RESUMO
The tumor-associated antigen STEAP1 is a potential therapeutic target that is expressed in most prostate tumors and at increased levels in metastatic castration-resistant prostate cancer (mCRPC). We developed a STEAP1-targeted XmAb 2+1 T-cell engager (TCE) molecule, AMG 509 (also designated xaluritamig), that is designed to redirect T cells to kill prostate cancer cells that express STEAP1. AMG 509 mediates potent T cell-dependent cytotoxicity of prostate cancer cell lines in vitro and promotes tumor regression in xenograft and syngeneic mouse models of prostate cancer in vivo. The avidity-driven activity of AMG 509 enables selectivity for tumor cells with high STEAP1 expression compared with normal cells. AMG 509 is the first STEAP1 TCE to advance to clinical testing, and we report a case study of a patient with mCRPC who achieved an objective response on AMG 509 treatment. SIGNIFICANCE: Immunotherapy in prostate cancer has met with limited success due to the immunosuppressive microenvironment and lack of tumor-specific targets. AMG 509 provides a targeted immunotherapy approach to engage a patient's T cells to kill STEAP1-expressing tumor cells and represents a new treatment option for mCRPC and potentially more broadly for prostate cancer. See related commentary by Hage Chehade et al., p. 20. See related article by Kelly et al., p. 76. This article is featured in Selected Articles from This Issue, p. 5.
Assuntos
Anticorpos Biespecíficos , Neoplasias de Próstata Resistentes à Castração , Masculino , Camundongos , Animais , Humanos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia , Linfócitos T , Imunoterapia , Anticorpos Biespecíficos/uso terapêutico , Microambiente Tumoral , Antígenos de Neoplasias , Oxirredutases/uso terapêuticoRESUMO
BACKGROUND: Checkpoint inhibitors targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) have demonstrated clinical efficacy in advanced melanoma, but only a subset of patients with inflamed tumors are responsive. Talimogene laherparepvec (T-VEC), a modified herpes simplex virus type 1 (HSV-1) expressing granulocyte-macrophage colony-stimulating factor (GM-CSF), is a first-in-class oncolytic immunotherapy approved for the treatment of melanoma and has been shown to inflame the tumor microenvironment. To evaluate the potential and mechanisms of T-VEC to elicit systemic antitumor immunity and overcome resistance to checkpoint inhibitors in murine tumor models, OncoVEXmGM-CSF was developed similarly to T-VEC, except the human GM-CSF transgene was replaced with murine GM-CSF. Previous work had demonstrated that OncoVEXmGM-CSF generated systemic antitumor immunity dependent on CD8+ T cells in an immune checkpoint-sensitive tumor cell model. METHODS: A novel B16F10 syngeneic tumor model with both HSV-1-permissive subcutaneous tumors and HSV-1-refractory experimental lung metastasis was used to study the local and systemic effects of OncoVEXmGM-CSF treatment alone or in combination with checkpoint inhibitors. RESULTS: Intratumoral injection of OncoVEXmGM-CSF in combination with an anti-CTLA-4 or anti-PD-1 blocking antibody led to increased tumor growth inhibition, a reduction in the number of lung metastases, and prolonged animal survival. OncoVEXmGM-CSF induced both neoantigen-specific and tumor antigen-specific T-cell responses. Furthermore, cured mice from the combination treatment of OncoVEXmGM-CSF and anti-CTLA-4 antibody rejected tumor rechallenges. CONCLUSIONS: These data support the concept that T-VEC and checkpoint inhibition may be an effective combination to treat patients with advanced melanoma.
Assuntos
Melanoma , Terapia Viral Oncolítica , Humanos , Animais , Camundongos , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Linfócitos T CD8-Positivos/patologia , Antígenos de Neoplasias , Microambiente TumoralRESUMO
Effective treatments for de novo and treatment-emergent small-cell/neuroendocrine (t-SCNC) prostate cancer represent an unmet need for this disease. Using metastatic biopsies from patients with advanced cancer, we demonstrate that delta-like ligand 3 (DLL3) is expressed in de novo and t-SCNC and is associated with reduced survival. We develop a PET agent, [89Zr]-DFO-DLL3-scFv, that detects DLL3 levels in mouse SCNC models. In multiple patient-derived xenograft models, AMG 757 (tarlatamab), a half-life-extended bispecific T-cell engager (BiTE) immunotherapy that redirects CD3-positive T cells to kill DLL3-expressing cells, exhibited potent and durable antitumor activity. Late relapsing tumors after AMG 757 treatment exhibited lower DLL3 levels, suggesting antigen loss as a resistance mechanism, particularly in tumors with heterogeneous DLL3 expression. These findings have been translated into an ongoing clinical trial of AMG 757 in de novo and t-SCNC, with a confirmed objective partial response in a patient with histologically confirmed SCNC. Overall, these results identify DLL3 as a therapeutic target in SCNC and demonstrate that DLL3-targeted BiTE immunotherapy has significant antitumor activity in this aggressive prostate cancer subtype. SIGNIFICANCE: The preclinical and clinical evaluation of DLL3-directed immunotherapy, AMG 757, and development of a PET radiotracer for noninvasive DLL3 detection demonstrate the potential of targeting DLL3 in SCNC prostate cancer.
