The proteasome inhibitors (PIs) bortezomib and carfilzomib, which target proteasome 20S subunit beta 5 (PSMB5) in cells, are widely used in multiple myeloma (MM) treatment. In this study, we demonstrated the role of interferon-stimulated 20 kDa exonuclease-like 2 (ISG20L2) in MM PI resistance. Gain- and loss-of-function studies showed that ISG20L2 suppressed MM cell sensitivity to PIs in vitro and in vivo. Patients with ISG20L2lo MM had a better response to PIs and a longer overall survival than patients with ISG20L2hi MM. Biotinylated bortezomib pull-down assays showed that ISG20L2 competed with PSMB5 in binding to bortezomib. The surface plasmon resonance assay confirmed the direct binding of bortezomib to ISG20L2. In ISG20L2hi MM cells, ISG20L2 attenuated the binding of bortezomib to PSMB5, resulting in lower inhibition of proteasome activity and therefore less bortezomib-induced cell death. Overall, we identified a potentially novel mechanism by which ISG20L2 conferred bortezomib resistance on MM. The expression of ISG20L2 correlated with MM PI responses and patient treatment outcomes.

Although bortezomib (BTZ) displays efficacy in treating multiple myeloma (MM), BTZ resistance in MM patients has been reported. Meanwhile, treating BTZ resistant MM cells with β-catenin inhibitors have demonstrated the ability to reserve BTZ resistance. Thus, the present study aimed to investigate the synergistic effect of the β-catenin inhibitors, ICG-001 and pyrvinium (PP), with BTZ in the treatment of BTZ-resistant MM cells. Different concentrations of ICG-001 (0-32 µM) or PP (0-32 nM) were used to treat the BTZ-resistant RPMI-8226 (RPMI-8226BR) and BTZ-resistant KMS-11 (KMS-11BR) cell lines, followed by a BTZ combination treatment. Subsequently, cell viability and apoptosis in these two cell lines were determined by CCK-8 assay and flow cytometry, respectively. The proteins involved in the Wnt/β-catenin signaling pathway were detected using western blotting. The Wnt/β-catenin signaling pathway was activated in the RPMI-8226BR and the KMS-11BR cells. In addition, the cell viability of RPMI-8226BR and KMS-11BR cells were decreased following β-catenin inhibitor (ICG-001 and PP) treatment alone. Furthermore, the β-catenin inhibitors, ICG-001 and PP, plus BTZ combination treatment revealed a notable decrease in cell viability and a marked increase in cell apoptosis rate, compared with that in cells treated with ICG-001, PP or BTZ alone in the RPMI-8226BR and KMS-11BR cell lines. In conclusion, the β-catenin inhibitors, ICG-001 and PP not only increased apoptosis, but also sensitized BTZ-resistant MM cells to BTZ, indicating their potential therapeutic application in MM.

HIV protease inhibitors (HIV-PI) are oral drugs for HIV treatment. HIV-PI have antitumor activity via induction of ER-stress, inhibition of phospho-AKT (p-AKT) and the proteasome, suggesting antimyeloma activity. We characterize the effects of all approved HIV-PI on myeloma cells. HIV-PI were compared regarding cytotoxicity, proteasome activity, ER-stress induction and AKT phosphorylation using myeloma cells in vitro. Nelfinavir is the HIV-PI with highest cytotoxic activity against primary myeloma cells and with an IC50 near therapeutic drug blood levels (8-14 μM), irrespective of bortezomib sensitivity. Only nelfinavir inhibited intracellular proteasome activity in situ at drug concentrations <40 μM. Ritonavir, saquinavir and lopinavir inhibited p-AKT comparable to nelfinavir, and showed similar synergistic cytotoxicity with bortezomib against bortezomib-sensitive cells. Nelfinavir had superior synergistic activity with bortezomib/carfilzomib in particular against bortezomib/carfilzomib-resistant myeloma cells. It inhibited not only the proteasomal β1/β5 active sites, similar to bortezomib/carfilzomib, but in addition the β2 proteasome activity not targeted by bortezomib/carfilzomib. Additional inhibition of β2 proteasome activity is known to sensitize cells for bortezomib and carfilzomib. Nelfinavir has unique proteasome inhibiting activity in particular on the bortezomib/carfilzomib-insensitive tryptic (β2) proteasome activity in intact myeloma cells, and is active against bortezomib/carfilzomib-resistant myeloma cells in vitro.

