HLA-Cw6 Alloreactive CTL Recognizing HLA-CW6+ Myeloma Cells but Not Non-Myeloma HLA-Cw6+ Cells Are Peptide Dependent.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2474-2474
Author(s):  
Alexandrine Geffroy ◽  
Agnes Moreau-Aubry ◽  
Regis Bataille ◽  
Catherine Pellat-Deceunynck
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cytotoxic T Lymphocytes ◽  
Myeloma Cell ◽  
Unrelated Donor ◽  
Myeloma Cell Line ◽  
Specific Lysis ◽  
Myeloma Cells ◽  
Target Ratio ◽  
Effector Target

Abstract We investigated the generation of myeloma-specific cytotoxic T lymphocytes (CTL) from unrelated normal donors non-HLA matched with the myeloma cell line SBN. The aim was to obtain alloreactive CTL specific of peptides to identify genes preferentially expressed by to myeloma cells. After coculture of SBN with PBL of unrelated donor, the T-cell line obtained was cloned and each CTL was assessed against SBN and SB-EBV (B-EBV cell line obtained by infecting B cells of SB patient with EBV) simultaneously. Only the CTL recognizing SBN but not SB-EBV were kept for further study. Among more than 200 clones screened, we isolated two different CTL CD8+ recognizing SBN only (60% of specific lysis at effector: target ratio 5). Their cytotoxicity was blocked by antibodies against HLA-I molecules and more precisely by mAb against HLA-B/Cw molecules. Both CTL recognized also other MM cell lines but only those matched with SBN for HLA-Cw0602 (XG6, BCN) suggesting an HLA-Cw6 recognition. Furthermore, antiserum directed against HLA-Cw6 abrogated the recognition of SBN, XG6 and BCN. Both CTL were not cytotoxic against BC-EBV nor against 3 other B-EBV cell lines derived from HLA-Cw0602 donors. This unreactivity was not restricted to B-EBV cell lines since PBL from 2 normal HLA-Cw0602 donors were also not recognized (cytotoxicity, TNF secretion). We directly measured cell surface HLA-Cw6 expression of both B-EBV and myeloma cells with a scFv directed against HLA-Cw6 and we showed that HLA-Cw6 was more expressed by B-EBV cells as compared to myeloma cells (2fold). So, the lack of recognition of B-EBV cells was not related to HLA-Cw6 expression level. We also looked for KIR expression by the CTL since KIR molecules could have blocked B-EBV recognition that expressed more HLA-Cw6 molecules as compared to myeloma cells. No KIR molecules (p58.1, p58.2, CD94) were found to be expressed by the CTL. To investigate whether the recognition of myeloma cells was peptide dependent, we performed two experiments i.e., acid elution and cold target competition. Acid elution (glycine 0.3M, pH=2.5) abrogated myeloma recognition and cold target competition showed that B-EBV cells were not recognized by the CTL. These data suggest that the CTL recognize a peptide or a set of peptides restricted to myeloma cells or that the CTL recognition is activated by tumor cells only. Anyway, such alloreactive CTL could be helpful for GVL reaction in allotransplanted patients with myeloma.

MYC Responsible for down-Regulation of CD33 Expression in Human Myeloma Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5116-5116
Author(s):  
Karim Shamsasenjan ◽  
Ken-ichiro Otsuyama ◽  
Mohd S. Iqbal ◽  
Maged S. Mahmoud ◽  
Michio M. Kawano
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Myeloma Cell Line ◽  
Down Regulation ◽  
Myeloma Cells ◽  
Expression Levels ◽  
Stat3 Phosphorylation ◽  
Serum Crp ◽  
Human Myeloma Cells

