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Potassium (K+) is one of the most abundant cations in the human body. Under normal conditions, the vast majority of K+ is found within cells, and the extracellular [K+] is tightly regulated to within 3.0 to 5.0 mM. However, it has recently been shown that high levels of localized necrosis can increase the extracellular concentration of K+ to above 50 mM. This raises the possibility that elevated extracellular K+ might influence a variety of biological processes that occur within regions of necrotic tissue. For example, K+ has been shown to play a central role in the replication cycles of numerous viral families, and in cases of lytic infection, localized regions containing large numbers of necrotic cells can be formed. Here, we show that the replication of the model poxvirus myxoma virus (MYXV) is delayed by elevated levels of extracellular K+. These increased K+ concentrations alter the cellular endocytic pathway, leading to increased phagocytosis but a loss of endosomal/lysosomal segregation. This slows the release of myxoma virus particles from the endosomes, resulting in delays in genome synthesis and infectious particle formation as well as reduced viral spread. Additionally, mathematical modeling predicts that the extracellular K+ concentrations required to impact myxoma virus replication can be reached in viral lesions under a variety of conditions. Taken together, these data suggest that the extracellular [K+] plays a role in determining the outcomes of myxoma infection and that this effect could be physiologically relevant during pathogenic infection. IMPORTANCE Intracellular K+ homeostasis has been shown to play a major role in the replication of numerous viral families. However, the potential impact of altered extracellular K+ concentrations is less well understood. Our work demonstrates that increased concentrations of extracellular K+ can delay the replication cycle of the model poxvirus MYXV by inhibiting virion release from the endosomes. Additionally, mathematical modeling predicts that the levels of extracellular K+ required to impact MYXV replication can likely be reached during pathogenic infection. These results suggest that localized viral infection can alter K+ homeostasis and that these alterations might directly affect viral pathogenesis.
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Myxoma virus , Humanos , Myxoma virus/genética , Potássio , Endossomos , Replicação Viral , VírionRESUMO
Lethal viral infections produce widespread inflammation with vascular leak, clotting, and bleeding (disseminated intravascular coagulation [DIC]), organ failure, and high mortality. Serine proteases in clot-forming (thrombotic) and clot-dissolving (thrombolytic) cascades are activated by an inflammatory cytokine storm and also can induce systemic inflammation with loss of normal serine protease inhibitor (serpin) regulation. Myxomavirus secretes a potent anti-inflammatory serpin, Serp-1, that inhibits clotting factor X (fX) and thrombolytic tissue- and urokinase-type plasminogen activators (tPA and uPA) with anti-inflammatory activity in multiple animal models. Purified serpin significantly improved survival in a murine gammaherpesvirus 68 (MHV68) infection in gamma interferon receptor (IFN-γR) knockout mice, a model for lethal inflammatory vasculitis. Treatment of MHV68-infected mice with neuroserpin, a mammalian serpin that inhibits only tPA and uPA, was ineffective. Serp-1 reduced virus load, lung hemorrhage, and aortic, lung, and colon inflammation in MHV68-infected mice and also reduced virus load. Neuroserpin suppressed a wide range of immune spleen cell responses after MHV68 infection, while Serp-1 selectively increased CD11c(+) splenocytes (macrophage and dendritic cells) and reduced CD11b(+) tissue macrophages. Serp-1 altered gene expression for coagulation and inflammatory responses, whereas neuroserpin did not. Serp-1 treatment was assessed in a second viral infection, mouse-adapted Zaire ebolavirus in wild-type BALB/c mice, with improved survival and reduced tissue necrosis. In summary, treatment with this unique myxomavirus-derived serpin suppresses systemic serine protease and innate immune responses caused by unrelated lethal viral infections (both RNA and DNA viruses), providing a potential new therapeutic approach for treatment of lethal viral sepsis.