Although proteasome inhibitors have emerged as the therapeutic backbone of multiple myeloma treatment, patients often relapse and become drug refractory. The combination between proteasome and histone deacetylase inhibitors has shown to be more efficient compared to monotherapy by enhancing the anti-myeloma activity and improving the patient's lifetime expectancy. Hybrid molecules, combining two drugs/pharmacophores in a single molecular entity, offer improved effectiveness by modulating more than one target and circumventing differences in the pharmacokinetic and pharmacodynamic profiles, which are the main disadvantages of combination therapy. Therefore, eleven histone deacetylase-proteasome inhibitor hybrids were synthesized, combining pharmacophores of entinostat and bortezomib. Compound 3 displayed the strongest antiproliferative activity with an IC50 value of 9.5 nM in the multiple myeloma cells RPMI 8226, 157.7 nM in the same cell line resistant to bortezomib, and 13.1 nM in a 3D spheroid model containing multiple myeloma and mesenchymal stem cells. Moreover, the compound inhibited 33% of histone deacetylase activity when RPMI 8226 cells were treated for 8 h at 10 µM. It also inhibited the proteasome activity with an IC50 value of 23.6 nM.

Chemotherapy-induced taste disorder is one of the critical issues in cancer therapy. Bortezomib, a proteasome inhibitor, is a key agent in multiple myeloma therapy, but it induces a taste disorder. In this study, we investigated the characteristics of bortezomib-induced taste disorder and the underlying mechanism in mice. Among the five basic tastes, the sour taste sensitivity of mice was significantly increased by bortezomib administration. In bortezomib-administered mice, protein expression of PKD2L1 was increased. The increased sour taste sensitivity induced by bortezomib returned to the control level on cessation of its administration. These results suggest that an increase in protein expression of PKD2L1 enhances the sour taste sensitivity in bortezomib-administered mice, and this alteration is reversed on cessation of its administration.

Bortezomib (Velcade™) is a reversible proteasome inhibitor that is approved for the treatment of multiple myeloma (MM). Despite its demonstrated clinical success, some patients are deprived of treatment due to primary refractoriness or development of resistance during therapy. To investigate the role of the duration of proteasome inhibition in the anti-tumor response of bortezomib, we established clonal isolates of HT-29 adenocarcinoma cells adapted to continuous exposure of bortezomib. These cells were ~30-fold resistant to bortezomib. Two novel and distinct mutations in the β5 subunit, Cys63Phe, located distal to the binding site in a helix critical for drug binding, and Arg24Cys, found in the propeptide region were found in all resistant clones. The latter mutation is a natural variant found to be elevated in frequency in patients with MM. Proteasome activity and levels of both the constitutive and immunoproteasome were increased in resistant cells, which correlated to an increase in subunit gene expression. These changes correlated with a more rapid recovery of proteasome activity following brief exposure to bortezomib. Increased recovery rate was not due to increased proteasome turnover as similar findings were seen in cells co-treated with cycloheximide. When we exposed resistant cells to the irreversible proteasome inhibitor carfilzomib we noted a slower rate of recovery of proteasome activity as compared to bortezomib in both parental and resistant cells. Importantly, carfilzomib maintained its cytotoxic potential in the bortezomib resistant cell lines. Therefore, resistance to bortezomib, can be overcome with irreversible inhibitors, suggesting prolonged proteasome inhibition induces a more potent anti-tumor response.

Although the introduction of bortezomib as a therapeutic strategy has improved the overall survival of multiple myeloma (MM) patients, 15-20% of high-risk patients do not respond to bortezomib over time or become resistant to treatment. Therefore, the development of new therapeutic strategies, such as combination therapies, is urgently needed.

Daratumumab, a CD38 human monoclonal antibody, demonstrated significant clinical activity in combination with bortezomib and dexamethasone versus bortezomib and dexamethasone alone in the primary analysis of CASTOR, a phase 3 study in relapsed and/or refractory multiple myeloma. A post hoc analysis based on treatment history and longer follow up is presented. After 19.4 (range: 0-27.7) months of median follow up, daratumumab plus bortezomib and dexamethasone prolonged progression-free survival (median: 16.7 versus 7.1 months; hazard ratio, 0.31; 95% confidence interval, 0.24-0.39; P<0.0001) and improved the overall response rate (83.8% versus 63.2%; P<0.0001) compared with bortezomib and dexamethasone alone. The progression-free survival benefit of daratumumab plus bortezomib and dexamethasone was most apparent in patients with 1 prior line of therapy (median: not reached versus 7.9 months; hazard ratio, 0.19; 95% con fidence interval, 0.12-0.29; P<0.0001). Daratumumab plus bortezomib and dexamethasone was also superior to bortezomib and dexamethasone alone in subgroups based on prior treatment exposure (bortezomib, thalidomide, or lenalidomide), lenalidomide-refractory status, time since last therapy (≤12, >12, ≤6, or >6 months), or cytogenetic risk. Minimal residual disease-negative rates were >2.5-fold higher with daratumumab across subgroups. The safety profile of daratumumab plus bortezomib and dexamethasone remained consistent with longer follow up. Daratumumab plus bortezomib and dexamethasone demonstrated significant clinical activity across clinically relevant subgroups and provided the greatest benefit to patients treated at first relapse. Trial registration: clinicaltrials.gov identifier: 02136134.

