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MOTIVATION: Huntington's disease (HD) may evolve through gene deregulation. However, the impact of gene deregulation on the dynamics of genetic cooperativity in HD remains poorly understood. Here, we built a multi-layer network model of temporal dynamics of genetic cooperativity in the brain of HD knock-in mice (allelic series of Hdh mice). To enhance biological precision and gene prioritization, we integrated three complementary families of source networks, all inferred from the same RNA-seq time series data in Hdh mice, into weighted-edge networks where an edge recapitulates path-length variation across source-networks and age-points. RESULTS: Weighted edge networks identify two consecutive waves of tight genetic cooperativity enriched in deregulated genes (critical phases), pre-symptomatically in the cortex, implicating neurotransmission, and symptomatically in the striatum, implicating cell survival (e.g. Hipk4) intertwined with cell proliferation (e.g. Scn4b) and cellular senescence (e.g. Cdkn2a products) responses. Top striatal weighted edges are enriched in modulators of defective behavior in invertebrate models of HD pathogenesis, validating their relevance to neuronal dysfunction in vivo. Collectively, these findings reveal highly dynamic temporal features of genetic cooperativity in the brain of Hdh mice where a 2-step logic highlights the importance of cellular maintenance and senescence in the striatum of symptomatic mice, providing highly prioritized targets. AVAILABILITY AND IMPLEMENTATION: Weighted edge network analysis (WENA) data and source codes for performing spectral decomposition of the signal (SDS) and WENA analysis, both written using Python, are available at http://www.broca.inserm.fr/HD-WENA/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Corpo Estriado , Doença de Huntington , Modelos Genéticos , Animais , Sobrevivência Celular , Corpo Estriado/citologia , Corpo Estriado/fisiopatologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Doença de Huntington/genética , Doença de Huntington/fisiopatologia , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Neurônios/patologiaRESUMO
BACKGROUND: MicroRNA (miRNA) regulation is associated with several diseases, including neurodegenerative diseases. Several approaches can be used for modeling miRNA regulation. However, their precision may be limited for analyzing multidimensional data. Here, we addressed this question by integrating shape analysis and feature selection into miRAMINT, a methodology that we used for analyzing multidimensional RNA-seq and proteomic data from a knock-in mouse model (Hdh mice) of Huntington's disease (HD), a disease caused by CAG repeat expansion in huntingtin (htt). This dataset covers 6 CAG repeat alleles and 3 age points in the striatum and cortex of Hdh mice. RESULTS: Remarkably, compared to previous analyzes of this multidimensional dataset, the miRAMINT approach retained only 31 explanatory striatal miRNA-mRNA pairs that are precisely associated with the shape of CAG repeat dependence over time, among which 5 pairs with a strong change of target expression levels. Several of these pairs were previously associated with neuronal homeostasis or HD pathogenesis, or both. Such miRNA-mRNA pairs were not detected in cortex. CONCLUSIONS: These data suggest that miRNA regulation has a limited global role in HD while providing accurately-selected miRNA-target pairs to study how the brain may compute molecular responses to HD over time. These data also provide a methodological framework for researchers to explore how shape analysis can enhance multidimensional data analytics in biology and disease.
Assuntos
Doença de Huntington/genética , Aprendizado de Máquina , MicroRNAs/metabolismo , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Humanos , Proteína Huntingtina/genética , Doença de Huntington/metabolismo , Camundongos , Neurônios/metabolismo , Proteômica , RNA Mensageiro/metabolismo , RNA-Seq , Repetições de TrinucleotídeosRESUMO
Transcriptional changes occur presymptomatically and throughout Huntington's disease (HD), motivating the study of transcriptional regulatory networks (TRNs) in HD We reconstructed a genome-scale model for the target genes of 718 transcription factors (TFs) in the mouse striatum by integrating a model of genomic binding sites with transcriptome profiling of striatal tissue from HD mouse models. We identified 48 differentially expressed TF-target gene modules associated with age- and CAG repeat length-dependent gene expression changes in Htt CAG knock-in mouse striatum and replicated many of these associations in independent transcriptomic and proteomic datasets. Thirteen of 48 of these predicted TF-target gene modules were also differentially expressed in striatal tissue from human disease. We experimentally validated a specific model prediction that SMAD3 regulates HD-related gene expression changes using chromatin immunoprecipitation and deep sequencing (ChIP-seq) of mouse striatum. We found CAG repeat length-dependent changes in the genomic occupancy of SMAD3 and confirmed our model's prediction that many SMAD3 target genes are downregulated early in HD.
Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Doença de Huntington/genética , Proteína Smad3/genética , Animais , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Doença de Huntington/metabolismo , Camundongos , Mapas de Interação de Proteínas , Proteômica , Proteína Smad3/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Cardiac remodelling and contractile dysfunction occur during both acute and chronic disease processes including the accumulation of insoluble aggregates of misfolded amyloid proteins that are typical features of Alzheimer's, Parkinson's and Huntington's disease (HD). While HD has been described mainly as a neurological disease, multiple epidemiological studies have shown that HD patients exhibit a high incidence of cardiovascular events leading to heart failure, and that this is the second highest cause of death. Given that huntingtin is ubiquitously expressed, cardiomyocytes may be at risk of an HD-related dysfunction. In mice, the forced expression of an expanded polyQ repeat under the control of a cardiac specific promoter led to severe heart failure followed by reduced lifespan. However the mechanism leading to cardiac dysfunction in the clinical and pre-clinical HD settings remains unknown. To unravel this mechanism, we employed the R6/2 transgenic and HdhQ150 knock-in mouse models of HD. We found that pre-symptomatic animals developed connexin-43 relocation and a significant deregulation of hypertrophic markers and Bdnf transcripts. In the symptomatic animals, pronounced functional changes were visualised by cardiac MRI revealing a contractile dysfunction, which might be a part of dilatated cardiomyopathy (DCM). This was accompanied by the re-expression of foetal genes, apoptotic cardiomyocyte loss and a moderate degree of interstitial fibrosis. To our surprise, we could identify neither mutant HTT aggregates in cardiac tissue nor a HD-specific transcriptional dysregulation, even at the end stage of disease. We postulate that the HD-related cardiomyopathy is caused by altered central autonomic pathways although the pathogenic effects of mutant HTT acting intrinsically in the heart may also be a contributing factor.
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Cardiomiopatia Dilatada/genética , Doença de Huntington/genética , Contração Miocárdica/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Animais , Cardiomiopatia Dilatada/patologia , Conexina 43/genética , Modelos Animais de Doenças , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Humanos , Proteína Huntingtina , Doença de Huntington/fisiopatologia , Camundongos , Proteínas do Tecido Nervoso/biossíntese , Proteínas Nucleares/biossíntese , Remodelação VentricularRESUMO
Modifiers of Huntington's disease (HD) include mismatch repair (MMR) genes; however, their underlying disease-altering mechanisms remain unresolved. Knockout (KO) alleles for 9 HD GWAS modifiers/MMR genes were crossed to the Q140 Huntingtin (mHtt) knock-in mice to probe such mechanisms. Four KO mice strongly ( Msh3 and Pms1 ) or moderately ( Msh2 and Mlh1 ) rescue a triad of adult-onset, striatal medium-spiny-neuron (MSN)-selective phenotypes: somatic Htt DNA CAG-repeat expansion, transcriptionopathy, and mHtt protein aggregation. Comparatively, Q140 cortex also exhibits an analogous, but later-onset, pathogenic triad that is Msh3 -dependent. Remarkably, Q140/homozygous Msh3-KO lacks visible mHtt aggregates in the brain, even at advanced ages (20-months). Moreover, Msh3 -deficiency prevents striatal synaptic marker loss, astrogliosis, and locomotor impairment in HD mice. Purified Q140 MSN nuclei exhibit highly linear age-dependent mHtt DNA repeat expansion (i.e. repeat migration), with modal-CAG increasing at +8.8 repeats/month (R 2 =0.98). This linear rate is reduced to 2.3 and 0.3 repeats/month in Q140 with Msh3 heterozygous and homozygous alleles, respectively. Our study defines somatic Htt CAG-repeat thresholds below which there are no detectable mHtt nuclear or neuropil aggregates. Mild transcriptionopathy can still occur in Q140 mice with stabilized Htt 140-CAG repeats, but the majority of transcriptomic changes are due to somatic repeat expansion. Our analysis reveals 479 genes with expression levels highly correlated with modal-CAG length in MSNs. Thus, our study mechanistically connects HD GWAS genes to selective neuronal vulnerability in HD, in which Msh3 and Pms1 set the linear rate of neuronal mHtt CAG-repeat migration to drive repeat-length dependent pathogenesis; and provides a preclinical platform for targeting these genes for HD suppression across brain regions. One Sentence Summary: Msh3 and Pms1 are genetic drivers of sequential striatal and cortical pathogenesis in Q140 mice by mediating selective CAG-repeat migration in HD vulnerable neurons.
