RESUMEN
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma occurring in children and carries a dismal prognosis when metastatic disease is detected. Our previous work has suggested the cytokine receptor IL4Rα may play a role in contributing to metastasis in the alveolar subtype of rhabdomyosarcoma (aRMS), and thus could present a therapeutic target. The IL4 signaling axis has been characterized in various adult cancers as well; however, pediatric trials often follow similar adult trials and the role of the IL4Rα receptor has not been explored in the context of a mediator of metastasis in adult disease. Here, we demonstrate that the impact of IL4Rα blockade in an orthotopic allograft model of aRMS is not mediated by a macrophage response. We further examine the effect of IL4 blockade in adult colon, breast, and prostate cancers and find that inhibition of IL4Rα signaling modulates in vitro cell viability of HCT-116 colon carcinoma cells; however, this finding did not translate to an autocrine-related in vivo difference in tumor burden or lung metastasis. Our results suggest that if humanized IL4 mouse host strains are not available (or not ideal due to the need for immunosuppressing the host innate immune response for xenograft systems), then genetically-engineered mice and mouse allograft studies may be the best indicator of therapeutic targeting efficacy.
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Macrófagos/metabolismo , Receptores de Interleucina-4/antagonistas & inhibidores , Rabdomiosarcoma/genética , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Metástasis de la Neoplasia , Rabdomiosarcoma/patologíaRESUMEN
The innate immune response to cytosolic DNA involves transcriptional activation of type I interferons (IFN-I) and proinflammatory cytokines. This represents the culmination of intracellular signaling pathways that are initiated by pattern recognition receptors that engage DNA and require the adaptor protein Stimulator of Interferon Genes (STING). These responses lead to the generation of cellular and tissue states that impair microbial replication and facilitate the establishment of long-lived, antigen-specific adaptive immunity. Ultimately this can lead to immune-mediated protection from infection but also to the cytotoxic T cell-mediated clearance of tumor cells. Intriguingly, pharmacologic activation of STING-dependent phenotypes is known to enhance both vaccine-associated immunogenicity and immune-based anti-tumor therapies. Unfortunately, the STING protein exists as multiple variant forms in the human population that exhibit differences in their reactivity to chemical stimuli and in the intensity of molecular signaling they induce. In light of this, STING-targeting drug discovery efforts require an accounting of protein variant-specific activity. Herein we describe a small molecule termed M04 that behaves as a novel agonist of human STING. Importantly, we find that the molecule exhibits a differential ability to activate STING based on the allelic variant examined. Furthermore, while M04 is inactive in mice, expression of human STING in mouse cells rescues reactivity to the compound. Using primary human cells in ex vivo assays we were also able to show that M04 is capable of simulating innate responses important for adaptive immune activation such as cytokine secretion, dendritic cell maturation, and T cell cross-priming. Collectively, this work demonstrates the conceivable utility of a novel agonist of human STING both as a research tool for exploring STING biology and as an immune potentiating molecule.
Asunto(s)
Inmunidad Innata/efectos de los fármacos , Factores Inmunológicos/farmacología , Proteínas de la Membrana/agonistas , Alelos , Animales , Descubrimiento de Drogas , Humanos , Inmunidad Innata/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , RatonesRESUMEN
BACKGROUND: Cancer patients with advanced disease routinely exhaust available clinical regimens and lack actionable genomic medicine results, leaving a large patient population without effective treatments options when their disease inevitably progresses. To address the unmet clinical need for evidence-based therapy assignment when standard clinical approaches have failed, we have developed a probabilistic computational modeling approach which integrates molecular sequencing data with functional assay data to develop patient-specific combination cancer treatments. METHODS: Tissue taken from a murine model of alveolar rhabdomyosarcoma was used to perform single agent drug screening and DNA/RNA sequencing experiments; results integrated via our computational modeling approach identified a synergistic personalized two-drug combination. Cells derived from the primary murine tumor were allografted into mouse models and used to validate the personalized two-drug combination. Computational modeling of single agent drug screening and RNA sequencing of multiple heterogenous sites from a single patient's epithelioid sarcoma identified a personalized two-drug combination effective across all tumor regions. The heterogeneity-consensus combination was validated in a xenograft model derived from the patient's primary tumor. Cell cultures derived from human and canine undifferentiated pleomorphic sarcoma were assayed by drug screen; computational modeling identified a resistance-abrogating two-drug combination common to both cell cultures. This combination was validated in vitro via a cell regrowth assay. RESULTS: Our computational modeling approach addresses three major challenges in personalized cancer therapy: synergistic drug combination predictions (validated in vitro and in vivo in a genetically engineered murine cancer model), identification of unifying therapeutic targets to overcome intra-tumor heterogeneity (validated in vivo in a human cancer xenograft), and mitigation of cancer cell resistance and rewiring mechanisms (validated in vitro in a human and canine cancer model). CONCLUSIONS: These proof-of-concept studies support the use of an integrative functional approach to personalized combination therapy prediction for the population of high-risk cancer patients lacking viable clinical options and without actionable DNA sequencing-based therapy.