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Hemorragia/tratamento farmacológico , Doença pelo Vírus Ebola/tratamento farmacológico , Infecções por Herpesviridae/tratamento farmacológico , Infecções por Herpesviridae/mortalidade , Proteínas de Membrana/farmacologia , Myxoma virus/química , Animais , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Modelos Animais de Doenças , Ebolavirus , Fator X/antagonistas & inibidores , Fator X/metabolismo , Gammaherpesvirinae , Hemorragia/mortalidade , Hemorragia/patologia , Hemorragia/virologia , Doença pelo Vírus Ebola/mortalidade , Doença pelo Vírus Ebola/patologia , Doença pelo Vírus Ebola/virologia , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Inflamação/tratamento farmacológico , Inflamação/mortalidade , Inflamação/patologia , Inflamação/virologia , Interferon gama/deficiência , Interferon gama/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Proteínas de Membrana/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Myxoma virus/fisiologia , Neuropeptídeos/farmacologia , Serpinas/farmacologia , Análise de Sobrevida , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Vasculite/tratamento farmacológico , Vasculite/mortalidade , Vasculite/patologia , Vasculite/virologia , NeuroserpinaRESUMO
Oncolytic viruses are being heavily investigated as novel methods to treat cancers; however, predicting their therapeutic efficacy remains challenging. The most commonly used predictive tests involve determining the in vitro susceptibility of a tumor's malignant cells to infection with an oncolytic agent. Whether these tests are truly predictive of in vivo efficacy, however, remains unclear. Here we demonstrate that a recombinant, oncolytic myxoma virus shows efficacy in two murine models of triple negative breast cancer despite extremely low permissivity of these models to viral infection. These data demonstrate that in vitro infectivity studies are not an accurate surrogate for therapeutic efficacy and suggest that other tests need to be developed.
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BACKGROUND: Arginine (Arg) is a semiessential amino acid whose bioavailability is required for the in vitro replication of several oncolytic viruses. In vivo, Arg bioavailability is regulated by a combination of dietary intake, protein catabolism, and limited biosynthesis through portions of the urea cycle. Interestingly, despite the importance of bioavailable Arg to support cellular proliferation, many forms of cancer are functionally auxotrophic for this amino acid due to the epigenetic silencing of argininosuccinate synthetase 1 (ASS1), an enzyme responsible for the conversion of citrulline and aspartate into the Arg precursor argininosuccinate. The impact of this silencing on oncolytic virotherapy (OV), however, has never been examined. METHODS: To address this gap in knowledge, we generated tumor cells lacking ASS1 and examined how loss of this enzyme impacted the in vivo replication and therapeutic efficacy of oncolytic myxoma virus (MYXV). We also generated a series of recombinant MYXV constructs expressing exogenous ASS1 to evaluate the therapeutic benefit of virally reconstituting Arg biosynthesis in ASS1-/- tumors. RESULTS: Our results show that the in vitro replication of oncolytic MYXV is dependent on the presence of bioavailable Arg. This dependence can be overcome by the addition of the metabolic precursor citrulline, however, this rescue requires expression of ASS1. Because of this, tumors formed from functionally ASS1-/- cells display significantly reduced MYXV replication as well as poorer therapeutic responses. Critically, both defects could be partially rescued by expressing exogenous ASS1 from recombinant oncolytic MYXVs. CONCLUSIONS: These results demonstrate that intratumoral defects to Arg metabolism can serve as a novel barrier to virally induced immunotherapy and that the exogenous expression of ASS1 can improve the efficacy of OV in Arg-auxotrophic tumors.
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Myxoma virus , Neoplasias , Vírus Oncolíticos , Humanos , Vírus Oncolíticos/metabolismo , Myxoma virus/genética , Citrulina , Neoplasias/patologia , Arginina/metabolismoRESUMO
T-cell immunoglobulin and mucin domain 3 (TIM3) is emerging as a potential target for antibody-based checkpoint blockade. However, the efficacy of TIM3 blockade in combination with other treatment modalities, has not been extensively studied. In the current work we combined TIM3 blockade with myxoma virus-based oncolytic virotherapy (OV). Our results demonstrate that myxoma virus's ability to initiate an immense antitumor immune response complements the ability of TIM3 blockade to shift the tumor microenvironment to a more proinflammatory state. As a result, the combination of TIM3 blockade and OV is able to completely eradicate established disease, while neither monotherapy is effective. These data represent the first demonstration that OV can enhance the efficacy of TIM3 blockade and suggest that this treatment may need to be incorporated into more aggressive, combinatorial regimens in order to fulfill its potential as an immunotherapeutic.
