Cytotoxic Effects Of Bortezomib Research Articles

Clarithromycin overcomes stromal cell-mediated drug resistance against proteasome inhibitors in myeloma cells via autophagy flux blockage leading to high NOXA expression.

We previously reported that macrolide antibiotics, such as clarithromycin (CAM), blocked autophagy flux, and simultaneous proteasome and autophagy inhibition by bortezomib (BTZ) plus CAM resulted in enhanced apoptosis induction in multiple myeloma (MM) cells via increased endoplasmic reticulum (ER) stress loading. However, in actual therapeutic settings, cell adhesion-mediated drug resistance between bone marrow stromal cells (BMSC) and MM cells has been known to be a barrier to treatment. To investigate whether CAM could enhance BTZ-induced cytotoxicity in MM cells under direct cell adhesion with BMSC, we established a co-culture system of EGFP-labeled MM cells with BMSC. The cytotoxic effect of BTZ on MM cells was diminished by its interaction with BMSC; however, the attenuated cytotoxicity was recovered by the co-administration of CAM, which upregulates ER stress loading and NOXA expression. Knockout of NOXA in MM cells canceled the enhanced cell death by CAM, indicating that NOXA is a key molecule for cell death induction by the co-administration of CAM. Since NOXA is degraded by autophagy as well as proteasomes, blocking autophagy with CAM resulted in the sustained upregulation of NOXA in MM cells co-cultured with BMSC in the presence of BTZ. Our data suggest that BMSC-associated BTZ resistance is mediated by the attenuation of ER stress loading. However, the addition of CAM overcomes BMSC-associated resistance via upregulation of NOXA by concomitantly blocking autophagy-mediated NOXA degradation and transcriptional activation of NOXA by ER stress loading.

Ex vivo propagation in a novel 3D high-throughput co-culture system for multiple myeloma

PurposeMultiple myeloma (MM) remains an incurable hematologic malignancy which ultimately develops drug resistance and evades treatment. Despite substantial therapeutic advances over the past years, the clinical failure rate of preclinically promising anti-MM drugs remains substantial. More realistic in vitro models are thus required to better predict clinical efficacy of a preclinically active compound.MethodsHere, we report on the establishment of a conical agarose 3D co-culture platform for the preclinical propagation of primary MM cells ex vivo. Cell growth was compared to yet established 2D and liquid overlay systems. MM cell lines (MMCL: RPMI-8226, U266, OPM-2) and primary patient specimens were tested. Drug sensitivity was examined by exploring the cytotoxic effect of bortezomib and the deubiquitinase inhibitor auranofin under various conditions.ResultsIn contrast to 2D and liquid overlay, cell proliferation in the 3D array followed a sigmoidal curve characterized by an initial growth delay but more durable proliferation of MMCL over 12 days of culture. Primary MM specimens did not expand in ex vivo monoculture, but required co-culture support by a human stromal cell line (HS-5, MSP-1). HS-5 induced a > fivefold increase in cluster volume and maintained long-term viability of primary MM cells for up to 21 days. Bortezomib and auranofin induced less cytotoxicity under 3D vs. 2D condition and in co- vs. monoculture, respectively.ConclusionsThis study introduces a novel model that is capable of long-term propagation and drug testing of primary MM specimens ex vivo overcoming some of the pitfalls of currently available in vitro models.

