The proteasome and its inhibitors in immune regulation and immune disorders

The ubiquitin-proteasome pathway is a well-characterized mechanism deputed to the degradation of intracellular proteins. Proteasomal degradation intervenes in the regulation of numerous cellular functions including signal transduction, apoptosis, cell cycle, and antigen presentation. In vitro and in vivo studies have shown that both normal and malignant cells of the immune system are exquisitely affected by inhibition of proteasome activity. This property is currently exploited in the treatment of multiple myeloma and mantle cell lymphoma, two B-cell malignancies that respond to treatment with the proteasome inhibitor bortezomib. Pharmacological inhibitors of the proteasome also affect function and survival of B and T lymphocytes and of dendritic cells and were shown to reduce autoimmune and inflammatory manifestations in several models of immune-mediated disorders. The present review offers an overview of the mechanisms implicated in the immunomodulatory effects of proteasome inhibitors and discusses prospective future applications for these small molecules in immune and inflammatory diseases.     

Comparison of antiproliferative and apoptotic effects of a novel proteasome inhibitor MLN2238 with bortezomib on K562 chronic myeloid leukemia cells

Inhibition of the proteasome has emerged as a clinically effective anticancer therapeutic approach in recent years. Bortezomib (Velcade®) showed extremely high potency against a wide range of cancer cell lines. Ixazomib (MLN9708-MLN2238), the second-generation proteasome inhibitor, selectivity and potency were similar to that of bortezomib, is currently being investigated in phase I studies. It shows superior antitumor activity in hematologic malignancy, especially multiple myelomas. In this study, for the first time, we evaluated and compared the antiproliferative and apoptotic effects of the novel proteasome inhibitor MLN2238 (the active form of MLN9708) with bortezomib using in vitro chronic myeloid leukemia. Cytotoxic and apoptotic effects of MLN2238 and bortezomib were determined by trypan blue dye exclusion assays, WST-1 cell proliferation assay, increased AnnexinV-PI binding capacity, changes in caspase-3 activity and loss of mitochondrial membrane potential (JC-1). Associated with proteasome pathway NFκB1 and c-myc mRNA expression levels were examined by the qRT-PCR method. We observed that cytotoxic and apoptotic effects on K562 cells were started at 5?μm of MLN2238 and 1?μm of bortezomib after 24 and 48?h. Also, MLN2238 and bortezomib downregulated NFκB1 and c-myc mRNA expression at 24?h. Our result revealed that MLN22238 and bortezomib had significant cytotoxic and apoptotic effects on K562 cells. Here, we first demonstrate in vitro data that support the development of MLN2238, by direct comparison with bortezomib on K562 cells.     

Dendritic cell maturation stage determines susceptibility to the proteasome inhibitor bortezomib

The proteasome inhibitor bortezomib has been used successfully in the treatment of non-Hodgkin lymphomas in humans, and in the treatment of graft versus host disease (GVHD) and autoimmune diseases in animal models. The mechanism of growth inhibition and immunosuppression is only partly understood. Here, we have evaluated the differential effect of bortezomib on human monocyte derived immature and mature dendritic cells (DCs) as the maturation stage of DCs determines their function. We found bortezomib to induce apoptotic cell death in immature DCs and to a much lesser extent, in mature DCs. Furthermore, cytokine-induced maturation of immature DCs was inhibited by bortezomib, whereas already matured DCs remained unaffected as seen by phenotype and allo-stimulatory capacity. This corresponded to a decreased NF-kappaB activity in immature DCs, whereas NF-kappaB activity of mature DCs was not affected. In conclusion, our data expand on previous reports on the effects of proteasome inhibitors on human monocyte-derived DCs by demonstrating a differential effect of bortezomib on immature versus mature DCs. Our findings suggest a potential role of bortezomib in modulating immune responses in humans through inhibition of DC maturation.     

Treatment with proteasome inhibitor bortezomib enhances antigen-specific CD8+ T-cell-mediated antitumor immunity induced by DNA vaccination

There is an urgent need to develop new innovative therapies for the control of cancer. Antigen-specific immunotherapy and the employment of proteasome inhibitors have emerged as two potentially plausible approaches for the control of cancer. In the current study, we explored the combination of the DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 E7 antigen (CRT/E7) with the proteasome inhibitor, bortezomib, for their ability to generate E7-specific immune responses and antitumor effects in vaccinated mice. We found that the combination of treatment with bortezomib and CRT/E7(detox) DNA generated more potent E7-specific CD8+ T cell immune responses and better therapeutic effects against TC-1 tumors in tumor-bearing mice compared to monotherapy. Furthermore, we found that treatment with bortezomib led to increased apoptosis of TC-1 tumor cells and could render the TC-1 tumor cells more susceptible to lysis by E7-specific CD8+ T cells. Our data have significant implications for future clinical translation.     

