Multiple Myeloma Cells Adhere to Netrin-1 Via Heparin-Sulphate Moieties

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3952-3952
Author(s):  
Jeesun Park ◽  
David R Fooksman ◽  
Amitabha Mazumder ◽  
Michael L Dustin
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Adhesion ◽  
Conflicts Of Interest ◽  
Cell Spreading ◽  
Myeloma Cell ◽  
Axonal Guidance ◽  
Intercellular Adhesion Molecule ◽  
Myeloma Cells ◽  
Guidance Cue

Abstract Abstract 3952 A major obstacle to the treatment of Multiple Myeloma (MM) is the localization of myeloma cells to the bone marrowstroma, enabling drug resistance. The exact mechanisms of adhesion of myeloma cells to the bone marrow are not known, but adhesion molecules and chemokine signals, in particular vascular cell adhesion protein 1 (VCAM-1) and C-X-C chemokine 12 (CXCL12) which control bone marrow tropism, are thought to be the main players. Netrin-1, which acts as an axonal guidance cue, plays a role in leukocyte migration in lymph nodes and in atherosclerotic lesions, but has not been tested as a substrate for myeloma cell adhesion previously. Based on expression of the netrin-1 receptor Deleted in Colorectal Cancer (DCC) on activated human B cells, we tested the ability of myeloma cells to adhere to netrin-1, an axonal guidance cue. Using interference reflection microscopy (IRM) which employs the method of interference of light reflected from nearby surfaces to measure cell-substratum distances and cell-substratum adhesion, we assessed cell adhesion and cell spreading on substrates immobilized on glass. Here, we used this technique to assess myeloma cell adhesion and migration on various substrates and found netrin-1 to be an exceptional adhesion ligand for myeloma cells. We prepared glass substrates coated with the recombinants ligands intercellular adhesion molecule,ICAM-1(50μM), and VCAM-1(50μM), with or without chemokine ligand, CXCL12(0.1mg/mL), which have been implicated in plasma cells and myeloma cell migration, previously. We used freshly purified, fluorescently-labeled primary myeloma cells from newly diagnosed patients, prior to any treatment. Using IRM, we imaged the cell contacting the substrate in order to measure adhesion and differentiate crawling versus fluid flow movement. Based on the IRM image, we could calculate the fraction of cells in the field that were adhered to the substrate, and compared between conditions and for various patient samples. We observed that myeloma cells can adhere and migrate slowly on VCAM-1 in the presence of CXCL12, but are unable to adhere to ICAM-1 with or without chemokines. We tested myeloma cell binding to netrin-1 and saw a strong adhesion 60–90% of cells in 7 out of 9 patients samples tested. The cell spreading on netrin-1 was more than 3 times larger than on VCAM-1 with CXCL12 substrates. Expression of netrin-1 in the bone marrow has not been determined yet nor its role in MM. Heparin-like molecule, SST0001, has been tested in myeloma studies, in an attempt to interfere with heparinase activity and syndecan-1 shedding, and tumor growth. We tested pre-blocking netrin-1 substrates with heparin and observed elimination of greater than 95% of myeloma cell adhesion in all patients samples tested. Treating patients with heparin-like molecules may have additional functions, by blocking binding to netrin-1 and soluble signals that contain heparin-binding domains. Reciprocally, blocking heparin-sulfated groups with netrin-1 may block myeloma cell adhesion and can be used to targeting strategy for chemotherapeutic drugs as well. Disclosures: No relevant conflicts of interest to declare.