Pivotal Role of OCL-Derived IGF1 in Drug Resistance and Bone Destruction in MM

Multiple myeloma (MM) is largely incurable, and is characterized by devastating bone destruction caused by increased osteoclast (OCL) differentiation and bone resorption in more than 85% of MM patients. OCLs in MM not only promote bone resorption but also increase MM cell growth and drug resistance. Despite recent advances in anti-myeloma treatment, development of anti-MM drug resistance is a major limitation of MM therapy. Therefore, new treatment modalities are urgently needed to overcome drug resistance and decrease bone resorption. IGF1 is a crucial factor for tumor cell growth and survival of malignant cells, especially in MM. IGFI also contributes to development of drug resistance of MM cells to anti-MM agents, including proteasome inhibitors and immunomodulatory agents, but how OCLs contribute to drug resistance is still not clearly delineated. We found that IGF1 was highly expressed in OCLs attached to bone and bone marrow myeloid cells in vivo, and the expression levels of IGF1 in OCLs from MM bearing mice is higher than in normal OCLs. Intriguingly, OCLs produced more IGF1 (0.8 ng/ml/protein) than MM cells (not detected) and bone marrow stromal cells (BMSCs) (0.4 ng/ml/protein) in vitro. In addition, IGF1 protein expression in OCLs was upregulated (1.8 fold) by treatment with conditioned media (CM) from 5TGM1 murine MM cells, TNF-α or IL-6, major paracrine factors that are increased in the bone marrow microenvironment in MM. These results suggest that OCLs are a major source of local IGF1 in the MM bone marrow microenvironment. To further characterize the role of OCL-derived IGF1, we generated a novel mouse with targeted deletion of Igf1 in OCLs (IGF1-/--OCL), and assessed the role of OCL-derived IGF1 in drug resistance of MM cells and bone destruction. Treatment of 5TGM1 cells with bortezomib (BTZ) (3 nM, 48 hours) decreased the viability of 5TGM1 cells by 50%. Importantly, the cytotoxic effects of BTZ on MM cells were decreased (by 5%) when MM cells were cocultured with OCLs from wild type (WT) mice. In contrast, coculture of MM cells with IGF1-/--OCLs or WT-OCLs treated with IGF1 neutralizing antibody (IGF1-ab) did not block BTZ's effects on MM cell death. Consistent with these results, coculture of MM cells with IGF1-/--OCLs or WT-OCLs treated with IGF1-ab resulted in BTZ-induced caspase-dependent apoptosis in MM cells. We next examined the effects of OCLs on the signaling pathways responsible for MM cell survival. WT-OCL-CM promptly induced the phosphorylation of Akt and activation of p38, ERK and NF-κB in MM cells. However, these pathways were not activated by MM cells treated with IGF1-/--OCL-CM or IGF1-ab-treated WT-OCL-CM. Since adhesion of MM cells to BMSCs via interaction of VLA-4 and VCAM-1 plays a critical role in cell adhesion-mediated drug resistance (CAMDR) in MM, we tested if treatment of human BMSCs with human OCL-CM upregulated VCAM-1 expression. We found that OCL-CM upregulated VCAM-1 expression on BMSCs (x fold). In contrast, treatment of BMSCs with OCLs treated with IGF1-ab blocked VCAM-1 induction. These data suggest that OCL-derived IGF1 can contribute to MM cell drug resistance in the bone marrow microenvironment. We then examined the role of IGF1 inhibition on osteoclastogenesis and the bone resorption capacity of OCLs. RANK ligand induced the expression of cathepsin K and NFATc1 in CD11b+ bone marrow cells from WT mice, differentiation markers of OCLs, and the formation of TRAP-positive multinucleated OCLs. However, OCLs formed by RANK ligand treatment of CD11b+ bone marrow cells from IGF1-/- mice had markedly decreased cathepsin K and NFATc1 expression and OCL formation. Next, we tested the bone resorption capacity of OCLs formed by CD11b+ bone marrow cells from IGF1-/- mice vs. WT mice. Similar numbers of OCLs were cultured with RANK ligand on bone slices for 72 hours. The bone resorption activity of Igf1-/--OCLs was significantly decreased (70%) compared with WT-OCLs. These results suggest that OCL-derived IGF1 plays a critical role in MM drug resistance and bone destruction, and that inhibition of the effect of IGF1 in OCLs should decrease MM drug resistance and bone destruction. Disclosures Roodman: Amgen trial of Denosumab versus Zoledronate: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

3232 Synergistic cytotoxic effects of bortezomib and ck2 inhibitor cx-4945 in acute lymphoblastic leukemia: turning off the prosurvival er chaperone BIP/GRP78 and turning on the proapoptotic NF-κb

3232 Synergistic cytotoxic effects of bortezomib and ck2 inhibitor cx-4945 in acute lymphoblastic leukemia: turning off the prosurvival er chaperone BIP/GRP78 and turning on the proapoptotic NF-κb

Expression of GITR Enhances Multiple Myeloma Cell Sensitivity to Bortezomib.

