ABSTRACT
BACKGROUND: Resistance to chemotherapy is a major problem in the treatment of patients with triple-negative breast cancer (TNBC). Preclinical data suggest that TNBC is dependent on proteasomes; however, clinical observations indicate that the efficacy of proteasome inhibitors in TNBC may be limited, suggesting the need for combination therapies. METHODS: We compared bortezomib and carfilzomib and their combinations with nelfinavir and lopinavir in TNBC cell lines and primary cells with regard to their cytotoxic activity, functional proteasome inhibition, and induction of the unfolded protein response (UPR). Furthermore, we evaluated the involvement of sXBP1, ABCB1, and ABCG2 in the cytotoxic activity of drug combinations. RESULTS: Carfilzomib, via proteasome ß5 + ß2 inhibition, is more cytotoxic in TNBC than bortezomib, which inhibits ß5 + ß1 proteasome subunits. The cytotoxicity of carfilzomib was significantly potentiated by nelfinavir or lopinavir. Carfilzomib with lopinavir induced endoplasmic reticulum stress and pro-apoptotic UPR through the accumulation of excess proteasomal substrate protein in TNBC in vitro. Moreover, lopinavir increased the intracellular availability of carfilzomib by inhibiting carfilzomib export from cells that express high levels and activity of ABCB1, but not ABCG2. CONCLUSION: Proteasome inhibition by carfilzomib combined with nelfinavir/lopinavir represents a potential treatment option for TNBC, warranting further investigation.
ABSTRACT
Cancer incidence is rising and this global challenge is further exacerbated by tumour resistance to available medicines. A promising approach to meet the need for improved cancer treatment is drug repurposing. Here we highlight the potential for repurposing disulfiram (also known by the trade name Antabuse), an old alcohol-aversion drug that has been shown to be effective against diverse cancer types in preclinical studies. Our nationwide epidemiological study reveals that patients who continuously used disulfiram have a lower risk of death from cancer compared to those who stopped using the drug at their diagnosis. Moreover, we identify the ditiocarb-copper complex as the metabolite of disulfiram that is responsible for its anti-cancer effects, and provide methods to detect preferential accumulation of the complex in tumours and candidate biomarkers to analyse its effect on cells and tissues. Finally, our functional and biophysical analyses reveal the molecular target of disulfiram's tumour-suppressing effects as NPL4, an adaptor of p97 (also known as VCP) segregase, which is essential for the turnover of proteins involved in multiple regulatory and stress-response pathways in cells.
Subject(s)
Alcohol Deterrents , Alcoholism/drug therapy , Antineoplastic Agents , Disulfiram/pharmacology , Disulfiram/therapeutic use , Drug Repositioning , Neoplasms/drug therapy , Nuclear Proteins/metabolism , Adult , Alcohol Deterrents/pharmacology , Alcohol Deterrents/therapeutic use , Alcoholism/epidemiology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Copper/chemistry , Denmark/epidemiology , Disulfiram/chemistry , Female , Heat-Shock Response/drug effects , Humans , Male , Mice , Middle Aged , Molecular Targeted Therapy , Neoplasms/metabolism , Neoplasms/mortality , Neoplasms/pathology , Nuclear Proteins/chemistry , Protein Aggregates , Protein Binding/drug effects , Proteolysis/drug effectsABSTRACT