Effects of Bortezomib on the Immune System: A Focus on Immune Regulation

Bortezomib, the first-in-class proteasome inhibitor, has become one of the standard treatments in multiple myeloma. The agent is thought to exert its antimyeloma effects through the inhibition of NF-κB. However, evidence suggests that bortezomib also affects additional cell survival pathways, such as the p44/42 mitogen-activated protein kinase pathway, and inhibitory effects on IL-6, TNF-α, and vascular endothelial growth factor have also been demonstrated. These diverse effects have prompted the investigation of bortezomib's activity in various immune and inflammatory processes. This review summarizes the data reported with bortezomib in the prevention of graft-versus-host disease, antibody-mediated graft rejection, and anti-angiogenesis and in the treatment of rheumatoid arthritis, multiple sclerosis, and other inflammatory diseases. The positive results obtained suggest a role for bortezomib in these different indications, and therefore further investigations are warranted.     

The expression of the endoplasmic reticulum stress sensor BiP/GRP78 predicts response to chemotherapy and determines the efficacy of proteasome inhibitors in diffuse large b-cell lymphoma

Activation of the endoplasmic reticulum (ER) stress pathway is associated with poor response to doxorubicin-containing regimens, such as rituximab, cyclophosphamide, hydroxydaunorubicin (doxorubicin), vincristine and prednisone (R-CHOP), in patients with diffuse large B-cell lymphoma (DLBCL). Bortezomib, a proteasome inhibitor, interferes with ER responses and improves survival in patients with aggressive hematologic malignant tumors, although its use in DLBCL patients remains controversial. The 78-kDa glucose-regulated protein (GRP78), also known as immunoglobulin heavy chain binding protein (BiP), is an ER stress sensor involved in the resistance to doxorubicin and bortezomib, but its role in the response to chemotherapy in DLBCL has not been explored before. We show that high BiP/GRP78 expression is related to worse overall survival (median overall survival, 5.2 versus 3.4 years). Moreover, cell death after R-CHOP in DLCBL cell lines is associated with decreased BiP/GRP78 expression. Conversely, DLBCL cell lines are primarily resistant to bortezomib, probably owing to BiP/GRP78 overexpression. Small-interfering RNA silencing of BiP/GRP78 renders all cell lines sensitive to bortezomib. R-CHOP with bortezomib (R-CHOP-BZ) reduces BiP/GRP78 expression and overcomes bortezomib resistance, mimicking the small-interfering RNA silencing of BiP/GRP78. Accordingly, R-CHOP-BZ is the most effective treatment, providing a rationale for the use of this combinational therapy to improve DLBCL patient survival. Moreover, this study provides preclinical evidence that the germinal center B-cell-like subtype DLBCL is sensitive to bortezomib combined with immunochemotherapy.     

A pilot study of bortezomib in Korean patients with relapsed or refractory myeloma

Recent clinical trials showed that bortezomib, a novel proteasome inhibitor, had therapeutic activity in multiple myeloma. However, there was no data about the feasibility of bortezomib in Korean patients. We performed a pilot study of bortezomib in patients with relapsed or refractory myeloma (1.3 mg/m2 twice weekly for 2 week in a 3-week cycle). Seven patients were enrolled. The median age of patients was 59 yr. All patients previously received VAD (vincristine, doxorubicin and dexamethasone) and thalidomide chemotherapy. Three patients previously received alkylator-containing chemotherapy and 4 patients, autologous stem cell transplantation. Bortezomib monotherapy resulted in 3 partial remissions (43%), 3 no changes (43%) and 1 progressive disease (14%). One patient who had no response to bortezomib monotherapy experienced partial remission after addition of dexamethasone to bortezomib. The most common serious toxicity was thrombocytopenia (grade 3/4, 10 of 20 cycles (50%)) and grade 3 peripheral neuropathy was developed in 2 of 20 cycles (10%). Drug-related adverse event led to discontinuation of bortezomib in 1 patient. There was no treatment related mortality. Overall, bortezomib seems to be effective and feasible. Conduction of larger clinical studies on Korean patients is necessary to characterize clinical efficacy and safety of bortezomib more precisely.     

