RESUMEN
Cyclic dinucleotide (CDN) agonists of the STimulator of InterferoN Genes (STING) pathway have shown immune activation and tumor clearance in pre-clinical models. However, CDNs administered intratumorally also promote STING activation leading to direct cytotoxicity of many cell types in the tumor microenvironment (TME), systemic inflammation due to rapid tumor extravasation of the CDN, and immune ablation in the TME. These result in a failure to establish immunological memory. ExoSTING, an engineered extracellular vesicle (EV) exogenously loaded with CDN, enhances the potency of CDN and preferentially activates antigen presenting cells in the TME. Following intratumoral injection, exoSTING was retained within the tumor, enhanced local Th1 responses and recruitment of CD8+ T cells, and generated systemic anti-tumor immunity to the tumor. ExoSTING at therapeutically active doses did not induce systemic inflammatory cytokines, resulting in an enhanced therapeutic window. ExoSTING is a novel, differentiated therapeutic candidate that leverages the natural biology of EVs to enhance the activity of CDNs.
Asunto(s)
Vesículas Extracelulares/fisiología , Vigilancia Inmunológica , Microambiente Tumoral/fisiología , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Prodrugs engineered for preferential activation in diseased versus normal tissues offer immense potential to improve the therapeutic indexes (TIs) of preclinical and clinical-stage active pharmaceutical ingredients that either cannot be developed otherwise or whose efficacy or tolerability it is highly desirable to improve. Such approaches, however, often suffer from trial-and-error design, precluding predictive synthesis and optimization. Here, using bromodomain and extra-terminal (BET) protein inhibitors (BETi)-a class of epigenetic regulators with proven anticancer potential but clinical development hindered in large part by narrow TIs-we introduce a macromolecular prodrug platform that overcomes these challenges. Through tuning of traceless linkers appended to a "bottlebrush prodrug" scaffold, we demonstrate correlation of in vitro prodrug activation kinetics with in vivo tumor pharmacokinetics, enabling the predictive design of novel BETi prodrugs with enhanced antitumor efficacies and devoid of dose-limiting toxicities in a syngeneic triple-negative breast cancer murine model. This work may have immediate clinical implications, introducing a platform for predictive prodrug design and potentially overcoming hurdles in drug development.
Asunto(s)
Antineoplásicos/farmacología , Diseño de Fármacos , Profármacos/farmacología , Proteínas/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Sustancias Macromoleculares/síntesis química , Sustancias Macromoleculares/química , Sustancias Macromoleculares/farmacología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Estructura Molecular , Profármacos/síntesis química , Profármacos/química , Proteínas/metabolismoRESUMEN
The promise of IL12 as a cancer treatment has yet to be fulfilled with multiple tested approaches being limited by unwanted systemic exposure and unpredictable pharmacology. To address these limitations, we generated exoIL12, a novel, engineered exosome therapeutic that displays functional IL12 on the surface of an exosome. IL12 exosomal surface expression was achieved via fusion to the abundant exosomal surface protein PTGFRN resulting in equivalent potency in vitro to recombinant IL12 (rIL12) as demonstrated by IFNγ production. Following intratumoral injection, exoIL12 exhibited prolonged tumor retention and greater antitumor activity than rIL12. Moreover, exoIL12 was significantly more potent than rIL12 in tumor growth inhibition. In the MC38 model, complete responses were observed in 63% of mice treated with exoIL12; in contrast, rIL12 resulted in 0% complete responses at an equivalent IL12 dose. This correlated with dose-dependent increases in tumor antigen-specific CD8+ T cells. Rechallenge studies of exoIL12 complete responder mice showed no tumor regrowth, and depletion of CD8+ T cells completely abrogated antitumor activity of exoIL12. Following intratumoral administration, exoIL12 exhibited 10-fold higher intratumoral exposure than rIL12 and prolonged IFNγ production up to 48 hours. Retained local pharmacology of exoIL12 was further confirmed using subcutaneous injections in nonhuman primates. This work demonstrates that tumor-restricted pharmacology of exoIL12 results in superior in vivo efficacy and immune memory without systemic IL12 exposure and related toxicity. ExoIL12 is a novel cancer therapeutic candidate that overcomes key limitations of rIL12 and thereby creates a therapeutic window for this potent cytokine.
Asunto(s)
Exosomas/metabolismo , Interleucina-12/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Macaca fascicularis , RatonesRESUMEN
Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two "scaffold" proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily. Molecules were loaded into EVs at high density and exhibited potent in vitro activity when fused to full-length or truncated forms of PTGFRN or BASP1. Furthermore, these engineered EVs retained pharmacodynamic activity in a variety of animal models. This engineering platform provides a simple approach to functionalize EVs with topologically diverse macromolecules and represents a significant advance toward unlocking the therapeutic potential of EVs.