Assuntos
Proteínas de Membrana , Neoplasias da Próstata , Animais , Humanos , Masculino , Camundongos , Anticorpos Monoclonais , Imunoterapia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligantes , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Tomografia por Emissão de Pósitrons , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/terapia , Neoplasias da Próstata/patologia , Zircônio , Tumores Neuroendócrinos/diagnóstico por imagem , Tumores Neuroendócrinos/patologia , Tumores Neuroendócrinos/terapiaRESUMO
Bispecific T-cell engager (BiTE) molecules have great potential to treat cancer. Nevertheless, dependent on the targeted tumor antigen, the mechanism of action that drives efficacy may also contribute to on-target/off-tumor toxicities. In this study, we characterize an anti-CD70 half-life extended BiTE molecule (termed N6P) which targets CD70, a TNF family protein detected in several cancers. First, the therapeutic potential of N6P was demonstrated using in vitro cytotoxicity assays and an orthotopic xenograft mouse study resulting in potent killing of CD70+ cancer cells. Next, in vitro characterization demonstrated specificity for CD70 and equipotent activity against human and cynomolgus monkey CD70+ cells. To understand the potential for on-target toxicity, a tissue expression analysis was performed and indicated CD70 is primarily restricted to lymphocytes in normal healthy tissues and cells. Therefore, no on-target toxicity was expected to be associated with N6P. However, in a repeat-dose toxicology study using cynomolgus monkeys, adverse N6P-mediated inflammation was identified in multiple tissues frequently involving the mesothelium and epithelium. Follow-up immunohistochemistry analysis revealed CD70 expression in mesothelial and epithelial cells in some tissues with N6P-mediated injury, but not in control tissues or those without injury. Collectively, the data indicate that for some target antigens such as CD70, BiTE molecules may exhibit activity in tissues with very low antigen expression or the antigen may be upregulated under stress enabling molecule activity. This work illustrates how a thorough understanding of expression and upregulation is needed to fully address putative liabilities associated with on-target/off-tumor activity of CD3 bispecific molecules.
Assuntos
Anticorpos Biespecíficos , Neoplasias , Animais , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Complexo CD3/metabolismo , Meia-Vida , Humanos , Macaca fascicularis , Camundongos , Neoplasias/metabolismo , Linfócitos TRESUMO
PURPOSE: Small-cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with a high relapse rate, limited therapeutic options, and poor prognosis. We investigated the antitumor activity of AMG 757, a half-life extended bispecific T-cell engager molecule targeting delta-like ligand 3 (DLL3)-a target that is selectively expressed in SCLC tumors, but with minimal normal tissue expression. EXPERIMENTAL DESIGN: AMG 757 efficacy was evaluated in SCLC cell lines and in orthotopic and patient-derived xenograft (PDX) mouse SCLC models. Following AMG 757 administration, changes in tumor volume, pharmacodynamic changes in tumor-infiltrating T cells (TILs), and the spatial relationship between the appearance of TILs and tumor histology were examined. Tolerability was assessed in nonhuman primates (NHPs). RESULTS: AMG 757 showed potent and specific killing of even those SCLC cell lines with very low DLL3 expression (<1,000 molecules per cell). AMG 757 effectively engaged systemically administered human T cells, induced T-cell activation, and redirected T cells to lyse tumor cells to promote significant tumor regression and complete responses in PDX models of SCLC and in orthotopic models of established primary lung SCLC and metastatic liver lesions. AMG 757 was well tolerated with no AMG 757-related adverse findings up to the highest tested dose (4.5 mg/kg weekly) in NHP. AMG 757 exhibits an extended half-life in NHP, which is projected to enable intermittent administration in patients. CONCLUSIONS: AMG 757 has a compelling safety and efficacy profile in preclinical studies making it a viable option for targeting DLL3-expressing SCLC tumors in the clinical setting.