Therapeutic Effects of a NEDD8-Activating Enzyme Inhibitor,Pevonedistat, on Sclerodermatous Graft-versus-Host Disease in Mice

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the sole treatment option for highly malignant hematologic disease; however, the major complication—graft-versus-host disease (GVHD)—still hinders its clinical application. In addition, chronic GVHD remains the major cause of long-term morbidity and mortality after allo-HSCT. Previously we showed that bortezomib, a proteasome inhibitor, can ameliorate the sclerodermatous GVHD response while maintaining graft-versus-tumor (GVT) effects. Here we report that pevonedistat (MLN4924), an inhibitor of the Nedd8-activating enzyme, which functions upstream of the proteasome in the ubiquitin-proteasome pathway, can also show similar protective effects. Recipient mice treated with pevonedistat demonstrated inhibitory effects on sclerodermatous GVHD pathogenesis. The beneficial effect of pevonedistat was observed to be temporally dependent. Whereas treatment given at the time of allo-HSCT administration or before the onset of symptoms worsened the scleroderma response, therapeutic administration starting at 20 days post-transplantation ameliorated the sclerodermatous GVHD. Flow cytometry analysis revealed differential effects on immune subsets, with inhibition of only antigen-presenting cells and not of donor T cells. Finally, pevonedistat preserved GVT effects in a sclerodermatous murine model of B cell lymphoma. Taken together, these data suggest that inhibition of neddylation with pevonedistat can serve as an alternative approach for the treatment of GVHD while maintaining GVT effects in a murine model of sclerodermatous GVHD.     

The promising role of monoclonal antibodies for immunotherapy of the HIV-associated cancer,non-Hodgkin lymphoma

Association between HIV/AIDS and some of the cancers such as lymphomais is well known. Relative risk for developing non-Hodgkin lymphoma (NHL) increases 60–200 folds in HIV-infected individuals. Diffuse large B cell lymphoma (DLBCL), primary effusion lymphoma (PEL), Burkitt's lymphoma (BL) and Plasmablastic Lymphoma (PBL) are among the most frequent subtypes. During the last century, scientists found that the immune system could potentially detect and destroy cancer cells. Therefore, they started a new field of study, which is named immunotherapy. There are different immunotherapeutic methods, among which therapeutic antibodies, such as Brentuximabvedotin (Adcetris), Ibritumomabtiuxetan (Zevalin) and rituximab (Rituxan), used for treatment of NHLs showed promising results. In this article, we will review the immunotherapeutic option, monoclonal antibodies, for treatment of HIV-associated NHLs as well as their recent clinical status. We will also discuss the selective monoclonal antibody for each subtype of NHLs.     

Sensitizing human multiple myeloma cells to the proteasome inhibitor bortezomib by novel curcumin analogs

The proteasome plays a vital role in the degradation of proteins involved in several pathways including the cell cycle, cellular proliferation and apoptosis and is a validated target in cancer treatment. Bortezomib (Velcade?, PS-341) is the first US FDA approved proteasome inhibitor anticancer drug used in the treatment of refractory multiple myeloma. In spite of its improved efficacy compared to alternative therapies, about 60% of patients do not respond to bortezomib due to the emergence of resistance. We hypothesized that novel small molecules could enhance the proteasome-inhibitory and anticancer activities of bortezomib in resistant multiple myeloma cells in vitro and in vivo. The dietary polyphenol curcumin has been shown to exert anti-cancer activity in several cancer cell lines, but the effects of curcumin in solid tumors have been modest primarily due to poor water solubility and poor bioavailability in tissues remote from the gastrointestinal tract. Here we show that the water-soluble analog of curcumin #12, but not curcumin, in combination with bortezomib could enhance the proteasome-inhibitory effect in multiple myeloma cells. Furthermore, the sensitivity of the myeloma cells to cytotoxic killing in the presence of otherwise sublethal concentrations of bortezomib was enhanced by incubation with the curcumin analog #12. These findings justify further investigation into those combinations that may yield potential therapeutic benefit.     

Critical analysis and diagnostic usefulness of limited immunophenotyping of B-cell non-Hodgkin lymphomas by flow cytometry

We report the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of various immunophenotypes characteristic of each class of B-cell non-Hodgkin lymphoma (NHL) based on analysis of 352 morphologically well-characterized B-cell NHLs and 175 benign lymph nodes (LNs) using 2-color flow cytometry. All B-cell NHLs that exhibited a characteristic immunophenotype (except diffuse large B-cell lymphoma) had a high NPV. The immunophenotypes of small lymphocytic lymphoma and mantle cell lymphoma showed high specificity, but only small lymphocytic lymphoma also showed a high PPV. One third of follicular lymphomas coexpressed CD23 and CD10. Diffuse large B-cell NHL showed no consistent immunophenotype. About 90% of all benign LNs expressed no substantial amounts of CD5, CD10, or CD23. Most benign LNs also failed to express substantial amounts of immunoglobulin heavy chains. In contrast, about 90% of NHLs showed expression of 1 or 2 heavy chains. The expression pattern of immunoglobulin light chains was not found helpful in favoring one lymphoma type over another. The usefulness of each immunophenotype for each lymphoma group is of particular diagnostic importance in limited specimens, such as fine-needle aspiration biopsies, small core biopsies, body effusions, extranodal sites, and nodal tissues with various artifacts.     