OBJECTIVES: To assess the potential of second-generation proteasome inhibition by carfilzomib and its combination with the human immunodeficiency virus (HIV) protease inhibitors (HIV-PIs) lopinavir and nelfinavir in vitro for improved treatment of clear cell renal cell cancer (ccRCC). MATERIALS AND METHODS: Cytotoxicity, reactive oxygen species (ROS) production, and unfolded protein response (UPR) activation of proteasome inhibitors, HIV-PIs, and their combination were assessed in three cell lines and primary cells derived from three ccRCC tumours by MTS assay, flow cytometry, quantitative reverse transcriptase-polymerase chain reaction and western blot, respectively. Proteasome activity was determined by activity based probes. Flow cytometry was used to assess apoptosis by annexin V/propidium iodide assay and ATP-binding cassette sub-family B member 1 (ABCB1) activity by MitoTracker™ Green FM efflux assay (Thermo Fisher Scientific, MA, USA). RESULTS: Lopinavir and nelfinavir significantly increased the cytotoxic effect of carfilzomib in all cell lines and primary cells. ABCB1 efflux pump inhibition, induction of ROS production, and UPR pre-activation by lopinavir were identified as underlying mechanisms of this strong synergistic effect. Combined treatment led to unresolved protein stress, increased activation of pro-apoptotic UPR pathway, and a significant increase in apoptosis. CONCLUSION: The combination of the proteasome inhibitor carfilzomib and the HIV-PIs lopinavir and nelfinavir has a strong synergistic cytotoxic activity against ccRCCin vitro at therapeutically relevant drug concentrations. This effect is most likely explained by synergistic UPR triggering and ABCB1-modulation caused by HIV-PIs. Our findings suggest that combined treatment of second-generation proteasome inhibitors and HIV-PIs should be investigated in patients with metastatic RCC within a clinical trial.
Subject(s)
Antineoplastic Agents/therapeutic use , Carcinoma, Renal Cell/drug therapy , HIV Protease Inhibitors/therapeutic use , Kidney Neoplasms/drug therapy , Lopinavir/therapeutic use , Nelfinavir/therapeutic use , Proteasome Inhibitors/therapeutic use , Cell Line, Tumor , Drug Resistance, Neoplasm , Endoplasmic Reticulum Stress/drug effects , HumansABSTRACT
Downregulation of the unfolded protein response mediates proteasome inhibitor resistance in multiple myeloma. The Human Immunodeficieny Virus protease inhibitor nelfinavir activates the unfolded protein response in vitro. We determined dose-limiting toxicity and recommended dose for phase II of nelfinavir in combination with the proteasome inhibitor bortezomib. Twelve patients with advanced hematologic malignancies were treated with nelfinavir (2500-5000 mg/day p.o., days 1-14, 3+3 dose escalation) and bortezomib (1.3 mg/m(2), days 1, 4, 8, 11; 21-day cycles). A run in phase with nelfinavir monotherapy allowed pharmakokinetic/pharmakodynamic assessment of nelfinavir in the presence or absence of concomittant bortezomib. End points included dose-limiting toxicity, activation of the unfolded protein response, proteasome activity, toxicity and response to trial treatment. Nelfinavir 2×2500 mg was the recommended phase II dose identified. Nelfinavir alone significantly up-regulated expression of proteins related to the unfolded protein response in peripheral blood mononuclear cells and inhibited proteasome activity. Of 10 evaluable patients in the dose escalation cohort, 3 achieved a partial response, 4 stable disease for 2 cycles or more, while 3 had progressive disease as best response. In an exploratory extension cohort with 6 relapsed, bortezomib-refractory, lenalidomide-resistant myeloma patients treated at the recommended phase II dose, 3 reached a partial response, 2 a minor response, and one progressive disease. The combination of nelfinavir with bortezomib is safe and shows promising activity in advanced, bortezomib-refractory multiple myeloma. Induction of the unfolded protein response by nelfinavir may overcome the biological features of proteasome inhibitor resistance. (clinicaltrials.gov identifier: 01164709).