Bortezomib, a first-generation proteasome inhibitor widely used in chemotherapy for hematologic malignancy, has effective anti-cancer activity but often causes severe peripheral neuropathy. Although bortezomib-induced peripheral neuropathy (BIPN) is a dose-limiting toxicity, there are no recommended therapeutics for its prevention or treatment. One of the most critical problems is a lack of knowledge about pathological mechanisms of BIPN. Here, we summarize the known mechanisms of BIPN based on preclinical evidence, including morphological abnormalities, involvement of non-neuronal cells, oxidative stress, and alterations of transcriptional programs in both the peripheral and central nervous systems. Moreover, we describe the necessity of advancing studies that identify the potential efficacy of approved drugs on the basis of pathological mechanisms, as this is a convincing strategy for rapid translation to patients with cancer and BIPN.

Bortezomib, a proteasome-specific inhibitor, has emerged as a promising cancer therapeutic agent. However, development of resistance to bortezomib may pose a challenge to effective anticancer therapy. Therefore, characterization of cellular mechanisms involved in bortezomib resistance and development of effective strategies to overcome this resistance represent important steps in the advancement of bortezomib-mediated cancer therapy.

Mechanisms underlying interactions between a novel, clinically relevant circularized tumor necrosis factor-related apoptosis inducing ligand (TRAIL) agonist, circularly permuted TRAIL (CPT) have been examined in multiple myeloma (MM) cells sensitive or resistant to bortezomib (BTZ). Various MM cell lines for example, U266, including those resistant to bortezomib-resistant U266 cells were exposed to low nanomolar concentrations of bortezomib ± CPT and apoptosis monitored. Circularly permuted TRAIL and bortezomib synergistically induced apoptosis in both BTZ-naïve and -resistant cells. The regimen up-regulated DR4 receptor internalization in MM cells, known to modulate both NF-κB and extrinsic apoptotic pathways. CPT/BTZ disrupted the non-canonical NF-κB pathway, reflected by tumor necrosis factor (TNF) receptor associated factors 3 (TRAF3) up-regulation, NF-κB inducing kinase down-regulation, diminished p52 and p50 processing, and B-cell lymphoma-extra large (BCL-XL) down-regulation, but failed to inactivate the canonical NF-κB pathway, reflected by unchanged or increased expression of phospho-p65. The regimen also sharply increased extrinsic apoptotic pathway activation. Cells exhibiting TRAF3 knock-down, dominant-negative Fas-associated protein with death domain, knock-down of caspase-8, BCL-2/BCL-XL, or exposure to a caspase-9 inhibitor displayed markedly reduced CPT/BTZ sensitivity. Concordant results were observed in bortezomib-resistant cells. The regimen was also active in the presence of stromal cells and was relatively sparing toward normal CD34+ hematopoietic cells. Finally, ex vivo results revealed synergism in primary MM primary cells, including those BTZ, and the CPT/BTZ regimen significantly decreased tumor growth in a patient-derived MM xenograft model. These results indicate that the CPT/BTZ regimen acts via the non-canonical NF-κB as well as intrinsic/extrinsic apoptotic pathways to induce cell death in MM cells, and may represent an effective strategy in the setting of bortezomib resistance.

IgA nephropathy is the most common glomerulonephritis in the world. We conducted a pilot trial (NCT01103778) to test the effect of bortezomib in patients with IgA nephropathy and significant proteinuria.

Bortezomib is a proteasome inhibitor that has shown impressive efficacy in the treatment of multiple myeloma. In mice, the addition of dextran sulfate sodium (DSS) to drinking water leads to acute colitis that can serve as an experimental animal model for human ulcerative colitis.

