed the frequency and the prognostic value of the immunophenotypic detection of CD81 surface expression in MM-PC of high-risk smoldering (SMM) and symptomatic MM patients at diagnosis and its role as a potential therapeutic target. The study included 230 elderly MM patients treated according to the Spanish GEM05>65y trial, and a validation set based on 325 transplant candidates MM patients enrolled in the GEM05<65y trial was used. In addition we analyzed a total of 56 high-risk SMM patients. The median follow-up was 32 and 22 months for the MM and SMM, respectively. Expression of CD81 on MM-PC was assessed by multiparameter flow cytometry (MFC). FISH was performed at baseline in immunomagnetic-enriched PCs from 211 of the 230 elderly MM patients, and in a subset of these patients (N=23) mRNA gene expression profiling (GEP) was also performed. MFC immunophenotyping showed the presence of CD81+ MM-PC in 20 of 56 (36%) SMM, and 90 of 230 (39%) MM patients. CD81+ SMM cases had a shorter TTP to symptomatic disease than CD81− patients (NR vs 37 months, P =.02). Similarly, CD81+symptomatic MM patients showed shorter PFS (3y: 29% vs 48%, P <.001) and OS (3y: 64% vs 76%, P =.008) rates compared to CD81− cases. Multivariate analysis including other baseline variables showed that the best combination of independent predictive parameters for PFS were: CD81+ expression on MM-PC (HR=1.8; P =.004), high-risk cytogenetics (HR=1.8; P =.02) and percentage of MM-PC in S-phase (>2%; HR=1.7; P =.02); in turn for OS, CD81+ expression (HR=2.2; P =.005), high-risk cytogenetics (HR=2.2; P =.006) and age (≥75 years; HR=1.8; P =.03) were selected. To validate the adverse impact of CD81+ expression, we explored whether this new marker would retain its prognostic value in a series of 325 transplant candidates, symptomatic MM patients. CD81+ cases (138 out of 325, 42%) showed shorter PFS (3y: 44% vs 62%, P <.001) and OS (3y: 74% vs 89%, P =.004) rates as compared to CD81− cases. The prognostic influence of CD81 expression prompted us to investigate its potential role as a therapeutic target in MM cell lines. 5 out of the 13 cell lines analyzed were homogeneously positive for CD81 (RPMI-8226, RPMI-LR5, NCI-H929, OPM-2, JJN3) while the others exhibited no expression; these results were further validated by western blotting. We then tested the effect of two different anti-CD81 antibodies on cell proliferation on 2 CD81+ cell lines (RPMI-8226 and JJN3) as well as in 1 CD81− (MM1S) MM cell line. Interestingly, the two antibodies tested decreased cell proliferation (around 30%, P ≤.005) in the two CD81+ cell lines, whereas no differences were found for the CD81− MM1S cell line. To assess whether anti-CD81 antibodies facilitate immune effector cell function, we performed in vitro ADCC. However, no effect was noted in the CD81+ RPMI-8226 cell line, using either PBMCs or the macrophage cell line RAW264.7, and at different cell ratios. Similar results were also found upon using CDC assay in CD81+ RPMI-8226 and JJN3 cell lines. Finally, we explored in a subgroup of MM patients (n=23) in which information was available, the relationship between positivity for CD81 by MFC and its genomic expression. The expression level of CD81 mRNA was significantly decreased in CD81− MM cases (P <.001) vs. both healthy adults and CD81+ patients (P <.001), with no significant differences between these two latter subgroups. Moreover, we found a highly significant correlation between the expression of CD81 mRNA by GEP and the percentage of CD81+ MM-PC by MFC (r=.812; P <.001). We further investigated by GEP, differences in other genes involved in the CD81 signaling pathway, and CD81+MM patients showed significantly higher levels of CD79A (P =.03) and SYK (P =.02) mRNA than CD81−cases. In summary, our findings show the existence of a phenotypic-genomic correlation of CD81 expression in patients with myeloma. Expression of CD81 in MM-PC is an independent prognostic factor for patients with symptomatic MM and a marker for risk of progression in SMM. Blockage of CD81 with anti-CD81 antibodies does not show significant anti-myeloma activity; therefore, the precise mechanism of CD81 activation of MM-PC deserves further investigations. Disclosures: Paiva: Celgene: Honoraria; Janssen: Honoraria. Rosiñol:Janssen: Honoraria; Celgene: Honoraria. Mateos:Janssen: Honoraria; Celgene: Honoraria. Lahuerta:Janssen: Honoraria; Celgene: Honoraria. Blade:Janssen: Honoraria; Celgene: Honoraria. San Miguel:Janssen-Cilag: Honoraria; Celgene: Honoraria.