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Bortezomib/therapeutic use , Leukemia/drug therapy , Lymphoma/drug therapy , Multiple Myeloma/drug therapy , Nelfinavir/therapeutic use , Aged , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/pharmacokinetics , Bortezomib/pharmacokinetics , Drug Administration Schedule , Drug Combinations , Drug Resistance, Neoplasm/drug effects , Female , HIV Protease Inhibitors/pharmacokinetics , HIV Protease Inhibitors/therapeutic use , Humans , Leukemia/diagnosis , Leukemia/genetics , Leukemia/pathology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/pathology , Lymphoma/diagnosis , Lymphoma/genetics , Lymphoma/pathology , Male , Middle Aged , Multiple Myeloma/diagnosis , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Nelfinavir/pharmacokinetics , Proteasome Endopeptidase Complex/drug effects , Treatment Outcome , Unfolded Protein Response/drug effectsABSTRACT
Proteasomes are therapeutic targets for various cancers and autoimmune diseases. Constitutively expressed proteasomes have three active sites, ß1c, ß2c, and ß5c. Lymphoid tissues also express the immunoproteasome subunits ß1i, ß2i, and ß5i. Rapid and simultaneous measurement of the activity of these catalytic subunits would assist in the discovery of new inhibitors, improve analysis of proteasome inhibitors in clinical trials, and simplify analysis of subunit expression. In this work, we present a cocktail of activity-based probes that enables simultaneous gel-based detection of all six catalytic human proteasome subunits. We used this cocktail to develop specific inhibitors for ß1c, ß2c, ß5c, and ß2i, to compare the active-site specificity of clinical proteasome inhibitors, and to demonstrate that many hematologic malignancies predominantly express immunoproteasomes. Furthermore, we show that selective and complete inhibition of ß5i and ß1i is cytotoxic to primary cells from acute lymphocytic leukemia (ALL) patients.
Subject(s)
Molecular Probes/chemistry , Proteasome Endopeptidase Complex/metabolism , Catalytic Domain , HumansABSTRACT
Proteasome inhibitor resistance is a challenge for myeloma therapy. Bortezomib targets the ß5 and ß1 activity, but not the ß2 activity of the proteasome. Bortezomib-resistant myeloma cells down-regulate the activation status of the unfolded protein response, and up-regulate ß2 proteasome activity. To improve proteasome inhibition in bortezomib-resistant myeloma and to achieve more efficient UPR activation, we have developed LU-102, a selective inhibitor of the ß2 proteasome activity. LU-102 inhibited the ß2 activity in intact myeloma cells at low micromolar concentrations without relevant co-inhibition of ß1 and ß5 proteasome subunits. In proteasome inhibitor-resistant myeloma cells, significantly more potent proteasome inhibition was achieved by bortezomib or carfilzomib in combination with LU-102, compared to bortezomib/carfilzomib alone, resulting in highly synergistic cytotoxic activity of the drug combination via endoplasmatic reticulum stress-induced apoptosis. Combining bortezomib/carfilzomib with LU-102 significantly prolonged proteasome inhibition and increased activation of the unfolded protein response and IRE1-a activity. IRE1-α has recently been shown to control myeloma cell differentiation and bortezomib sensitivity (Leung-Hagesteijn, Cancer Cell 24:3, 289-304). Thus, ß2-selective proteasome inhibition by LU-102 in combination with bortezomib or carfilzomib results in synergistic proteasome inhibition, activation of the unfolded protein response, and cytotoxicity, and overcomes bortezomib/carfilzomib resistance in myeloma cells in vitro.