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Neoplasias , Humanos , Neoplasias/terapia , Microambiente TumoralRESUMO
BACKGROUND: Oncolytic virotherapy (OV) represents a method to treat a variety of solid tumors by inducing antitumor immune responses. While this therapy has been extremely efficacious in preclinical models, translating these successes into human patients has proven challenging. One of the major reasons for these failures is the existence of immune-regulatory mechanisms, which dampen the efficacy of virally induced antitumor immunity. Unfortunately, the full extent of these immune-regulatory pathways remains unclear. METHODS: To address this issue, we generated a doubly recombinant, oncolytic myxoma virus which expresses both a soluble fragment of programmed cell death protein 1 (PD1) and an interleukin 12 (IL-12) fusion protein (vPD1/IL-12 (virus-expressing PD1 and IL-12)). We then tested the molecular impact and therapeutic efficacy of this construct in multiple models of disseminated disease to identify novel pathways, which are associated with poor therapeutic outcomes. RESULTS: Our results demonstrate that vPD1/IL-12 causes robust inflammation during therapy including inducing high levels of tumor necrosis factor (TNF). Surprisingly, although expression of TNF has generally been assumed to be beneficial to OV, the presence of this TNF appears to inhibit therapeutic efficacy by reducing intratumoral T-cell viability. Likely because of this, disruption of the TNF pathway, either through genetic knockout or antibody-based blockade, significantly enhances the overall outcomes of vPD1/IL-12-based therapy that allows for the generation of complete cures in normally non-responsive models. CONCLUSIONS: These data suggest that some aspects of OV-induced inflammation might represent a double-edged sword during therapy and that specific blockade of TNF might enhance the efficacy of these treatments.
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Myxoma virus , Terapia Viral Oncolítica , Vírus Oncolíticos , Fator de Necrose Tumoral alfa , Humanos , Inflamação , Interleucina-12/genética , Interleucina-12/metabolismo , Myxoma virus/genética , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Human Cu-ATPases ATP7A and ATP7B maintain copper homeostasis through regulated trafficking between intracellular compartments. Inactivation of these transporters causes Menkes disease and Wilson disease, respectively. In Menkes disease, copper accumulates in kidneys and causes tubular damage, indicating that the renal ATP7B does not compensate for the loss of ATP7A function. We show that this is likely due to a kidney-specific regulation of ATP7B. Unlike ATP7A (or hepatic ATP7B) which traffics from the TGN to export copper, renal ATP7B does not traffic and therefore is unlikely to mediate copper export. The lack of ATP7B trafficking is not on account of the loss of a kinase-mediated phosphorylation or simultaneous presence of ATP7A in renal cells. Rather, the renal ATP7B appears 2-3 kDa smaller than hepatic ATP7B. Recombinant ATP7B expressed in renal cells is similar to hepatic protein in size and trafficking. The analysis of ATP7B mRNA revealed a complex behavior of exon 1 upon amplification, suggesting that it could be inefficiently translated. Recombinant ATP7B lacking exon 1 traffics differently in renal and hepatic cells, but does not fully recapitulate the endogenous phenotype. We discuss factors that may contribute to cell-specific behavior of ATP7B and propose a role for renal ATP7B in intracellular copper storage.