Recently tumor necrosis factor receptor super family member 18 (TNFRSF18, also called GITR) has been identified as a novel tumor suppressor gene in Multiple Myeloma (MM), undergoing aberrant DNA methylation-mediated gene expression silencing. Furthermore, the expression of GITR blocks canonical NF-κB activation in MM cells in response to TNFα. Bortezomib, a proteasome inhibitor, can induce NF-κB activation, which may significantly influence the drug response in MM patients. In this study, we aim to elucidate if GITR status is associated with response to Bortezomib in MM cells through regulating GITR mediated NF-κB blockade. We found that GITR was significantly downregulated in MM patients and cell lines. Overexpression of GITR inhibited non-canonical NF-κB activation induced by TNFα. Moreover, NF-κB inhibitor induced apoptosis in GITR-deficient MM cells in response to TNFα. In addition, overexpression of GITR could inhibit Bortezomib-induced NF-κB activation and enhance the cytotoxicity of Bortezomib in GITR-deficient MM cell line (MM1.S). In contrast, knockdown of GITR attenuated the cytotoxic effect of Bortezomib on GITR proficient MM (RPMI) cell line and increased NF-κB activation. Finally, overexpression of GITR enhanced the sensitivity to Bortezomib in co-culture with bone marrow stromal cells and significantly reduced the tumor growth in MM1.S xenograft mice. In conclusion, we demonstrated that GITR expression can enhance the sensitivity to Bortezomib by inhibiting Bortezomib-induced NF-κB activation.

Modelling the efficacy of hyperthermia treatment

Multimodal oncological strategies which combine chemotherapy or radiotherapy with hyperthermia, have a potential of improving the efficacy of the non-surgical methods of cancer treatment. Hyperthermia engages the heat-shock response (HSR) mechanism, the main component of which are heat-shock proteins. Cancer cells have already partially activated HSR, thereby hyperthermia may be more toxic to them relative to normal cells. On the other hand, HSR triggers thermotolerance, i.e. hyperthermia-treated cells show an impairment in their susceptibility to a subsequent heat-induced stress. This poses questions about efficacy and optimal strategy for anti-cancer therapy combined with hyperthermia treatment. To address these questions, we adapt our previous HSR model and propose its stochastic extension. We formalize the notion of a HSP-induced thermotolerance. Next, we estimate the intensity and the duration of the thermotolerance. Finally, we quantify the effect of a multimodal therapy based on hyperthermia and a cytotoxic effect of bortezomib, a clinically approved proteasome inhibitor. Consequently, we propose an optimal strategy for combining hyperthermia and proteasome inhibition modalities. In summary, by a mathematical analysis of HSR, we are able to support the common belief that the combination of cancer treatment strategies increases therapy efficacy.

Cytotoxic effects of bortezomib combined with fludarabine in T-cell lymphoma cell line HUT-78 in vitro

Cytotoxic effects of bortezomib combined with fludarabine in T-cell lymphoma cell line HUT-78 in vitro

Abstract 1518: Multiple myeloma cells cross talk with bone marrow stroma lead to induction of DKK1 expression and produce bortezomib resistance

Abstract The importance of the micro-environment in tumor progression is now well established. Dynamic interaction between multiple myeloma (MM) cells and bone marrow stromal cells (BMSCs) plays critical role in the progression of MM including drug response and bone erosion. Firstly, we found that DKK1 expression was statistically increased in MM cells (MOLP8, KMS12BM, KMS12PE, NCIH929, LP1, MOLP2, EJM, U266, RPMI8226, and 536MM, respectively) co-cultured with BMSCs obtained from MM patients when compared to MM cells alone (p<0.05). Also, MM cells co-cultured with BMSCs were significantly more resistant against 25 nM bortezomib than MM cells alone (p<0.05). We, therefore, established a bortezomib-resistant cell line using U266 cells (U266/velR), and explored the characteristics of U266/velR cells. Cytotoxic effect of bortezomib in U266/velR was 1.5 folds lower than U266 cells, and the cross-resistance against thalidomide was observed. DKK1 expression in U266/velR was higher than that in parental cells. We found that elevated levels of p-p65 were detected in U266/velR, and the degree of p65 and I-κB expression levels reduced by bortezomib was different between U266/velR and U266. Elevated levels of p-p65/p65 were effectively suppressed by treating with NF-κB activator inhibitor (6-aminoquinazoline). Also, combined treatment of bortezomib and 6-aminoquinazoline reduced the expression of DKK1 in co-culture of U266 cells and BMSCs. The expression of HGF in CD138− fractions of MM bone marrow cells was higher than that in its CD138+ fractions. Whereas, IL-6 and OPN in CD138+ fraction of MM bone marrow cells was higher compared to its CD138− fractions. In co-culture of U266 and BMSCs, IL-6, OPN and HGF was dramatically increased compared to U266 alone and BMSC alone (p<0.01; p<0.05). Taken together, cytokines and growth factor regulated by dynamic interplay between MM cells and BMSCs contributes the bortezomib response and bone erosion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1518. doi:1538-7445.AM2012-1518