Asunto(s)
Biología Computacional/métodos , Evaluación Preclínica de Medicamentos/métodos , Quimioterapia Combinada/métodos , Modelos Estadísticos , Medicina de Precisión/métodos , Rabdomiosarcoma Alveolar/tratamiento farmacológico , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Perros , Sinergismo Farmacológico , Femenino , Xenoinjertos , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos NODRESUMEN
BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in the pediatric cancer population. Survival among metastatic RMS patients has remained dismal yet unimproved for years. We previously identified the class I-specific histone deacetylase inhibitor, entinostat (ENT), as a pharmacological agent that transcriptionally suppresses the PAX3:FOXO1 tumor-initiating fusion gene found in alveolar rhabdomyosarcoma (aRMS), and we further investigated the mechanism by which ENT suppresses PAX3:FOXO1 oncogene and demonstrated the preclinical efficacy of ENT in RMS orthotopic allograft and patient-derived xenograft (PDX) models. In this study, we investigated whether ENT also has antitumor activity in fusion-negative eRMS orthotopic allografts and PDX models either as a single agent or in combination with vincristine (VCR). METHODS: We tested the efficacy of ENT and VCR as single agents and in combination in orthotopic allograft and PDX mouse models of eRMS. We then performed CRISPR screening to identify which HDAC among the class I HDACs is responsible for tumor growth inhibition in eRMS. To analyze whether ENT treatment as a single agent or in combination with VCR induces myogenic differentiation, we performed hematoxylin and eosin (H&E) staining in tumors. RESULTS: ENT in combination with the chemotherapy VCR has synergistic antitumor activity in a subset of fusion-negative eRMS in orthotopic "allografts," although PDX mouse models were too hypersensitive to the VCR dose used to detect synergy. Mechanistic studies involving CRISPR suggest that HDAC3 inhibition is the primary mechanism of cell-autonomous cytoreduction in eRMS. Following cytoreduction in vivo, residual tumor cells in the allograft models treated with chemotherapy undergo a dramatic, entinostat-induced (70-100%) conversion to non-proliferative rhabdomyoblasts. CONCLUSION: Our results suggest that the targeting class I HDACs may provide a therapeutic benefit for selected patients with eRMS. ENT's preclinical in vivo efficacy makes ENT a rational drug candidate in a phase II clinical trial for eRMS.
Asunto(s)
Benzamidas/uso terapéutico , Inhibidores de Histona Desacetilasas/uso terapéutico , Piridinas/uso terapéutico , Rabdomiosarcoma Embrionario/tratamiento farmacológico , Adolescente , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Benzamidas/administración & dosificación , Sistemas CRISPR-Cas , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Reprogramación Celular/efectos de los fármacos , Reprogramación Celular/genética , Niño , Preescolar , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 1/genética , Inhibidores de Histona Desacetilasas/administración & dosificación , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Piridinas/administración & dosificación , RNA-Seq , Rabdomiosarcoma Alveolar/tratamiento farmacológico , Rabdomiosarcoma Alveolar/enzimología , Rabdomiosarcoma Alveolar/patología , Rabdomiosarcoma Embrionario/enzimología , Rabdomiosarcoma Embrionario/patología , Carga Tumoral/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Vincristina/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Secretion of interleukin-1ß (IL-1ß) represents a fundamental innate immune response to microbial infection that, at the molecular level, occurs following activation of proteolytic caspases that cleave the immature protein into a secretable form. Human cytomegalovirus (HCMV) is the archetypal betaherpesvirus that is invariably capable of lifelong infection through the activity of numerous virally encoded immune evasion phenotypes. Innate immune pathways responsive