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Adenosina Trifosfatases/fisiologia , Proteínas de Transporte de Cátions/fisiologia , Cobre/metabolismo , Rim/fisiologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular , ATPases Transportadoras de Cobre , Éxons , Humanos , Rim/metabolismo , Dados de Sequência Molecular , Fosforilação , Transporte Proteico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
Poxviruses are large enveloped viruses that replicate exclusively in the cytoplasm. Like all viruses, their replication cycle begins with virion adsorption to the cell surface. Unlike most other viral families, however, no unique poxviral receptor has ever been identified. In the absence of a unique receptor, poxviruses are instead thought to adhere to the cell surface primarily through electrostatic interactions between the positively charged viral envelope proteins and the negatively charged sulfate groups on cellular glycosaminoglycans (GAGs). While these negatively charged GAGs are an integral part of all eukaryotic membranes, their specific expression and sulfation patterns differ between cell types. Critically, while poxviral binding has been extensively studied using virally centered genetic strategies, the impact of cell-intrinsic changes to GAG charge has never been examined. Here we show that loss of heparin sulfation, accomplished by deleting the enzyme N-Deacetylase and N-Sulfotransferase-1 (NDST1) which is essential for GAG sulfation, significantly reduces the binding affinity of both vaccinia and myxoma viruses to the cell surface. Strikingly, however, while this lowered binding affinity inhibits the subsequent spread of myxoma virus, it actually enhances the overall spread of vaccinia by generating more diffuse regions of infection. These data indicate that cell-intrinsic GAG sulfation plays a major role in poxviral infection, however, this role varies significantly between different members of the poxviridae.
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Poxviridae/fisiologia , Replicação Viral , Animais , Linhagem Celular , Heparina/metabolismo , Espaço Intracelular/metabolismo , Camundongos , Poxviridae/metabolismo , Sulfotransferases/deficiênciaRESUMO
Cu-ATPase ATP7B (Wilson's disease protein) transports copper into the trans-Golgi network for biosynthetic incorporation into ceruloplasmin and sequesters excess copper to endocytic vesicles for further export out of the cell. The activity and intracellular location of ATP7B are regulated by copper levels; the trafficking of ATP7B between cellular compartments is coupled to changes in the level of protein phosphorylation. Neither the nature of the kinase(s) phosphorylating ATP7B nor the location of phosphorylation sites is known. We demonstrate that the membrane-bound ATP7B is phosphorylated by an ATP-dependent, GTP-independent kinase that can be either soluble or membrane-associated. Mg(2+) or Mn(2+) is necessary for kinase activity. We further show that the recombinant N-terminal domain of ATP7B (N-ATP7B) is a specific target for a kinase-mediated phosphorylation in vitro and in cells. Although exogenous addition of copper is not required for kinase activity, copper binding to N-ATP7B markedly alters the exposure of loops connecting the metal-binding subdomains (MBDs) to proteolysis and facilitates phosphorylation by 25-30%. MBD1-2 and MBD4-5 linkers become protected, while MBD2-3 and MBD3-4 regions remain exposed. A significant, 5-fold increase in the level of phosphorylation is also observed for the ATP7B variant that lacks the 29 kDa N-terminal fragment (mostly likely comprised of MBD1-3). Analysis of phosphorylated peptides by two-dimensional gel electrophoresis and mass spectrometry points to the loop connecting MBD3 and MBD4 as a region of phosphorylation. Altogether, the results suggest a mechanism in which kinase-mediated phosphorylation of ATP7B is controlled by a conformational state of N-ATP7B.
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Adenosina Trifosfatases/química , Proteínas de Transporte de Cátions/química , Proteínas Quinases/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Sítios de Ligação , Catálise , Proteínas de Transporte de Cátions/metabolismo , Células Cultivadas , Cobre/química , Cobre/metabolismo , ATPases Transportadoras de Cobre , Guanosina Trifosfato/metabolismo , Humanos , Magnésio/química , Magnésio/metabolismo , Manganês/química , Manganês/metabolismo , Modelos Biológicos , Fosforilação , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rede trans-Golgi/metabolismoRESUMO
Serine proteinase inhibitors, also called serpins, are an ancient grouping of proteins found in primitive organisms from bacteria, protozoa and horseshoe crabs and thus likely present at the time of the dinosaurs, up to all mammals living today. The innate or inflammatory immune system is also an ancient metazoan regulatory system, providing the first line of defense against infection or injury. The innate inflammatory defense response evolved long before acquired, antibody dependent immunity. Viruses have developed highly effective stratagems that undermine and block a wide variety of host inflammatory and immune responses. Some of the most potent of these immune modifying strategies utilize serpins that have also been developed over millions of years, including the hijacking by some viruses for defense against host immune attacks. Serpins represent up to 2-10 percent of circulating plasma proteins, regulating actions as wide ranging as thrombosis, inflammation, blood pressure control and even hormone transport. Targeting serpin-regulated immune or inflammatory pathways makes evolutionary sense for viral defense and many of these virus-derived inhibitory proteins have proven to be highly effective, working at very low concentrations--even down to the femptomolar to picomolar range. We are studying these viral anti-inflammatory proteins as a new class of immunomodulatory therapeutic agents derived from their native viral source. One such viral serpin, Serp-1 is now in clinical trial (conducted by VIRON Therapeutics, Inc.) for acute unstable coronary syndromes (unstable angina and small heart attacks), representing a 'first in class' therapeutic study. Several other viral serpins are also currently under investigation as anti-inflammatory or anti-immune therapeutics. This chapter describes these original studies and the ongoing analysis of viral serpins as a new class of virus-derived immunotherapeutic.