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Peroxisome Proliferator-Activated Receptor Gamma Promotes the Survival of Human T Lymphoma Cells through Its Regulation of Cellular Metabolism.

Blood ◽

10.1182/blood.v108.11.2392.2392 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2392-2392

Author(s):

Chunyan Yang ◽

Zibo Song ◽

Seung-Hee Jo ◽

Balazs Csernus ◽

Amy Chadburn ◽

...

Keyword(s):

Growth Factor ◽

Cell Survival ◽

Cell Line ◽

Cell Lines ◽

Ppar Gamma ◽

Lymphoma Cell ◽

Peroxisome Proliferator ◽

Lymphoma Cell Line ◽

Peroxisome Proliferator Activated Receptor ◽

T Lymphoma

Abstract Peroxisome proliferator-activated receptor gamma is a metabolic regulator involved in maintaining glucose and fatty acid homeostasis. Besides its metabolic functions, the receptor has also been implicated in tumorigenesis. Ligands of PPAR gamma have been found to induce apoptosis in a variety of tumor cell lines including lymphomas. However, apoptosis induction may not depend on the receptor since high doses of PPAR gamma agonists are required for this process. Using cells containing or lacking PPAR gamma, we reported previously that PPAR gamma attenuates apoptosis induced by growth factor withdrawal in a murine lymphocytic cell line via a receptor dependent mechanism. PPAR gamma exerts this effect by enhancing ability of cells to maintain their mitochondrial membrane potential during growth factor deprivation. In the current study, we demonstrate that PPAR gamma is expressed in human primary T lymphoma tissues and activation of PPAR gamma protects cells from serum starvation-induced apoptosis in human T lymphoma cell lines. Further, we show that the survival effect of PPAR gamma is mediated through its actions on cellular metabolic activities. In serum-deprived cells, PPAR gamma attenuates the decline in cellular ATP and suppresses accumulation of reactive oxygen species (ROS) in favor of cell survival. Moreover, PPAR gamma regulates ROS through its coordinated transcriptional control of proteins and enzymes involved in ROS production and scavenge. Introduction of PPAR gamma into a PPAR gamma-null T lymphoma cell line leads to increased cell survival. Meanwhile, knocking down the receptor in a PPAR gamma-positive lymphoma cell line reduces cell survival rate. Our studies identify cell survival promotion as a novel activity of PPAR gamma and suggest that high expression of PPAR gamma in lymphoma cells confers on them a survival advantage that renders cells resistant to growth factor and nutrient deprivation. These findings highlight the need for further investigation into the role of PPAR gamma in lymphoma and other types of cancer prior to widespread use of its agonists as anticancer therapeutics.