Subject(s)
Antineoplastic Agents/pharmacology , Bortezomib/pharmacology , Drug Resistance, Neoplasm , Oligopeptides/pharmacology , Proteasome Inhibitors/pharmacology , Animals , Cell Line, Tumor , Disease Models, Animal , Drug Synergism , Humans , Mice , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Xenograft Model Antitumor AssaysSubject(s)
Drug Resistance, Neoplasm , Mitochondria/metabolism , Multiple Myeloma/drug therapy , Proteasome Inhibitors/pharmacology , Sphingomyelins/biosynthesis , Aged , Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Bortezomib/pharmacology , Cell Line, Tumor , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Homeostasis , Humans , Hydrogen Peroxide/chemistry , Lipids/chemistry , Metabolomics , Middle Aged , Oligopeptides/pharmacology , Proteasome Endopeptidase Complex/metabolism , Protein Folding , Reactive Oxygen Species/metabolismABSTRACT
Chemotherapy resistance is still a major problem in the treatment of patients with non-small-cell-lung carcinoma (NSCLC), and novel concepts for the induction of cytotoxicity in NSCLC are highly warranted. Proteotoxicity, the induction of cytotoxicity by targeting the ubiquitin proteasome system, represents an appealing innovative strategy. The combination of the proteasome inhibitor bortezomib (BTZ) and the proteotoxic stress-inducing HIV drug nelfinavir (NFV) synergistically induces proteotoxicity and shows encouraging preclinical efficacy in NSCLC. The second-generation proteasome inhibitor carfilzomib (CFZ) is superior to BTZ and overcomes BTZ resistance in multiple myeloma patients. Here, we show that CFZ together with NFV is superior to the BTZ + NFV combination in inducing endoplasmic reticulum stress and proteotoxicity through the accumulation of excess proteasomal substrate protein in NSCLC in vitro and ex vivo. Interestingly, NFV increases the intracellular availability of CFZ through inhibition of CFZ export from NSCLC cells that express multidrug resistance (MDR) protein. Combining CFZ with NFV may therefore represent a future treatment option for NSCLC, which warrants further investigation.
Subject(s)
Antineoplastic Agents , Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Multiple Myeloma , Humans , Bortezomib/pharmacology , Bortezomib/therapeutic use , Nelfinavir/pharmacology , Nelfinavir/therapeutic use , Proteasome Inhibitors/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Multiple Myeloma/pathology , Proteasome Endopeptidase Complex , Lung Neoplasms/drug therapy , ApoptosisABSTRACT
Targeting proteasome with proteasome inhibitors (PIs) is an approved treatment strategy in multiple myeloma that has also been explored pre-clinically and clinically in other hematological malignancies. The approved PIs target both the constitutive and the immunoproteasome, the latter being present predominantly in cells of lymphoid origin. Therapeutic targeting of the immunoproteasome in cells with sole immunoproteasome activity may be selectively cytotoxic in malignant cells, while sparing the non-lymphoid tissues from the on-target PIs toxicity. Using activity-based probes to assess the proteasome activity profile and correlating it with the cytotoxicity assays, we identified B-cell chronic lymphocytic leukemia (B-CLL) to express predominantly immunoproteasome activity, which is associated with high sensitivity to approved proteasome inhibitors and, more importantly, to the immunoproteasome selective inhibitors LU005i and LU035i, targeting all immunoproteasome active subunits or only the immunoproteasome ß5i, respectively. At the same time, LU102, a proteasome ß2 inhibitor, sensitized B-CLL or immunoproteasome inhibitor-inherently resistant primary cells of acute myeloid leukemia, B-cell acute lymphoblastic leukemia, multiple myeloma and plasma cell leukemia to low doses of LU035i. The immunoproteasome thus represents a novel therapeutic target, which warrants further testing with clinical stage immunoproteasome inhibitors in monotherapy or in combinations.
Subject(s)
Antineoplastic Agents , Leukemia, Lymphocytic, Chronic, B-Cell , Multiple Myeloma , Antineoplastic Agents/pharmacology , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Multiple Myeloma/drug therapy , Proteasome Endopeptidase Complex , Proteasome Inhibitors/pharmacology , Proteasome Inhibitors/therapeutic useABSTRACT
Dendritic cells (DCs) play a key role in the pathogenesis of HIV infection. HIV interacts with these cells through 2 pathways in 2 temporal phases, initially via endocytosis and then via de novo replication. Here the transcriptional response of human DCs to HIV-1 was studied in these phases and at different stages of the virus replication cycle using purified HIV-1 envelope proteins, and inactivated and viable HIV-1. No differential gene expression was detected in response to envelope. However, more than 100 genes were differentially expressed in response to entry of viable and inactivated HIV-1 in the first phase. A completely different set of genes was differentially expressed in the second phase, predominantly in response to viable HIV-1, including up-regulation of immune regulation genes, whereas genes encoding lysosomal enzymes were down-regulated. Cathepsins B, C, S, and Z RNA and protein decreased, whereas cathepsin L was increased, probably reflecting a concomitant decrease in cystatin C. The net effect was markedly diminished cathepsin activity likely to result in enhanced HIV-1 survival and transfer to contacting T lymphocytes but decreased HIV-1 antigen processing and presentation to these T cells.