Abstract Human myeloma cells from about 10% of cases with multiple myeloma expressed CD33 and have monocytoid morphology with convoluted nuclei, and all these patients had no increase in serum CRP values. In CD33(+) myeloma cells as well as myeloma cell lines, CD33 expression levels were correlated with the increased expression levels of CEBPA (C/EBPα) gene. This correlation was confirmed by the finding that transfection with the CEBPA gene induced CD33 expression in a CD33(−) myeloma cell line. As suggested by the lack of an increase in serum CRP values in CD33(+) myelomas, IL-6 down-regulated the expression of CD33 in CD33(+) myeloma cell lines along with the down-regulation of CEBPA gene expression. Cucurbitacin I (STAT3 inhibitor) but not U0126 (MAPK inhibitor) could abolish the effect of IL-6. Furthermore, IL-6 up-regulated the expression of MYC via STAT3 phosphorylation and MYC bound to the promoter region of CEBPA gene followed by the down-regulation of the CEBPA expression. It was confirmed that introduction of shRNA for MYC into a CD33(+) myeloma cell line blocked the IL6-induced down-regulation of CD33 and CEBPA expression. Therefore, these results indicate that IL-6 can reverse the expression level of CD33 by up-regulating MYC followed by the down-regulation of CEBPA expression.

Sphingolipids as a novel target for the treatment of multiple myeloma.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e19534-e19534
Author(s):  
Yubin Kang ◽  
Jagadish Kummetha Venketa
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Line ◽  
Cell Lines ◽  
Apoptotic Cell ◽  
Myeloma Cell ◽  
Sphingolipid Metabolism ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Novel Target

e19534 Background: Multiple myeloma (MM) is the second most common hematological malignancy in the United States and accounts for ~10,600 deaths annually. MM remains an incurable disease and almost all patients will eventually relapse and become refractory to currently available therapeutic agents. There is an unmet need for better understanding the disease’s molecular pathways and for identifying novel therapeutic targets. Sphingolipid metabolism is being increasingly recognized as a key pathway in tumor cell proliferation and in tumor sensitivity to anticancer drugs. We hypothesize that altered sphingolipid metabolism plays an important role in the pathogenesis of MM, thus providing a novel target in the treatment of MM. Methods: We first assayed sphingolipid metabolism including sphingolipid metabolites and sphingolipid metabolizing genes in myeloma cell lines, in freshly isolated human primary CD138+myeloma cells, and in publically available dataset. We then tested the efficacy of the selective SK2 inhibitor (ABC294640) and the SK2 shRNA in killing myeloma cells in vitro. Results: 1) Compared to immortalized B cells, the levels of pro-apoptotic ceramides were decreased whereas the proliferative sphingosine 1-phosphate (S1P) was increased in myeloma cell lines. 2) The expression of several key sphingolipid-metabolizing genes including sphingosine kinase (SK) 1 and 2 was altered in freshly isolated human primary bone marrow myeloma cells and in publically available microarray dataset. 3) The selective SK2 inhibitor (ABC294640) induces apoptotic cell death and inhibits myeloma cell growth with an IC50of ~20 μM in 9 myeloma cell lines. 4) Interestingly, OPM-1 myeloma cell line was extremely sensitive to ABC294640 with an IC50of <5 µM whereas U266 myeloma cell line was resistant to ABC294640. SK2 shRNA induced apoptotic cell death in OPM-1, but not in U266 cells. We are currently investigating the molecular mechanisms underlying the resistance of U266 myeloma cells to ABC294640. Conclusions: Our data demonstrated that sphingolipid metabolism provides an attractive target in the treatment of refractory/relapased multiple myeloma.

Molecular Mechanism of CD56 Expression in Human Myeloma Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5130-5130
Author(s):  
Mohd S. Iqbal ◽  
Ken-ichiro Otsuyama ◽  
Karim Shamsasenjan ◽  
Michio M. Kawano
Keyword(s):  
Transcription Factors ◽  
Cell Line ◽  
Cell Lines ◽  
Promoter Region ◽  
Cell Lineage ◽  
Myeloma Cell ◽  
Myeloma Cell Line ◽  
Down Regulation ◽  
Myeloma Cells ◽  
Cd56 Expression