Asunto(s)
Vesículas Extracelulares/trasplante , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas/administración & dosificación , Proteínas Represoras/metabolismo , Animales , Comunicación Celular , Sistemas de Liberación de Medicamentos , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Femenino , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Ratones , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Proteínas Represoras/genéticaRESUMEN
The neuromodulatory gene DISC1 is disrupted by a t(1;11) translocation that is highly penetrant for schizophrenia and affective disorders, but how this translocation affects DISC1 function is incompletely understood. N-methyl-D-aspartate receptors (NMDAR) play a central role in synaptic plasticity and cognition, and are implicated in the pathophysiology of schizophrenia through genetic and functional studies. We show that the NMDAR subunit GluN2B complexes with DISC1-associated trafficking factor TRAK1, while DISC1 interacts with the GluN1 subunit and regulates dendritic NMDAR motility in cultured mouse neurons. Moreover, in the first mutant mouse that models DISC1 disruption by the translocation, the pool of NMDAR transport vesicles and surface/synaptic NMDAR expression are increased. Since NMDAR cell surface/synaptic expression is tightly regulated to ensure correct function, these changes in the mutant mouse are likely to affect NMDAR signalling and synaptic plasticity. Consistent with these observations, RNASeq analysis of the translocation carrier-derived human neurons indicates abnormalities of excitatory synapses and vesicle dynamics. RNASeq analysis of the human neurons also identifies many differentially expressed genes previously highlighted as putative schizophrenia and/or depression risk factors through large-scale genome-wide association and copy number variant studies, indicating that the translocation triggers common disease pathways that are shared with unrelated psychiatric patients. Altogether, our findings suggest that translocation-induced disease mechanisms are likely to be relevant to mental illness in general, and that such disease mechanisms include altered NMDAR dynamics and excitatory synapse function. This could contribute to the cognitive disorders displayed by translocation carriers.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Translocación Genética , Proteínas Adaptadoras del Transporte Vesicular , Animales , Proteínas Portadoras/genética , Estudio de Asociación del Genoma Completo , Humanos , Ratones , Modelos Animales , Trastornos del Humor/genética , Mutación , Proteínas del Tejido Nervioso/genética , Plasticidad Neuronal , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Esquizofrenia/genética , Análisis de Secuencia de ARN , Sinapsis/metabolismoRESUMEN
At present there are no drugs for the treatment of chronic liver fibrosis that have been approved by the Food and Drug administration of the United States. Telmisartan, a small-molecule antihypertensive drug, displays antifibrotic activity, but its clinical use is limited because it causes systemic hypotension. Here, we report the scalable and convergent synthesis of macromolecular telmisartan prodrugs optimized for preferential release in diseased liver tissue. We optimized the release of active telmisartan in fibrotic liver to be depot-like (that is, a constant therapeutic concentration) through the molecular design of telmisartan brush-arm star polymers, and show that these lead to improved efficacy and to the avoidance of dose-limiting hypotension in both metabolically and chemically induced mouse models of hepatic fibrosis, as determined by histopathology, enzyme levels in the liver, intact-tissue protein markers, hepatocyte necrosis protection, and gene-expression analyses. In rats and dogs, the prodrugs are retained long-term in liver tissue and have a well-tolerated safety profile. Our findings support the further development of telmisartan prodrugs that enable infrequent dosing in the treatment of liver fibrosis.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Diseño de Fármacos , Cirrosis Hepática/tratamiento farmacológico , Profármacos/uso terapéutico , Telmisartán/uso terapéutico , Bloqueadores del Receptor Tipo 1 de Angiotensina II/química , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacocinética , Animales , Tetracloruro de Carbono/toxicidad , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Semivida , Hígado/metabolismo , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Polímeros/química , Profármacos/química , Profármacos/farmacocinética , Ratas , Ratas Sprague-Dawley , Telmisartán/químicaRESUMEN
In the version of this Article originally published, the author Peter Blume-Jensen was not denoted as a corresponding author; this has now been amended and the author's email address has been added. The 'Correspondence and requests for materials' statement was similarly affected and has now been updated with the author's initials 'P.B-J.'
