Expression of Heat Shock Protein 90 and the Effects of HSP90 Inhibitor (17-AAG) in Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4916-4916
Author(s):  
Serhan Alkan ◽  
Jan Duus ◽  
Ismail H. Bahar ◽  
Keith F. Izban ◽  
Hytham Al-Masri ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Cell Line ◽  
Flow Cytometric Analysis ◽  
Heat Shock Protein 90 ◽  
Myeloma Cell ◽  
Immunoperoxidase Staining ◽  
Myeloma Cells ◽  
Innovative Strategy

Abstract Heat Shock Protein 90 (HSP90) is required for structural folding and maintenance of conformational integrity of various proteins, including several associated with cellular signaling. Recent studies utilizing 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90, demonstrated antitumor effect in solid tumors. In order to test whether HSP90 could be targeted in multiple myeloma (MM) patients, we first investigated expression of HSP90 by immunofluorescence and flow cytometric analysis in a myeloma cell line (U266) and primary myeloma cells. Following demonstration of HSP90 expression in myeloma cells, archival samples of 32 MM patients were analyzed by immunoperoxidase staining. Myeloma cells in all patients showed strong cytoplasmic expression of HSP90 in all samples and 55 % also demonstrated concurrent nuclear immunopositivity. Treatment of U266 and primary MM cells with 17AAG resulted in significantly increased apoptosis compared to untreated control cells. Analysis of anti-apoptotic BCL2 family proteins in MM cells incubated with 17-AAG revealed down-regulation of BCL-2, BCL-XL and MCL-1. Furthermore, while low concentration of bortezomib had no cell death, combination of 17AAG and bortezomib treatment revealed a synergistic apoptotic effect on the U266 cell line. These data suggest that targeted inhibition of HSP90 may prove to be a valid and innovative strategy for the development of future therapeutic options for MM patients.

Depletion of the Intracellular Akt Kinase, and Its Downstream Signaling, Mediates Synergy between the Proteasome Inhibitor MG-132 and the Heat Shock Protein 90 Inhibitor 17-AAG in the Multiple Myeloma Cell Line U266.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3364-3364
Author(s):  
Christopher Maisel ◽  
Nizar Bahlis ◽  
Yanling Miao ◽  
Lili Liu ◽  
Stanton Gerson
Keyword(s):  
Multiple Myeloma ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Cell Line ◽  
Proteasome Inhibitor ◽  
Dose Range ◽  
Proteasome Inhibitors ◽  
Heat Shock Protein 90 ◽  
Hsp 70 ◽  
Hsp 90

Abstract While proteasome inhibitors are effective therapy for multiple myeloma (MM), their efficacy could be improved by synergistic targeting of apoptosis. The intracellular serine/threonine kinase Akt has been demonstrated to have a central role in MM cell growth, survival, and drug resistance. Akt is activated by extracellular cytokines such as IGF-1 and IL-6, and contributes to MM resistance by ameliorating the apoptotic effects of proteasome inhibition. Akt requires chaperone proteins for proper stability and function, including the 90 kD molecule Heat Shock Protein 90 (HSP-90). HSP-90 function is abrogated by geldanamycin and its derivative, 17-allylamino-17-demethoxygeldanamycin (17-AAG). In the U266 MM cell line, the IC50 of the proteasome inhibitor MG-132 and 17-AAG was 100 nM and 800 nM, respectively. Following exposure of U266 MM cells to either drug alone, or the combination at a fixed-ratio of their IC50s (1:8), apoptosis was determined by Annexin V staining and FACScan analysis. Synergy analysis was performed using Calcusyn (Biosoft, Cambridge, UK). We found that the combination index (CI) was synergistic (CI<1) throughout the dose range, with a CI = 0.449 ± 0.298 at the combination IC50 (highly significant). For example, the apoptotic effect of 50 nM MG-132 and 400 nM 17-AAG was 6 ±2 % and 23 ±3 %, respectively, whereas the 50:400 nM combination produced apoptosis in 68 ± 2 % of the cells. To analyze effects on Akt and its substrates, we incubated U266 MM cells with MG-132 (50 nM), 17-AAG (400 nM), or the combination. We harvested lysates after zero, two, six, and 24 hours incubation, and Western blot analysis was performed. Co-incubation with MG-132 and 17-AAG, but not either alone, depleted Akt by 24 hours post-therapy. Co-treatment also produced significantly greater upregulation of HSP-90 and HSP-70 than 17-AAG alone, thus demonstrating greater functional inhibition of Akt. The combination also demonstrated the greatest abrogation of Akt-mediated effects on mitochondrial apoptosis: Co-treatment produced the greatest expression of BAD, decreased BCL-XL expression, reduced phosphorylation of GSK-3, and produced the greatest activation of caspase 3. Monotherapy with 17-AAG upregulated HSP-90 and HSP-70, reduced BCL-XL expression, and activated caspase 9. MG-132 monotherapy produced none of these effects. These findings demonstrate that synergy between proteasome inhibitors and 17-AAG is mediated by Akt depletion and abrogation of Akt signaling, predominantly by MG-132 augmentation of 17-AAG-mediated decay of Akt. Down-regulation of Akt-mediated resistance allows dual-apoptotic signaling and synergistic effect of combination therapy. These findings demonstrate a mechanistic rationale for utilizing heat shock protein inhibitors in combination with proteasome inhibitors as therapy for MM.