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Anti-Inflamatórios/uso terapêutico , Doenças do Sistema Imunitário/terapia , Inibidores de Serina Proteinase/uso terapêutico , Serpinas/uso terapêutico , Proteínas Virais/uso terapêutico , Viroses/terapia , Animais , HumanosRESUMO
INTRODUCTION: Cancer has become one of the most critical health issues of modern times. To overcome the ineffectiveness of current treatment options, research is being done to explore new therapeutic modalities. One such novel treatment is oncolytic virotherapy (OV) which uses tumor tropic viruses to specifically target and kill malignant cells. While OV has shown significant promise in recent clinical trials, the therapeutic use of viruses poses a number of unique challenges. In particular, obtaining effective viral spread throughout the tumor microenvironment remains problematic. Previous work has suggested this can be overcome by forcing oncolytic viruses to induce syncytia formation. METHODS: In the current work, we generated a series of recombinant myxoma viruses expressing exogenous fusion proteins from other viral genomes and examined their therapeutic potential in vitro and in vivo. RESULTS: Similar to previous studies, we observed that the expression of these fusion proteins during myxoma infection induced the formation of multinucleated syncytia which increased viral spread and lytic potential compared to non-fusogenic controls. Contrary to expectations, however, the treatment of established tumors with these viruses resulted in decreased therapeutic efficacy which corresponded with reduced viral persistence. DISCUSSION: These findings indicate that enhanced viral spread caused by syncytia formation can actually reduce the efficacy of OV and supports a number of previous works suggesting that the in vitro properties of viruses frequently fail to predict their in vivo efficacy.
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Expression of PDL1 on the surface of tumor cells can blunt the efficacy of many cancer immunotherapies. For example, our lab has previously shown that tumors derived from malignant cells incapable of expressing PDL1 are highly susceptible to immunotherapy induced by oncolytic virus treatment while tumors derived from PDL1 capable cells are highly resistant. In patient biopsies, however, expression of PDL1 on malignant cells is often not uniform with some cells expressing PDL1 while others do not. Importantly, how this partial PDL1 positivity influences the outcomes of immunotherapy remains largely unknown. In the current work, we expand on our previous findings by generating partially PDL1 positive tumors in immune competent animals and asking what percentage of tumor cells must express PDL1 for a tumor to become functionally resistant to oncolytic treatment. Our results indicate that the responsiveness of partially PDL1+ tumors correlates linearly with the percentage of PDL1 capable cells present at the initiation of treatment. Additionally, we observe that tumors which relapse after treatment display a significant increase in the numbers of PDL1 capable cells present suggesting that specific editing of mixed tumors might play a role in disease relapse. These data indicate that varying levels of PDL1 expression can play a significant role in the outcomes of oncolytic immunotherapy and challenges the concept that tumors should be viewed as simply PDL1+ or PDL1-.