Subject(s)
Dendritic Cells/virology , HIV-1/genetics , HIV-1/pathogenicity , Antigen Presentation , Base Sequence , Cathepsins/genetics , Cathepsins/metabolism , Cystatins/genetics , Cystatins/metabolism , DNA Primers/genetics , Dendritic Cells/immunology , Dendritic Cells/metabolism , Gene Expression , Genes, Viral , HIV Envelope Protein gp120/physiology , HIV-1/physiology , Humans , In Vitro Techniques , Kinetics , Lysosomes/enzymology , Lysosomes/genetics , Models, Biological , Oligonucleotide Array Sequence Analysis , T-Lymphocytes/virology , Virus Internalization , Virus ReplicationABSTRACT
Introduction of the proteasome inhibitor bortezomib has dramatically improved clinical outcomes in multiple myeloma. However, most patients become refractory to bortezomib-based therapies. On the molecular level, development of resistance to bortezomib in myeloma cells is accompanied by complex metabolic changes resulting in increased protein folding capacity, and less dependency on the proteasome. In this study, we show that aminopeptidase B, encoded by the RNPEP gene, is upregulated in bortezomib-resistant myeloma cell lines, and in a murine in vivo model. Moreover, increased RNPEP expression is associated with shorter survival in multiple myeloma patients previously treated with bortezomib-containing regimens. Additionally, expression is increased in plasma cell precursors, a B-lymphoid compartment previously associated with myeloma stem cells. We hypothesized that increased aminopeptidase B expression in aggressive myeloma clones may be used therapeutically toward elimination of the cells via the use of a novel peptide-drug conjugate, melphalan flufenamide (melflufen). Melflufen, a substrate of aminopeptidase B, efficiently eliminates bortezomib-resistant myeloma cells in vitro and in vivo, and completely suppresses clonogenic myeloma growth in vitro at subphysiological concentrations. Thus, melflufen represents a novel treatment option that is able to eradicate drug-resistant myeloma clones characterized by elevated aminopeptidase B expression.
ABSTRACT
Proteasome inhibitors (PIs) are approved backbone treatments in multiple myeloma. More recently, inhibition of proteasome activity with the PI bortezomib has been clinically evaluated as a novel treatment strategy in pediatric acute lymphoblastic leukemia (ALL). However, we lack a marker that could identify ALL patients responding to PI-based therapy. By using a set of activity-based proteasome probes in conjunction with cytotoxicity assays, we show that B-cell precursor ALL (BCP-ALL), in contrast to T-ALL, demonstrates an increased activity of immunoproteasome over constitutive proteasome, which correlates with high ex vivo sensitivity to the PIs bortezomib and ixazomib. The novel selective PI LU015i-targeting immunoproteasome ß5i induces cytotoxicity in BCP-ALL containing high ß5i activity, confirming immunoproteasome activity as a novel therapeutic target in BCP-ALL. At the same time, cotreatment with ß2-selective proteasome inhibitors can sensitize T-ALL to currently available PIs, as well as to ß5i selective PI. In addition, levels of total and spliced forms of XBP1 differ between BCP-ALL and T-ALL, and only in BCP-ALL does high-spliced XBP1 correlate with sensitivity to bortezomib. Thus, in BCP-ALL, high immunoproteasome activity may serve as a predictive marker for PI-based treatment options, potentially combined with XBP1 analyses.
