RESUMO
BACKGROUND: Pivekimab sunirine (IMGN632) is a first-in-class antibody-drug conjugate comprising a high-affinity CD123 antibody, cleavable linker, and novel indolinobenzodiazepine pseudodimer payload. CD123 is overexpressed in several haematological malignancies, including acute myeloid leukaemia. We present clinical data on pivekimab sunirine in relapsed or refractory acute myeloid leukaemia. METHODS: This first-in-human, phase 1/2 dose-escalation and dose-expansion study enrolled participants aged 18 years or older at nine hospitals in France, Italy, Spain, and the USA with CD123+ haematological malignancies (Eastern Cooperative Oncology Group performance status of 0-1); participants reported here were in a cohort of participants with acute myeloid leukaemia who were refractory to or had relapsed on one or more previous treatments for acute myeloid leukaemia. The 3â+â3 dose-escalation phase evaluated two dosing schedules: schedule A (once every 3 weeks, on day 1 of a 3-week cycle) and fractionated schedule B (days 1, 4, and 8 of a 3-week cycle). The dose-expansion phase evaluated two cohorts: one cohort given 0·045 mg/kg of bodyweight (schedule A) and one cohort given 0·090 mg/kg of bodyweight (schedule A). The primary endpoints were the maximum tolerated dose and the recommended phase 2 dose. Antileukaemia activity (overall response and a composite complete remission assessment) was a secondary endpoint. The study is ongoing and registered with ClinicalTrials.gov, NCT03386513. FINDINGS: Between Dec 29, 2017, and May 27, 2020, 91 participants were enrolled (schedule A, n=68; schedule B, n=23). 30 (44%) of schedule A participants were female and 38 (56%) were male; 60 (88%) were White, six (9%) were Black or African American, and two (3%) were other races. Pivekimab sunirine at doses of 0·015 mg/kg to 0·450 mg/kg in schedule A was administered in six escalating doses with no maximum tolerated dose defined; three dose-limiting toxicities were observed (reversible veno-occlusive disease; 0·180 mg/kg, n=1 and 0·450 mg/kg, n=1; and neutropenia; 0·300 mg/kg, n=1). Schedule B was not pursued further on the basis of comparative safety and antileukaemia findings with schedule A. The recommended phase 2 dose was selected as 0·045 mg/kg once every 3 weeks. At the recommended phase 2 dose (n=29), the most common grade 3 or worse treatment-related adverse events were febrile neutropenia (three [10%]), infusion-related reactions (two [7%]), and anaemia (two [7%]). Treatment-related serious adverse events occurring in 5% or more of participants treated at the recommended phase 2 dose were febrile neutropenia (two [7%]) and infusion-related reactions (two [7%]). Among 68 participants who received schedule A, one death (1%) was considered to be treatment-related (cause unknown; 0·300 mg/kg cohort). At the recommended phase 2 dose, the overall response rate was 21% (95% CI 8-40; six of 29) and the composite complete remission rate was 17% (95% CI 6-36; five of 29). INTERPRETATION: Pivekimab sunirine showed single-agent activity across multiple doses, with a recommended phase 2 dose of 0·045 mg/kg once every 3 weeks. These findings led to a phase 1b/2 study of pivekimab sunirine plus azacitidine and venetoclax in patients with CD123-positive acute myeloid leukaemia. FUNDING: ImmunoGen.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Neutropenia Febril , Neoplasias Hematológicas , Imunoconjugados , Leucemia Mieloide Aguda , Humanos , Feminino , Masculino , Imunoconjugados/efeitos adversos , Subunidade alfa de Receptor de Interleucina-3 , Leucemia Mieloide Aguda/tratamento farmacológicoRESUMO
AIMS: Mirvetuximab soravtansine is a first-in-class antibody-drug conjugate recently approved for the treatment of folate receptor-α positive ovarian cancer. The aim of this study was to develop a population pharmacokinetic model to describe the concentration-time profiles of mirvetuximab soravtansine, the payload (DM4) and a metabolite (S-methyl-DM4). METHODS: Mirvetuximab soravtansine was administered intravenously from 0.15 to 7 mg/kg to 543 patients with predominantly platinum-resistant ovarian cancer in 3 clinical studies, and the plasma drug concentrations were analysed using a nonlinear mixed-effects modelling approach. Stepwise covariate modelling was performed to identify covariates. RESULTS: We developed a semi-mechanistic population pharmacokinetic model that included linear and nonlinear routes for the elimination of mirvetuximab soravtansine and a target compartment for the formation and disposition of the payload and metabolite in tumour cells. The clearance and volume of the central compartment were 0.0153 L/h and 2.63 L for mirvetuximab soravtansine, 8.83 L/h and 3.67 L for DM4, and 2.04 L/h and 6.3 L for S-methyl-DM4, respectively. Body weight, serum albumin and age were identified as statistically significant covariates. Exposures in patients with renal or hepatic impairment and who used concomitant cytochrome P450 (CYP) 3A4 inhibitors were estimated. CONCLUSION: There is no need for dose adjustment due to covariate effects for mirvetuximab soravtansine administered at the recommended dose of 6 mg/kg based on adjusted ideal body weight. Dose adjustment is not required for patients with mild or moderate renal impairment, mild hepatic impairment, or when concomitant weak and moderate CYP3A4 inhibitors are used.