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Expression of the chemokine receptor CCR1 promotes the dissemination of multiple myeloma plasma cells in vivo

Haematologica ◽

10.3324/haematol.2020.253526 ◽

2020 ◽

pp. 0-0 ◽

Cited By ~ 1

Author(s):

Mara N. Zeissig ◽

Duncan R. Hewett ◽

Vasilios Panagopoulos ◽

Krzysztof M. Mrozik ◽

L. Bik To ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Line ◽

Plasma Cells ◽

Prognostic Indicator ◽

Myeloma Cell ◽

Nsg Mice ◽

Human Myeloma Cell Line ◽

Chemokine Cxcl12 ◽

Rpmi 8226

Multiple myeloma (MM) disease progression is dependent on the ability of MM plasma cells (PCs) to egress from the bone marrow (BM), enter the circulation and disseminate to distal BM sites. Expression of the chemokine CXCL12 by BM stromal cells is crucial for MM PC retention within the BM. However, the mechanisms which overcome CXCL12-mediated retention to enable dissemination are poorly understood. We have previously identified that treatment with the CCR1 ligand CCL3 inhibits the response to CXCL12 in MM cell lines, suggesting that CCL3/CCR1 signalling may enable egress of MM PC from the BM. Here, we demonstrated that CCR1 expression was an independent prognostic indicator in newly diagnosed MM patients. Furthermore, we showed that CCR1 is a crucial driver of dissemination in vivo, with CCR1 expression in the murine MM cell line 5TGM1 being associated with an increased incidence of bone and splenic disseminated tumours in C57BL/KaLwRij mice. Furthermore, we demonstrated that CCR1 knockout in the human myeloma cell line OPM2 resulted in a >95% reduction in circulating MM PC numbers and BM and splenic tumour dissemination following intratibial injection in NSG mice. Therapeutic targeting of CCR1 with the inhibitor CCX9588 significantly reduced OPM2 or RPMI-8226 dissemination in intratibial xenograft models. Collectively, our findings suggest a novel role for CCR1 as a critical driver of BM egress of MM PCs during tumour dissemination. Furthermore, these data suggest that CCR1 may represent a potential therapeutic target for the prevention of MM tumour dissemination.

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Induction of mitochondrial changes in myeloma cells by imexon

Blood ◽

10.1182/blood.v97.11.3544 ◽

2001 ◽

Vol 97 (11) ◽

pp. 3544-3551 ◽

Cited By ~ 45

Author(s):

Katerina Dvorakova ◽

Caroline N. Waltmire ◽

Claire M. Payne ◽

Margaret E. Tome ◽

Margaret M. Briehl ◽

...

Keyword(s):

Cell Line ◽

Acute Promyelocytic Leukemia ◽

Cell Lines ◽

Nuclear Dna ◽

Leukemia Cell ◽

Myeloma Cell ◽

Promyelocytic Leukemia ◽

Leukemia Cell Line ◽

Myeloma Cells ◽

Rpmi 8226

Imexon is a cyanoaziridine derivative that has antitumor activity in multiple myeloma. Previous studies have shown that imexon induces oxidative stress and apoptosis in the RPMI 8226 myeloma cell line. This study reports that imexon has cytotoxic activity in other malignant cell lines including NCI-H929 myeloma cells and NB-4 acute promyelocytic leukemia cells, whereas normal lymphocytes and U266 myeloma cells are substantially less sensitive. Flow cytometric experiments have shown that imexon treatment is associated with the formation of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential (Δψm) in imexon-sensitive myeloma cell lines and NB-4 cells. In contrast, reduction of Δψm and increased levels of ROS were not observed in imexon-resistant U266 cells. Treatment of imexon-sensitive RPMI 8226 cells with the antioxidant N-acetyl-l-cysteine (NAC) protects cells against these effects of imexon. Mitochondrial swelling was observed by electron microscopy in RPMI 8226 myeloma cells treated with 180 μM imexon as early as 4 hours. Damage to mitochondrial DNA was detected by a semiquantitative polymerase chain reaction assay in imexon-treated RPMI 8226 cells; however, nuclear DNA was not affected. Finally, partial protection of RPMI 8226 cells against the imexon effects was achieved by treatment with theonyltrifluoroacetone, an inhibitor of superoxide production at mitochondrial complex II. These changes are consistent with mitochondrial oxidation and apoptotic signaling as mediators of the growth inhibitory effects of imexon. Interestingly, oxidative damage and decrease of Δψm induced by imexon highly correlates with sensitivity to imexon in several myeloma cell lines and an acute promyelocytic leukemia cell line.