Abstract 558: Bortezomib induction of the unfolded protein response mediates Epstein-Barr virus lytic activation but is separable from bortezomib-induced cytotoxicity

Abstract Epstein-Barr virus (EBV) is associated with many human cancers. Several therapeutic strategies involving the pharmacologic activation of the EBV lytic cycle for tumor killing have been described (1-2). We previously identified bortezomib, a proteasome inhibitor approved for the treatment of multiple myeloma and mantle cell lymphoma, as a potent activator of EBV lytic gene expression. The mechanism of action of bortezomib as either a viral lytic activator or an anticancer drug remains poorly understood. However, induction of the unfolded protein response (UPR) has emerged as a central theme in both areas of investigation (3). In order to further evaluate the role of the UPR in lytic activation, we treated lymphoid and epithelial tumor cell lines with bortezomib and assessed induction of the UPR and EBV lytic gene expression. We found dose-dependent parallel induction of UPR and EBV lytic infection across cell lines. However, an EBV-immortalized lymphoblastoid cell line was relatively resistant to both bortezomib-induced UPR and induction of EBV lytic gene expression, but not resistant to bortezomib-induced cell killing. To further investigate, we assessed induction of the UPR by thapsigargin which leads to depletion of Ca2+ in the ER. Thapsigargin induced the UPR and EBV lytic gene expression across all the cell lines tested. Blocking ER stress and UPR activation, by cycloheximide treatment or by BiP knockdown, diminished EBV lytic gene expression but had no effect on cellular toxicity. Additionally, regulated knockdown of UPR associated transcription factors (XBP1(s), c-Jun) blocked lytic activation by both agents. Taken together, these results suggest that induction of the UPR by bortezomib is tightly linked to EBV lytic activation but may be separable from the cytotoxic effects of bortezomib.

Inhibition of IRAK1 As a Novel Therapeutic Strategy in Acute Myeloid Leukemia and Myelodysplastic Syndrome