Issue Information ‐ TOC

The proteasome inhibitor bortezomib has been widely used to treat patients with multiple myeloma (MM). However, some patients show primary or secondary resistance. In recent work published in The Journal of Pathology, Beyar‐Katz et al demonstrate that bortezomib treatment stimulates a host inflammatory response, which in turn promotes MM cell migration, viability, and proliferation. These effects appear to be mediated by pro‐inflammatory M1‐like stromal macrophages partly via secretion of cytokine IL‐16. These unexpected findings imply that the binary M1/M2 definition of macrophages may not accurately describe the complexity and heterogeneity of macrophages associated with MM tumour growth and progression, and further suggest that bortezomib treatment stimulates host‐driven tumour‐promoting activity in addition to its cytotoxic activity, thus leading to potential bortezomib resistance in MM patients. Understanding the underlying mechanisms may identify novel targets to overcome or prevent bortezomib resistance. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Complete atrioventricular block secondary to bortezomib use in multiple myeloma

Bortezomib is an inhibitor of 26S proteasome, which is an effective treatment for multiple myeloma. The common adverse effects of bortezomib are asthenic conditions, gastrointestinal disturbances, and peripheral neuropathy. Here we describe a patient with dyspnea and general weakness because of complete atrioventricular block while receiving bortezomib. We immediately stopped bortezomib, and after inserting a permanent VDD pacemaker, the patients' symptoms disappeared.     

Proteasome inhibition with bortezomib induces a therapeutically relevant depletion of plasma cells in SLE but does not target their precursors

Long‐lived plasma cells (PCs) not only provide protective humoral immunity, they are also an essential component of the autoreactive immunologic memory that may drive chronic immune responses in systemic autoimmunity, such as systemic lupus erythematosus (SLE). The therapeutic relevance of their targeting has been demonstrated in preclinical models and severe, treatment‐refractory cases of autoimmune diseases using the proteasome inhibitor bortezomib. Herein, we describe in detail the dynamic serologic changes and effects on immune effector cells in eight SLE patients receiving a median two cycles of 1.3 mg/m² intravenous bortezomib. Upon proteasome inhibition, immunoglobulin levels gradually declined by ～30%, associated with a significant reduction of autoantibodies, and serum complement whereas B‐cell activation factor levels increased. While proteasome inhibition was associated with a significant depletion of short‐ and long‐lived PCs in peripheral blood and bone marrow by ～50%, including those with a distinctly mature CD19^? phenotype, their precursor B cells and T cells largely remained unaffected, resulting in a rapid repopulation of short‐lived PCs after bortezomib withdrawal, accompanied by increasing autoantibody levels. Collectively, these findings identify proteasome inhibitors as a promising treatment option for refractory SLE, but also indicate that PC depletion needs to be combined with targeted B‐cell therapies for sustained responses in systemic autoimmunity.     

Expression of Smac/DIABLO in B-cell non-Hodgkin and Hodgkin lymphomas

Inhibitor of apoptosis proteins (IAPs) are upregulated in cancers and suppress cell death, in part, through their ability to directly inhibit caspases. Inhibitor of apoptosis proteins are differentially expressed in B-cell lymphomas. The functions of some IAPs are counteracted by the cell death inducer, second mitochondrial-derived activator of caspases/direct IAP binding protein with low pI (Smac/DIABLO). In this study, we investigated the expression levels of Smac/DIABLO in 14 lymphoma cell lines by Western blot analysis. We also assessed 247 B-cell non-Hodgkin's lymphoma (NHL) and 40 Hodgkin's lymphoma (HL) tumors using immunohistochemical methods. Smac/DIABLO was expressed in most NHL and all HL cell lines. In NHL, Smac/DIABLO was expressed in 117 (47%) tumors and was differentially expressed in various NHL types. In most NHLs, from 29% to 68% of tumors were positive; however, Smac/DIABLO was not detected in small lymphocytic lymphoma/chronic lymphocytic leukemia and Burkitt lymphoma, and was rare in extranodal marginal zone B-cell lymphoma. In HL, Smac/DIABLO was positive in 25 (63%) tumors. Unlike NHL, all types of HL were positive for Smac/DIABLO, although nodular sclerosis was least often positive. The differential expression of Smac/DIABLO in NHLs suggests that apoptotic mechanisms are differentially involved in their pathogenesis. These results may also have implications for using Smac/DIABLO or its agonists as therapeutic agents.     