Ovarian cancer is associated with increased expression of the pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8), which induces tumor cell proliferation, angiogenesis, and metastasis. Even though bortezomib (BZ) has shown remarkable anti-tumor activity in hematological malignancies, it has been less effective in ovarian cancer; however, the mechanisms are not understood. We have recently shown that BZ unexpectedly induces the expression of IL-8 in ovarian cancer cells in vitro, by IκB kinase (IKK)-dependent mechanism. Here, we tested the hypothesis that IKK inhibition reduces the IL-8 production and increases BZ effectiveness in reducing ovarian tumor growth in vivo. Our results demonstrate that the combination of BZ and the IKK inhibitor Bay 117085 significantly reduces the growth of ovarian tumor xenografts in nude mice when compared to either drug alone. Mice treated with the BZ/Bay 117085 combination exhibit smallest tumors, and lowest levels of IL-8. Furthermore, the reduced tumor growth in the combination group is associated with decreased tumor levels of S536P-p65 NFκB and its decreased recruitment to IL-8 promoter in tumor tissues. These data provide the first in vivo evidence that combining BZ with IKK inhibitor is effective, and suggest that using IKK inhibitors may increase BZ effectiveness in ovarian cancer treatment.

Bortezomib (BTZ) is a proteasome inhibitor as approved by US FDA for the treatment of multiple myeloma. It exhibits significant anti-cancer properties, against solid tumors; but lacks aqueous solubility, chemical stability which hinders its successful formulation development. The present study is an attempt to deliver BTZ using N-(2-hydroxypropyl) methacrylamide (HPMA) based copolymeric conjugates and biotinylated PNPs in an effective manner. Study describes a systematic synthetic pathway to synthesize functional polymeric conjugates such as HPMA-Biotin (HP-BT) HPMA-Polylactic acid (HPLA) and HPMA-PLA-Biotin (HPLA-BT) followed by exhaustive characterization both spectroscopically and microscopically. Our strategy yielded polymeric nanoparticles (PNPs) of narrow size range of 199.7 ± 1.32 nm. Release studies were performed at pH 7.4 and 5.6. PNPs were 2-folds less hemolytic (p < 0.0001) than pure drug. BTZ loaded PNPs of HPLA-BT demonstrated significant anti-cancer activity against MCF-7 cells. IC50 value of these PNPs was 56.06 ± 0.12 nM, which was approximately two folds less than BTZ (p < 0.0001). Cellular uptake study confirmed that higher uptake of formulations might be an outcome of biotin surface tethering characteristics that enhanced selectivity and targeting of formulations efficiently. In vivo pharmacokinetics evidenced increased bioavailability (AUC0 t-∞) of DL-HPLA-BT PNPs (drug loaded) than BTZ with an improved half-life. Overall the developed PNPs led to the improved and effective BTZ delivery.

Multiple myeloma (myeloma in short) is an incurable cancer of antibody-producing plasma cells that comprise 13% of all hematological malignancies. The proteasome inhibitor bortezomib has improved treatment significantly, but inherent and acquired resistance to the drug remains a problem. We here show that bortezomib-induced cytotoxicity was completely dampened when cells were supplemented with cysteine or its derivative, glutathione (GSH) in ANBL-6 and INA-6 myeloma cell lines. GSH is a major component of the antioxidative defense in eukaryotic cells. Increasing intracellular GSH levels fully abolished bortezomib-induced cytotoxicity and transcriptional changes. Elevated intracellular GSH levels blocked bortezomib-induced nuclear factor erythroid 2-related factor 2 (NFE2L2, NRF2)-associated stress responses, including upregulation of the xCT subunit of the Xc- cystine-glutamate antiporter. INA-6 cells conditioned to increasing bortezomib doses displayed reduced bortezomib sensitivity and elevated xCT levels. Inhibiting Xc- activity potentiated bortezomib-induced cytotoxicity in myeloma cell lines and primary cells, and re-established sensitivity to bortezomib in bortezomib-conditioned cells. We propose that intracellular GSH level is the main determinant of bortezomib-induced cytotoxicity in a subset of myeloma cells, and that combined targeting of the proteasome and the Xc- cystine-glutamate antiporter can circumvent bortezomib resistance.

The BH3 mimetic ABT-737 is a potent inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L), and Bcl-w. The Bcl-2 family modulates sensitivity to anticancer drugs in many cancers, including melanomas. In this study, we examined whether ABT-737 is effective in killing melanoma cells either alone or in combination with a proteasome inhibitor already in clinical use (Bortezomib) in vitro and in vivo, and further evaluated the mechanisms of action. Results showed that ABT-737 alone induced modest cytotoxicity in melanoma cells, but only at higher doses. Knock-down of the anti-apoptotic proteins Bcl-2, Bcl-X(L), or Mcl-1 with siRNAs demonstrated that Mcl-1 is the critical mediator of melanoma's resistance to ABT-737 treatment. However, ABT-737 displayed strong synergistic lethality when combined with Bortezomib. Immunoblot analyses demonstrated that Bortezomib increased expression of Noxa, a pro-apoptotic Bcl-2 member that antagonizes Mcl-1. Additionally, siRNA-mediated inhibition of Noxa expression protected melanoma cells from cytotoxicity induced by the combination treatment. These results demonstrate that Bortezomib synergizes with ABT-737 by neutralizing Mcl-1's function via increased levels of Noxa. In a xenograft mouse model, although drug doses were limited due to toxicity, ABT-737 or Bortezomib slowed melanoma tumor growth compared to the control, and the drug combination significantly decreased growth compared to either drug alone. These data imply that less toxic drugs fulfilling a function similar to Bortezomib to neutralize Mcl-1 are promising candidates for combination with ABT-737 for treating melanomas.