Assuntos
Anticorpos Monoclonais Humanizados , Imunoconjugados , Maitansina , Neoplasias Ovarianas , Humanos , Feminino , Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas/tratamento farmacológico , Imunoconjugados/efeitos adversos , Ácido Fólico/farmacologia , Ácido Fólico/uso terapêutico , Maitansina/análogos & derivadosRESUMO
Antibody-drug conjugates (ADC) are a novel way to deliver potent cytotoxic compounds to cells expressing a specific antigen. Four ADC targeting CD19, including SAR3419 (coltuximab ravtansine), have entered clinical development. Here, we present huB4-DGN462, a novel ADC based on the SAR3419 anti-CD19 antibody linked via sulfo-SPDB to the potent DNA-alkylating agent DGN462. huB4-DGN462 had improved in vitro anti-proliferative and cytotoxic activity compared to SAR3419 across multiple B-cell lymphoma and human acute lymphoblastic leukemia cell lines. In vivo experiments using lymphoma xenografts models confirmed the in vitro data. The response of B-cell lymphoma lines to huB4-DGN462 was not correlated with CD19 expression, the presence of BCL2 or MYC translocations, TP53 inactivation or lymphoma histology. In conclusion, huB4-DGN462 is an attractive candidate for clinical investigation in patients with B-cell malignancies.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antígenos CD19/metabolismo , Antineoplásicos/farmacologia , Imunoconjugados/farmacologia , Leucemia/metabolismo , Linfoma/metabolismo , Maitansina/análogos & derivados , Animais , Anticorpos Monoclonais Humanizados/química , Antineoplásicos/química , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Humanos , Imunoconjugados/química , Leucemia/tratamento farmacológico , Leucemia/patologia , Linfoma/tratamento farmacológico , Linfoma/patologia , Maitansina/química , Maitansina/farmacologia , Camundongos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CONTEXT.: Folate receptor-α (FRα, encoded by the FOLR1 gene) is overexpressed in several solid tumor types, including epithelial ovarian cancer (EOC), making it an attractive biomarker and target for FRα-based therapy in ovarian cancer. OBJECTIVE.: To describe the development, analytic verification, and clinical performance of the VENTANA FOLR1 Assay (Ventana Medical Systems Inc) in EOC. DESIGN.: We used industry standard studies to establish the analytic verification of the VENTANA FOLR1 Assay. Furthermore, the VENTANA FOLR1 Assay was used in the ImmunoGen Inc-sponsored SORAYA study to select patients for treatment with mirvetuximab soravtansine (MIRV) in platinum-resistant EOC. RESULTS.: The VENTANA FOLR1 Assay is highly reproducible, demonstrated by a greater than 98% overall percent agreement (OPA) for repeatability and intermediate precision studies, greater than 93% OPA for interreader and greater than 96% for intrareader studies, and greater than 90% OPA across all observations in the interlaboratory reproducibility study. The performance of the VENTANA FOLR1 Assay in the SORAYA study was evaluated by the overall staining acceptability rate, which was calculated using the number of patient specimens that were tested with the VENTANA FOLR1 Assay that had an evaluable result. In the SORAYA trial, data in patients who received MIRV demonstrated clinically meaningful efficacy, and the overall staining acceptability rate of the assay was 98.4%, demonstrating that the VENTANA FOLR1 Assay is safe and effective for selecting patients who may benefit from MIRV. Together, these data showed that the assay is highly reliable, consistently producing evaluable results in the clinical setting. CONCLUSIONS.: The VENTANA FOLR1 Assay is a robust and reproducible assay for detecting FRα expression and identifying a patient population that derived clinically meaningful benefit from MIRV in the SORAYA study.