Assuntos
Anticorpos Biespecíficos , Anticorpos Monoclonais , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Pulmonares , Proteínas de Membrana , Carcinoma de Pequenas Células do Pulmão , Linfócitos T , Animais , Feminino , Humanos , Camundongos , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Apoptose , Proliferação de Células , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Membrana/antagonistas & inibidores , Camundongos Endogâmicos NOD , Camundongos SCID , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/imunologia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours1,2. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity3-5. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Piridinas/farmacologia , Piridinas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Imunoterapia , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Fosforilação/efeitos dos fármacos , Piperazinas/administração & dosagem , Piperazinas/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Piridinas/administração & dosagem , Piridinas/química , Pirimidinas/administração & dosagem , Pirimidinas/química , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Purpose: Talimogene laherparepvec, a new oncolytic immunotherapy, has been recently approved for the treatment of melanoma. Using a murine version of the virus, we characterized local and systemic antitumor immune responses driving efficacy in murine syngeneic models.Experimental Design: The activity of talimogene laherparepvec was characterized against melanoma cell lines using an in vitro viability assay. Efficacy of OncoVEXmGM-CSF (talimogene laherparepvec with the mouse granulocyte-macrophage colony-stimulating factor transgene) alone or in combination with checkpoint blockade was characterized in A20 and CT-26 contralateral murine tumor models. CD8+ depletion, adoptive T-cell transfers, and Enzyme-Linked ImmunoSpot assays were used to study the mechanism of action (MOA) of systemic immune responses.Results: Treatment with OncoVEXmGM-CSF cured all injected A20 tumors and half of contralateral tumors. Viral presence was limited to injected tumors and was not responsible for systemic efficacy. A significant increase in T cells (CD3+/CD8+) was observed in injected and contralateral tumors at 168 hours. Ex vivo analyses showed these cytotoxic T lymphocytes were tumor-specific. Increased neutrophils, monocytes, and chemokines were observed in injected tumors only. Importantly, depletion of CD8+ T cells abolished all systemic efficacy and significantly decreased local efficacy. In addition, immune cell transfer from OncoVEXmGM-CSF-cured mice significantly protected from tumor challenge. Finally, combination of OncoVEXmGM-CSF and checkpoint blockade resulted in increased tumor-specific CD8+ anti-AH1 T cells and systemic efficacy.Conclusions: The data support a dual MOA for OncoVEXmGM-CSF that involves direct oncolysis of injected tumors and activation of a CD8+-dependent systemic response that clears injected and contralateral tumors when combined with checkpoint inhibition. Clin Cancer Res; 23(20); 6190-202. ©2017 AACR.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Imunoterapia , Neoplasias/imunologia , Neoplasias/metabolismo , Terapia Viral Oncolítica , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Adenoviridae/genética , Transferência Adotiva , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Imunomodulação , Imunoterapia/métodos , Estimativa de Kaplan-Meier , Depleção Linfocítica , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/patologia , Melanoma/terapia , Camundongos , Neoplasias/patologia , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Transgenes , Carga Tumoral , Replicação Viral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Therapeutic resistance is a major obstacle to achieving durable clinical responses with targeted therapies, highlighting a need to elucidate the underlying mechanisms responsible for resistance and identify strategies to overcome this challenge. An emerging body of data implicates the tyrosine kinase MET in mediating resistance to BRAF inhibitors in BRAFV600E mutant melanoma. In this study we observed a dominant role for the HGF/MET axis in mediating resistance to BRAF and MEK inhibitors in models of BRAFV600E and NRAS mutant melanoma. In addition, we showed that MAPK pathway inhibition induced rapid increases in MET and GAB1 levels, providing novel mechanistic insight into how BRAFV600E mutant melanoma is primed for HGF-mediated rescue. We also determined that tumor-derived HGF, not systemic HGF, may be required to convey resistance to BRAF inhibition in vivo and that resistance could be reversed following treatment with AMG 337, a selective MET inhibitor. In summary, these findings support the clinical evaluation of MET-directed targeted therapy to circumvent resistance to BRAF and MEK inhibitors in BRAFV600E mutant melanoma. In addition, the induction of MET following treatment with BRAF and MEK inhibitors has the potential to serve as a predictive biomarker for identifying patients best suited for MET inhibitor combination therapy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/farmacologia , Fator de Crescimento de Hepatócito/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Indóis/farmacologia , Melanoma/genética , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Piridonas/farmacologia , Sulfonamidas/farmacologia , Triazóis/farmacologia , Vemurafenib , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In nonsmall cell lung cancer (NSCLC), the threonine(790)-methionine(790) (T790M) point mutation of EGFR kinase is one of the leading causes of acquired resistance to the first generation tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. Herein, we describe the optimization of a series of 7-oxopyrido[2,3-d]pyrimidinyl-derived irreversible inhibitors of EGFR kinase. This led to the discovery of compound 24 which potently inhibits gefitinib-resistant EGFR(L858R,T790M) with 100-fold selectivity over wild-type EGFR. Compound 24 displays strong antiproliferative activity against the H1975 nonsmall cell lung cancer cell line, the first line mutant HCC827 cell line, and promising antitumor activity in an EGFR(L858R,T790M) driven H1975 xenograft model sparing the side effects associated with the inhibition of wild-type EGFR.