Bortezomib‐induced pro‐inflammatory macrophages as a potential factor limiting anti‐tumour efficacy

Multiple myeloma (MM) is a chronic progressive malignancy of plasma cells. Although treatment with the novel proteasome inhibitor, bortezomib, significantly improves patient survival, some patients fail to respond due to the development of de novo resistance. We have previously shown that cytotoxic drugs can induce pro‐tumorigenic host‐mediated effects which contribute to tumour re‐growth and metastasis, and thus limit anti‐tumour efficacy. However, such effects and their impact on tumour cell aggressiveness have not been investigated using cytostatic agents such as bortezomib. Here we show that plasma from bortezomib‐treated mice significantly increases migration, viability and proliferation of MM cells in vitro, compared to plasma from vehicle treated mice. In vivo, bortezomib induces the mobilization of pro‐angiogenic bone marrow cells. Furthermore, mice treated with bortezomib and subsequently were used as recipients for an injection of MM cells succumb to MM earlier than mice treated with the vehicle. We show that bortezomib promotes pro‐inflammatory macrophages which account for MM cell aggressiveness, an effect which is partially mediated by interleukin‐16. Accordingly, co‐inoculation of MM cells with pro‐inflammatory macrophages from bortezomib‐treated mice accelerates MM disease progression. Taken together, our results suggest that, in addition to the known effective anti‐tumour activity of bortezomib, host‐driven pro‐tumorigenic effects generated in response to treatment can promote MM aggressiveness, and thus may contribute to the overall limited efficacy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Aberrant expression of T-cell-associated antigens on B-cell non-Hodgkin lymphomas

We describe 9 well-characterized cases of B-cell non-Hodgkin lymphoma (NHL) that showed aberrant expression of T-cell-associated antigens by 2-color flow cytometry. Cases were as follows: chronic lymphocytic leukemia/small lymphocytic lymphoma, 4; follicle center cell lymphoma, 2; mantle cell lymphoma, 1; and diffuse large B-cell lymphoma, 2. CD2 was the most commonly expressed antigen (5 cases). CD8 and CD7 were identified in 2 cases each, including 1 case that expressed both CD7 and CD4. The disease course and response to treatment were compatible with the type and stage of lymphoma. No unusually aggressive behavior was noted in any case. A control group of 59 cases of benign lymph nodes analyzed during the same period showed no aberrant expression of T-cell-associated antigens; thus, such expression is not a feature of benign lymphoid proliferations. Study of these B-cell lymphomas may prove invaluable to study aberrant activation of silent or repressed T-cell differentiation genes. CD2-expressing B-cell NHLs may represent clonal expansion of CD2+ B lymphocytes that normally constitute a small fraction of peripheral B lymphocytes and should not be confused with composite B- and T-cell lymphomas. Unless aggressive behavior is noted consistently, no aggressive treatment is justified.     

Small-molecule inhibition of proteasome and silencing by vascular endothelial cell growth factor-specific siRNA induce additive antitumor activity in multiple myeloma

Angiogenesis plays an important role in the pathogenesis and progression in multiple myeloma (MM), and MM cells secrete vascular endothelial growth factor (VEGF), which further promotes proliferation of the tumor cells. Therefore, we evaluated the anti-myeloma effect of VEGF small interfering RNA (siRNA) silencing in MM cells and whether it can be augmented by the additional application of bortezomib directed against the 26S proteasome. After transfection with VEGF siRNA, we observed a reduction of VEGF expression in all studied cell lines: OPM-2, RPMI-8226, INA-6, Jurkat, Raji, and Karpas-299, as well as in cells of MM and lymphoma patients. VEGF siRNA significantly induced apoptosis and inhibited proliferation in OPM-2 cells (P<0.0001), RPMI-8226 (P<0.0001), and INA-6 (P<0.01) versus controls. Cotreatment with VEGF siRNA and bortezomib in MM cells resulted in an exaggerated inhibition of proliferation and induction of apoptosis compared with VEGF siRNA or bortezomib alone (P<0.001). In addition, the combination of VEGF siRNA and bortezomib significantly (P<0.01) reversed multidrug resistance gene 1-dependent resistance of MM cells. Our data suggest that small-molecule inhibition of proteasome and silencing by VEGF-specific siRNA may be associated with an additive antitumor activity and might be a suitable target for new, therapeutic strategies using RNA interference in MM.