Abstract CD56 (NCAM1), one of the non-B cell lineage markers, is frequently detected on primary myeloma cells from more than 80% patients with overt myeloma. Microarray analysis of the CD56(+) myeloma cell lines showed markedly increased expressions of transcription factors involved in the neuronal cell lineage compared to the CD56(−) myeloma cell lines. There are three important binding sites of transcription factors such as HOX, SOX and PAX, present in the CD56 promoter region. Furthermore, ChIP analysis showed the specific bindings of HOXB9 and SOX1 in the CD56 promoter region of both the CD56(+) and CD56(−) myeloma cell line, while there was no significant difference observed in the PAX8 expression between the CD56(+) and CD56(−) myeloma cell lines. RT-PCR and western blot analysis confirmed that HOXB3 and HOXB9 as well as some of their potential cofactors were up-regulated in the CD56(+) myeloma cell lines with the concomitant down-regulation of the SOX1 and OCT3/4. First, we focused on the role of down-regulation of SOX1 in the CD56(−) myeloma cell line by the shRNA transfection. However, shRNA against SOX1 failed to induce CD56 expression in CD56(−) myeloma cell line. Therefore, these results indicate that the down-regulation of SOX1 alone may not be enough for the CD56 induction which might also require the overexpression of either HOXB9 or HOXB3, and the further study in the molecular mechanism of CD56 expression might contribute to the understanding of complex network of the interaction of transcription factors in human myeloma cells.

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.

Enforced CD19 Expression Leads to Growth Inhibition and Reduced Tumorigenicity

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3551-3558 ◽  
Author(s):  
Maged S. Mahmoud ◽  
Ryuichi Fujii ◽  
Hideaki Ishikawa ◽  
Michio M. Kawano
Keyword(s):  
Growth Inhibition ◽  
Cell Line ◽  
Myeloma Cell ◽  
Inhibitory Effect ◽  
Myeloma Cell Line ◽  
Growth Inhibitory Effect ◽  
Myeloma Cells ◽  
Growth Inhibitory ◽  
Human Myeloma Cell Line

In multiple myeloma (MM), the cell surface protein, CD19, is specifically lost while it continues to be expressed on normal plasma cells. To examine the biological significance of loss of CD19 in human myeloma, we have generated CD19 transfectants of a tumorigenic human myeloma cell line (KMS-5). The CD19 transfectants showed slower growth rate in vitro than that of control transfectants. They also showed a lower capability for colony formation as evaluated by anchorage-independent growth in soft agar assay. The CD19 transfectants also had reduced tumorigenicity in vivo when subcutaneously implanted into severe combined immunodeficiency (SCID)-human interleukin-6 (hIL-6) transgenic mice. The growth-inhibitory effect was CD19-specific and probably due to CD19 signaling because this effect was not observed in cells transfected with a truncated form of CD19 that lacks the cytoplasmic signaling domain. The in vitro growth-inhibitory effect was confirmed in a nontumorigenic human myeloma cell line (U-266). However, introduction of the CD19 gene into a human erythroleukemia cell line (K-562) also induced growth inhibition, suggesting that this effect is CD19-specific, but not restricted to myeloma cells. These data suggest that the specific and generalized loss of CD19 in human myeloma cells could be an important factor contributing to the proliferation of the malignant plasma cell clones in this disease.

Induction of Myeloma Cell Line-specific Cytotoxic T Lymphocytes using Monocyte-derived Dendritic Cells Pulsed with Myeloma Cell Line Lysates

2006 ◽  
Vol 41 (3) ◽  
pp. 186
Author(s):  
Myong-Suk Park ◽  
Jung-Sun Park ◽  
Hyun-Kyu Kang ◽  
Sang-Ki Kim ◽  
Jong-Ho Won ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
T Lymphocytes ◽  
Cell Line ◽  
Cytotoxic T Lymphocytes ◽  
Myeloma Cell ◽  
Myeloma Cell Line

Induction of Multiple Myeloma-Specific Cytotoxic T Lymphocytes Using HLA-A2.1-Specific CD19 and CD20 Peptides.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2477-2477
Author(s):  
Jooeun Bae ◽  
Jeff A. Martinson ◽  
Hans G. Klingemann ◽  
Steven Treon ◽  
Kenneth C. Anderson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Lymphocytes ◽  
Cytotoxic Activity ◽  
Cell Lines ◽  
Cytotoxic T Lymphocytes ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Specific Ctls ◽  
Ifn Γ ◽  
Hla A2.1