RESUMO
Anemia of inflammation (AI) is commonly observed in chronic inflammatory states and may hinder patient recovery and survival. Induction of hepcidin, mediated by interleukin 6, leads to iron-restricted erythropoiesis and anemia. Several translational studies have been directed at neutralizing hepcidin overexpression as a therapeutic strategy against AI. However, additional hepcidin-independent mechanisms contribute to AI, which are likely mediated by a direct effect of inflammatory cytokines on erythropoiesis. In this study, we used wild-type, hepcidin knockout (Hamp-KO) and interleukin 6 knockout (IL-6-KO) mice as models of AI. AI was induced with heat-killed Brucella abortus (BA). The distinct roles of iron metabolism and inflammation triggered by interleukin 6 and hepcidin were investigated. BA-treated wild-type mice showed increased expression of hepcidin and inflammatory cytokines, as well as transitory suppression of erythropoiesis and shortened red blood cell lifespan, all of which contributed to the severe anemia of these mice. In contrast, BA-treated Hamp-KO or IL-6-KO mice showed milder anemia and faster recovery compared with normal mice. Moreover, they exhibited different patterns in the development and resolution of anemia, supporting the notion that interleukin 6 and hepcidin play distinct roles in modulating erythropoiesis in AI.
Assuntos
Anemia/imunologia , Brucella abortus , Brucelose/imunologia , Hepcidinas/imunologia , Interleucina-6/imunologia , Anemia/genética , Anemia/microbiologia , Animais , Medula Óssea/imunologia , Brucelose/complicações , Modelos Animais de Doenças , Eritropoese/imunologia , Feminino , Hepcidinas/genética , Temperatura Alta , Interleucina-6/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Recuperação de Função Fisiológica/imunologiaRESUMO
Iron maldistribution has been implicated in the etiology of many diseases including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino-acid peptide. Hepcidin is induced by inflammation and causes iron to be sequestered within cells of the reticuloendothelial system, suppressing erythropoiesis and blunting the activity of erythropoiesis stimulating agents (ESAs). For this reason, neutralization of hepcidin has been proposed as a therapeutic treatment of AI. The aim of the current work was to generate fully human anti-hepcidin antibodies (Abs) as a potential human therapeutic for the treatment of AI and other iron maldistribution disorders. An enzyme-linked immunosorbent assay was established using these Abs to identify patients likely to benefit from either ESAs or anti-hepcidin agents. Using human hepcidin knock-in mice, the mechanism of action of the Abs was shown to be due to an increase in available serum iron leading to enhanced red cell hemoglobinization. One of the Abs, 12B9m, was validated in a mouse model of AI and demonstrated to modulate serum iron in cynomolgus monkeys. The 12B9m Ab was deemed to be an appropriate candidate for use as a potential therapeutic to treat AI in patients with kidney disease or cancer.