to cytoplasmic double-stranded DNA (dsDNA) are known to be activated in response to contact between HCMV and host cells. Here, we used clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) genome editing to demonstrate that the dsDNA receptor absent in melanoma 2 (AIM2) is required for secretion of IL-1ß following HCMV infection. Furthermore, dsDNA-responsive innate signaling induced by HCMV infection that leads to activation of the type I interferon response is also shown, unexpectedly, to play a contributory role in IL-1ß secretion. Importantly, we also show that rendering virus particles inactive by UV exposure leads to substantially increased IL-1ß processing and secretion and that live HCMV can inhibit this, suggesting the virus encodes factors that confer an inhibitory effect on this response. Further examination revealed that ectopic expression of the immediate early (IE) 86-kDa protein (IE86) is actually associated with a block in transcription of the pro-IL-1ß gene and, independently, diminishment of the immature protein. Overall, these results reveal two new and distinct phenotypes conferred by the HCMV IE86 protein, as well as an unusual circumstance in which a single herpesviral protein exhibits inhibitory effects on multiple molecular processes within the same innate immune response.IMPORTANCE Persistent infection with HCMV is associated with the operation of diverse evasion phenotypes directed at antiviral immunity. Obstruction of intrinsic and innate immune responses is typically conferred by viral proteins either associated with the viral particle or expressed immediately after entry. In line with this, numerous phenotypes are attributed to the HCMV IE86 protein that involve interference with innate immune processes via transcriptional and protein-directed mechanisms. We describe novel IE86-mediated phenotypes aimed at virus-induced secretion of IL-1ß. Intriguingly, while many viruses target the function of the molecular scaffold required for IL-1ß maturation to prevent this response, we find that HCMV and IE86 target the IL-1ß protein specifically. Moreover, we show that IE86 impairs both the synthesis of the IL-1ß transcript and the stability of the immature protein. This indicates an unusual phenomenon in which a single viral protein exhibits two molecularly separate evasion phenotypes directed at a single innate cytokine.
Asunto(s)
Citomegalovirus/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Evasión Inmune , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Humanos , Proteolisis , Células THP-1RESUMEN
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood with an unmet clinical need for decades. A single oncogenic fusion gene is associated with treatment resistance and a 40 to 45% decrease in overall survival. We previously showed that expression of this PAX3:FOXO1 fusion oncogene in alveolar RMS (aRMS) mediates tolerance to chemotherapy and radiotherapy and that the class I-specific histone deacetylase (HDAC) inhibitor entinostat reduces PAX3:FOXO1 protein abundance. Here, we established the antitumor efficacy of entinostat with chemotherapy in various preclinical cell and mouse models and found that HDAC3 inhibition was the primary mechanism of entinostat-induced suppression of PAX3:FOXO1 abundance. HDAC3 inhibition by entinostat decreased the activity of the chromatin remodeling enzyme SMARCA4, which, in turn, derepressed the microRNA miR-27a. This reexpression of miR-27a led to PAX3:FOXO1 mRNA destabilization and chemotherapy sensitization in aRMS cells in culture and in vivo. Furthermore, a phase 1 clinical trial (ADVL1513) has shown that entinostat is tolerable in children with relapsed or refractory solid tumors and is planned for phase 1B cohort expansion or phase 2 clinical trials. Together, these results implicate an HDAC3-SMARCA4-miR-27a-PAX3:FOXO1 circuit as a driver of chemoresistant aRMS and suggest that targeting this pathway with entinostat may be therapeutically effective in patients.