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Antígeno B7-H1/genética , Biomarcadores Tumorais , Expressão Gênica , Imunomodulação , Imunoterapia , Terapia Viral Oncolítica , Vírus Oncolíticos , Animais , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Imunomodulação/genética , Imunofenotipagem , Melanoma Experimental , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Oncolytic virotherapy represents an attractive option for the treatment of a variety of aggressive or refractory tumors. While this therapy is effective at rapidly debulking directly injected tumor masses, achieving complete eradication of established disease has proven difficult. One method to overcome this challenge is to use oncolytic viruses to induce secondary antitumor immune responses. Unfortunately, while the initial induction of these immune responses is typically robust, their subsequent efficacy is often inhibited through a variety of immunoregulatory mechanisms, including the PD1/PDL1 T-cell checkpoint pathway. To overcome this inhibition, we generated a novel recombinant myxoma virus (vPD1), which inhibits the PD1/PDL1 pathway specifically within the tumor microenvironment by secreting a soluble form of PD1 from infected cells. This virus both induced and maintained antitumor CD8+ T-cell responses within directly treated tumors and proved safer and more effective than combination therapy using unmodified myxoma and systemic αPD1 antibodies. Localized vPD1 treatment combined with systemic elimination of regulatory T cells had potent synergistic effects against metastatic disease that was already established in secondary solid organs. These results demonstrate that tumor-localized inhibition of the PD1/PDL1 pathway can significantly improve outcomes during oncolytic virotherapy. Furthermore, they establish a feasible path to translate these findings against clinically relevant disease. Cancer Res; 77(11); 2952-63. ©2017 AACR.
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Melanoma Experimental/imunologia , Terapia Viral Oncolítica/métodos , Receptor de Morte Celular Programada 1/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microambiente TumoralRESUMO
INTRODUCTION: Multiple myeloma is a clonal malignancy of plasma B cells. Although recent advances have improved overall prognosis, virtually all myeloma patients still succumb to relapsing disease. Therefore, novel therapies to treat this disease remain urgently needed. We have recently shown that treatment of human multiple myeloma cells with an oncolytic virus known as myxoma results in rapid cell death even in the absence of viral replication; however, the specific mechanisms and pathways involved remain unknown. MATERIALS AND METHODS: To determine how myxoma virus eliminates human multiple myeloma cells, we queried the apoptotic pathways that were activated after viral infection using immunoblot analysis and other cell biology approaches. RESULTS: Our results indicate that myxoma virus infection initiates apoptosis in multiple myeloma cells through activation of the extrinsic initiator caspase-8. Caspase-8 activation subsequently results in cleavage of BH3 interacting-domain death agonist and loss of mitochondrial membrane potential causing secondary activation of caspase-9. Activation of caspase-8 appears to be independent of extrinsic death ligands and instead correlates with depletion of cellular inhibitors of apoptosis. We hypothesize that this depletion results from virally mediated host-protein shutoff because a myxoma construct that overexpresses the viral decapping enzymes displays improved oncolytic potential. CONCLUSION: Taken together, these results suggest that myxoma virus eliminates human multiple myeloma cells through a pathway unique to oncolytic poxviruses, making it an excellent therapeutic option for the treatment of relapsed or refractory patients.
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Mieloma Múltiplo/genética , Myxoma virus/genética , Apoptose , Morte Celular , Humanos , Mieloma Múltiplo/patologia , Transdução de SinaisRESUMO
Multiple myeloma is an incurable malignancy of plasma B-cells. Traditional chemotherapeutic regimes often induce initial tumor regression; however, virtually all patients eventually succumb to relapse caused by either reintroduction of disease during autologous transplant or expansion of chemotherapy resistant minimal residual disease. It has been previously demonstrated that an oncolytic virus known as myxoma can completely prevent myeloma relapse caused by reintroduction of malignant cells during autologous transplant. The ability of this virus to treat established residual disease in vivo, however, remained unknown. Here we demonstrate that intravenous administration of myxoma virus into mice bearing disseminated myeloma results in the elimination of 70-90% of malignant cells within 24 hours. This rapid debulking was dependent on direct contact of myxoma virus with residual myeloma and did not occur through destruction of the hematopoietic bone marrow niche. Importantly, systemic myxoma therapy also induced potent antimyeloma CD8+ T cell responses which localized to the bone marrow and were capable of completely eradicating established myeloma in some animals. These results demonstrate that oncolytic myxoma virus is not only effective at preventing relapse caused by reinfusion of tumor cells during stem cell transplant, but is also potentially curative for patients bearing established minimal residual disease.