Assuntos
Anemia Ferropriva/tratamento farmacológico , Anticorpos Neutralizantes/farmacologia , Eritrócitos/efeitos dos fármacos , Hemoglobinas/biossíntese , Ferro/sangue , Anemia Ferropriva/sangue , Anemia Ferropriva/patologia , Animais , Anticorpos Neutralizantes/biossíntese , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Eritrócitos/metabolismo , Eritrócitos/patologia , Eritropoese/efeitos dos fármacos , Feminino , Hematínicos/farmacologia , Humanos , Inflamação/prevenção & controle , Macaca fascicularis , Masculino , CamundongosRESUMO
Iron is a trace element important for the proper folding and function of various proteins. Physiological regulation of iron stores is of critical importance for RBC production and antimicrobial defense. Hepcidin is a key regulator of iron levels within the body. Under conditions of iron deficiency, hepcidin expression is reduced to promote increased iron uptake from the diet and release from cells, whereas during conditions of iron excess, induction of hepcidin restricts iron uptake and movement within the body. The cytokine IL-6 is well established as an important inducer of hepcidin. The presence of this cytokine during inflammatory states can induce hepcidin production, iron deficiency, and anemia. In this study, we show that IL-22 also influences hepcidin production in vivo. Injection of mice with exogenous mouse IgG1 Fc fused to the N terminus of mouse IL-22 (Fc-IL-22), an IL-22R agonist with prolonged and enhanced functional potency, induced hepcidin production, with a subsequent decrease in circulating serum iron and hemoglobin levels and a concomitant increase in iron accumulation within the spleen. This response was independent of IL-6 and was attenuated in the absence of the IL-22R-associated signaling kinase, Tyk2. Ab-mediated blockade of hepcidin partially reversed the effects on iron biology caused by IL-22R stimulation. Taken together, these data suggest that exogenous IL-22 regulates hepcidin production to physiologically influence iron usage.
Assuntos
Hepcidinas/fisiologia , Interleucinas/fisiologia , Ferro/metabolismo , Sequência de Aminoácidos , Anemia Ferropriva/sangue , Anemia Ferropriva/induzido quimicamente , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Células Cultivadas , Feminino , Hepatócitos/metabolismo , Hepcidinas/antagonistas & inibidores , Hepcidinas/biossíntese , Hepcidinas/genética , Hepcidinas/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Interleucina-6/fisiologia , Interleucinas/genética , Interleucinas/farmacologia , Interleucinas/toxicidade , Ferro/sangue , Deficiências de Ferro , Síndrome de Job/metabolismo , Fígado/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Dados de Sequência Molecular , Fosforilação , Processamento de Proteína Pós-Traducional , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de IgG/deficiência , Receptores de Interleucina/agonistas , Receptores de Interleucina/fisiologia , Proteínas Recombinantes de Fusão/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Baço/metabolismo , Baço/patologia , TYK2 Quinase/deficiência , TYK2 Quinase/metabolismo , Interleucina 22RESUMO
Ferroportin is the primary means of cellular iron efflux and a key component of iron metabolism. Hepcidin regulates Fpn activity by inducing its internalization and degradation. The mechanism of internalization is reported to require JAK2 activation, phosphorylation of Fpn tyrosine residues 302 and 303, and initiation of transcription through STAT3 phosphorylation. These findings suggest Fpn may be a target for therapeutic intervention through JAK2 modulation. To evaluate the proposed mechanism, Fpn internalization was assessed using several techniques combined with reagents that specifically recognized cell-surface Fpn. In vitro results demonstrated that Hepc-induced Fpn internalization did not require JAK2 or phosphorylation of Fpn residues 302 and 303, nor did it induce JAK-STAT signaling. In vivo, inhibition of JAK2 had no effect on Hepc-induced hypoferremia. However, internalization was delayed by mutation of two Fpn lysine residues that may be targets of ubiquitination.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Janus Quinase 2/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Fatores de Transcrição STAT/metabolismo , Tirosina/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Peptídeos Catiônicos Antimicrobianos , Proteínas de Transporte de Cátions/genética , Células HEK293 , Hepcidinas , Humanos , Janus Quinase 2/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Transporte Proteico , Transdução de Sinais , UbiquitinaçãoRESUMO
Iron maldistribution has been implicated in multiple diseases, including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino acid peptide. Hepcidin is induced by inflammation, causes iron to be sequestered, and thus, potentially contributes to AI. Human hepcidin (hHepc) overexpression in mice caused an iron-deficient phenotype, including stunted growth, hair loss, and iron-deficient erythropoiesis. It also caused resistance to supraphysiologic levels of erythropoiesis-stimulating agent, supporting the hypothesis that hepcidin may influence response to treatment in AI. To explore the role of hepcidin in inflammatory anemia, a mouse AI model was developed with heat-killed Brucella abortus treatment. Suppression of hepcidin mRNA was a successful anemia treatment in this model. High-affinity antibodies specific for hHepc were generated, and hHepc knock-in mice were produced to enable antibody testing. Antibody treatment neutralized hHepc in vitro and in vivo and facilitated anemia treatment in hHepc knock-in mice with AI. These data indicate that antihepcidin antibodies may be an effective treatment for patients with inflammatory anemia. The ability to manipulate iron metabolism in vivo may also allow investigation of the role of iron in a number of other pathologic conditions.