Asunto(s)
ADN Helicasas/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , MicroARNs/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción Paired Box/metabolismo , Rabdomiosarcoma Alveolar/metabolismo , Factores de Transcripción/metabolismo , Animales , Antineoplásicos/farmacología , Benzamidas/farmacología , Línea Celular Tumoral , Biología Computacional , Resistencia a Antineoplásicos , Epigénesis Genética , Femenino , Transferencia Resonante de Energía de Fluorescencia , Proteína Forkhead Box O1/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Ratones , Trasplante de Neoplasias , Factor de Transcripción PAX3/metabolismo , Piridinas/farmacología , Análisis de Secuencia de ARN , Vincristina/farmacologíaRESUMEN
The type I interferon (IFN) system represents an essential innate immune response that renders cells resistant to virus growth via the molecular actions of IFN-induced effector proteins. IFN-mediated cellular states inhibit growth of numerous and diverse virus types, including those of known pathogenicity as well as potentially emerging agents. As such, targeted pharmacologic activation of the IFN response may represent a novel therapeutic strategy to prevent infection or spread of clinically impactful viruses. In light of this, we employed a high-throughput screen to identify small molecules capable of permeating the cell and of activating IFN-dependent signaling processes. Here we report the identification and characterization of N-(methylcarbamoyl)-2-{[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-2-phenylacetamide (referred to as C11), a novel compound capable of inducing IFN secretion from human cells. Using reverse genetics-based loss-of-function assays, we show that C11 activates the type I IFN response in a manner that requires the adaptor protein STING but not the alternative adaptors MAVS and TRIF. Importantly, treatment of cells with C11 generated a cellular state that potently blocked replication of multiple emerging alphavirus types, including chikungunya, Ross River, Venezuelan equine encephalitis, Mayaro, and O'nyong-nyong viruses. The antiviral effects of C11 were subsequently abrogated in cells lacking STING or the type I IFN receptor, indicating that they are mediated, at least predominantly, by way of STING-mediated IFN secretion and subsequent autocrine/paracrine signaling. This work also allowed characterization of differential antiviral roles of innate immune signaling adaptors and IFN-mediated responses and identified MAVS as being crucial to cellular resistance to alphavirus infection.IMPORTANCE Due to the increase in emerging arthropod-borne viruses, such as chikungunya virus, that lack FDA-approved therapeutics and vaccines, it is important to better understand the signaling pathways that lead to clearance of virus. Here we show that C11 treatment makes human cells refractory to replication of a number of these viruses, which supports its value in increasing our understanding of the immune response and viral pathogenesis required to establish host infection. We also show that C11 depends on signaling through STING to produce antiviral type I interferon, which further supports its potential as a therapeutic drug or research tool.
Asunto(s)
Alphavirus/metabolismo , Antivirales/farmacología , Fibroblastos/metabolismo , Proteínas de la Membrana/agonistas , Transducción de Señal/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Comunicación Autocrina/efectos de los fármacos , Comunicación Autocrina/genética , Fibroblastos/patología , Fibroblastos/virología , Humanos , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Comunicación Paracrina/efectos de los fármacos , Comunicación Paracrina/genética , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Transducción de Señal/genéticaRESUMEN
The ongoing concurrent outbreaks of Zika, Chikungunya, and dengue viruses in Latin America and the Caribbean highlight the need for development of broad-spectrum antiviral treatments. The type I interferon (IFN) system has evolved in vertebrates to generate tissue responses that actively block replication of multiple known and potentially zoonotic viruses. As such, its control and activation through pharmacological agents may represent a novel therapeutic strategy for simultaneously impairing growth of multiple virus types and rendering host populations resistant to virus spread. In light of this strategy's potential, we undertook a screen to identify novel interferon-activating small molecules. Here, we describe 1-(2-fluorophenyl)-2-(5-isopropyl-1,3,4-thiadiazol-2-yl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione, which we termed AV-C. Treatment of human cells with AV-C activates innate and interferon-associated responses that strongly inhibit replication of Zika, Chikungunya, and dengue viruses. By utilizing genome editing, we investigated the host proteins essential to AV-C-induced cellular states. This showed that the compound requires a TRIF-dependent signaling cascade that culminates in IFN regulatory factor 3 (IRF3)-dependent expression and secretion of type I interferon to elicit antiviral responses. The other canonical IRF3-terminal adaptor proteins STING and IPS-1/MAVS were dispensable for AV-C-induced phenotypes. However, our work revealed an important inhibitory role for IPS-1/MAVS, but not TRIF, in flavivirus replication, implying that TRIF-directed viral evasion may not occur. Additionally, we show that in response to AV-C, primary human peripheral blood mononuclear cells secrete proinflammatory cytokines that are linked with establishment of adaptive immunity to viral pathogens. Ultimately, synthetic innate immune activators such as AV-C may serve multiple therapeutic purposes, including direct antimicrobial responses and facilitation of pathogen-directed adaptive immunity.IMPORTANCE The type I interferon system is part of the innate immune response that has evolved in vertebrates as a first line of broad-spectrum immunological defense against an unknowable diversity of microbial, especially viral, pathogens. Here, we characterize a novel small molecule that artificially activates this response and in so doing generates a cellular state antagonistic to growth of currently emerging viruses: Zika virus, Chikungunya virus, and dengue virus. We also show that this molecule is capable of eliciting cellular responses that are predictive of establishment of adaptive immunity. As such, this agent may represent a powerful and multipronged therapeutic tool to combat emerging and other viral diseases.