RESUMO
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.
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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.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Mieloma Múltiplo , Humanos , Bortezomib/farmacologia , Bortezomib/uso terapêutico , Nelfinavir/farmacologia , Nelfinavir/uso terapêutico , Inibidores de Proteassoma/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Mieloma Múltiplo/patologia , Complexo de Endopeptidases do Proteassoma , Neoplasias Pulmonares/tratamento farmacológico , ApoptoseRESUMO
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.
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Antineoplásicos , Leucemia Linfocítica Crônica de Células B , Mieloma Múltiplo , Antineoplásicos/farmacologia , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Mieloma Múltiplo/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêuticoRESUMO
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.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Complexo de Endopeptidases do Proteassoma/imunologia , Inibidores de Proteassoma/farmacologia , Proteína 1 de Ligação a X-Box/metabolismo , Compostos de Boro/farmacologia , Bortezomib/farmacologia , Morte Celular/efeitos dos fármacos , Criança , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína 1 de Ligação a X-Box/genéticaRESUMO
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.
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Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mieloma Múltiplo , Proteínas de Neoplasias/biossíntese , Oligopeptídeos/uso terapêutico , Regulação para Cima/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologiaRESUMO
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.
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Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Modelos Biológicos , Mutação , Inibidores de Proteassoma/farmacologia , Células A549 , Substituição de Aminoácidos , Compostos de Boro/farmacologia , Bortezomib/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica , Glicina/análogos & derivados , Glicina/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Indóis/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Oligopeptídeos/farmacologia , Panobinostat/farmacologia , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirimidinas/farmacologia , Tiazóis/farmacologiaAssuntos
Resistencia a Medicamentos Antineoplásicos , Mitocôndrias/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteassoma/farmacologia , Esfingomielinas/biossíntese , Idoso , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Bortezomib/farmacologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Homeostase , Humanos , Peróxido de Hidrogênio/química , Lipídeos/química , Metabolômica , Pessoa de Meia-Idade , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.
Assuntos
Antineoplásicos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Idoso , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Bortezomib/metabolismo , Bortezomib/farmacologia , Bortezomib/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
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.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Inibidores da Protease de HIV/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Lopinavir/uso terapêutico , Nelfinavir/uso terapêutico , Inibidores de Proteassoma/uso terapêutico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , HumanosRESUMO
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.
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Dissuasores de Álcool , Alcoolismo/tratamento farmacológico , Antineoplásicos , Dissulfiram/farmacologia , Dissulfiram/uso terapêutico , Reposicionamento de Medicamentos , Neoplasias/tratamento farmacológico , Proteínas Nucleares/metabolismo , Adulto , Dissuasores de Álcool/farmacologia , Dissuasores de Álcool/uso terapêutico , Alcoolismo/epidemiologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Cobre/química , Dinamarca/epidemiologia , Dissulfiram/química , Feminino , Resposta ao Choque Térmico/efeitos dos fármacos , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Neoplasias/metabolismo , Neoplasias/mortalidade , Neoplasias/patologia , Proteínas Nucleares/química , Agregados Proteicos , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacosRESUMO
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.
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Adenosina Trifosfatases/genética , Indóis/administração & dosagem , Mieloma Múltiplo/tratamento farmacológico , Proteínas Nucleares/genética , Fator 1 Nuclear Respiratório/genética , Inibidores de Proteassoma/administração & dosagem , Pirimidinas/administração & dosagem , Adenosina Trifosfatases/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Proteínas Nucleares/antagonistas & inibidores , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Ubiquitina/genética , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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).
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Bortezomib/uso terapêutico , Leucemia/tratamento farmacológico , Linfoma/tratamento farmacológico , Mieloma Múltiplo/tratamento farmacológico , Nelfinavir/uso terapêutico , Idoso , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Bortezomib/farmacocinética , Esquema de Medicação , Combinação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Inibidores da Protease de HIV/farmacocinética , Inibidores da Protease de HIV/uso terapêutico , Humanos , Leucemia/diagnóstico , Leucemia/genética , Leucemia/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Linfoma/diagnóstico , Linfoma/genética , Linfoma/patologia , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Nelfinavir/farmacocinética , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Resultado do Tratamento , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
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.