Subject(s)
Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Proteasome Endopeptidase Complex/immunology , Proteasome Inhibitors/pharmacology , X-Box Binding Protein 1/metabolism , Boron Compounds/pharmacology , Bortezomib/pharmacology , Cell Death/drug effects , Child , Gene Expression Regulation, Leukemic/drug effects , Glycine/analogs & derivatives , Glycine/pharmacology , Humans , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Proteasome Endopeptidase Complex/metabolism , Protein Subunits/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , X-Box Binding Protein 1/geneticsABSTRACT
The natural product syringolin A (SylA) is a potent proteasome inhibitor with promising anticancer activities. To further investigate its potential as a lead structure, selectivity profiling with cell lysates was performed. At therapeutic concentrations, a rhodamine-tagged SylA derivative selectively bound to the 20 S proteasome active sites without detectable off-target labelling. Additional profiling with lysates of wild-type and bortezomib-adapted leukaemic cell lines demonstrated the retention of this proteasome target and subsite selectivity as well as potency even in clinically relevant cell lines. Our studies, therefore, propose that further development of SylA might indeed result in an improved small molecule for the treatment of leukaemia.
Subject(s)
Boronic Acids/administration & dosage , Leukemia, Experimental/pathology , Peptides, Cyclic/pharmacology , Proteasome Endopeptidase Complex/metabolism , Pyrazines/administration & dosage , Animals , Bortezomib , Cell Line, Tumor , Leukemia, Experimental/enzymology , Magnetic Resonance Spectroscopy , Mice , Spectrometry, Mass, Electrospray IonizationABSTRACT
The biosynthesis of immunoglobulin leads to constitutive endoplasmic reticulum (ER) stress in myeloma cells, which activates the unfolded protein response (UPR). The UPR promotes protein folding by chaperones and increases proteasomal degradation of misfolded protein. Excessive ER stress induces apoptosis and represents a molecular basis for the bortezomib sensitivity of myeloma. Most solid malignancies such as sarcoma, by contrast, are poorly bortezomib sensitive and display low levels of ER stress. We hypothesized that pharmacologic induction of ER stress might sensitize malignancies to bortezomib treatment. We show that the HIV protease inhibitor ritonavir induces ER stress in bortezomib-resistant sarcoma cells. Ritonavir triggered the UPR, decreased the degradation of newly synthesized protein, but did not directly inhibit proteasomal active sites in the therapeutic dose range in contrast to bortezomib. Whereas neither bortezomib nor ritonavir monotherapy translated into significant apoptosis at therapeutic drug levels, the combination strongly increased the level of ER stress and activated PERK, IRE1, and ATF6, synergistically induced CHOP, JNK, caspase-4, and caspase-9, and resulted in >90% apoptosis. In summary, ritonavir increases the level of ER stress induced by bortezomib, which sensitizes bortezomib-resistant cells to bortezomib-induced apoptosis. Ritonavir may therefore be tested clinically to improve the sensitivity of solid malignancies toward bortezomib treatment.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Boronic Acids/pharmacology , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/pathology , Pyrazines/pharmacology , Ritonavir/pharmacology , Sarcoma/pathology , Binding Sites , Bortezomib , Cell Death/drug effects , Cell Line, Tumor , Drug Screening Assays, Antitumor , G2 Phase/drug effects , Humans , Neoplasm Proteins/biosynthesis , Proteasome Inhibitors , Protein Biosynthesis/drug effects , Protein Folding , Protein Subunits/metabolismABSTRACT
Experimental data on resistance mechanisms of multiple myeloma (MM) to ixazomib (IXA), a second-generation proteasome inhibitor (PI), are currently lacking. We generated MM cell lines with a 10-fold higher resistance to IXA as their sensitive counterparts, and observed cross-resistance towards the PIs carfilzomib (CFZ) and bortezomib (BTZ). Analyses of the IXA-binding proteasome subunits PSMB5 and PSMB1 show increased PSMB5 expression and activity in all IXA-resistant MM cells, and upregulated PSMB1 expression in IXA-resistant AMO1 cells. In addition, sequence analysis of PSMB5 revealed a p.Thr21Ala mutation in IXA-resistant MM1.S cells, and a p.Ala50Val mutation in IXA-resistant L363 cells, whereas IXA-resistant AMO1 cells lack PSMB5 mutations. IXA-resistant cells retain their sensitivity to therapeutic agents that mediate cytotoxic effects via induction of proteotoxic stress. Induction of ER stress and apoptosis by the p97 inhibitor CB-5083 was strongly enhanced in combination with the PI3Kα inhibitor BYL-719 or the HDAC inhibitor panobinostat suggesting potential therapeutic strategies to circumvent IXA resistance in MM. Taken together, our newly established IXA-resistant cell lines provide first insights into resistance mechanisms and overcoming treatment strategies, and represent suitable models to further study IXA resistance in MM.