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/agonistas , Antivirales/farmacología , Benzopiranos/farmacología , Virus Chikungunya/fisiología , Virus del Dengue/fisiología , Tiadiazoles/farmacología , Replicación Viral , Virus Zika/fisiología , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Antivirales/química , Antivirales/aislamiento & purificación , Benzopiranos/química , Benzopiranos/aislamiento & purificación , Línea Celular , Fiebre Chikungunya/tratamiento farmacológico , Virus Chikungunya/efectos de los fármacos , Citocinas/biosíntesis , Replicación del ADN/efectos de los fármacos , Dengue/tratamiento farmacológico , Virus del Dengue/efectos de los fármacos , Virus del Dengue/metabolismo , Descubrimiento de Drogas , Edición Génica , Interacciones Huésped-Patógeno , Humanos , Evasión Inmune , Inmunidad Innata/efectos de los fármacos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/efectos de los fármacos , Interferón Tipo I/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Tiadiazoles/química , Tiadiazoles/aislamiento & purificación , Virus Zika/efectos de los fármacosRESUMEN
Risk or presence of metastasis in medulloblastoma causes substantial treatment-related morbidity and overall mortality. Through the comparison of cytokines and growth factors in the cerebrospinal fluid (CSF) of metastatic medulloblastoma patients with factors also in conditioned media of metastatic MYC amplified medulloblastoma or leptomeningeal cells, we were led to explore the bioactivity of IGF1 in medulloblastoma by elevated CSF levels of IGF1, IGF-sequestering IGFBP3, IGFBP3-cleaving proteases (MMP and tPA), and protease modulators (TIMP1 and PAI-1). IGF1 led not only to receptor phosphorylation but also accelerated migration/adhesion in MYC amplified medulloblastoma cells in the context of appropriate matrix or meningothelial cells. Clinical correlation suggests a peri-/sub-meningothelial source of IGF-liberating proteases that could facilitate leptomeningeal metastasis. In parallel, studies of key factors responsible for cell autonomous growth in MYC amplified medulloblastoma prioritized IGF1R inhibitors. Together, our studies identify IGF1R as a high value target for clinical trials in high risk medulloblastoma.
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Neoplasias Cerebelosas/líquido cefalorraquídeo , Meduloblastoma/líquido cefalorraquídeo , Neoplasias Meníngeas/líquido cefalorraquídeo , Receptores de Somatomedina/metabolismo , Adolescente , Antineoplásicos/farmacología , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/líquido cefalorraquídeo , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/patología , Niño , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Expresión Génica , Humanos , Concentración 50 Inhibidora , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/líquido cefalorraquídeo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/líquido cefalorraquídeo , Factor I del Crecimiento Similar a la Insulina/genética , Masculino , Metaloproteinasa 9 de la Matriz/líquido cefalorraquídeo , Metaloproteinasa 9 de la Matriz/genética , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/secundario , Neoplasias Meníngeas/tratamiento farmacológico , Neoplasias Meníngeas/secundario , Terapia Molecular Dirigida , Inhibidor 1 de Activador Plasminogénico/líquido cefalorraquídeo , Inhibidor 1 de Activador Plasminogénico/genética , Receptor IGF Tipo 1 , Receptores de Somatomedina/antagonistas & inhibidores , Receptores de Somatomedina/genética , Inhibidor Tisular de Metaloproteinasa-1/líquido cefalorraquídeo , Inhibidor Tisular de Metaloproteinasa-1/genéticaRESUMEN
Pharmacologic stimulation of innate immune processes represents an attractive strategy to achieve multiple therapeutic outcomes including inhibition of virus replication, boosting antitumor immunity, and enhancing vaccine immunogenicity. In light of this we sought to identify small molecules capable of activating the type I interferon (IFN) response by way of the transcription factor IFN regulatory factor 3 (IRF3). A high throughput in vitro screen yielded 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (referred to herein as G10), which was found to trigger IRF3/IFN-associated transcription in human fibroblasts. Further examination of the cellular response to this molecule revealed expression of multiple IRF3-dependent antiviral effector genes as well as type I and III IFN subtypes. This led to the establishment of a cellular state that prevented replication of emerging Alphavirus species including Chikungunya virus, Venezuelan Equine Encephalitis virus, and Sindbis virus. To define cellular proteins essential to elicitation of the antiviral activity by the compound we employed a reverse genetics approach that utilized genome editing via CRISPR/Cas9 technology. This allowed the identification of IRF3, the IRF3-activating adaptor molecule STING, and the IFN-associated transcription factor STAT1 as required for observed gene induction and antiviral effects. Biochemical analysis indicates that G10 does not bind to STING directly, however. Thus the compound may represent the first synthetic small molecule characterized as an indirect activator of human STING-dependent phenotypes. In vivo stimulation of STING-dependent activity by an unrelated small molecule in a mouse model of Chikungunya virus infection blocked viremia demonstrating that pharmacologic activation of this signaling pathway may represent a feasible strategy for combating emerging Alphaviruses.