Abstract 612Recent work has shown that acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients exhibit downregulation of miR-146a, a miRNA that negatively regulates the innate immune pathway by targeting IRAK1 and TRAF6. Mice lacking miR-146a show elevated IRAK1 protein expression, and develop AML and MDS-like features resembling the human diseases. Prior to this study, the role of IRAK1 in human myeloid malignancies was unknown. We conducted a comparison of gene expression profiles of 136 cases of MDS CD34+ cells with 17 normal CD34+ cells obtained from ArrayExpress (E-GEOD-19429; Pellagatti et al., Leukemia, 2010). According to this data set, we observed IRAK1 overexpression in MDS patients (P = 0.017). IRAK1 is a serine/threonine kinase, and after phosphorylation on threonine-209 (T209), its kinase activity is induced, thus allowing for subsequent activation of TRAF6 and eventually NF-kB. Interestingly, we observed higher basal levels of phospho-IRAK1 at T209 in MDS and AML samples as compared to normal human CD34+ cells. To investigate the potential role of IRAK1 in AML and MDS, we used genetic and pharmacological approaches to suppress IRAK1 activity in MDS/AML cell lines and bone marrow cells from MDS patients. RNAi-mediated knockdown of IRAK1 in MDS and AML samples resulted in impaired growth of malignant hematopoietic stem/progenitor cells in methylcellulose assays and rapid apoptosis in vitro. In addition, we used a small-molecule inhibitor (benzimidazole analog; Amgen Inc.) to potently inhibit IRAK1 kinase activity. MDS/AML cell lines and MDS patient samples cultured with the IRAK1 inhibitor exhibited impaired growth and increased apoptosis, which coincided with decreased phospho-IRAK1 at T209, and active versions of TRAF6 and NF-kB. Importantly, the inhibition of IRAK1 kinase function is selectively detrimental to MDS and AML samples while preserving normal CD34+ cell viability and function. Given this novel requirement of IRAK1 in MDS and AML, we examined whether Lenalidomide or Bortezomib, two treatment options for MDS/AML and reported immunosuppressors, exhibit anti-leukemic activity in part by targeting IRAK1. We observed that Bortezomib, but not Lenalidomide, inhibits IRAK1 mRNA and protein expression in MDS/AML cells. The cytotoxic effect of Bortezomib can be partly rescued by forced expression of IRAK1 in these cells. To determine the molecular and cellular basis of cell death following loss of IRAK1 function or expression, we applied microarrays to MDS cells treated with IRAK1 inhibitor or transduced with a lentiviral vector encoding an shRNA targeting IRAK1. An overlap of commonly deregulated genes imposed by loss of IRAK1 expression or by the IRAK1 inhibitor revealed unique pathways relevant to the survival of MDS and AML cells. In summary, these findings are the first to implicate IRAK1 in the maintenance of myeloid malignancies and describe the effectiveness of an IRAK1 inhibitor on suppressing MDS and AML viability. Disclosures:Oliva:Celgene: Consultancy.

NF-κB and MCL-1 Are Important Determinants for the Effectiveness of Bortezomib In CD34+ AML Versus CD34− AML Cells

Abstract 1420AML patients still have an unfavorable outcome despite the treatment with intensive chemotherapy. New therapeutic options are explored for targeting the limited population of leukemic stem cells that is responsible for the sustained leukemic growth. In particular combination of agents are tested that trigger separate cellular cytotoxic pathways.In leukemic cell lines it has been shown that the cytokine rhTrail (recombinant human Tumor necrosis factor- related apoptosis inducing ligand) is an effective cytotoxic agent and synergy in effectiveness has been observed with the proteasome inhibitor bortezomib. However, limited information is available about the efficacy of both agents in patient AML cells and more specifically in the CD34+ enriched AML stem cell fraction. Firstly, we demonstrated cytotoxic effects of Trail in the leukemic cell line HL60 and resistance in Oci-AML3 cells. We observed synergism in HL60 cells and sensitization towards Trail in Oci-AML3 cells upon bortezomib addition. To investigate the efficacy of both agents in primary AML cells, AML mononuclear cells (n=17) were treated with Trail (100ng/mL) and bortezomib (20nM) and cultured in short- and long-term cultures. Cytotoxic effects of bortezomib were observed in 65% of the patients, whereas Trail was not effective, neither in the absence nor presence of bortezomib despite the expression of the death receptors. To study whether bortezomib also affected the leukemic enriched stem cell fraction, AML mononuclear cells were sorted in CD34+ AML and CD34− AML fractions and exposed to bortezomib (n=5). Our results demonstrated that CD34− AML cell fractions were most prone for the effects of bortezomib (survival CD34− versus CD34+ = 45% ± 18% versus 75% ± 18%, p = 0.03) which were confirmed with the long-term culture assay. These results could not be ascribed to bortezomib being less effective in non-dividing cells. HL60 cells were used as a model system and exposed to CFSE for making a distinction between dividing and non-dividing cells. CFSE bright and dim cells were equally sensitive to the effects of bortezomib.Since NF-κB is an important downstream target of bortezomib, EMSA’s were performed on CD34+ AML and CD34− AML cells. Preliminary data indicates that the highest NF-κB activity is present in the CD34− AML cells. These findings are in line with results of a micro-array study (de Jonge et al, Leukemia 2011) with a separate set of 49 AMLs sorted in CD34+ AML versus CD34− AML cells. Cytokines and chemokines with NF-κB binding sites in their promoter region are significantly upregulated in CD34− AML cells versus CD34+ AML cells.The limited effectiveness of bortezomib in CD34+ AML cells might be related to an inappropriate downregulation of anti-apoptotic genes in part controlled by NF-κB. It has been shown that MCL-1 downregulation, mediated by bortezomib, has an essential role in the bortezomib-mediated apoptosis. Microarray analysis showed that MCL-1 is significantly elevated in CD34+ AML cells compared to normal CD34+ cells (p=0.0002). However, western blots demonstrated that these high levels of MCL-1 expression did not decrease in AMLs upon bortezomib exposure. To demonstrate whether downregulation of MCL-1 sensitize AML cells for the effects of bortezomib, Oci-AML3 cells were transduced with MCL-1 shRNA and displayed a 4-fold increased sensitivity to bortezomib (IC50 = 2.7nM versus 10nM). Results in primary AML cells are in progress.In conclusion, these results demonstrated that CD34− AML cells are more prone for the effects of bortezomib than CD34+ AML cells likely due to higher NF-κB activity; the limited effectiveness in CD34+ AML cells might be overcome by modulation of the anti-apoptotic protein MCL-1. Disclosures:Quax:Triskel Therapeutics: Employment, Membership on an entity's Board of Directors or advisory committees.