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/drug effects , Models, Biological , Mutation , Proteasome Inhibitors/pharmacology , A549 Cells , Amino Acid Substitution , Boron Compounds/pharmacology , Bortezomib/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/genetics , Gene Expression , Glycine/analogs & derivatives , Glycine/pharmacology , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Humans , Indoles/pharmacology , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Oligopeptides/pharmacology , Panobinostat/pharmacology , Phosphatidylinositol 3-Kinase/genetics , Phosphatidylinositol 3-Kinase/metabolism , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Pyrimidines/pharmacology , Thiazoles/pharmacologyABSTRACT
Proteasome inhibitors (PIs) are a backbone of multiple myeloma (MM) therapy. The proteasome harbors six proteolytically active subunits (ß1, ß2, ß5), while ß5 was identified as rate-limiting and is a primary target of clinically available PIs. The most effective pattern of subunit inhibition provided by these PIs for cytotoxic activity in MM is unknown. A head-to-head comparison of clinically available PIs shows that in the clinically relevant setting only the co-inhibition of ß1 or ß2 with ß5 activity achieves meaningful functional proteasome inhibition and cytotoxicity, while the selective ß2/ß5 inhibition of both constitutive and immunoproteasome is the most cytotoxic. In the long-term setting, selective inhibition of ß5 subunit is sufficient to induce cytotoxicity in PI-sensitive, but not in PI-resistant MM, and the ß5/ß2 co-inhibition is the most cytotoxic in PI-resistant MM. These results give a rational basis for selecting individual PIs for the treatment of MM.
Subject(s)
Antineoplastic Agents/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/metabolism , Aged , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Bortezomib/metabolism , Bortezomib/pharmacology , Bortezomib/therapeutic use , Drug Resistance, Neoplasm/drug effects , Female , Humans , Male , Mice , Mice, Inbred BALB C , Middle Aged , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Oligopeptides/metabolism , Oligopeptides/pharmacology , Oligopeptides/therapeutic use , Proteasome Endopeptidase Complex/chemistry , Proteasome Inhibitors/pharmacology , Proteasome Inhibitors/therapeutic use , Protein Subunits/antagonists & inhibitors , Protein Subunits/metabolism , Tissue Distribution , Tumor Cells, CulturedABSTRACT
The way the MHC II-associated proteolytic system of APC handles exogenous antigen is key to the stimulation of the T cell in infections and immunotherapy settings. Using a cell-impermeable, activity-based probe (ABP) for papain cathepsins, the most abundant type of endocytic proteases, we have simulated the encounter between exogenous antigen and endocytic proteases in live human monocyte-derived dendritic cells (MO-DC). Although cathepsin S (CatS), -B, -H, and -X were active in DC-derived endocytic fractions in vitro, the peptide-size tracer was routed selectively to active CatS after internalization by macropinocytosis. Blocking of the vacuolar adenosine triphosphatase abolished this CatS-selective targeting, and LPS-induced maturation of DC resulted in degradation of active CatS. Conjugation of the ABP to a protein facilitated the delivery to endocytic proteases and resulted in labeling of sizable amounts of CatB and CatX, although CatS still remained the major protease reached by this construct. Conjugation of the probe to a cell-penetrating peptide (CPP) routed the tracer to the entire panel of intracellular cathepsins, independently from endocytosis or LPS stimulation. Thus, different means of internalization result in differential targeting of active cathepsins in live MO-DC. CPP may serve as vehicles to target antigen more efficiently to protease-containing endocytic compartments.