Influence of Targeted Therapy in Redefining High-Risk Myeloma.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. sci-7-sci-7
Author(s):  
Lori Hazlehurst ◽  
William S. Dalton ◽  
Danielle Yarde ◽  
Yulia Nefedova ◽  
Dmitry Gabrilovich
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Drug Resistance ◽  
Clinical Trials ◽  
Cell Adhesion ◽  
Tumor Microenvironment ◽  
Residual Disease ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Acquired Drug Resistance

Abstract Multiple myeloma is a disease that typically responds to initial treatment; however, the disease is not cured by chemotherapy, and drug resistance ultimately develops. Most studies investigating the problem of drug resistance have focused on acquired resistance or resistance that occurs after response to prior therapy as a result of residual disease. Intrinsic factors, such as reduced drug uptake, enhanced damage response (i.e., DNA repair), altered drug metabolism, or inhibition of programmed cell death pathways are known to contribute to acquired drug resistance. For example, it was recently reported that the acquired melphalan resistant phenotype in myeloma cell lines was associated with over-expression of the Fanconi anemia (FA)/BRCA pathway genes. Enhanced interstrand cross-link (ICL) repair via the FA/BRCA pathway was causally related to melphalan resistance and disruption of this pathway using knock-down techniques reversed drug resistance. Furthermore, bortezomib (Velcade) has been reported to enhance melphalan treatment, and recent pre-clinical data has shown that bortezomib reduces FA/BRCA gene expression and function. Clinical trials are necessary to determine the role of the FA/ BRCA pathway in acquired drug resistance for myeloma patients and whether targeting this pathway enables prevention of or the ability to overcome acquired melphalan resistance in myeloma patients. Conversely, factors that promote tumor cell survival and drug resistance that are external to the tumor cell itself might exist. Evidence supporting the importance of understanding the influence of the tumor microenvironment on drug sensitivity has been reported by several investigators. The tumor microenvironment for hematologic malignancies, including myeloma, is principally the bone marrow. The bone marrow contains candidate components that contribute to reduced drug activity, minimal residual disease, and emergence of drug resistant cells. Cell adhesion molecules expressed by myeloma cells, including the β integrins, bind to fibronectin and other extracellular matrix components of the bone marrow, and this interaction contributes to a reversible, de novo drug resistance phenotype called “cell adhesion mediated drug resistance” or CAMDR. Adhesion via integrins is known to activate a network of signal transduction pathways that influence cell survival, growth, and differentiation. Several targets that are influenced by integrin adhesion and may contribute to CAM-DR include the following: reduced proapoptotic Bim levels, alterations in nuclear topoisomerase II levels, increased p27 kip1 levels, and changes in FLIP1 levels. In addition, myeloma cell adhesion to bone marrow stroma (BMS) involves other adhesion molecules and signaling events that promote CAMDR. For example, Notch1 receptors expressed on multiple myeloma cells when stimulated by Jagged causes growth arrest and protection from drug-induced apoptosis. Recently, approaches to inhibit integrin and Notch signaling associated with CAM-DR have been examined pre-clinically. Clinical trials are necessary to determine if these approaches will prevent or overcome CAM-DR in patients.

scholarly journals The type III transforming growth factor-β receptor inhibits proliferation, migration, and adhesion in human myeloma cells

2011 ◽  
Vol 22 (9) ◽  
pp. 1463-1472 ◽  
Author(s):  
Kathleen E. Lambert ◽  
Huang Huang ◽  
Karthikeyan Mythreye ◽  
Gerard C. Blobe
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Adhesion ◽  
Growth Factor ◽  
Cell Growth ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Β Receptor

Transforming growth factor-β (TGF-β) plays an important role in regulating hematopoiesis, inhibiting proliferation while stimulating differentiation when appropriate. We previously demonstrated that the type III TGF-β receptor (TβRIII, or betaglycan) serves as a novel suppressor of cancer progression in epithelial tumors; however, its role in hematologic malignancies is unknown. Here we demonstrate that TβRIII protein expression is decreased or lost in the majority of human multiple myeloma specimens. Functionally, restoring TβRIII expression in myeloma cells significantly inhibited cell growth, proliferation, and motility, largely independent of its ligand presentation role. In a reciprocal fashion, shRNA-mediated silencing of endogenous TβRIII expression enhanced cell growth, proliferation, and motility. Although apoptosis was not affected, TβRIII inhibited proliferation through induction of the cyclin-dependent kinase inhibitors p21 and p27. TβRIII further regulated myeloma cell adhesion, increasing homotypic myeloma cell adhesion while decreasing myeloma heterotropic adhesion to bone marrow stromal cells. Mechanistically, live cell imaging of myeloma and stroma cell cocultures revealed that TβRIII-mediated inhibition of heterotropic adhesion was associated with decreased duration of myeloma/bone marrow stromal cell interaction. These results suggest that loss of TβRIII expression during multiple myeloma progression contributes to disease progression through its functional effects on increased cell growth, proliferation, motility, and adhesion.