Abstract We have identified novel CD19 and CD20 antigen-derived HLA-A2.1-specific immunogenic peptides, CD19150–158 (KLMSPKLYV) and CD20188–196 (SLFLGILSV), for generating cytotoxic T lymphocytes (CTLs) against malignant B-cell diseases. Initial testing showed that the CTLs displayed antigen-specific and HLA-A2.1-restriced cytotoxic activity against both Burkitt’s lymphoma and chronic lymphoid leukemia cell lines. The observed cytotoxic activity of the CTLs was shown to be specific to the CD19150–158 or the CD20188–196 peptides. Additionally, the CTLs displayed a distinct phenotype (majority CD69+/CD45RO+) along with a significant (p&lt;0.05) increase in cell proliferation and IFN-γ release following re-stimulation with HLA-A2.1+/CD19+/CD20+ tumor cell lines. Based on emerging information that clonogenic myeloma cells express CD19 and/or CD20, we evaluated the activity of the CD19 and CD20 peptide specific-CTLs against several multiple myeloma cell lines. Five of 10 myeloma cell lines evaluated were HLA-A2.1-positive and expressed both CD19 and CD20 antigens. CD19 peptide specific-CTLs generated from normal donors were able to specifically lyse CD19+/HLA-A2.1+ MM cell lines (30% lysis; 10:1 E:T ratio) but did not lyse CD19−/HLA-A2.1+ or CD19+/HLA-A2.1− cell lines. Similarly, the CD20-specific CTLs generated from normal donors lysed CD20+/HLA-A2.1+ MM cell lines (25% lysis; 10:1 E:T ratio), in a manner restricted to HLA-A2.1 and specific to antigens. We next showed IFN-γ production by the CTLs after exposure to CD19+/HLA-A2.1+ or CD20+/HLA-A2.1+ MM cells. Moreover, we have demonstrated the ability to expand CD20-CTLs under serum-free culture conditions while maintaining their cytotoxic activity (28–49%). In ongoing studies, we are evaluating the ability of CD19- and CD20-specific CTLs to eliminate clonogenic myeloma cells in vitro and in vivo in a SCID mouse model of myeloma. These preclinical studies strongly suggest that immunogenic CD19 and CD20 peptide-based vaccines represent a promising immunotherapeutic approach in myeloma.

Expression of TRAIL Receptors on the Multiple Myeloma Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4868-4868
Author(s):  
Juan Li ◽  
Junhe Li ◽  
Shaokai Luo ◽  
Yin Zhao
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
Mononuclear Cells ◽  
Myeloma Cell ◽  
Myeloma Cell Line ◽  
Killing Effect ◽  
Myeloma Cells ◽  
Decoy Receptors ◽  
Trail Receptors ◽  
Chemotherapy Agents

Abstract Objective To study the different expression of death receptors and decoy receptors on mononuclear cells from patients with multiple myeloma and myeloma cell line KM3 and compare the different expression of TRAIL receptors after chemotherapy or exposure to doxorubicin, to explore the mechanisms by which TRAIL selectively kills tumor cells. Methods Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry was used to investigate the expression of four receptors on mononuclear cells in 23 multiple myeloma patients and myeloma cell line KM3 and 15 controls, we furthermore compared the changes of expression mode after chemotherapy and incubation of KM3 cell with sub-clinical concentration of Doxorubicin. Results There finds only DR4 and DR5 on KM3 cell line without the expression of DcR1 and DcR2. Expression of DR4 and DR5 on mononuclear cells of MM patients is higher than that of controls (P&lt;0.05), but DcR1 and DcR2 expression was lower than that of controls (P&lt;0.05), after chemotherapy and exposure to Doxorubicin, the expression of DR5 on MM cells was up-regulated (P&lt;0.05) Conclusions The expression of four receptors on myeloma cells and normal controls was significantly different, which might account for the selective killing effect of TRAIL on MM cells. DR5 was up-regulated on KM3 when incubating with Doxorubicin and after chemotherapy which suggests chemotherapy agents might enhance the apopotosis of MM cells through up-regulating of DR5 receptor.