RESUMEN
Cannabinoid receptor CB1 is expressed abundantly in the brain and presumably in the peripheral tissues responsible for energy metabolism. It is unclear if the antiobesity effects of rimonabant, a CB1 antagonist, are mediated through the central or the peripheral CB1 receptors. To address this question, we generated transgenic mice with central nervous system (CNS)-specific knockdown (KD) of CB1, by expressing an artificial microRNA (AMIR) under the control of the neuronal Thy1.2 promoter. In the mutant mice, CB1 expression was reduced in the brain and spinal cord, whereas no change was observed in the superior cervical ganglia (SCG), sympathetic trunk, enteric nervous system, and pancreatic ganglia. In contrast to the neuronal tissues, CB1 was undetectable in the brown adipose tissue (BAT) or the liver. Consistent with the selective loss of central CB1, agonist-induced hypothermia was attenuated in the mutant mice, but the agonist-induced delay of gastrointestinal transit (GIT), a primarily peripheral nervous system-mediated effect, was not. Compared to wild-type (WT) littermates, the mutant mice displayed reduced body weight (BW), adiposity, and feeding efficiency, and when fed a high-fat diet (HFD), showed decreased plasma insulin, leptin, cholesterol, and triglyceride levels, and elevated adiponectin levels. Furthermore, the therapeutic effects of rimonabant on food intake (FI), BW, and serum parameters were markedly reduced and correlated with the degree of CB1 KD. Thus, KD of CB1 in the CNS recapitulates the metabolic phenotype of CB1 knockout (KO) mice and diminishes rimonabant's efficacy, indicating that blockade of central CB1 is required for rimonabant's antiobesity actions.
Asunto(s)
Fármacos Antiobesidad/farmacología , Peso Corporal/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Ingestión de Energía/efectos de los fármacos , Obesidad/metabolismo , Piperidinas/farmacología , Pirazoles/farmacología , Receptor Cannabinoide CB1/metabolismo , Adiponectina/sangre , Adiposidad/efectos de los fármacos , Adiposidad/genética , Animales , Fármacos Antiobesidad/uso terapéutico , Biomarcadores/sangre , Peso Corporal/genética , Sistema Nervioso Central/efectos de los fármacos , Colesterol/sangre , Dieta Alta en Grasa/efectos adversos , Ingestión de Energía/genética , Tránsito Gastrointestinal/fisiología , Hipotermia/prevención & control , Insulina/sangre , Leptina/sangre , Ratones , Ratones Noqueados , Ratones Transgénicos , MicroARNs , Mutación , Obesidad/tratamiento farmacológico , Obesidad/genética , Sistema Nervioso Periférico/efectos de los fármacos , Sistema Nervioso Periférico/metabolismo , Fenotipo , Piperidinas/uso terapéutico , Regiones Promotoras Genéticas , Pirazoles/uso terapéutico , Receptor Cannabinoide CB1/genética , Rimonabant , Triglicéridos/sangreRESUMEN
In epithelial tissues, the lineage relationship between normal progenitor cells and cell type(s) of origin for cancer has been poorly understood. Here we show that a known regulator of prostate epithelial differentiation, the homeobox gene Nkx3-1, marks a stem cell population that functions during prostate regeneration. Genetic lineage-marking demonstrates that rare luminal cells that express Nkx3-1 in the absence of testicular androgens (castration-resistant Nkx3-1-expressing cells, CARNs) are bipotential and can self-renew in vivo, and single-cell transplantation assays show that CARNs can reconstitute prostate ducts in renal grafts. Functional assays of Nkx3-1 mutant mice in serial prostate regeneration suggest that Nkx3-1 is required for stem cell maintenance. Furthermore, targeted deletion of the Pten tumour suppressor gene in CARNs results in rapid carcinoma formation after androgen-mediated regeneration. These observations indicate that CARNs represent a new luminal stem cell population that is an efficient target for oncogenic transformation in prostate cancer.
Asunto(s)
Linaje de la Célula , Células Epiteliales/patología , Células Madre Neoplásicas/patología , Neoplasias de la Próstata/patología , Andrógenos/deficiencia , Andrógenos/metabolismo , Animales , Castración , Diferenciación Celular , División Celular , Transformación Celular Neoplásica , Células Epiteliales/metabolismo , Células Epiteliales/trasplante , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Riñón , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/trasplante , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Regeneración , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The mechanisms by which homeoproteins bind selectively to target genes in vivo have long remained unresolved. Here we report that PIAS1 confers DNA-binding specificity on the Msx1 homeoprotein by regulating its subnuclear localization and proximity to target genes. We demonstrate that the interaction of Msx1 with PIAS1, but not its sumoylation, is required for Msx1 to function as an inhibitor of myoblast differentiation through repression of myogenic regulatory genes, such as MyoD. We find that PIAS1 enables Msx1 to bind selectively to a key regulatory element in MyoD, the CER, in myoblast cells and to distinguish the CER from other nonregulatory TAAT-containing sequences. We show that PIAS1 is required for the appropriate localization and retention of Msx1 at the nuclear periphery in myoblast cells. Furthermore, we demonstrate that myogenic regulatory genes that are repressed by Msx1, namely MyoD and Myf5, are located at the nuclear periphery in myoblast cells. We propose that a key regulatory event for DNA-binding specificity by homeoproteins in vivo is their appropriate targeting to subnuclear compartments where their target genes are located, which can be achieved by cofactors such as PIAS1.