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Sondas Moleculares/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínio Catalítico , HumanosRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Bortezomib/farmacologia , Resistencia a Medicamentos Antineoplásicos , Oligopeptídeos/farmacologia , Inibidores de Proteassoma/farmacologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Proteasome-inhibiting drugs (PI) are gaining importance in hematologic oncology. The proteasome carries three proteolytically active subunits (ß1, ß2, ß5). All established PI (bortezomib and carfilzomib), as well as experimental drugs in the field (dalanzomib, oprozomib, and ixazomib), by design target the rate-limiting ß5 subunit. It is unknown whether ß2-selective proteasome inhibition can also be exploited toward anticancer treatment. Combining PI with the pan B-cell-directed Bruton tyrosine kinase inhibitor ibrutinib appears a natural option for future improved treatment of multiple myeloma (MM) and B-cell lymphomas. However, bortezomib induces phosphorylation of IκB and activation of NF-κB in MM cells, while ibrutinib inhibits the IκB/NF-κB axis, suggesting antagonistic signaling. A ß2-selective proteasome inhibitor may lack such antagonistic signaling effects. METHODS: We recently introduced LU-102, the first ß2-selective PI available for preclinical testing. We here compare bortezomib with carfilzomib and LU-102 in MM and MCL in vitro with regard to their effects on pIκB/NF-κB signaling and their cytotoxic activity in combination with ibrutinib. RESULTS: LU-102 reduced phosphorylation of IκB, in contrast to bortezomib and carfilzomib, and was a superior inhibitor of NF-κB activation in MM cells. This translated into highly synergistic cytotoxicity between LU-102 and ibrutinib, which was able to overcome BTZ resistance and CFZ resistance. By contrast, BTZ lacked consistent synergistic cytotoxicity with ibrutinib. CONCLUSION: Ibrutinib is highly synergistic with ß2-selective proteasome inhibition against MM and MCL in vitro. Novel ß2-selective proteasome inhibitors may be exploited to overcome bortezomib/carfilzomib resistance and boost the activity of BTK inhibitors against B-cell-derived malignancies.
Assuntos
Antineoplásicos/farmacologia , Proteínas I-kappa B/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Oligopeptídeos/farmacologia , Inibidores de Proteassoma/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Adenina/análogos & derivados , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Linfoma de Célula do Manto/tratamento farmacológico , Fosforilação , Piperidinas , Pirazinas/farmacologiaRESUMO
BACKGROUND: Protein metabolism is an innovative potential therapeutic target for AML. Proteotoxic stress (PS) sensitizes malignant cells for proteasome inhibitor treatment. Some HIV protease inhibitors (HIV-PI) induce PS and may therefore be combined with proteasome inhibitors to achieve PS-targeted therapy of AML. METHODS: We investigated the effects of all nine approved HIV-PI alone and in combination with proteasome inhibitors on AML cell lines and primary cells in vitro. RESULTS: Ritonavir induced cytotoxicity and PS at clinically achievable concentrations, and induced synergistic PS-triggered apoptosis with bortezomib. Saquinavir, nelfinavir and lopinavir were likewise cytotoxic against primary AML cells, triggered PS-induced apoptosis, inhibited AKT-phosphorylation and showed synergistic cytotoxicity with bortezomib and carfilzomib at low micromolar concentrations. Exclusively nelfinavir inhibited intracellular proteasome activity, including the ß2 proteasome activity that is not targeted by bortezomib/carfilzomib. CONCLUSIONS: Of the nine currently approved HIV-PI, ritonavir, saquinavir, nelfinavir and lopinavir can sensitize AML primary cells for proteasome inhibitor treatment at low micromolar concentrations and may therefore be tested clinically toward a proteotoxic stress targeted therapy of AML.