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.

CD56 Expression Is the Potent Prognostic Marker of the Thalidomide Efficacy in the Patients with Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5142-5142
Author(s):  
Akio Mori ◽  
Yutaka Tsutsumi ◽  
Satoshi Hashino ◽  
Hiroe Kanamori ◽  
Makoto Ibata ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Adhesion ◽  
Adhesion Molecules ◽  
Prognostic Marker ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Myeloma Cells ◽  
Cd56 Expression

Abstract Thalidomide (Thal) alone or in combination with steroids achieves responses even in the setting of refractory multiple myeloma (MM), however, responses are still limited. The precise mechanism of Thal action is unknown, further, no distinct marker, which could prognosticate the efficacy of Thal, is known. Therefore, we evaluated the correlation between the efficacy of Thal and the potent prognostic factors in patients with refractory MM. Ten patients with refractory MM received Thal at doses of 50 or 100 mg per day and steroids, either dexamethasone (Dex) or prednisolone (PSL). Dex was administrated 20 mg per day, 4 days every 28 days, and PSL was administrated 10 mg per day. The median age was 71.5 years (range, 62–79 years) and 20 % were man, and all patients were diagnosed as clinical stage IIIA based on the Durie and Salmon classification. The therapeutic response was assessed according to the modified criteria of Southwest Oncology Group (SWOG). Among 10 patients, 7 patients were the responders; 2 had complete remission, 3 had partial remission, and 2 had minimal remission. There were no differences in the pretreatment characteristics of responders and nonresponders (age, sex, type and concentration of serum and/or urine monoclonal component, international prognostic index, presence of bone lesion, and chromosomal abnormalities). However, flow cytometric evaluation of the myeloma cells revealed that CD56, which is one of the adhesion molecules N-CAM, expressed more than 45 % in all responders, while those expressed less than 5 % in all nonresponders (84 ± 19 (±SD) % v/s 4 ± 2 %, P=0.017). Furthermore, CD56 expression of the myeloma cells was reduced from 84% to 70 ± 32 % after Thal therapy in all evaluated responders (P =0.048). These results suggest that CD56 expression of the myeloma cells could be the potent prognostic marker of the Thal efficacy. Moreover, it was reported that Thal reduced the expression of cell adhesion molecules, such as LFA-1 and ICAM-1, and abrogated the binding of MM cells to bone marrow stromal cells, that triggered the secretion of interleukin-6 and vascular endothelial growth factor. Taken together, it was suggested that Thal reduced the expression of CD56 and altered the MM cell adhesion to bone marrow stromal cells, and that could be one of the pathogenesis of anti-MM activity of Thal.

Multiple Myeloma: Interleukin-15 Antagonizes Bone Morphogenetic Protein-Induced Apoptosis and Growth Inhibition.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3444-3444
Author(s):  
Magne Rekvig ◽  
Anne-Tove Brenne ◽  
Torstein Baade Ro ◽  
Anders Waage ◽  
Magne Borset ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Growth Inhibition ◽  
Cell Line ◽  
Caspase 3 ◽  
Myeloma Cell ◽  
Stimulus Figure ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Induced Apoptosis