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The recent development of chemotherapeutic proteasome inhibitors, such as bortezomib, has improved the outcomes of patients suffering from the plasma cell malignancy multiple myeloma. Unfortunately, many patients treated with these drugs still suffer relapsing disease due to treatment-induced upregulation of the antiapoptotic protein Mcl1. We have recently demonstrated that an oncolytic poxvirus, known as myxoma, can rapidly eliminate primary myeloma cells by inducing cellular apoptosis. The efficacy of myxoma treatment on proteasome inhibitor-relapsed or -refractory myeloma, however, remains unknown. We now demonstrate that myxoma-based elimination of myeloma is not affected by cellular resistance to proteasome inhibitors. Additionally, myxoma virus infection specifically prevents expression of Mcl1 following induction of the unfolded protein response, by blocking translation of the unfolded protein response activating transcription factor (ATF)4. These results suggest that myxoma-based oncolytic therapy represents an attractive option for myeloma patients whose disease is refractory to chemotherapeutic proteasome inhibitors due to upregulation of Mcl1.
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Modification of the tumor microenvironment by inflammatory cells represents a newly recognized driving force in cancer with critical roles in tumor invasion, growth, angiogenesis, and metastasis. Increased thrombolytic cascade serine proteases, specifically urokinase-type plasminogen activator and its receptor, correlate with inflammatory cell migration, pancreatic cancer growth, invasion and unfavorable outcomes. Inflammation in pancreatic cancer is linked with myeloid-derived suppressor cell (MDSC) activity and cancer progression. Myxomavirus is a complex DNA virus encoding highly potent immune modulators. Serp-1 and M-T7 are two such secreted anti-inflammatory myxomaviral proteins. Serp-1 inhibits uPA, plasmin and coagulation factor X while M-T7 inhibits C, CC, and CXC chemokines. We have explored the potential use of these viral proteins for treatment of a range of human cancer isolates engrafted in severe combined immunodeficient (SCID) mice. Engrafted tumors were treated with either Serp-1, neuroserpin, a related mammalian serpin that inhibits thrombolytic proteases, or M-T7. Serp-1 and neuroserpin inhibited growth of the pancreatic cancer cell line Hs766t (P=0.03 and P=0.01, respectively) at 4 weeks after implantation. Serp-1 also inhibited growth of a second pancreatic cancer cell line MIA PaCa-2 in mice (P=0.02). Growth of the human breast cancer line MDA231 was not inhibited by Serp-1. M-T7, in contrast, did not alter growth of any of the cancer cell lines tested after implant into SCID mice. Serpin inhibition of pancreatic tumor growth was associated with a significant decrease in splenocyte MDSC counts by flow cytometry (P=0.009), without detected change in other splenocyte subpopulations. Serp-1 and NSP treatment also significantly reduced macrophage infiltration in tumors (P=0.001). In summary two anti-inflammatory serpins reduced inflammatory macrophage invasion and pancreatic tumor cell growth, suggesting potential therapeutic efficacy.
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Inflammatory responses now have a defined central role in cancer cell growth, invasion, and metastases. Anti-inflammatory proteins from viruses target key stages in immune response pathways and have potential as novel therapeutics for cancer, including highly potent virus-derived inhibitors of protease, chemokine, cytokine, and apoptotic cascades that have been identified. Serine proteases, in addition to their conventional roles in thrombosis, thrombolysis, and apoptotic pathways, are essential regulators of inflammation and are associated with developing cancers. Chemokines drive other inflammatory response pathways with central roles in cell invasion and activation as well as establishing the microenvironment of tumors, modulating immune cell infiltration, cancer cell proliferation, metastasis, and angiogenesis. This review focuses on the mechanisms of action and potential for application of viral immunomodulatory proteins as anticancer therapeutics.