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

In Vitro Efficacy of Agonistic Antibody to TRAIL-R1 (Mapatumumab) and Low Dose Bortezomib in Multiple Myeloma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5169-5169
Author(s):  
Paul Neeson ◽  
Karen Chen ◽  
Tsin Tai ◽  
Amanda Shin ◽  
Joanne Davis ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Cell Line ◽  
Cell Lines ◽  
Plasma Cell ◽  
Surface Expression ◽  
Myeloma Cell ◽  
Myeloma Cell Line ◽  
Human Myeloma Cell Line ◽  
Data Points ◽  
Dose Response Study

Abstract The proteasome inhibitor Bortezomib (Bz) is a potent inducer of plasma cell apoptosis via suppression of NFkB activity. Bz is also associated with the dose limiting induction of thrombocytopenia as well as inhibition of dendritic cell (DC) function, which may limit the induction of autologous T cell responses to myeloma antigens in vivo. We hypothesized that the dose, and therefore the adverse effects of Bz, may be reduced by the addition of the agonistic anti-TRAIL-R1 antibody Mapatumumab (Mp). A dose of Bz that did not result in detectable apoptosis was determined for each of the cultured human plasma cell lines (RPMI8226, U266, LP-1, NCI-H929, OPM-2 and JJN3) via titration experiments in which each cell line was cultured for 24–48 hrs in Bz concentrations from 0.1–10nM. Cells were stained with annexin V-FITC and the viability dye 7-AAD and analyzed on a BD LSRII flow cytometer. Plasma cell surface expression of TRAIL-R-1 and -2 were also assessed and correlated with sensitivity to Mp induced apoptosis. Bz monotherapy at 10nM produced 85–98% apoptosis in all plasma cell lines. In contrast 4/6 cell lines were sensitive to Mp alone, RPMI8226 at 0.06ug/ml, U266 and OPM-2 at 1ug/ml and LP-1 at 10ug/ml. When non-apoptosis inducing Bz doses were used in combination with titrated doses of Mp (from 0.01 to 50ug/ml) apoptosis of RPMI8226, U266 and OPM-2 was enhanced by 32%, 30% and 15% respectively compared to Mp alone (p=&lt;0.05 for each cell line). The particular sensitivity of RPMI8226 to Bz/Mp therapy correlated with this cell line’s higher level of TRAIL-R 1 surface expression in comparison to U266. In contrast, the addition of Bz to Mp did not result in any additional apoptosis induction over Mp alone in LP-1, JJN3 and NCI-H929. Conversely, 24 hrs of Bz pretreatment resulted in subsequent enhanced sensitivity of LP-1 to Mp but not NCI-H929. In conclusion, the effective dose of Bz may be significantly reduced when used in combination with Mp. The Bz+Mp combination therapy may be more efficacious than Bz alone and allow dose modification of Bz. Reduction in Bz dose may in-turn reduce side-effects whilst allowing the preservation or promotion of endogenous immune responses. Figure 1. Combination bortezomib and Mapatumamab therapy induce synergistic death of the human myeloma cell line RPMI8226. A dose response study for (A) bortezomib (B) Mapatumamab was performed on RPMI8226 cells by treating the cells with titrated doses of the monotherapy for 48 hours in triplicate wells. Subsequently RPMI8226 cells were treated with combination bortezomib and Mapatumamab therapy (C). Bortezomib dose (1nM) did not induce apoptosis above untreated controls, Mp was added at doses from 0.03 to 0.15ug/ml. A statistical comparison was made between apoptosis with Mp monotherapy versus Bz plus Mp using student t-test, * represents data points where combination therapy is statistically different to Mp alone, p&lt;0.05. Results are representative of two separate experiments. Figure 1. Combination bortezomib and Mapatumamab therapy induce synergistic death of the human myeloma cell line RPMI8226. A dose response study for (A) bortezomib (B) Mapatumamab was performed on RPMI8226 cells by treating the cells with titrated doses of the monotherapy for 48 hours in triplicate wells. Subsequently RPMI8226 cells were treated with combination bortezomib and Mapatumamab therapy (C). Bortezomib dose (1nM) did not induce apoptosis above untreated controls, Mp was added at doses from 0.03 to 0.15ug/ml. A statistical comparison was made between apoptosis with Mp monotherapy versus Bz plus Mp using student t-test, * represents data points where combination therapy is statistically different to Mp alone, p&lt;0.05. Results are representative of two separate experiments.

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