Abstract Multiple myeloma has two distinct features: Expansion of malignant plasma cells within the bone marrow accompanied by skeletal destruction. Bone morphogenetic proteins (BMPs) have been shown to induce apoptosis and inhibit growth in myeloma cells. BMPs are members of the TGF-β superfamily of proteins capable of inducing bone formation, and regulate proliferation, differentiation and apoptosis. We have investigated myeloma cell apoptosis and proliferation with BMP-4 and −6 in concert with the myeloma cell growth factors interleukin (IL)-2, IL-6, IL-10, IL-15, IL-21, tumor necrosis factor (TNF)-α and insulin-like growth factor (IGF)-1. Eight samples of highly purified myeloma cells from patients and a human myeloma cell line, IH-1 (Brenne AT et al. Blood. 2002 May 15;99(10):3756–62.), were used in this study. Cytokine concentrations used in the referred experiments were for BMP-4 20ng/ml, BMP-6 250ng/ml, IL-15 20ng/ml and IL-6 0,1ng/ml, respectively. Growth inhibition was measured in a proliferation assay by methyl-[3H]-thymidine incorporation and apoptosis by annexin V- FITC-binding/PI-uptake on flow cytometry. IL-15 antagonized growth inhibition (Figure A) and prevented apoptosis induced by BMP-4 (Figure B) and BMP-6 in the myeloma cell line IH-1. IL-15 also antagonized the growth inhibition induced by BMP-4 and/or BMP-6 in three out of eight patient samples. Neither IL-6, nor any of the other investigated cytokines were able to rescue the myeloma cells from growth inhibition and apoptosis induced by BMP-4 and -6. Among the investigated cytokines, we found that IL-15 has a unique capability to antagonize BMP- induced apoptosis and growth inhibition in myeloma cells. We examined cleavage of the proapoptotic protein caspase-3 and found that BMP-4 activated caspase-3 in the IH-1 cell line. This activation of caspase-3 was blocked by IL-15 but not by IL-6. We have demonstrated a possible mechanism for myeloma cells to escape apoptosis and growth-inhibition within the bone marrow. Intramedullar levels of IL-15 and BMPs may play a role in the pathogenesis of multiple myeloma. Figure A. Proliferation in response to BMP-4 stimulus Figure A. Proliferation in response to BMP-4 stimulus Figure B. Apoptosis in response to BMP-4 stimulus Figure B. Apoptosis in response to BMP-4 stimulus

Myeloma Cell Survival and Importance of Crosstalk Between Notch1-Jagged2 and CD28-B7 Pathways In Dendritic Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1741-1741
Author(s):  
Chandana Koorella ◽  
Jayakumar Nair ◽  
Louise Carlson ◽  
Megan Murray ◽  
Cheryl H Rozanski ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Notch Signaling ◽  
Cell Biology ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Negative Regulator ◽  
Soluble Form ◽  
Myeloma Cells ◽  
Notch1 Signaling

