scholarly journals Morphogens and growth factor signalling in the myeloma bone-lining niche

2021 ◽  
Author(s):  
Emma V. Morris ◽  
Claire M. Edwards
Keyword(s):  
Bone Marrow ◽  
Cell Biology ◽  
Plasma Cells ◽  
Cell Types ◽  
Myeloma Cell ◽  
Bone Cell ◽  
Signalling Pathways ◽  
Perivascular Niche ◽  
Myeloma Cells ◽  
Endosteal Niche

AbstractMultiple myeloma is a malignancy caused by the clonal expansion of abnormal plasma cells. Myeloma cells have proven to be incredibly successful at manipulating their microenvironment to promote growth and to evade modern therapies. They have evolved to utilise the integral signalling pathways of the bone and bone marrow to drive disease progression. The bone marrow is often described in the context of a single structure that fills the bone cavity and supports normal haematopoiesis. However, within that structure exists two anatomically different niches, the perivascular niche and the endosteal niche. These contain different cell types functioning to support normal immune and blood cell production as well as healthy bone. These cells secrete numerous signalling molecules that can influence myeloma cell biology and behaviour. The endosteal niche is home to specific bone cell lineages and plays a pivotal role in myeloma cell establishment and survival. This review will concentrate on some of the signalling pathways that are hijacked by myeloma cells to shape a favourable environment, and the different influences myeloma cells are exposed to depending on their spatial location within the bone marrow.

Expression of CD21 antigen on myeloma cells and its involvement in their adhesion to bone marrow stromal cells

Blood ◽  
1995 ◽  
Vol 85 (12) ◽  
pp. 3704-3712 ◽  
Author(s):  
N Huang ◽  
MM Kawano ◽  
MS Mahmoud ◽  
K Mihara ◽  
T Tsujimoto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cells ◽  
Plasma Cells ◽  
Cell Types ◽  
Myeloma Cell ◽  
Adhesion Assay ◽  
Myeloma Cells ◽  
Stromal Cell Line

The mature myeloma cells express very late antigen 5 (VLA-5) and MPC-1 antigens on their surface and adhere to bone marrow (BM) stromal cells more tightly than the VLA-5-MPC-1-immature myeloma cells in vitro. The VLA-5 and MPC-1 antigens possibly function as two of the molecules responsible for interaction of mature myeloma cells with BM stromal cells. However, the immature myeloma cells do interact with BM stromal cells, and it is unclear which adhesion molecules mediate their interaction. In this study, we found that both immature and mature myeloma cells expressed CD21, an adhesion molecule known to bind to CD23. CD21 was also detected on normal plasma cells. To evaluate the role of CD21 expression on myeloma cells, two myeloma cell lines, NOP-2 (VLA-5-MPC-1-) and KMS-5 (VLA-5+MPC-1+), were used as representatives of immature and mature myeloma cell types, respectively, and an adhesion assay was performed between the myeloma cell lines and BM stromal cells. Antibody-blocking results showed that adhesion of the mature type KMS-5 to KM102, a human BM-derived stromal cell line, or to short-term cultured BM primary stromal cells was inhibited by monoclonal antibodies (MoAbs) against CD21, VLA-5, and MPC-1, and inhibition of adhesion of the immature type NOP-2 to KM102 by the anti-CD21 MoAb was observed as well. Furthermore, CD23 was detected on KM102. Treatment of KM102 with an anti-CD23 MoAb also inhibited adhesion of either KMS-5 or NOP-2 to KM102. Therefore, we propose that CD21 expressed on myeloma cells likely functions as a molecule responsible for the interaction of immature myeloma cells as well as mature myeloma cells with BM stromal cells, and CD23 may be the ligand on the stromal cells for the CD21-mediated adhesion.

scholarly journals Heterogeneous expression of a novel MPC-1 antigen on myeloma cells: possible involvement of MPC-1 antigen in the adhesion of mature myeloma cells to bone marrow stromal cells

Blood ◽  
1993 ◽  
Vol 82 (12) ◽  
pp. 3721-3729 ◽  
Author(s):  
N Huang ◽  
MM Kawano ◽  
H Harada ◽  
Y Harada ◽  
A Sakai ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Cell Line ◽  
Bone Marrow Stromal Cells ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Human Myeloma Cell Line ◽  
Heterogeneous Expression