Delayed treatment with vitamin C and N-acetyl-l-cysteine protects Schwann cells without compromising the anti-myeloma activity of bortezomib

Bortezomib-induced peripheral neuropathy (BIPN) emerges as a disabling adverse effect. As rat models for BIPN have demonstrated damage in nerve Schwann cells, we screened for cytoprotective agents to devise a method of rescuing Schwann cells from the cytotoxic effects of bortezomib without compromising its anti-myeloma effects. Schwann cells underwent macroautophagy along with cytoplasmic inclusion body and vacuole formation, and appeared much less susceptible to bortezomib-induced cytotoxicity than did myeloma cells. Vitamin C or N-acetyl-L-cysteine (NAC) achieved near-complete rescue of Schwann cells treated with bortezomib at 30 nM or less, and these agents in combination are able to cooperatively inhibit the morphological changes and the cytotoxicity in Schwann cells with higher doses of bortezomib. The delayed addition of vitamin C and/or NAC after the exposure to bortezomib alleviated the cytotoxicity in Schwann cells but not myeloma cells. These results suggest that delayed treatment with these agents may be instrumental in prophylaxis of BIPN.

Abstract LB-2: Insulin-like growth factor 1 potentiates the cytotoxic activity of bortezomib against myeloma cells

Abstract Multiple Myeloma (MM) is a clonal plasma cell disorder whose growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type 1 (IGF-1). Bortezomib, the first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma and has been suggested to induce apoptotsis. We analyzed the impact of recombinant IGF-1 combined with the proteasome inhibitor bortezomib on human plasma cell lines in vitro and in vivo and on fresh human myeloma cells ex vivo. We found that IGF-1 enhanced the cytotoxic activity of bortezomib in vitro against the LP1, RPMI8226, U266 and MM1.S lines. This potentiating effect was confirmed on MM1.S cells using a flow cytometric analysis of annexin V staining, and showed that the enhanced toxicity could be inhibited by the presence of a monoclonal antibody directed against the IGF-1 receptor (IGF1-R). IGF-1 was also found to enhance the cytotoxic activity of other proteasome inhibitors against MM1.S cells, including MG115, MG132, PSI and epoximicin. In vivo studies were performed in SCID mice bearing MM1.S xenografts. The co-administration of IGF-1 bortezomib significantly delayed tumor growth in comparison to that observed in mice treated with bortezomib alone. Fresh human myeloma cells exposed to bortezomib ex vivo displayed a larger annexin V positive fraction when they were co-incubated with IGF-1 then when they were exposed to bortezomib alone. This effect, which could be observed in subpopulations of CD45 hi and CD45 lo cells, could be reversed by an antibody directed against IGF-1R. Thus in each of these situations, IGF-1 increased the sensitivity of multiple myeloma cells to the cytotoxic effect of bortezomib. Analysis of pro- and anti-apoptotic proteins in MM1.S cells by immunoblotting showed that the addition of IGF-1 to bortezomib significantly enhanced the content of Bax, Bad and Bak and significantly reduced the content of Bcl2, BclX-L and Bfl-1. Other observations made with the IGF-1/bortezomib combination include an increase in the content of cleaved caspase 3 and in P21 protein. Preliminary data showed an increased content of CHOP protein, suggesting that the IGF-1/bortezomib combination might enhance reticulum stress in MM1.S cells, thus leading to an Unfolded Protein Response (UPR) and to cell death. These results suggest that IGF-1 sensitizes myeloma cells to proteasome inhibitors by contributing to the enhancement of the reticulum stress. Overall these results suggest that exposure of myeloma cells to one of their key growth factors, IGF-1, significantly enhanced their sensitivity to bortezomib as well as to other proteasome inhibitors. This phenomenon appears to involve several pathways and may be dependent on the high baseline level of reticulum stress present in myeloma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-2. doi:10.1158/1538-7445.AM2011-LB-2