Abstract Abstract 1741 Multiple myeloma is a neoplasm of bone marrow resident plasma cells characterized by critical interactions between myeloma cells and bone marrow stromal cells. This interaction leads to production of IL-6, an important factor in myeloma cell biology. However, the molecular and cellular components involved in myeloma induced IL-6 production remain largely uncharacterized. While at the cellular level, dendritic cells (DC)-expressing CD80/CD86 (collectively called B7, ligands with short cytoplasmic tails and signaling partners of CD28 expressed on myeloma cells) - in the bone marrow microenvironment have been implicated as being an important component, at the molecular level the CD28-B7 and Notch1-Jagged2 pathways were separately implicated by us (in DC) and others in myeloma induced IL-6 production. Although Notch signaling leading to IL-6 production in DC is well understood, the mechanism of “backsignaling” via B7 is largely uncharacterized. To better understand downstream B7 signaling leading to IL-6 production, DC were stimulated with CD28-Ig (a soluble form of CD28 which mimicks myeloma cell-bound CD28) in the presence or absence of an inhibitor of Notch signaling, gamma secretase inhibitor (GSI). DC treated with CD28-Ig alone produced significantly (p< 0.001) higher levels of IL-6 when compared to DC treated with CD28-Ig and GSI. GSI specifically targeted Notch signaling as observed by decreased expression of Notch gene targets: Hes-1 (2 fold decrease) and Deltex-4 (4 fold decrease). Also, decreased IL-6 levels in presence of GSI were not due to the decrease in B7 expression on DC. To specifically implicate the importance of Notch1 and Jagged2, we blocked Notch1 signaling using blocking antibodies and observed a similar decrease in IL-6 production upon blocking Notch1 signaling. Our results suggest that CD28 mediated IL-6 production is dependent on Notch1 signaling and crosstalk between the Notch1-Jagged2 and CD28-B7 pathways leads to IL-6 production by DC. The model of crosstalk between CD28-B7 and Notch1-Jagged2 pathways was also observed in murine bone marrow derived dendritic cells (BMDC), where a significant (p<0.001) down regulation of IL-6 was observed upon blocking Notch signaling. One possible mechanism of crosstalk involves direct effect of B7 crosslinking by CD28-Ig on Notch expression/signaling leading to increase in IL-6 production. We tested for this possibility in DC and found no significant change in Notch expression/signaling. We thus hypothesized that the mechanism of crosstalk involves molecules downstream to Notch and/or B7. Notch signaling has been reported to be involved in the regulation of PTEN (a negative regulator of the PI3K/Akt pathway). Previous studies have also shown the importance of FoxO3a-a transcription factor tightly regulated by Akt- in regulating IL-6 production in BMDC upon B7 crosslinking. We therefore tested the possible involvement of PTEN (molecule downstream of Notch signaling), Akt and FoxO3a (molecules downstream of B7) in crosstalk between the two pathways aforementioned by testing the effect of GSI on their regulation at the protein level. We observed an approximate 2 fold decrease in phospho-PTEN/PTEN ratio in DC treated with GSI and remained so even after B7 crosslinking at an early time point (15 min. post CD28-Ig treatment.) Further, phospho-Akt/Akt ratio decreased by 1.6 fold in DC treated with both GSI and CD28-Ig compared to CD28-Ig alone at 30 min. We therefore hypothesize a model of crosstalk involving Notch mediated regulation of PTEN leading to IL-6 production via regulation of Akt and possibly FoxO3a upon B7 crosslinking. Interestingly enough “backsignaling” via B7 in myeloma-induced IL-6 production seems to involve molecules well characterized in CD28 signaling of T-cells. Targeting IL-6 induced by crosstalk between these two pathways prompts not only clinical evaluation to improve MM patient outcome but also extends to advancing knowledge in T-cell and normal plasma cell biology as well. Disclosures: No relevant conflicts of interest to declare.

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.

A Novel Small Molecule with Epigenetic Activity Prolongs Survival in a Mouse Model of Multiple Myeloma Refractory to Standard Drugs

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5121-5121
Author(s):  
Sergei Vatolin ◽  
Khan Nazeer Shahper ◽  
Yvonne Parker ◽  
Daniel Lindner ◽  
Frederic J. Reu
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Chromatin Condensation ◽  
Xenograft Model ◽  
Myeloma Cells ◽  
Tail Vein ◽  
Tail Vein Injection ◽  
Mouse Xenograft Model ◽  
Number Of Patients

Abstract Abstract 5121 Multiple myeloma refractory to bortezomib, IMiDs™, and conventional therapies represents an unmet medical need. An increasing number of patients progress to this stage since treatment related mortality has decreased. To test promising compounds for activity in this setting we established an NSG mouse xenograft model with serial transplantation by tail vein injection of myeloma cells from a patient with IgG kappa myeloma relapsed and refractory to all standard drugs. Eight days after tail vein injection monoclonal human IgG can be detected in serum. Bone marrow engraftment in young (6–12 weeks) NSG mice after sublethal radiation (275cGy) is close to 100% (n=32). Untreated mice die within less than 2 months, usually with liver and spleen metastasis (anti-human CD138 flow cytometry). In a drug screen that used a novel method developed in our lab, chromatin condensation PCR, we identified a non nucleoside compound (4I3) that potently (1mM) reactivated expression of epigenetically silenced genes and displayed cancer-specific growth and survival inhibition in myeloma cell lines but not normal cells. Normal bone marrow cells continued to divide at doses 10x higher than required to kill 80% of myeloma cells. 4I3 suppressed DNMT1 protein but rapid cell kill (within 1–2 days) suggested additional mechanisms which we currently investigate. Given IV to mice after documentation of engraftment by IgG serum immunoblots, it prolonged survival in an ongoing experiment. Updated results will be presented at the meeting. 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 Fate of Ikaros in Multiple Myeloma Cells upon Treatment with Lenalidomide and Proteasome Inhibitor