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We have developed an inducible Huntington's disease (HD) mouse model that allows temporal control of whole-body allele-specific mutant huntingtin (mHtt) expression. We asked whether moderate global lowering of mHtt (~50%) was sufficient for long-term amelioration of HD-related deficits and, if so, whether early mHtt lowering (before measurable deficits) was required. Both early and late mHtt lowering delayed behavioral dysfunction and mHTT protein aggregation, as measured biochemically. However, long-term follow-up revealed that the benefits, in all mHtt-lowering groups, attenuated by 12 months of age. While early mHtt lowering attenuated cortical and striatal transcriptional dysregulation evaluated at 6 months of age, the benefits diminished by 12 months of age, and late mHtt lowering did not ameliorate striatal transcriptional dysregulation at 12 months of age. Only early mHtt lowering delayed the elevation in cerebrospinal fluid neurofilament light chain that we observed in our model starting at 9 months of age. As small-molecule HTT-lowering therapeutics progress to the clinic, our findings suggest that moderate mHtt lowering allows disease progression to continue, albeit at a slower rate, and could be relevant to the degree of mHTT lowering required to sustain long-term benefits in humans.
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Doença de Huntington , Camundongos , Humanos , Animais , Lactente , Doença de Huntington/tratamento farmacológico , Doença de Huntington/genética , Agregados Proteicos , Proteína Huntingtina/genética , Proteína Huntingtina/líquido cefalorraquidiano , Modelos Animais de Doenças , Corpo Estriado/metabolismo , Progressão da DoençaRESUMO
Brain tissue transcriptomes may be organized into gene coexpression networks, but their underlying biological drivers remain incompletely understood. Here, we undertook a large-scale transcriptomic study using 508 wild-type mouse striatal tissue samples dissected exclusively in the afternoons to define 38 highly reproducible gene coexpression modules. We found that 13 and 11 modules are enriched in cell-type and molecular complex markers, respectively. Importantly, 18 modules are highly enriched in daily rhythmically expressed genes that peak or trough with distinct temporal kinetics, revealing the underlying biology of striatal diurnal gene networks. Moreover, the diurnal coexpression networks are a dominant feature of daytime transcriptomes in the mouse cortex. We next employed the striatal coexpression modules to decipher the striatal transcriptomic signatures from Huntington's disease models and heterozygous null mice for 52 genes, uncovering novel functions for Prkcq and Kdm4b in oligodendrocyte differentiation and bipolar disorder-associated Trank1 in regulating anxiety-like behaviors and nocturnal locomotion.
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Doença de Huntington , Transcriptoma , Animais , Camundongos , Proteína Quinase C-theta/genética , Redes Reguladoras de Genes , Doença de Huntington/genética , EncéfaloRESUMO
HDinHD (Huntington's Disease in High Definition; HDinHD.org) is an open online portal for the HD research community that presents a synthesized view of HD-related scientific data. Here, we present a broad overview of HDinHD and highlight the newly launched HDinHD Explorer tool that enables researchers to discover and explore a wide range of diverse yet interconnected HD-related data. We demonstrate the utility of HDinHD Explorer through data mining of a single collection of newly released in vivo therapeutic intervention study reports alongside previously published reports.
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Doença de Huntington , Humanos , Doença de Huntington/genéticaRESUMO
Loss of cellular homeostasis has been implicated in the etiology of several neurodegenerative diseases (NDs). However, the molecular mechanisms that underlie this loss remain poorly understood on a systems level in each case. Here, using a novel computational approach to integrate dimensional RNA-seq and in vivo neuron survival data, we map the temporal dynamics of homeostatic and pathogenic responses in four striatal cell types of Huntington's disease (HD) model mice. This map shows that most pathogenic responses are mitigated and most homeostatic responses are decreased over time, suggesting that neuronal death in HD is primarily driven by the loss of homeostatic responses. Moreover, different cell types may lose similar homeostatic processes, for example, endosome biogenesis and mitochondrial quality control in Drd1-expressing neurons and astrocytes. HD relevance is validated by human stem cell, genome-wide association study, and post-mortem brain data. These findings provide a new paradigm and framework for therapeutic discovery in HD and other NDs.