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Anti-Inflamatórios/uso terapêutico , Neoplasias/tratamento farmacológico , Proteínas Virais/uso terapêutico , Animais , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/metabolismo , Quimiocinas/metabolismo , Humanos , Imunomodulação , Camundongos , Neoplasias/imunologia , Neoplasias/metabolismo , Ligação Proteica , Receptores de Quimiocinas/imunologia , Receptores de Quimiocinas/metabolismo , Receptores de Quimiocinas/uso terapêutico , Serpinas/imunologia , Serpinas/metabolismo , Serpinas/uso terapêutico , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Vírus/imunologia , Vírus/metabolismoRESUMO
Poxviruses express highly active inhibitors, including serine proteinase inhibitors (serpins), designed to target host immune defense pathways. Recent work has demonstrated clinical efficacy for a secreted, myxomaviral serpin, Serp-1, which targets the thrombotic and thrombolytic proteases, suggesting that other viral serpins may have therapeutic application. Serp-2 and CrmA are intracellular cross-class poxviral serpins, with entirely distinct functions from the Serp-1 protein. Serp-2 and CrmA block the serine protease granzyme B (GzmB) and cysteine proteases, caspases 1 and 8, in apoptotic pathways, but have not been examined for extracellular anti-inflammatory activity. We examined the ability of these cross-class serpins to inhibit plaque growth after arterial damage or transplant and to reduce leukocyte apoptosis. We observed that purified Serp-2, but not CrmA, given as a systemic infusion after angioplasty, transplant, or cuff-compression injury markedly reduced plaque growth in mouse and rat models in vivo. Plaque growth was inhibited both locally at sites of surgical trauma, angioplasty or transplant, and systemically at non-injured sites in ApoE-deficient hyperlipidemic mice. With analysis in vitro of human cells in culture, Serp-2 selectively inhibited T cell caspase activity and blocked cytotoxic T cell (CTL) mediated killing of T lymphocytes (termed fratricide). Conversely, both Serp-2 and CrmA inhibited monocyte apoptosis. Serp-2 inhibitory activity was significantly compromised either in vitro with GzmB antibody or in vivo in ApoE/GzmB double knockout mice. Conclusions The viral cross-class serpin, Serp-2, that targets both apoptotic and inflammatory pathways, reduces vascular inflammation in a GzmB-dependent fashion in vivo, and inhibits human T cell apoptosis in vitro. These findings indicate that therapies targeting Granzyme B and/or T cell apoptosis may be used to inhibit T lymphocyte apoptosis and inflammation in response to arterial injury.
Assuntos
Aorta/efeitos dos fármacos , Estenose das Carótidas/tratamento farmacológico , Citotoxicidade Imunológica/efeitos dos fármacos , Inflamação/tratamento farmacológico , Serpinas/farmacologia , Linfócitos T/efeitos dos fármacos , Proteínas Virais/farmacologia , Angioplastia/efeitos adversos , Animais , Aorta/imunologia , Aorta/transplante , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Estenose das Carótidas/etiologia , Estenose das Carótidas/imunologia , Estenose das Carótidas/patologia , Caspase 1/metabolismo , Caspase 8/metabolismo , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Granzimas/antagonistas & inibidores , Granzimas/metabolismo , Humanos , Inflamação/etiologia , Inflamação/imunologia , Inflamação/patologia , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Serpinas/genética , Serpinas/isolamento & purificação , Linfócitos T/imunologia , Proteínas Virais/genética , Proteínas Virais/isolamento & purificaçãoRESUMO
Chemokines are important for activation of a host of cellular immune and inflammatory responses including cell signaling, activation, and communication. M-T7, a myxoma virus protein, inhibits the activity of chemokines by direct binding to chemokines and/or with glycosaminoglycans (GAGs). To study the effects of this chemokine-modulating protein (CMP), we use a variety of in vitro and in vivo techniques to evaluate M-T7 inhibition of inflammatory cells. To quickly analyze the effects of M-T7, changes in cell adhesion and membrane fluidity are measured as well as cell migration in mouse ascites. For more physiological analyses, an aortic transplant model in rodents is used to assess change in inflammatory cell infiltrates and vascular plaque growth (rejection). Utilization of the combination of these in vitro and in vivo techniques allows for a more complete study of the chemokine-modulating activity of M-T7, and can be used to study other immune and inflammation-modulating proteins.