RESUMO
In this study we generated a novel dual specific phosphatase 4 (DUSP4) deletion mouse using a targeted deletion strategy in order to examine the role of MAP kinase phosphatase-2 (MKP-2) in immune responses. Lipopolysaccharide (LPS) induced a rapid, time and concentration-dependent increase in MKP-2 protein expression in bone marrow-derived macrophages from MKP-2(+/+) but not from MKP-2(-/-) mice. LPS-induced JNK and p38 MAP kinase phosphorylation was significantly increased and prolonged in MKP-2(-/-) macrophages whilst ERK phosphorylation was unaffected. MKP-2 deletion also potentiated LPS-stimulated induction of the inflammatory cytokines, IL-6, IL-12p40, TNF-α, and also COX-2 derived PGE(2) production. However surprisingly, in MKP-2(-/-) macrophages, there was a marked reduction in LPS or IFNγ-induced iNOS and nitric oxide release and enhanced basal expression of arginase-1, suggesting that MKP-2 may have an additional regulatory function significant in pathogen-mediated immunity. Indeed, following infection with the intracellular parasite Leishmania mexicana, MKP-2(-/-) mice displayed increased lesion size and parasite burden, and a significantly modified Th1/Th2 bias compared with wild-type counterparts. However, there was no intrinsic defect in MKP-2(-/-) T cell function as measured by anti-CD3 induced IFN-γ production. Rather, MKP-2(-/-) bone marrow-derived macrophages were found to be inherently more susceptible to infection with Leishmania mexicana, an effect reversed following treatment with the arginase inhibitor nor-NOHA. These findings show for the first time a role for MKP-2 in vivo and demonstrate that MKP-2 may be essential in orchestrating protection against intracellular infection at the level of the macrophage.
Assuntos
Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Leishmania mexicana/patogenicidade , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/prevenção & controle , Macrófagos/imunologia , Proteínas Tirosina Fosfatases/fisiologia , Animais , Arginase/metabolismo , Western Blotting , Medula Óssea/imunologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Células Cultivadas , Feminino , Leishmaniose Cutânea/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Óxido Nítrico/metabolismo , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The mechanisms underpinning the coupling of GPCRs, such as PAR-2, to the phosphorylation of p65 NFkappaB have not been investigated. In the current study we found that trypsin and the selective PAR-2 activating peptide, 2f-LIGKV-OH, stimulated large and sustained increases in the serine 536 phosphorylation of p65/RelA in a transfected skin epithelial cell line and primary keratinocytes. Parallel experiments showed that in both cell types, p65 NFkappaB phosphorylation is mediated through the selective activation of IKK2. Treatment with PKC inhibitor GF109203X or PKCalpha siRNA reduced phosphorylation at 15 min but not 30 min, whilst rottlerin, a selective PKCdelta inhibitor and PKCdelta siRNA reduced the response at both time points. Pre-treatment of cells with the novel Gq/11 inhibitor YM-254890 and Gq/11 siRNA caused a similar pattern of inhibition and also reduced PAR-2-mediated NFkappaB transcriptional activity. Furthermore, stimulation of cells through a novel PAR-2 mutant PAR-2(34-43), delayed p65 phosphorylation but was without effect on the kinetics of ERK activation. Inhibition of Gi or G12/13 pathways by pertussis toxin pre-treatment or over-expression of the RGS mutant Lsc, also did not effect NFkappaB phosphorylation. Taken together these data indicate dependency for Gq/11 in early phosphorylation of p65 NFkappaB and this subsequently affects initial NFkappaB-dependent gene transcriptional activity, however later regulation of p65 is unaffected. Overall these novel data demonstrate an IKK2-dependent, predominantly G-protein-independent pathway involved in PAR-2 regulation of NFkappaB phosphorylation in keratinocytes.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Queratinócitos/metabolismo , Fosfosserina/metabolismo , Receptor PAR-2/metabolismo , Fator de Transcrição RelA/metabolismo , Acetofenonas/farmacologia , Benzopiranos/farmacologia , Células Cultivadas , Células Clonais , Genes Reporter , Humanos , Proteínas I-kappa B/metabolismo , Indóis/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/enzimologia , Maleimidas/farmacologia , Proteínas Mutantes/metabolismo , Peptídeos Cíclicos/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-delta/metabolismo , Interferência de RNA/efeitos dos fármacosRESUMO