Generation of Tumor-Specific Cytotoxic T Lymphocytes in Multiple Myeloma Using Dendritic Cells Pulsed with Tumor-Derived Heat Shock Protein gp96.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2451-2451
Author(s):  
Qing Yi ◽  
Jianfei Qian ◽  
Jin Xie ◽  
Siqing Wang ◽  
Muta E. Freeman ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Dendritic Cells ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Cytotoxic T Lymphocytes ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Specific Ctls ◽  
Heat Shock Protein Gp96

Abstract To develop more effective immunotherapy strategies for patients with multiple myeloma (MM), it is important to identify novel tumor antigens. Heat shock protein gp96, a highly conserved glycoprotein, is a chaperon molecule that carries intracellularly processed peptides from proteins synthesized by the cells, including those of tumor-specific or -associated proteins. Recent studies in solid tumors have shown that tumor-derived gp96 is immunogenic and potent at stimulating the generation of tumor-specific cytotoxic T lymphocytes (CTLs). In this study, we examined whether myeloma-derived gp96 can be used as potent myeloma antigen. To generate myeloma-specific CTLs, immature dendritic cells (DCs), obtained from cultures of blood monocytes from HLA-A2+ healthy individuals or patients, were pulsed with gp96 protein purified from the myeloma cell line U266 (A2+), and induced maturation using the cytokine cocktail. Autologous T cells were then stimulated every two weeks with these DCs, and cytotoxicity was examined against gp96-pusled DCs, myeloma cell lines, and primary myeloma cells isolated from patients. After several cycles of in vitro stimulation, specific CTL lines were obtained, which consisted of both CD4+ and CD8+ T cells. These CTLs demonstrated not only specific cytotoxicity against gp96-pusled DCs and the cell line U266, but also significantly killed A2+ primary myeloma cells. No killing was observed against A2+ normal lymphocytes including B cells or A2− myeloma cell lines and primary myeloma cells from patients. Using the cold target inhibition assay, we showed that the same CTLs mediated the killing of both gp96-pulsed DCs and myeloma tumor cells. The cytotoxicity was MHC class I- and, to a lesser extent, class II-restricted, indicating that the gp96 contained both class I- and II-restricted tumor-derived peptides and that myeloma cells naturally present these peptides in the context of their surface MHC molecules. Upon antigen stimulation, these CTLs secreted predominantly interferon-g, detected by the ELISPOT assay and intracellular staining, indicating that they belong to the type-1 T-cell subsets. Furthermore, the CTLs lysed the target cells mainly through the perforin-mediated pathway because Concanamycin A but not Brefeldin-A, the selective inhibitors for perforin- or Fas-, respectively, mediated pathways, abrogated the cytolytic activity of the cells. These results therefore show that myeloma-derived gp96-specific CTLs are able to lyse myeloma tumor cells including primary myeloma plasma cells from patients and, thus, provide a rationale for gp96-based immunotherapy in MM.

scholarly journals Heat Shock Protein-90 Inhibitors Increase MHC Class I-Related Chain A and B Ligand Expression on Multiple Myeloma Cells and Their Ability to Trigger NK Cell Degranulation

2009 ◽  
Vol 183 (7) ◽  
pp. 4385-4394 ◽  
Author(s):  
Cinzia Fionda ◽  
Alessandra Soriani ◽  
Giulia Malgarini ◽  
Maria Luisa Iannitto ◽  
Angela Santoni ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Mhc Class I ◽  
Nk Cell ◽  
Heat Shock Protein 90 ◽  
Class I ◽  
Myeloma Cells ◽  
Ligand Expression

New Molecular and Biological Mechanism of Antitumor Activities of KW-2478, a Novel Nonansamycin Heat Shock Protein 90 Inhibitor, in Multiple Myeloma Cells

2010 ◽  
Vol 16 (10) ◽  
pp. 2792-2802 ◽  
Author(s):  
Takayuki Nakashima ◽  
Toshihiko Ishii ◽  
Hisashi Tagaya ◽  
Toshihiro Seike ◽  
Hiroshi Nakagawa ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 90 ◽  
Biological Mechanism ◽  
Antitumor Activities ◽  
Myeloma Cells