Asunto(s)
Antivirales/farmacología , Fiebre Chikungunya/inmunología , Proteínas de la Membrana/agonistas , Transducción de Señal/inmunología , Tiazinas/farmacología , Alphavirus/inmunología , Infecciones por Alphavirus/inmunología , Animales , Células Cultivadas , Virus Chikungunya/inmunología , Ensayos Analíticos de Alto Rendimiento , Humanos , Immunoblotting , Factor 3 Regulador del Interferón/inmunología , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood cancer. We performed a chemical screen in patient-derived DIPG cultures along with RNA-seq analyses and integrated computational modeling to identify potentially effective therapeutic strategies. The multi-histone deacetylase inhibitor panobinostat demonstrated therapeutic efficacy both in vitro and in DIPG orthotopic xenograft models. Combination testing of panobinostat and the histone demethylase inhibitor GSK-J4 revealed that the two had synergistic effects. Together, these data suggest a promising therapeutic strategy for DIPG.
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Benzazepinas/administración & dosificación , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Glioma/tratamiento farmacológico , Ácidos Hidroxámicos/administración & dosificación , Indoles/administración & dosificación , Pirimidinas/administración & dosificación , Animales , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/patología , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Glioma/genética , Glioma/patología , Humanos , Panobinostat , Análisis de Secuencia de ARN , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Rhabdomyosarcomas of the parotid and submandibular glands have the histological appearance of a skeletal muscle tumor yet can be found in tissue with no striated muscular elements. We examine the potential cell-of-origin for rhabdomyosarcoma and whether salivary tumors represent primary malignancy or metastasis. We have previously established genetically engineered mouse models of rhabdomyosarcoma. In these mice, rhabdomyosarcoma is only induced when a Pax3:Foxo1 fusion oncogene is activated with concurrent loss of p53 function (for alveolar rhabdomyosarcoma) or loss of p53 function alone (for embryonal rhabdomyosarcoma) using Cre-lox technology. These mutations are only activated under the control of promoters specific for selected cell lineages, previously thought to be myogenesis-restricted. RT-PCR and immunohistochemistry for lineage-specific promoter gene products reveal these promoters are active in wild-type mouse salivary gland. Given that mouse rhabdomyosarcoma frequently originates in the salivary glands and these myogenic-related promoters are normally expressed in salivary tissue, a high likelihood exists that the salivary gland contains a cell-of-origin of this muscle-related cancer.
RESUMEN
Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1.
Asunto(s)
Antineoplásicos/farmacología , Benzamidas/farmacología , Piridinas/farmacología , Rabdomiosarcoma Alveolar/patología , Animales , Línea Celular Tumoral , Linaje de la Célula , Modelos Animales de Enfermedad , Epigénesis Genética/efectos de los fármacos , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Alveolar rhabdomyosarcoma (aRMS) is an aggressive myogenic childhood malignancy, not infrequently presenting as incurable metastatic disease. To identify therapeutic targets, we performed an unbiased tyrosine kinome RNA interference screen in primary cell cultures from a genetically engineered, conditional mouse model of aRMS. We identified ephrin receptor B4 (EphB4) as a target that is widely expressed in human aRMS and that portends a poor clinical outcome in an expression level-dependent manner. We also uncovered cross-talk of this ephrin receptor with another receptor tyrosine kinase, PDGFRß, which facilitates PDGF ligand-dependent, ephrin ligand-independent activation of EphB4 converging on the Akt and Erk1/2 pathways. Conversely, EphB4 activation by its cognate ligand, EphrinB2, did not stimulate PDGFRß; instead, apoptosis was paradoxically induced. Finally, we showed that small-molecule inhibition of both PDGFRß and EphB4 by dasatinib resulted in a significant decrease in tumor cell viability in vitro, as well as decreased tumor growth rate and significantly prolonged survival in vivo. To our knowledge, these results are the first to identify EphB4 and its cross-talk with PDGFRß as unexpected vital determinants of tumor cell survival in aRMS, with EphB4 at the crux of a bivalent signaling node that is either mitogenic or proapoptotic.