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Insulin-Like Growth Factor Binding Protein-3 (IGFBP3) Gene Expression In Myeloma Cell Lines and Patients

Blood ◽

10.1182/blood.v116.21.2489.2489 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2489-2489

Author(s):

Alessandra Perego ◽

Prerna Tewari ◽

Mark Lawler ◽

Paul Browne ◽

Stephen Drain ◽

...

Keyword(s):

Gene Expression ◽

Growth Factor ◽

Cell Line ◽

Cell Lines ◽

Classification System ◽

Myeloma Cell ◽

Functional Categories ◽

U266 Cell ◽

U266 Cell Line ◽

Igfbp 3

Abstract Abstract 2489 Introduction: Epigenetic aberrations play an important role in the development and progression of Multiple Myeloma (MM). 5-aza-2-deoxycytidine (5Aza-dC) and Trichostatin A (TSA) have been studied in reactivating the expression of epigenetically-silenced genes. IGFBP3 gene is a member of the insulin-like growth factor binding protein (IGFBP) family and can regulate cell growth and death by the ability to bind insulin-like growth factors (IGFs) as well as its IGF-independent effects involving binding to other molecules. Several in vitro, in vivo studies as well as clinical evidence point to IGFBP-3 as an anti-cancer molecule. In our study we analyzed global changes in gene expression profiles of MM cell lines, responding to 5Aza-dC and TSA and we evaluated the IGFBP3 expression in three myeloma cell lines and in samples from myeloma patients. Methods: Human MM cell lines U266 and H929 were treated either with 0.5 micromol/L 5Aza-dC for 7 days or with 100 ng/mL TSA for 24 h or with the combination of 0.5 micromol/L 5Aza-dC for 7 days and 100 ng/mL TSA for additional 24 h. Control cells received no drug treatment. Applied Biosystems microarray platform ABI 1300 was used for carrying out microarray profiling and analysis. To classify up-regulated genes into functional categories the PANTHER Classification System was used (http://www.pantherdb.org). Stained or unstained bone marrow slides were collected from archival samples of 179 myeloma patients. For validation of microarray results, real-time reverse transcription-PCR (RT-PCR) was performed using Taqman Gene Expression Assays (Applied Biosystems). Results: After treatment with 5Aza-dC there was up regulation of gene expression in 698 genes in H929 cell line and 258 genes in U266 cell line. After treatment with TSA 719 genes were up regulated in H929 cell line and 742 genes in U266 cell line. The exposure to the combination of 5Aza-dC/TSA resulted in up-regulation of 921 genes in H929 cell line and 615 genes in U266 cell line. By using Panther classification system we classified up-regulated genes into functional categories and we identified several 5Aza-dC or TSA up-regulated genes that are involved in important cancer-related pathways including cell cycle, apoptosis, cell adhesion, oncogenesis and cell metabolism, including DNA repair and nucleosome assembly. Between these genes we particularly found interesting the expression changes of IGFBP-3 which was up regulated after treatment with 5-azacitidine in U266 cell line (level of induced expression was 5,16). The microarray data were validated in 3 MM cell lines and 179 patients samples by analysis of relative changes in IGFBP3 expression through ΔΔCt method. We found that the expression levels of IGFBP3 were significantly lower in U266 and RPMI myeloma cell lines, but not in H929 cell line and interestingly levels were lower in 54 percent of patients (Fig 1). It has been demonstrated decreased IGFBP-3 expression is associated with cancer progression and in our study we have shown that the down-regulation of this gene may be involved also in myeloma pathogenesis and mediate progression events, hence levels of IGFBP3 may be considered as a biomarker for disease staging. From a therapeutic point of view, up-regulation of IGFBP3 might be considered as target therapeutic strategy for monoclonal gammopathies/ smouldering Myelomas at high risk of progression to active myeloma. Disclosures: No relevant conflicts of interest to declare.

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