Congenital muscular dystrophy, caused by mutations in LAMA2 (the gene encoding laminin α2 chain), is a severe and incapacitating disease for which no therapy is yet available. We have recently demonstrated that proteasome activity is increased in laminin α2 chain-deficient muscle and that treatment with the nonpharmaceutical proteasome inhibitor MG-132 reduces muscle pathology in laminin α2 chain-deficient dy(3K)/dy(3K) mice. Here, we explore the use of the selective and therapeutic proteasome inhibitor bortezomib (currently used for treatment of relapsed multiple myeloma and mantle cell lymphoma) in dy(3K)/dy(3K) mice and in congenital muscular dystrophy type 1A muscle cells. Outcome measures included quantitative muscle morphology, gene and miRNA expression analyses, proteasome activity, motor activity, and survival. Bortezomib improved several histological hallmarks of disease, partially normalized miRNA expression (miR-1 and miR-133a), and enhanced body weight, locomotion, and survival of dy(3K)/dy(3K) mice. In addition, bortezomib reduced proteasome activity in congenital muscular dystrophy type 1A myoblasts and myotubes. These findings provide evidence that the proteasome inhibitor bortezomib partially reduces laminin α2 chain-deficient muscular dystrophy. Investigation of the clinical efficacy of bortezomib administration in congenital muscular dystrophy type 1A clinical trials may be warranted.

Many frontline chemotherapeutic agents produce robust neuropathy as a dose-limiting side effect; however, the persistence of chemotherapy-related sensory disturbances and pain are not well documented. We have previously investigated the qualities of bortezomib-induced pain, and now seek to determine the ongoing nature of this pain. Twenty-six control subjects and 11 patients who had previously been treated with bortezomib and who were experiencing ongoing pain consented to recurring quantitative sensory testing. A pilot immunohistochemistry study of skin innervation was also performed on patient-obtained biopsies. Psychophysical testing in patients revealed persistent changes including decreased skin temperature in the area of pain, diminished touch and sharpness detection, increased pegboard completion times, and decreased sensitivity to skin heating. Additionally, the intensity of pain, as captured by the use of a visual analog scale and pain descriptors, was reported by patients to be unchanged during the retest despite similar morphine equivalent daily doses. The patient skin biopsies displayed a marked decrease in the density of epidermal nerve fibers and Meissner's corpuscles. These results signify a persistent and severe impairment of Aβ, Aδ, and C fibers in patients with chronic bortezomib-induced chemoneuropathy. Further, this study reports a loss of both epidermal nerve fibers and Meissner's corpuscles.

Although multiple myeloma (MM) patients benefit from standard bortezomib (BTZ) chemotherapy, they develop drug resistance, resulting in relapse. We investigated whether histone deacetylase 6 (HDAC6) inhibitor A452 overcomes bortezomib resistance in MM. We show that HDAC6-selective inhibitor A452 significantly decreases the activation of BTZ-resistant markers, such as extracellular signal-regulated kinases (ERK) and nuclear factor kappa B (NF-κB), in acquired BTZ-resistant MM cells. Combination treatment of A452 and BTZ or carfilzomib (CFZ) synergistically reduces BTZ-resistant markers. Additionally, A452 synergizes with BTZ or CFZ to inhibit the activation of NF-κB and signal transducer and activator of transcription 3 (STAT3), resulting in decreased expressions of low-molecular-mass polypeptide 2 (LMP2) and LMP7. Furthermore, combining A452 with BTZ or CFZ leads to synergistic cancer cell growth inhibition, viability decreases, and apoptosis induction in the BTZ-resistant MM cells. Overall, the synergistic effect of A452 with CFZ is more potent than that of A452 with BTZ in BTZ-resistant U266 cells. Thus, our findings reveal the HDAC6-selective inhibitor as a promising therapy for BTZ-chemoresistant MM.