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Receptor EphB4/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Rabdomiosarcoma Alveolar/metabolismo , Rabdomiosarcoma Alveolar/patología , Transducción de Señal , Animales , Apoptosis/efectos de los fármacos , Becaplermina , Benzamidas/farmacología , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dasatinib , Efrina-B2/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Humanos , Mesilato de Imatinib , Ratones , Factores de Transcripción Paired Box/metabolismo , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Pronóstico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-sis/farmacología , Pirimidinas/farmacología , Interferencia de ARN/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Receptor Cross-Talk/efectos de los fármacos , Receptor EphB4/antagonistas & inhibidores , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Rabdomiosarcoma Alveolar/enzimología , Transducción de Señal/efectos de los fármacos , Tiazoles/farmacologíaRESUMEN
BACKGROUND: Alveolar rhabdomyosarcoma (aRMS) is a myogenic childhood sarcoma frequently associated with a translocation-mediated fusion gene, Pax3:Foxo1a. METHODS: We investigated the complementary role of Rb1 loss in aRMS tumor initiation and progression using conditional mouse models. RESULTS: Rb1 loss was not a necessary and sufficient mutational event for rhabdomyosarcomagenesis, nor a strong cooperative initiating mutation. Instead, Rb1 loss was a modifier of progression and increased anaplasia and pleomorphism. Whereas Pax3:Foxo1a expression was unaltered, biomarkers of aRMS versus embryonal rhabdomyosarcoma were both increased, questioning whether these diagnostic markers are reliable in the context of Rb1 loss. Genome-wide gene expression in Pax3:Foxo1a,Rb1 tumors more closely approximated aRMS than embryonal rhabdomyosarcoma. Intrinsic loss of pRb function in aRMS was evidenced by insensitivity to a Cdk4/6 inhibitor regardless of whether Rb1 was intact or null. This loss of function could be attributed to low baseline Rb1, pRb and phospho-pRb expression in aRMS tumors for which the Rb1 locus was intact. Pax3:Foxo1a RNA interference did not increase pRb or improve Cdk inhibitor sensitivity. Human aRMS shared the feature of low and/or heterogeneous tumor cell pRb expression. CONCLUSIONS: Rb1 loss from an already low pRb baseline is a significant disease modifier, raising the possibility that some cases of pleomorphic rhabdomyosarcoma may in fact be Pax3:Foxo1a-expressing aRMS with Rb1 or pRb loss of function.
RESUMEN
UNLABELLED: Since the advent of tyrosine kinase inhibitors as targeted therapies in cancer, several receptor tyrosine kinases (RTK) have been identified as operationally important for disease progression. Rhabdomyosarcoma (RMS) is a malignancy in need of new treatment options; therefore, better understanding of the heterogeneity of RTKs would advance this goal. Here, alveolar RMS (aRMS) tumor cells derived from a transgenic mouse model expressing two such RTKs, platelet-derived growth factor (PDGFR)α and insulin-like growth factor (IGF)-1R, were investigated by fluorescence-activated cell sorting (FACS). Sorted subpopulations that were positive or negative for PDGFRα and IGF-1R dynamically altered their cell surface RTK expression profiles as early as the first cell division. Interestingly, a difference in total PDGFRα expression and nuclear IGF-1R expression was conserved in populations. Nuclear IGF-1R expression was greater than cytoplasmic IGF-1R in cells with initially high cell surface IGF-1R, and cells with high nuclear IGF-1R established tumors more efficiently in vivo. RNA interference-mediated silencing of IGF-1R in the subpopulation of cells initially harboring higher cell surface and total IGF-1R resulted in significantly reduced anchorage-independent colony formation as compared with cells with initially lower cell surface and total IGF-1R expression. Finally, in accordance with the findings observed in murine aRMS, human aRMS also had robust expression of nuclear IGF-1R. IMPLICATIONS: RTK expression status and subcellular localization dynamics are important considerations for personalized medicine.