Abstract Recent immunophenotypic analysis has shown that the heterogeneous expression of the adhesion molecule VLA-5 classifies myeloma cells into VLA-5+ mature and VLA-5- immature subpopulations. To further clarify the two myeloma subpopulations, we generated a monoclonal antibody, MPC- 1, by immunizing mice with an adherent human myeloma cell line, KMS-5. The MPC-1 antibody recognized a 48-Kd surface antigen on KMS-5 but not on U-266, a nonadherent human myeloma cell line. Specificity characterization showed that MPC-1 antigen was expressed on mature myeloma cells, normal plasma cells, and mature B cells, whereas pre-B cells and germinal center B cells lacked its expression. Monocytes and a human bone marrow stromal cell line, KM102, also expressed this antigen. Two subclones of MPC-1+ VLA-5+ (KMS-5Ad) and MPC-1-VLA-5+ (KMS- 5NAd) were separated from the KMS-5 cell line. The KMS-5NAd adhered to KM102 more tightly than did the KMS-5NAd, and the U-266 (MPC-1-VLA-5-) displayed almost no adherence to the KM102. The adhesion of the KMS-5Ad was partially inhibited by the MPC-1 antibody. These results, taken together, suggest that the MPC-1 antigen serves as a differentiation marker for B-lineage cells, including plasma cells, and may function as an adhesion molecule involved in the interaction of mature myeloma cells with bone marrow stromal cells.

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

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3375-3383 ◽  
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.

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.

MDX-1097 Binds Specifically to Kappa Myeloma Cells and Anti-Tumour Activity Is Mediated by Multiple Effector Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1846-1846
Author(s):  
Mae Wong ◽  
Parisa Asvadi ◽  
Rosanne Dunn ◽  
Darren Jones ◽  
Douglas Campbell ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Human Tissue ◽  
Plasma Cells ◽  
Specific Binding ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Effector Cells ◽  
Anti Tumour Activity ◽  
Tumour Activity

Abstract Abstract 1846 Poster Board I-872 Previous studies have described a murine monoclonal antibody, mKap, that specifically recognizes a cell surface antigen expressed on kappa myeloma cells and not on normal lymphoid cells. This antigen has been identified and designated kappa myeloma antigen (KMA). KMA consists of free kappa light chains (kFLC) not associated with heavy chain and is present on plasma cells isolated from kappa myeloma (MMk) patient bone marrow aspirates, kappa myeloma cell lines and kappa macroglobulinemia. In vitro data demonstrated that mKap was able to inhibit cell growth and induce apoptosis in myeloma cell lines. In addition, pre-clinical studies demonstrated that mKap was well tolerated and showed significant efficacy in a SCID xenograft model of MM. MDX-1097 is a chimeric version of mKap that is currently in development for the treatment of kappa restricted multiple myeloma. The antibody retains the binding affinity and specificity of mKap. Specific binding of MDX-1097 to malignant plasma cells isolated from MMk patient bone marrow aspirates has recently been demonstrated by flow cytometry. In addition a human tissue cross-reactivity study was performed using immunohistochemistry to assess the potential binding of MDX-1097-FITC to cryosections taken from a human tissue panel of three normal donors. The results demonstrated that MDX-1097 bound to bone marrow plasma cells from two patients with kappa cell dyscrasia but did not bind to normal human tissue samples or to plasma cells from a patient with lambda plasmacytoma. The ability of serum kFLC to inhibit MDX-1097 binding to the myeloma cell line, JJN3, was assessed by flow cytometry using serum derived from 32 MMk patients. The results indicated that MDX-1097 at a concentration of 100μg/mL (equivalent to an estimated serum concentration of 5mg/kg dose) is capable of binding to myeloma cells in the presence of 0–250μg/mL of serum kFLC. In vitro functional studies have demonstrated that MDX-1097 engages Fc receptor bearing effector cells and induces antibody dependent cellular cytotoxicity (ADCC) in kappa myeloma cell lines in the presence of healthy donor peripheral blood mononuclear cells. Further investigations have verified that purified natural killer cells (NK) play a major role in MDX-1097 anti-tumour activity. Importantly, recent studies have demonstrated that antibody dependent cellular phagocytosis by macrophages contributes to the anti-tumour activity of several therapeutic monoclonal antibodies. Preliminary data indicates that MDX-1097 may be capable of inducing enhanced uptake by macrophages. In conclusion MDX-1097 showed specific binding to KMA on myeloma cells isolated from patient's bone marrow samples and antibody binding is observed in the presence of kFLC in patient serum. In addition MDX-1097 anti-tumour activity is probably mediated by multiple Fc receptor bearing effector cells. Disclosures: Wong: Immune System Therapeutics: Employment. Asvadi:Immune System Therapeutics: Employment. Dunn:Immune System Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Jones:Immune System Therapeutics: Employment. Campbell:Immune System Therapeutics: Employment.