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3281-3281
Author(s):  
Saravanan Ganesan ◽  
Nithya Balasundaram ◽  
Hamenth Kumar Palani ◽  
Ansu Abu Alex ◽  
Sachin David ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Proteasome Inhibition ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Ubiquitinated Proteins ◽  
Two Agents ◽  
Autophagy Pathway

Abstract Recent evidences suggests that the efficacy of Lenalidomide (LEN) depends upon its ability to degrade IKZF1 and IKZF3 proteins via cereblon dependent ubiquitin proteasome pathway [Science. 2014 Jan 17; 343(6168): 301-305]. Based on this model it would theoretically be antagonistic to combine LEN with proteasome inhibitors (PI). However, it is well recognized that there is significant synergism when LEN is combined with PI and this combination is routinely and effectively used in the clinic. The mechanism of synergy and the fate of IKZF1 and IKZF3 when these two agents are combined is poorly understood. We undertook a series of experiments to study the fate of IKZF1 when this combination of drugs was used in multiple myeloma cells. Combining LEN (1uM) along with bortezomib (BTZ; 1nM) a PI showed a significant kill on U266 cells (myeloma cell line) on day 5 post treatment (n=3; P=0.02) when compared to either of the agents alone. In an MTT assay, the synergism was well documented with a combination index of 0.5 (Fig 1a). Next we assessed the function of proteasome (chymotrypsin activity) when LEN was combined with PI. We observed that LEN alone does not interfere with proteasome activity. It was noted that BTZ alone at the concentration used (5 nM) was able to effectively inhibit the activity of proteasome (Fig 1b). It was also observed that combining these two agents does not interfere with BTZ action in inhibiting proteasome complex (Fig 1b). As a result of efficient proteasome inhibition, we observed an accumulation of ubiquitinated proteins in the BTZ and LEN + BTZ treated cells when compared to control and LEN alone treated cells (Fig 1c). Next, we looked for the fate of IKZF1 in U266 cells treated with LEN, BTZ and in combination of both the drugs. As reported, we observed a degradation of IKZF1 in U266 cells upon treatment with LEN. While we did not see any degradation of IKZF1 in BTZ alone treated cells. It was noted that in combination treated cells (LEN+BTZ) there was a degradation of IKZF1 (Fig 1c). In spite of significant proteasome complex inhibition, degradation of IKZF1 was observed which suggested a proteasome independent mechanism. It is well known that proteasome inhibition results in upregulation of the autophagy pathway which in turn can degrade the accumulated ubiquitinated proteins. We noted that upon treatment with BTZ or LEN+BTZ an induction of autophagy was observed, as evidenced by an increase in generation of LC3II bands on an immunoblot (Fig 1c). To support our hypothesis that IKZF1 is degraded by autophagy in the absence of proteasome complex, we pre-treated the U266 cells with an autophagy inhibitor (3-methyladenine) followed by treatment with LEN and BTZ and noted an accumulation of IKZF1 proteins (Fig 1d). We also observed a downregulation of IKZF1 target genes IRF4 and c-MYC by 12 and 24 hours in the combination treated cells (data not shown). Taken together this data demonstrates that there is (i) significant in-vitro synergism between the two agents (ii) the combination additively induces autophagy pathway (iii) IKZF1 protein can be degraded via this autophagy pathway in the presence of effective proteasome inhibition. While additional mechanisms of synergy between these two agents cannot be excluded, further enhancing autophagy pathway in these cells by drugs like sirolimus (autophagy inducer in myeloma cells) could potentially improve the synergy between these two drugs. Disclosures No relevant conflicts of interest to declare.

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