Asunto(s)
ADN/metabolismo , Factor de Transcripción MSX1/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Animales , Sitios de Unión/genética , Línea Celular , Núcleo Celular/metabolismo , ADN/genética , Humanos , Factor de Transcripción MSX1/genética , Ratones , Modelos Biológicos , Desarrollo de Músculos , Mioblastos/metabolismo , Proteínas Inhibidoras de STAT Activados/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Recent studies have shown that several loss-of-function mouse models of prostate carcinogenesis can develop a spectrum of precancerous lesions that resemble human prostatic intraepithelial neoplasia (PIN). Here, we have investigated the malignant potential of the high-grade PIN lesions that form in Nkx3.1(+/-); Pten(+/-) compound mutant mice and demonstrate their neoplastic progression in a serial transplantation/tissue recombination assay. Furthermore, we find that a majority of Nkx3.1(+/-); Pten(+/-) mice greater than 1 year of age develop invasive adenocarcinoma, which is frequently accompanied by metastases to lymph nodes. Finally, we observe androgen independence of high-grade PIN lesions after androgen ablation of Nkx3.1(+/-); Pten(+/-) mice. We conclude that Nkx3.1(+/-); Pten(+/-) mice recapitulate key features of advanced prostate cancer and represent a useful model for investigating associated molecular mechanisms and for evaluating therapeutic approaches.
Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/patología , Proteínas de Homeodominio/genética , Mutación , Monoéster Fosfórico Hidrolasas/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Adenocarcinoma/cirugía , Animales , Modelos Animales de Enfermedad , Humanos , Metástasis Linfática , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Invasividad Neoplásica , Trasplante de Neoplasias , Orquiectomía , Fosfohidrolasa PTEN , Neoplasias de la Próstata/cirugía , Ratas , Testosterona/sangreRESUMEN
The murine prostate is a structure that is made up of four distinct lobes; the dorsal and lateral prostates (often grouped together as the dorsolateral prostate), the anterior (coagulating gland) and the ventral prostate. Previous work has implicated Hox genes in the development of these structures, but how each lobe acquires unique identities for specific functions has not been addressed. In this study, the ventral prostate-specific function of Hoxb13 is described. Mice lacking Hoxb13 function show normal numbers of duct tips, but mice mutant for both Hoxb13 and Hoxd13 exhibit severe hypoplasia of the duct tips, revealing a role for Hoxb13 in ventral prostate morphogenesis. Additionally, a ventral lobe-specific defect was identified in Hoxb13 mutants wherein the epithelium is composed of simple cuboidal cells rather than of tall columnar cells. Ventral prostate ducts appear devoid of contents and do not express the ventral prostate-specific secretory proteins p12, a kazal-type protease inhibitor and p25, a spermine binding protein. These defects are not due to reduction of Nkx3.1 expression or to a global effect on androgen receptor signaling. These results suggest a specific role for Hoxb13 in a differentiation pathway that gives the ventral prostate epithelium a unique identity, as well as a more general role in ventral prostate morphogenesis that is redundant with other Hox13 paralogs.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas de Homeodominio/metabolismo , Próstata/crecimiento & desarrollo , Próstata/metabolismo , Proteínas de Secreción Prostática/metabolismo , Animales , Biomarcadores , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Proteínas de Homeodominio/genética , Humanos , Masculino , Ratones , Mutación , Fenotipo , Próstata/anatomía & histología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
To address the expression and function of Hoxb13, the 5' most Hox gene in the HoxB cluster, we have generated mice with loss-of-function and beta-galactosidase reporter insertion alleles of this gene. Mice homozygous for Hoxb13 loss-of-function mutations show overgrowth in all major structures derived from the tail bud, including the developing secondary neural tube (SNT), the caudal spinal ganglia, and the caudal vertebrae. Using the beta-galactosidase reporter allele of Hoxb13, also a loss-of-function allele, we found that the expression patterns of Hoxb13 in the developing spinal cord and caudal mesoderm are closely associated with overgrowth phenotypes in the tails of homozygous mutant animals. These phenotypes can be explained by the observed increased cell proliferation and decreased levels of apoptosis within the tail of homozygous mutant mice. This analysis of Hoxb13 function suggests that this 5' Hox gene may act as an inhibitor of neuronal cell proliferation, an activator of apoptotic pathways in the SNT, and as a general repressor of growth in the caudal vertebrae.