Proteasome Inhibitor Bortezomib Disrupts Tumor Necrosis Factor-Related Apoptosis-Inducing ligand (TRAIL) Expression and Natural Killer (NK) Cell Killing of TRAIL Receptor-Positive Multiple Myeloma Cells

AbstractAbstract 5021Bortezomib, a potent 26S proteasome inhibitor, is approved for the treatment of multiple myeloma (MM) and clinical trials are under way to evaluate its efficacy in other malignant diseases. However, cytotoxic effects of bortezomib on immune-competent cells have also been observed. The aim of this study was to investigate the putative effects of Bortezomib on activated human natural killer (NK) cell function. In this project, primary lymphocytes or NK cells were isolated or purified from peripheral blood mononuclear cells of healthy donors. Expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by interleukin (IL)-2-activated NK cells was quantified by flow cytometry and real time-PCR method. NK cell degranulation activity and cytotoxicity against MM cell lines were evaluated by CD107a surface translocation and 51Cr release assays, respectively. Our results demonstrate that bortezomib markedly downregulated cell surface expression of TRAIL on primary human IL-2-activated NK cells in a dose-dependent manner. Moreover, bortezomib reduced TRAIL mRNA expression in these cells, suggesting that bortezomib regulates TRAIL expression of activated NK cells at the transcriptional level. Interestingly, pharmacological inhibition of the transcription factor NF-κB also profoundly decreased TRAIL expression both at protein and mRNA levels, indicating a novel role of NF-κB in the regulation of TRAIL expression in activated human NK cells. Furthermore, perforin-independent killing of the human MM cell lines was significantly suppressed following bortezomib treatment. In addition, blocking cell surface-bound TRAIL with a TRAIL antibody impaired lymphokine-activated killer (LAK) cell-mediated lysis of the TRAIL-sensitive MM cell line, RPMI8226. Our study demonstrated that bortezomib decreases TRAIL expression by IL-2 activated NK cells in a dose-dependent manner and disrupts TRAIL-mediated killing of TRAIL-sensitive myeloma cells, suggesting that Bortezomib may potentially hamper NK-dependent immunosurveillance against tumors in patients treated with this drug. Disclosures:No relevant conflicts of interest to declare.

The proteasome inhibitor bortezomib disrupts tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and natural killer (NK) cell killing of TRAIL receptor-positive multiple myeloma cells

Bortezomib, a potent 26S proteasome inhibitor, is approved for the treatment of multiple myeloma (MM) and clinical trials are under way to evaluate its efficacy in other malignant diseases. However, cytotoxic effects of bortezomib on immune-competent cells have also been observed. In this study, we show that bortezomib downregulates cell surface expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on primary human interleukin (IL)-2-activated natural killer (NK) cells. Pharmacological inhibition of the transcription factor, NF-κB also profoundly decreased TRAIL expression, suggesting that NF-κB is involved in the regulation of TRAIL expression in activated human NK cells. Furthermore, perforin-independent killing of the human MM cell lines RPMI8226 and U266 by NK cells was markedly suppressed following bortezomib treatment. In addition, blocking cell surface-bound TRAIL with a TRAIL antibody impaired NK cell-mediated lysis of the TRAIL-sensitive MM cell line, RPMI8226. In conclusion, the proteasome is likely to be involved in the regulation of TRAIL expression in primary human IL-2-activated NK cells. Proteasome inhibition by bortezomib disrupts TRAIL expression and TRAIL dependent and/or independent pathway-mediated killing of myeloma cells, suggesting that bortezomib may potentially hamper NK-dependent immunosurveillance against tumors in patients treated with this drug.