PURPOSE: In the current study, we investigate the activation of antiapoptotic signaling pathways in response to proteasome inhibitor treatment in pancreatic cancer and evaluate the use of concomitant inhibition of these pathways to augment proteasome inhibitor treatment responses. EXPERIMENTAL DESIGN: Pancreatic cancer cell lines and mouse flank xenografts were treated with proteasome inhibitor alone or in combination with chemotherapeutic compounds (gemcitabine, erlotinib, and bevacizumab), induction of apoptosis and effects on tumor growth were assessed. The effect of bortezomib (a first-generation proteasome inhibitor) and NPI-0052 (a second-generation proteasome inhibitor) treatment on key pancreatic mitogenic and antiapoptotic pathways [epidermal growth factor receptor, extracellular signal-regulated kinase, and phosphoinositide-3-kinase (PI3K)/AKT] was determined and the ability of inhibitors of these pathways to enhance the effects of proteasome inhibition was assessed in vitro and in vivo. RESULTS: Our data showed that proteasome inhibitor treatment activates antiapoptotic and mitogenic signaling pathways (epidermal growth factor receptor, extracellular signal-regulated kinase, c-Jun-NH2-kinase, and PI3K/AKT) in pancreatic cancer. Additionally, we found that activation of these pathways impairs tumor response to proteasome inhibitor treatment and inhibition of the c-Jun-NH2-kinase and PI3K/AKT pathways increases the antitumor effects of proteasome inhibitor treatment. CONCLUSION: These preclinical studies suggest that targeting proteasome inhibitor-induced antiapoptotic signaling pathways in combination with proteasome inhibition may augment treatment response in highly resistant solid organ malignancies. Further evaluation of these novel treatment combinations in clinical trials is warranted.
Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Receptores ErbB/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteases/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Adenocarcinoma/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Bevacizumab , Western Blotting , Ácidos Borônicos/administração & dosagem , Bortezomib , Linhagem Celular Tumoral , Cetuximab , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Feminino , Humanos , Lactonas/administração & dosagem , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteassoma , Pirazinas/administração & dosagem , Pirróis/administração & dosagem , Quinazolinas/administração & dosagem , Transdução de Sinais/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
The myeloid differentiation antigen CD33 has long been exploited as a target for antibody-based therapeutic approaches in acute myeloid leukemia (AML). Validation of this strategy was provided with the approval of the CD33-targeting antibody-drug conjugate (ADC) gemtuzumab ozogamicin in 2000; the clinical utility of this agent, however, has been hampered by safety concerns. Thus, the full potential of CD33-directed therapy in AML remains to be realized, and considerable interest exists in the design and development of more effective ADCs that confer high therapeutic indices and favorable tolerability profiles. Here, we describe the preclinical characterization of a novel CD33-targeting ADC, IMGN779, which utilizes a unique DNA-alkylating payload to achieve potent antitumor effects with good tolerability. The payload, DGN462, is prototypical of a novel class of purpose-created indolinobenzodiazeprine pseudodimers, termed IGNs. With low picomolar potency, IMGN779 reduced viability in a panel of AML cell lines in vitro Mechanistically, the cytotoxic activity of IMGN779 involved DNA damage, cell-cycle arrest, and apoptosis consistent with the mode of action of DGN462. Moreover, IMGN779 was highly active against patient-derived AML cells, including those with adverse molecular abnormalities, and sensitivity correlated to CD33 expression levels. In vivo, IMGN779 displayed robust antitumor efficacy in multiple AML xenograft and disseminated disease models, as evidenced by durable tumor regressions and prolonged survival. Taken together, these findings identify IMGN779 as a promising new candidate for evaluation as a novel therapeutic in AML. Mol Cancer Ther; 17(6); 1271-9. ©2018 AACR.