Subject(s)
Biological Transport , Cathepsins/metabolism , Dendritic Cells/metabolism , Endocytosis , Leucine/analogs & derivatives , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/physiology , Animals , Cells, Cultured , Cysteine Endopeptidases/metabolism , Histocompatibility Antigens Class II/metabolism , Humans , Leucine/pharmacokinetics , Mice , Mice, Inbred BALB C , Microspheres , Molecular Probes/metabolism , Molecular Structure , Monocytes/metabolism , Monocytes/physiology , Peptides/pharmacokinetics , Phagocytosis , Protein Transport , Streptavidin/pharmacokinetics , Vacuoles/enzymologyABSTRACT
Inhibition of the AAA ATPase, p97, was recently shown to be a novel method for targeting the ubiquitin proteasome system, and CB-5083, a first-in-class inhibitor of p97, has demonstrated broad antitumor activity in a range of both hematologic and solid tumor models. Here, we show that CB-5083 has robust activity against multiple myeloma cell lines and a number of in vivo multiple myeloma models. Treatment with CB-5083 is associated with accumulation of ubiquitinated proteins, induction of the unfolded protein response, and apoptosis. CB-5083 decreases viability in multiple myeloma cell lines and patient-derived multiple myeloma cells, including those with background proteasome inhibitor (PI) resistance. CB-5083 has a unique mechanism of action that combines well with PIs, which is likely owing to the p97-dependent retro-translocation of the transcription factor, Nrf1, which transcribes proteasome subunit genes following exposure to a PI. In vivo studies using clinically relevant multiple myeloma models demonstrate that single-agent CB-5083 inhibits tumor growth and combines well with multiple myeloma standard-of-care agents. Our preclinical data demonstrate the efficacy of CB-5083 in several multiple myeloma disease models and provide the rationale for clinical evaluation as monotherapy and in combination in multiple myeloma. Mol Cancer Ther; 16(11); 2375-86. ©2017 AACR.
Subject(s)
Adenosine Triphosphatases/genetics , Indoles/administration & dosage , Multiple Myeloma/drug therapy , Nuclear Proteins/genetics , Nuclear Respiratory Factor 1/genetics , Proteasome Inhibitors/administration & dosage , Pyrimidines/administration & dosage , Adenosine Triphosphatases/antagonists & inhibitors , Animals , Apoptosis/drug effects , Cell Proliferation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Nuclear Proteins/antagonists & inhibitors , Proteasome Endopeptidase Complex/drug effects , Ubiquitin/genetics , Unfolded Protein Response/drug effects , Xenograft Model Antitumor AssaysSubject(s)
Drug Resistance, Multiple/drug effects , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Multiple Myeloma , Neoplasm Proteins/biosynthesis , Oligopeptides/therapeutic use , Up-Regulation/drug effects , ATP Binding Cassette Transporter, Subfamily B/biosynthesis , Humans , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Multiple Myeloma/pathologyABSTRACT
The synthesis and biological evaluation of platinum(II) amine complexes designed to act as inhibitors of the human cysteine protease cathepsin B, a thiol-dependent enzyme, is described. The complexes, composed of a cathepsin targeting ligand and a platinum(II) moiety with varying degrees of reactivity towards nucleophiles were characterized by physical-analytical methods and a proof of principle was illustrated in a model reaction. In biological tests for inhibitory activity against cathepsin B the presented compounds did not show significant inhibitory activity.