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.

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

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

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

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

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

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

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

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

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

Dual Src/Abl Kinase Targeted Inhibition in Myeloma Microenvironment Promotes Myeloma Cell Apoptosis Both in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2813-2813
Author(s):  
Karthik Ramasamy ◽  
Lee Macpherson ◽  
Ghulam J Mufti ◽  
Stephen Schey ◽  
Yolanda Calle
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Stromal Cells ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Abl Kinase ◽  
Osteoclast Function

Abstract Abstract 2813 Poster Board II-789 Osteoclast, in addition to eroding the bone resulting in lytic lesions, enhances plasma cell proliferation and survival via direct cell to cell contact. Src family protein tyrosine kinases (SFKs) and c-Abl kinase play important role downstream of integrin adhesion receptors, and regulate the cytoskeletal organisation, cell motility and gene expression in response to cell adhesion. We hypothesised targeting SFKs and Abl kinase with the small molecule tyrosine kinase inhibitor Dasatinib has potential to reduce adhesion of plasma cells to ECM proteins in the bone marrow and modify the microenvironment by inhibiting osteoclast function, specifically bone resorption. As a result, myeloma cells could be sensitised to drugs with cytotoxic properties such as dexamethasone. Osteoclasts were generated from primary bone marrow mononuclear cells of myeloma and MGUS patients (n=10). Using Immunofluorescence, we found that Dasatinib 100nM but not dexamethasone inhibited osteoclastogenesis and disrupted the actin cytoskeletal organisation with actin clusters formed in the periphery of the cell. There was absence of actin ring formation at sealing zones which is essential for bone resorption. This effect consistently led to impaired osteoclast function, evidenced by fewer resorption pits formed on rabbit dentine slices on toluidine blue staining. Experiments were repeated ≥ 3 times. In plasma cells, the combination of dexamethasone and Dasatinib synergistically (Calcusyn software) inhibited cell proliferation at clinically relevant concentrations and induced apoptosis of human and murine myeloma cell lines alone and in cocultures with human stromal cells ( p<.001). Dasatinib alone at 200 nM concentration does not inhibit plasma cell proliferation with maximal serum concentration achieved in Phase I CML trials being 180nM. Additionally, Dasatinib and Dexamethasone in combination inhibited secretion of IL-6 but not MIL -1 alpha in stromal cell cocultures. Dasatinib but not dexamethasone significantly inhibited adhesion of myeloma cell lines on Fibronectin despite integrin activation with Magnesium EGTA. This effect was mediated through down regulation of both Src and Abl phosphorylation. Both Dasatinib and Dexamethasone inhibited adhesion of PC on stromal cells and osteoclasts. Taken together, our in vitro results suggest that Dasatinib and dexamethasone could be an effective therapeutic combination with Dasatinib impairing adhesion of plasma cells to the bone marrow microenvironment as well as osteoclast function and resultant bone disease thereby sensitising myeloma cells to the cytotoxic effect of dexamethasone. We have also established that the combination of Dasatinib 75mg/kg and dexamethasone 1mg/kg is not toxic to C57BL/KaLwRij mice. The anti-myeloma efficacy of these drugs alone and in combination is being currently studied. The combination of Dasatinib 100 mg OD days 1-28 and Dexamethasone 20mg OD on Day 1-4, 15-18 has resulted in a partial response (EBMT criteria) in 2 multiply relapsed and steroid refractory myeloma patients without significant toxicity. Serum calcium levels fell commensurate with disease response and we are currently performing experiments to analyse the effect of the drug combination on osteoclast function in vivo. These findings warrant exploring this drug combination in steroid resistant myeloma and patients with extensive skeletal disease prospectively in a phase I/II trial. Disclosures: Off Label Use: Dasatinib is not licensed for Myeloma.

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