Combined effects of the proteasome inhibitor bortezomib and Hsp70 inhibitors on the B16F10 melanoma cell line.

Studies have shown that the 26S proteasome is involved in cell cycle control, transcription, DNA repair, immune response and protein synthesis. In the present study, we investigated the antiproliferative effects of the proteasome inhibitor bortezomib and heat shock protein (Hsp)70 inhibitors on the B16F10 melanoma cell line. The IC50 value of bortezomib was found to be 2.46 nM, while that of the Hsp70 inhibitor quercetin was 45 µM in the B16F10 cells. This indicates that bortezomib is more effective than quercetin in inhibiting cell growth. In response to treatment with 10 nM bortezomib for 24 h, cells underwent rounding, shrinkage and detachment. Unexpectedly, such morphological changes were not observed in cells treated with 20 µM quercetin alone, nor in cells treated with bortezomib + quercetin, indicating that quercetin inhibited the cytotoxic effects of bortezomib. Quantitation of cell viability also indicated that quercetin interfered with the cytotoxic effects of bortezomib. However, the combination of quercetin with another proteasome inhibitor, MG132, caused significant cell death as compared to single-agent treatment. A DNA ladder assay also confirmed the inhibitory effect of quercetin on the apoptosis-inducing effect of bortezomib. However, quercetin did not prevent the induction of apoptosis by MG132; on the contrary, it potentiated the apoptosis-inducing effect of MG132. These results suggest that the combination of quercetin with clinically beneficial proteasome inhibitors (except bortezomib) may have increased efficacy in the treatment of cancer. We also tested the combination of two other Hsp70 inhibitors, KNK-437 and schisandrin-B, in combination with bortezomib. Neither of these combinations was more effective than single-agent treatment.

Proteasome inhibition induces apoptosis in primary human natural killer cells and suppresses NKp46-mediated cytotoxicity

Bortezomib is a selective and potent inhibitor of the proteasome and has prominent effects in vitro and in vivo against tumors. Very recently, cytotoxic effects of bortezomib on immune-competent cells such as T cells and dendritic cells were also revealed. The aim of the study was to investigate the effects of this agent on natural killer cell survival and function. We investigated cytotoxic properties of bortezomib on natural killer cell apoptosis and function. Primary resting natural killer cells were purified from peripheral blood mononuclear cells of healthy donors by negative selection. The apoptotic cells were quantified by dual labeling of recombinant annexin V and propidium iodide. Mitochondrial membrane potential and expression of natural killer cell activating receptors were also quantified by flow cytometry. Natural killer cell cytotoxicity against murine and human tumor cells was tested by chromium 51 release assay. Our results demonstrate that bortezomib induces apoptosis in resting natural killer cells in a dose- and time-dependent manner. Glutathione, a reactive oxygen species scavenger, prevented the loss of mitochondrial membrane potential and conferred protection against bortezomib-induced apoptosis in resting natural killer cells, indicating a role for oxidative stress. Additionally, bortezomib significantly decreased expression of the natural killer activating receptor NKp46 in non-apoptotic resting natural killer cells in a dose-dependent manner, and as a result the redirected cytotoxicity mediated via NKp46 activation was diminished. Bay 11-7082, a pharmacological inhibitor of NF-kappaB activation, also reduced NKp46 expression and suppressed redirected cytotoxicity. Bortezomib induces apoptosis in primary resting natural killer cells in a dose- and time-dependent manner, and reduces NKp46 receptor expression as well as natural killer cell cytotoxicity mediated by the NKp46 activation pathway, suggesting that bortezomib may disrupt natural killer cell-mediated immunity through at least two different mechanisms: induction of natural killer cell apoptosis, and suppression of NKp46 receptor-mediated cytotoxicity.

Epidermal growth factor receptor inhibition sensitizes renal cell carcinoma cells to the cytotoxic effects of bortezomib

Epidermal growth factor receptor inhibition sensitizes renal cell carcinoma cells to the cytotoxic effects of bortezomib