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Proteína Huntingtina/genética , Doença de Huntington/genética , Mutação , Proteostase , Animais , Modelos Animais de Doenças , Feminino , Proteína Huntingtina/metabolismo , Masculino , CamundongosRESUMO
Although Huntington's disease (HD) is a well studied Mendelian genetic disorder, less is known about its associated epigenetic changes. Here, we characterize DNA methylation levels in six different tissues from 3 species: a mouse huntingtin (Htt) gene knock-in model, a transgenic HTT sheep model, and humans. Our epigenome-wide association study (EWAS) of human blood reveals that HD mutation status is significantly (p < 10-7) associated with 33 CpG sites, including the HTT gene (p = 6.5 × 10-26). These Htt/HTT associations were replicated in the Q175 Htt knock-in mouse model (p = 6.0 × 10-8) and in the transgenic sheep model (p = 2.4 × 10-88). We define a measure of HD motor score progression among manifest HD cases based on multiple clinical assessments. EWAS of motor progression in manifest HD cases exhibits significant (p < 10-7) associations with methylation levels at three loci: near PEX14 (p = 9.3 × 10-9), GRIK4 (p = 3.0 × 10-8), and COX4I2 (p = 6.5 × 10-8). We conclude that HD is accompanied by profound changes of DNA methylation levels in three mammalian species.
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Metilação de DNA , Epigênese Genética , Proteína Huntingtina/genética , Doença de Huntington/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Animais Geneticamente Modificados , Comportamento Animal , Ilhas de CpG/genética , Estudos Transversais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Seguimentos , Técnicas de Introdução de Genes , Loci Gênicos , Estudo de Associação Genômica Ampla , Carga Global da Doença , Humanos , Doença de Huntington/sangue , Doença de Huntington/diagnóstico , Doença de Huntington/epidemiologia , Estudos Longitudinais , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Estudos Prospectivos , Proteínas Recombinantes/genética , Sistema de Registros/estatística & dados numéricos , Índice de Gravidade de Doença , Ovinos , Adulto JovemRESUMO
In Huntington's disease (HD) patients and in model organisms, messenger RNA transcriptome has been extensively studied; in contrast, comparatively little is known about expression and potential role of microRNAs. Using RNA-sequencing, we have quantified microRNA expression in four brain regions and liver, at three different ages, from an allelic series of HD model mice with increasing CAG length in the endogenous Huntingtin gene. Our analyses reveal CAG length-dependent microRNA expression changes in brain, with 159 microRNAs selectively altered in striatum, 102 in cerebellum, 51 in hippocampus, and 45 in cortex. In contrast, a progressive CAG length-dependent microRNA dysregulation was not observed in liver. We further identify microRNAs whose transcriptomic response to CAG length expansion differs significantly among the brain regions and validate our findings in data from a second, independent cohort of mice. Using existing mRNA expression data from the same animals, we assess the possible relationships between microRNA and mRNA expression and highlight candidate microRNAs that are negatively correlated with, and whose predicted targets are enriched in, CAG-length dependent mRNA modules. Several of our top microRNAs (Mir212/Mir132, Mir218, Mir128 and others) have been previously associated with aspects of neuronal development and survival. This study provides an extensive resource for CAG length-dependent changes in microRNA expression in disease-vulnerable and -resistant brain regions in HD mice, and provides new insights for further investigation of microRNAs in HD pathogenesis and therapeutics.
Assuntos
Proteína Huntingtina/genética , Doença de Huntington/genética , MicroRNAs/genética , Repetições de Trinucleotídeos , Animais , Encéfalo/metabolismo , Humanos , Camundongos , TranscriptomaRESUMO
Huntington's disease (HD) is an inherited neurodegenerative disorder of which skeletal muscle atrophy is a common feature, and multiple lines of evidence support a muscle-based pathophysiology in HD mouse models. Inhibition of myostatin signaling increases muscle mass, and therapeutic approaches based on this are in clinical development. We have used a soluble ActRIIB decoy receptor (ACVR2B/Fc) to test the effects of myostatin/activin A inhibition in the R6/2 mouse model of HD. Weekly administration from 5 to 11 weeks of age prevented body weight loss, skeletal muscle atrophy, muscle weakness, contractile abnormalities, the loss of functional motor units in EDL muscles and delayed end-stage disease. Inhibition of myostatin/activin A signaling activated transcriptional profiles to increase muscle mass in wild type and R6/2 mice but did little to modulate the extensive Huntington's disease-associated transcriptional dysregulation, consistent with treatment having little impact on HTT aggregation levels. Modalities that inhibit myostatin signaling are currently in clinical trials for a variety of indications, the outcomes of which will present the opportunity to assess the potential benefits of targeting this pathway in HD patients.