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

Panobinostat Induces Upregulation of CD38 and Augments the Anti-Myeloma Efficacy of Daratumumab in Pre-Clinical Models

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4481-4481 ◽  
Author(s):  
Estefania Garcia-Guerrero ◽  
Tea Gogishvili ◽  
Sophia Danhof ◽  
Martin Schreder ◽  
Celine Pallaud ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Line ◽  
Myeloma Cell ◽  
Target Cells ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Cd38 Expression ◽  
Clinical Models ◽  
Pre Treatment

Abstract Background: Immunotherapy with monoclonal antibodies (mAbs) has recently entered the clinical arena in multiple myeloma, including Daratumumab that targets CD38 on malignant plasma cells. The efficacy of mAbs depends on antigen density and expression of accessory ligands on target cells to initiate cell- and complement-dependent effector mechanisms. Here, we investigate the use of the histone deacetylase inhibitor (HDACi) Panobinostat to modulate target antigen expression and ligand profile on myeloma in favor of potent mAb-mediated recognition and destruction. We show that Panobinostat augments CD38 expression specifically on myeloma cells and demonstrate powerful synergy with anti-CD38 mAb Daratumumab in pre-clinical models. Methods: The myeloma cell line MM1.S and primary myeloma cells were treated with titrated doses of Panobinostat (0, 10, 25 nM) and expression of CD38 and a panel of additional target molecules including B-cell maturation antigen (BCMA) and SLAMF7, as well as accessory ligands analyzed by flow cytometry at 24, 48 and 72 hours. Antibody-dependent cellular cytotoxicity (ADCC) against Panobinostat treated and untreated myeloma cells was analyzed at 4 and 20 hours after addition of PBMC at an effector to target ratio of 25:1 in the presence of Daratumumab (1, 10, 50 ug/mL) or an isotype control antibody. Results: We first treated the myeloma cell line MM1.S with Panobinostat and analyzed its direct cytotoxic anti-myeloma effect. Consistent with previous work, the percentage of live MM1.S myeloma cells had decreased to 85% and 50% after 48 hours of exposure to 10 and 25 nM respectively. We analyzed expression of CD38 on residual live, i.e. 7-AAD negative MM1.S cells by flow cytometry and observed a 1.5 (10 nM) and 2-fold (25 nM) increase of CD38 expression by mean fluorescence intensity (MFI) compared to baseline levels and untreated control cells. The increase in CD38 expression was already detectable after 24 hours and plateaued between 48 and 72 hours. We confirmed our observation in primary myeloma cells from multiple donors (n=4) and detected an even stronger increase to 2 (10 nM) and 4-fold (25 nM) higher CD38 expression compared to untreated cells at 48 hours. Interestingly, expression of BCMA and SLAMF7 was not increased after Panobinostat treatment at all tested concentrations and time points in both MM1.S and primary myeloma. We confirmed that Panobinostat-induced upregulation of CD38 specifically occurred in myeloma, and neither observed this phenomenon in a panel of leukemia and lymphoma cell lines including Raji (Burkitt) and JeKo-1 (mantle cell), nor on resting/activated primary CD8+ and CD4+ T cells that we isolated from peripheral blood of several donors (n=3). Next, we were interested in determining whether the increase in CD38 expression enabled superior anti-myeloma activity of the anti-CD38 mAb Daratumumab. Panobinostat pre-treatment was done for 48 hours at 10 nM as this is a clinically achievable serum level with currently approved regimens. Indeed, significantly higher ADCC was mediated by Daratumumab at all tested concentrations (1, 10 and 50 ug/mL) against MM1.S that we had exposed to Panobinostat. At 4 hours, ADCC was 45% and 25% in Panobinostat-treated and untreated MM1.S respectively, and at 20 hours, near-complete, >90% ADCC of Panobinostat-pre-treated MM1.S had occurred, whereas only 65% of MM1.S were eliminated by Daratumumab without Panobinostat pre-treatment. These data were confirmed in multiple experiments with MM1.S and PBMC from different donors, and with primary myeloma cells. Experiments to evaluate synergy of Panobinostat and Daratumumab therapy in a xenograft model (NSG/MM1.S) are ongoing. Conclusions: Our data demonstrate that the HDACi Panobinostat induces upregulation of CD38 on myeloma and a subsequent dramatic increase of Daratumumab-mediated ADCC in pre-clinical models. These data suggest that Panobinostat could be used synergistically with Daratumumab in a clinical setting to increase response rates and extend duration of responses to Daratumumab. Panobinostat has a known ability to modulate the transcriptional profile of myeloma cells and our data demonstrate for the first time that this ability can be utilized to augment the therapeutic index of antibody-based immunotherapy in multiple myeloma. Disclosures Pallaud: Novartis: Employment. Lehmann:Novartis: Employment. Hudecek:Novartis: Research Funding.

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