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Imunológicos/farmacologia , Imunoconjugados/farmacologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/antagonistas & inibidores , Animais , Antineoplásicos Alquilantes/química , Antineoplásicos Imunológicos/química , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Desenho de Fármacos , Humanos , Imunoconjugados/química , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos , Estrutura Molecular , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Naratuximab emtansine (IMGN529) is an investigational antibody-drug conjugate consisting of a CD37-targeting antibody conjugated to the maytansine-derived microtuble disruptor, DM1. IMGN529 has shown promising preclinical and clinical activity in non-Hodgkin lymphoma, including diffuse large B-cell lymphoma (DLBCL). Due to the aggressive nature of the disease, DLBCL is often treated with combination therapies to maximize clinical outcomes; therefore, we investigated the potential of combining IMGN529 with both standard-of-care and emerging therapies against multiple oncology-relevant targets and pathways. The strongest enhancement in potency was seen with anti-CD20 antibodies, including rituximab. The combination of IMGN529 and rituximab was more potent than either agent alone, and this combinatorial benefit was associated with increased apoptotic induction and cell death. Additional studies revealed that rituximab treatment increased the internalization and degradation of the CD37-targeting antibody moiety of IMGN529. The combination of IMGN529 and rituximab was highly efficacious in multiple xenograft models, with superior antitumor efficacy seen compared to either agent alone or treatment with R-CHOP therapy. These findings suggest a novel mechanism whereby the potency of IMGN529 can be enhanced by CD20 binding, which results in the increased internalization and degradation of IMGN529 leading to the generation of greater amounts of cytotoxic catabolite. Overall, these data provide a biological rationale for the enhanced activity of IMGN529 in combination with rituximab and support the ongoing clinical evaluation of IMGN529 in combination with rituximab in patients with relapsed and/or refractory DLBCL.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Linfoma não Hodgkin/tratamento farmacológico , Rituximab/farmacologia , Animais , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Humanos , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/metabolismo , Linfoma não Hodgkin/patologia , Camundongos , Terapia de Alvo Molecular , Proteólise , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MAP Kinase Phosphatase-2 (MKP-2) is a dual specific nuclear phosphatase which is selective for both ERK and JNK, MAP kinases implicated in the regulation of apoptosis in response to genotoxic stress. Here we report the conditional expression of MKP-2 in human embryonic kidney cells 293. We demonstrate that Flag-WT-MKP-2 is able to rescue cells from apoptotic commitment when subjected to UV-C or cisplatin treatment. We establish that upon stimulation all three major MAP kinase families (ERK, JNK and p38 MAP kinases) are activated. However, MKP-2 is surprisingly only able to deactivate JNK in vivo. Furthermore, whilst pre-treatment of cells with either the JNK inhibitor SP600125, or the MEK-1 inhibitor PD98059, also reverses UV-C and cisplatin-induced apoptosis, the anti-apoptotic effect of MKP-2 overexpression is not additive with SP600125 but is with PD098059, suggesting that MKP-2 is involved in specifically terminating JNK activity and not ERK. The inability of MKP-2 to dephosphorylate ERK in vivo is also not due to the inability of Flag-MKP-2 to bind both ERK and JNK; phosphorylated forms of each kinase are co-precipitated with both WT and CI-MKP-2. Immunofluorescence studies however demonstrate that ERK is exclusively cytosolic in origin and not translocated to the nucleus following UV-C and cisplatin treatment whilst JNK is principally nuclear. These studies demonstrate the in vivo specificity of MKP-2 for JNK and not ERK and show that nuclear-targeted JNK is involved in genotoxic stress-induced apoptosis.