Assuntos
Citotoxinas/farmacologia , Inibidores da Protease de HIV/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Proteassoma/farmacologia , Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Regulação Leucêmica da Expressão Gênica , Humanos , Lopinavir/farmacologia , Nelfinavir/farmacologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ritonavir/farmacologia , Saquinavir/farmacologia , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Inhibition of the proteasome is a widely used strategy for treating multiple myeloma that takes advantage of the heavy secretory load that multiple myeloma cells (MMCs) have to deal with. Resistance of MMCs to proteasome inhibition has been linked to incomplete disruption of proteasomal endoplasmic-reticulum (ER)-associated degradation (ERAD) and activation of non-proteasomal protein degradation pathways. The ATPase p97 (VCP/Cdc48) has key roles in mediating both ERAD and non-proteasomal protein degradation and can be targeted pharmacologically by small molecule inhibition. In this study, we compared the effects of p97 inhibition with Eeyarestatin 1 and DBeQ on the secretory apparatus of MMCs with the effects induced by the proteasome inhibitor bortezomib, and the effects caused by combined inhibition of p97 and the proteasome. We found that p97 inhibition elicits cellular responses that are different from those induced by proteasome inhibition, and that the responses differ considerably between MMC lines. Moreover, we found that dual inhibition of both p97 and the proteasome terminally disrupts ER configuration and intracellular protein metabolism in MMCs. Dual inhibition of p97 and the proteasome induced high levels of apoptosis in all of the MMC lines that we analysed, including bortezomib-adapted AMO-1 cells, and was also effective in killing primary MMCs. Only minor toxicity was observed in untransformed and non-secretory cells. Our observations highlight non-redundant roles of p97 and the proteasome in maintaining secretory homeostasis in MMCs and provide a preclinical conceptual framework for dual targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma.
Assuntos
Adenosina Trifosfatases/metabolismo , Inibidores Enzimáticos/farmacologia , Mieloma Múltiplo/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Inibidores Enzimáticos/toxicidade , Humanos , Proteínas Nucleares/antagonistas & inibidores , Inibidores de Proteassoma/toxicidade , Biossíntese de Proteínas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
PURPOSE: Bortezomib (Velcade), a dipeptide boronate 20S proteasome inhibitor and an approved treatment option for multiple myeloma, is associated with a treatment-emergent, painful peripheral neuropathy (PN) in more than 30% of patients. Carfilzomib, a tetrapeptide epoxyketone proteasome inhibitor, currently in clinical investigation in myeloma, is associated with low rates of PN. We sought to determine whether PN represents a target-mediated adverse drug reaction (ADR). EXPERIMENTAL DESIGN: Neurodegenerative effects of proteasome inhibitors were assessed in an in vitro model utilizing a differentiated neuronal cell line. Secondary targets of both inhibitors were identified by a multifaceted approach involving candidate screening, profiling with an activity-based probe, and database mining. Secondary target activity was measured in rats and patients receiving both inhibitors. RESULTS: Despite equivalent levels of proteasome inhibition, only bortezomib reduced neurite length, suggesting a nonproteasomal mechanism. In cell lysates, bortezomib, but not carfilzomib, significantly inhibited the serine proteases cathepsin G (CatG), cathepsin A, chymase, dipeptidyl peptidase II, and HtrA2/Omi at potencies near or equivalent to that for the proteasome. Inhibition of CatG was detected in splenocytes of rats receiving bortezomib and in peripheral blood mononuclear cells derived from bortezomib-treated patients. Levels of HtrA2/Omi, which is known to be involved in neuronal survival, were upregulated in neuronal cells exposed to both proteasome inhibitors but was inhibited only by bortezomib exposure. CONCLUSION: These data show that bortezomib-induced neurodegeneration in vitro occurs via a proteasome-independent mechanism and that bortezomib inhibits several nonproteasomal targets in vitro and in vivo, which may play a role in its clinical ADR profile.
Assuntos
Ácidos Borônicos/efeitos adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/etiologia , Oligopeptídeos/efeitos adversos , Inibidores de Proteassoma , Pirazinas/efeitos adversos , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Ácidos Borônicos/administração & dosagem , Bortezomib , Células Cultivadas , Cisteína Endopeptidases/administração & dosagem , Cisteína Endopeptidases/efeitos adversos , Sistemas de Liberação de Medicamentos , Células Hep G2 , Humanos , Masculino , Modelos Biológicos , Oligopeptídeos/administração & dosagem , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirazinas/administração & dosagem , Ratos , Ratos Sprague-DawleyRESUMO
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.