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Doença de Huntington/patologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Miostatina/antagonistas & inibidores , Receptores de Activinas Tipo II/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Força da Mão/fisiologia , Proteína Huntingtina/química , Doença de Huntington/complicações , Doença de Huntington/fisiopatologia , Masculino , Camundongos , Músculo Esquelético/patologia , Atrofia Muscular/complicações , Atrofia Muscular/prevenção & controle , Agregados Proteicos/efeitos dos fármacosRESUMO
Age of Huntington's disease (HD) motoric onset is strongly related to the number of CAG trinucleotide repeats in the huntingtin gene, suggesting that biological tissue age plays an important role in disease etiology. Recently, a DNA methylation based biomarker of tissue age has been advanced as an epigenetic aging clock. We sought to inquire if HD is associated with an accelerated epigenetic age. DNA methylation data was generated for 475 brain samples from various brain regions of 26 HD cases and 39 controls. Overall, brain regions from HD cases exhibit a significant epigenetic age acceleration effect (p=0.0012). A multivariate model analysis suggests that HD status increases biological age by 3.2 years. Accelerated epigenetic age can be observed in specific brain regions (frontal lobe, parietal lobe, and cingulate gyrus). After excluding controls, we observe a negative correlation (r=-0.41, p=5.5×10-8) between HD gene CAG repeat length and the epigenetic age of HD brain samples. Using correlation network analysis, we identify 11 co-methylation modules with a significant association with HD status across 3 broad cortical regions. In conclusion, HD is associated with an accelerated epigenetic age of specific brain regions and more broadly with substantial changes in brain methylation levels.
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Envelhecimento/genética , Encéfalo/metabolismo , Metilação de DNA , Doença de Huntington/genética , Adolescente , Adulto , Fatores Etários , Idade de Início , Idoso , Envelhecimento/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
To gain insight into how mutant huntingtin (mHtt) CAG repeat length modifies Huntington's disease (HD) pathogenesis, we profiled mRNA in over 600 brain and peripheral tissue samples from HD knock-in mice with increasing CAG repeat lengths. We found repeat length-dependent transcriptional signatures to be prominent in the striatum, less so in cortex, and minimal in the liver. Coexpression network analyses revealed 13 striatal and 5 cortical modules that correlated highly with CAG length and age, and that were preserved in HD models and sometimes in patients. Top striatal modules implicated mHtt CAG length and age in graded impairment in the expression of identity genes for striatal medium spiny neurons and in dysregulation of cyclic AMP signaling, cell death and protocadherin genes. We used proteomics to confirm 790 genes and 5 striatal modules with CAG length-dependent dysregulation at the protein level, and validated 22 striatal module genes as modifiers of mHtt toxicities in vivo.
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Redes Reguladoras de Genes/genética , Genômica/métodos , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Proteômica/métodos , Animais , Córtex Cerebral/patologia , Córtex Cerebral/fisiologia , Corpo Estriado/patologia , Corpo Estriado/fisiologia , Feminino , Técnicas de Introdução de Genes/métodos , Proteína Huntingtina , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Rapid technological advances for the frequent monitoring of health parameters have raised the intriguing possibility that an individual's genotype could be predicted from phenotypic data alone. Here we used a machine learning approach to analyze the phenotypic effects of polymorphic mutations in a mouse model of Huntington's disease that determine disease presentation and age of onset. The resulting model correlated variation across 3,086 behavioral traits with seven different CAG-repeat lengths in the huntingtin gene (Htt). We selected behavioral signatures for age and CAG-repeat length that most robustly distinguished between mouse lines and validated the model by correctly predicting the repeat length of a blinded mouse line. Sufficient discriminatory power to accurately predict genotype required combined analysis of >200 phenotypic features. Our results suggest that autosomal dominant disease-causing mutations could be predicted through the use of subtle behavioral signatures that emerge in large-scale, combinatorial analyses. Our work provides an open data platform that we now share with the research community to aid efforts focused on understanding the pathways that link behavioral consequences to genetic variation in Huntington's disease.