Assuntos
Apoptose/fisiologia , Dano ao DNA/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais/fisiologia , Antracenos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular , Núcleo Celular/metabolismo , Cisplatino/farmacologia , Doxiciclina/farmacologia , Fosfatases de Especificidade Dupla , Flavonoides/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Microscopia de Fluorescência , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno , Mutação/genética , Oligopeptídeos , Peptídeos/genética , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteína Fosfatase 2 , Proteínas Tirosina Fosfatases/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Transfecção , Raios Ultravioleta , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
MAP kinase phosphatase-2 (MKP-2) is a member of a family of dual specificity phosphatases (DSPs) that function in both the cytosol and nucleus to inactivate the MAP kinases. The mechanism that controls the subcellular distribution of these proteins is currently unclear. In this study, we have used site-directed mutagenesis to remove two novel nuclear localization sequences, NLS-1 and -2, either alone or in combination (DNLS). Loss of NLS-1 or NLS-2 alone did not alter the nuclear targeting of MKP-2 but mutation of both resulted in MKP-2 being retained within the cytosol. Furthermore, whilst expression of WT-MKP-2, NLS-1 or NLS-2 reduced both sorbitol- or UV-stimulated nuclear c-Jun N-terminal kinase (JNK) activity in HEK293 cells, this effect was absent in cells expressing DNLS-MKP-2. Similarly, transient transfection of WT-MKP-2, NLS-1 or NLS-2, but not DNLS-MKP-2 was able to substantially reduce agonist-stimulated ANF reporter activity in rat cardiac myocytes. Taken together, these results indicate that whilst both novel NLS participate in the nuclear localization of MKP-2, the expression of either sequence is sufficient to retain nuclear targeting.
Assuntos
Proteínas Tirosina Fosfatases/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/enzimologia , Citosol/enzimologia , Fosfatases de Especificidade Dupla , Humanos , Fosfatases da Proteína Quinase Ativada por Mitógeno , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sinais de Localização Nuclear , Proteína Fosfatase 2 , Proteínas Tirosina Fosfatases/análise , Proteínas Tirosina Fosfatases/metabolismo , RatosRESUMO
1 In this study, we examined the role of Ca2+ in linking proteinase-activated receptor-2 (PAR2) to the nuclear factor kappa B (NFkappaB) pathway in a skin epithelial cell line NCTC2544 stably expressing PAR2 (clone G). 2 In clone G, PAR2-mediated NFkappaB luciferase reporter activity and NFkappaB DNA-binding activity was reduced by preincubation with BAPTA-AM but not BAPTA. Trypsin stimulation of inhibitory kappa B kinases, IKKalpha and IKKbeta, was also inhibited following pretreatment with BAPTA-AM. 3 BAPTA/AM also prevented PAR2-mediated IKKalpha activation in cultured primary human keratinocytes. 4 The effect of BAPTA-AM was also selective for the IKK/NFkappaB signalling axis; PAR2 coupling to ERK, or p38 MAP kinase was unaffected. 5 Pharmacological inhibition of the Ca2+-dependent regulatory protein calcineurin did not inhibit trypsin-stimulated IKK activity or NFkappaB-DNA binding; however, inhibition of Ca2+-dependent protein kinase C isoforms or InsP3 formation using GF109203X or the phospholipase C inhibitor U73122, respectively, reduced both IKK activity and NFkappaB-DNA binding. 6 Mutation of PAR2 within the C-terminal to produce a mutant receptor, which does not couple to Ca2+ signalling, but is able to activate ERK, abrogated NFkappaB-DNA binding and IKK activity stimulated by trypsin. 7 These results suggest a predominant role for the InsP3/Ca2+ axis in the regulation of IKK signalling and NFkappaB transcriptional activation.
Assuntos
Cálcio/fisiologia , Queratinócitos/fisiologia , NF-kappa B/fisiologia , Receptor PAR-2/fisiologia , Transdução de Sinais/fisiologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Linhagem Celular , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Humanos , Quinase I-kappa B , Indóis/farmacologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Maleimidas/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Oligonucleotídeos/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pirrolidinonas/farmacologia , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tacrolimo/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Tripsina/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
Although EGFR is a validated therapeutic target across multiple cancer indications, the often modest clinical responses to current anti-EGFR agents suggest the need for improved therapeutics. Here, we demonstrate that signal amplification driven by high-affinity EGFR ligands limits the capacity of monoclonal anti-EGFR antibodies to block pathway signaling and cell proliferation and that these ligands are commonly coexpressed with low-affinity EGFR ligands in epithelial tumors. To develop an improved antibody therapeutic capable of overcoming high-affinity ligand-mediated signal amplification, we used a network biology approach comprised of signaling studies and computational modeling of receptor-antagonist interactions. Model simulations suggested that an oligoclonal antibody combination may overcome signal amplification within the EGFR:ERK pathway driven by all EGFR ligands. Based on this, we designed MM-151, a combination of three fully human IgG1 monoclonal antibodies that can simultaneously engage distinct, nonoverlapping epitopes on EGFR with subnanomolar affinities. In signaling studies, MM-151 antagonized high-affinity EGFR ligands more effectively than cetuximab, leading to an approximately 65-fold greater decrease in signal amplification to ERK. In cell viability studies, MM-151 demonstrated antiproliferative activity against high-affinity EGFR ligands, either singly or in combination, while cetuximab activity was largely abrogated under these conditions. We confirmed this finding both in vitro and in vivo in a cell line model of autocrine high-affinity ligand expression. Together, these preclinical studies provide rationale for the clinical study of MM-151 and suggest that high-affinity EGFR ligand expression may be a predictive response marker that distinguishes MM-151 from other anti-EGFR therapeutics.