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Núcleo Celular/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Rabdomiosarcoma Alveolar/genética , Animales , Línea Celular Tumoral , Núcleo Celular/genética , Modelos Animales de Enfermedad , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Ratones SCID , Ratones Transgénicos , Rabdomiosarcoma Alveolar/metabolismo , Rabdomiosarcoma Alveolar/patología , Análisis de Matrices TisularesRESUMEN
Tumor cells of the muscle-related cancer alveolar rhabdomyosarcoma (aRMS) have dysregulated terminal myogenic differentiation that is characterized by continuous proliferation, decreased capacity to express markers of terminal differentiation, and inability of tumor cells to fuse to one another in the manner seen for normal myoblasts. Whether aRMS tumor cells can fuse with normal myogenic progenitors such as skeletal muscle stem cells (satellite cells) or myoblasts is unknown, as is the biological effect of fusion events if the phenomenon occurs. To study this possibility, we isolated primary satellite cells harboring a lacZ Cre-LoxP reporter gene for coculture with murine aRMS primary tumor cells expressing Cre. Results of in vitro and in vivo experiments demonstrated tumor cell-muscle cell progenitor fusion events as well as accelerated rates of tumor establishment and progression when satellite cells and derived muscle progenitors were coinjected with tumor cells in an orthotopic allograft model. Interleukin 4 receptor (IL-4R) blocking antibody treatment reversed fusion events in vitro and blocked tumor initiation and progression in vivo. Taken together, this study supports a potential role of tumor cell-host cell fusion and the strong therapeutic potential of IL-4R blockade to prevent the establishment of RMS tumors at new anatomical sites.
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Receptores de Interleucina-4/antagonistas & inhibidores , Rabdomiosarcoma Alveolar/patología , Animales , Diferenciación Celular/fisiología , Fusión Celular , Línea Celular , Línea Celular Tumoral , Modelos Animales de Enfermedad , Citometría de Flujo , Inmunohistoquímica , Ratones , Células Satélite del Músculo Esquelético/patologíaRESUMEN
BACKGROUND: Effective targeted therapies are needed in sarcomas, but the biological heterogeneity of these tumors has presented a challenge to clinical integration of small molecule inhibitors in sarcoma treatment. Here we outline a process to personalize therapy for sarcomas through a case study of a canine with spontaneous osteosarcoma. PROCEDURE: Rapid establishment of a primary tumor cell culture is described, followed by efficient functional characterization of the tumor that identified the Src inhibitor dasatinib as the most effective targeted therapy for this individual dog. RESULTS: Adjuvant dasatinib was administered for a total of 26 weeks following treatment with chemotherapy. Pharmacokinetic studies confirm that a therapeutic serum concentration was achieved at a tolerable dose of 0.75 mg/kg/day. The canine patient remains without evidence of recurrent disease 24 months following initial diagnosis. CONCLUSIONS: The approach described through this illustrative case study is broadly applicable and might be used for other solid tumors in canines as well as in humans.
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Neoplasias Óseas , Enfermedades de los Perros/tratamiento farmacológico , Osteosarcoma , Inhibidores de Proteínas Quinasas , Pirimidinas , Tiazoles , Animales , Neoplasias Óseas/diagnóstico por imagen , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/veterinaria , Línea Celular Tumoral , Dasatinib , Enfermedades de los Perros/diagnóstico por imagen , Perros , Osteosarcoma/diagnóstico por imagen , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/veterinaria , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacocinética , Pirimidinas/administración & dosificación , Pirimidinas/farmacocinética , Radiografía , Tiazoles/administración & dosificación , Tiazoles/farmacocinética , Factores de Tiempo , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
Alveolar rhabdomyosarcoma (aRMS) is a very aggressive sarcoma of children and young adults. Our previous studies have shown that small molecule inhibition of Pdgfra is initially very effective in an aRMS mouse model. However, slowly evolving, acquired resistance to a narrow-spectrum kinase inhibitor (imatinib) was common. We identified Src family kinases (SFKs) to be potentiators of Pdgfra in murine aRMS primary cell cultures from mouse tumors with evolved resistance in vivo in comparison to untreated cultures. Treating the resistant primary cell cultures with a combination of Pdgfra and Src inhibitors had a strong additive effect on cell viability. In Pdgfra knockout tumors, however, the Src inhibitor had no effect on tumor cell viability. Sorafenib, whose targets include not only PDGFRA but also the Src downstream target Raf, was effective at inhibiting mouse and human tumor cell growth and halted progression of mouse aRMS tumors in vivo. These results suggest that an adaptive Src-Pdgfra-Raf-Mapk axis is relevant to PDGFRA inhibition in rhabdomyosarcoma.