Assuntos
Anticorpos Monoclonais/farmacologia , Receptores ErbB/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Epitopos/imunologia , Epitopos/metabolismo , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Feminino , Humanos , Ligantes , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos SCID , Microscopia Confocal , Terapia de Alvo Molecular , Neoplasias/imunologia , Neoplasias/metabolismoRESUMO
MAP kinase phosphatase-2 (MKP-2) is a member of the family of dual specificity phosphatases that functions to inactivate the ERK and JNK MAP kinase signalling pathways. Here, we identify a novel human MKP-2 variant (MKP-2-S) lacking the MAP kinase binding site but retaining the phosphatase catalytic domain. Endogenous MKP-2-S transcripts and proteins were found in PC3 prostate and MDA-MB-231 breast cancer cells and also human prostate biopsies. Cellular transfection of MKP-2-S gave rise to a nuclear protein of 33kDa which displayed phosphatase activity comparable to the formerly described long form of MKP-2 (MKP-2-L). Due to its lack of a kinase interacting motif (KIM), MKP-2-S did not bind to JNK or ERK; MKP-2-L bound ERK and to a lesser extent JNK. Protein turnover of adenoviral expressed MKP-2-S was accelerated relative to MKP-2-L, with a greater susceptibility to proteosomal-mediated degradation. MKP-2-S retained its ability to deactivate JNK in a similar manner as MKP-2-L and was an effective inhibitor of LPS-stimulated COX-2 induction. However, unlike MKP-2-L, MKP-2-S was unable to reverse serum-induced ERK activation or significantly inhibit endothelial cell proliferation. These findings reveal the occurrence of a novel splice variant of MKP-2 which is unable to bind ERK and may be significant in the dysregulation of MAP kinase activity in certain disease states, particularly in breast and prostate cancers.
Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Splicing de RNA , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Fosfatases de Especificidade Dupla/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HeLa , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Dados de Sequência Molecular , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Targeted therapy against the BRAF/mitogen-activated protein kinase (MAPK) pathway is a promising new therapeutic approach for the treatment of melanoma. Treatment with selective BRAF inhibitors results in a high initial response rate but limited duration of response. To counter this, investigators propose combining this therapy with other targeted agents, addressing the issue of redundancy and signaling through different oncogenic pathways. An alternative approach is combining BRAF/MAPK-targeted agents with immunotherapy. Preliminary evidence suggests that oncogenic BRAF (BRAF(V600E)) contributes to immune escape and that blocking its activity via MAPK pathway inhibition leads to increased expression of melanocyte differentiation antigens (MDA). Recognition of MDAs is a critical component of the immunologic response to melanoma, and several forms of immunotherapy capitalize on this recognition. Among the various approaches to inhibiting BRAF/MAPK, broad MAPK pathway inhibition may have deleterious effects on T lymphocyte function. Here, we corroborate the role of oncogenic BRAF in immune evasion by melanoma cells through suppression of MDAs. We show that inhibition of the MAPK pathway with MAPK/extracellular signal-regulated kinase kinase (MEK) inhibitors or a specific inhibitor of BRAF(V600E) in melanoma cell lines and tumor digests results in increased levels of MDAs, which is associated with improved recognition by antigen-specific T lymphocytes. However, treatment with MEK inhibitors impairs T lymphocyte function, whereas T-cell function is preserved after treatment with a specific inhibitor of BRAF(V600E). These findings suggest that immune evasion of melanomas mediated by oncogenic BRAF may be reversed by targeted BRAF inhibition without compromising T-cell function. These findings have important implications for combined kinase-targeted therapy plus immunotherapy for melanoma.