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Comportamento Animal , Genoma/genética , Proteína Huntingtina/genética , Doença de Huntington/genética , Camundongos/genética , Fenótipo , Animais , Mapeamento Cromossômico/métodos , Estudo de Associação Genômica Ampla/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos/classificação , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder. The debilitating choreic movements that plague HD patients have been attributed to striatal degeneration induced by the loss of cortically supplied brain-derived neurotrophic factor (BDNF). Here, we show that in mouse models of early symptomatic HD, BDNF delivery to the striatum and its activation of tyrosine-related kinase B (TrkB) receptors were normal. However, in striatal neurons responsible for movement suppression, TrkB receptors failed to properly engage postsynaptic signaling mechanisms controlling the induction of potentiation at corticostriatal synapses. Plasticity was rescued by inhibiting p75 neurotrophin receptor (p75NTR) signaling or its downstream target phosphatase-and-tensin-homolog-deleted-on-chromosome-10 (PTEN). Thus, corticostriatal synaptic dysfunction early in HD is attributable to a correctable defect in the response to BDNF, not its delivery.
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Córtex Cerebral/fisiopatologia , Corpo Estriado/fisiopatologia , Modelos Animais de Doenças , Doença de Huntington/fisiopatologia , Receptor trkB/deficiência , Transdução de Sinais/genética , Animais , Córtex Cerebral/patologia , Corpo Estriado/patologia , Técnicas de Introdução de Genes , Doença de Huntington/genética , Doença de Huntington/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Receptor trkB/antagonistas & inibidores , Receptor trkB/fisiologiaRESUMO
PURPOSE: To explore the role of TWEAK in tumor growth and antitumor immune response and the activity and mechanism of RG7212, an antagonistic anti-TWEAK antibody, in tumor models. EXPERIMENTAL DESIGN: TWEAK-induced signaling and gene expression were explored in tumor cell lines and inhibition of these effects and antitumor efficacy with RG7212 treatment was assessed in human tumor xenograft-, patient-derived xenograft, and syngeneic tumor models and phase I patients. Genetic features correlated with antitumor activity were characterized. RESULTS: In tumor cell lines, TWEAK induces proliferation, survival, and NF-κB signaling and gene expression that promote tumor growth and suppress antitumor immune responses. TWEAK-inducible CD274, CCL2, CXCL-10 and -11 modulate T-cell and monocyte recruitment, T-cell activation, and macrophage differentiation. These factors and TWEAK-induced signaling were decreased, and tumor, blood, and spleen immune cell composition was altered with RG7212 treatment in mice. RG7212 inhibits tumor growth in vivo in models with TWEAK receptor, Fn14, expression, and markers of pathway activation. In phase I testing, signs of tumor shrinkage and stable disease were observed without dose-limiting toxicity. In a patient with advanced, Fn14-positive, malignant melanoma with evidence of tumor regression, proliferation markers were dramatically reduced, tumor T-cell infiltration increased, and tumor macrophage content decreased. Antitumor activity, a lack of toxicity in humans and animals and no evidence of antagonism with standard of care or targeted agents in mice, suggests that RG7212 is a promising agent for use in combination therapies in patients with Fn14-positive tumors.
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Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Neoplasias/imunologia , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Inibidores do Fator de Necrose Tumoral , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocina TWEAK , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Neoplasias/genética , Neoplasias/patologia , Carga Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Fatores de Necrose Tumoral/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A high percentage of patients with BRAF(V600E) mutant melanomas respond to the selective RAF inhibitor vemurafenib (RG7204, PLX4032) but resistance eventually emerges. To better understand the mechanisms of resistance, we used chronic selection to establish BRAF(V600E) melanoma clones with acquired resistance to vemurafenib. These clones retained the V600E mutation and no second-site mutations were identified in the BRAF coding sequence. Further characterization showed that vemurafenib was not able to inhibit extracellular signal-regulated kinase phosphorylation, suggesting pathway reactivation. Importantly, resistance also correlated with increased levels of RAS-GTP, and sequencing of RAS genes revealed a rare activating mutation in KRAS, resulting in a K117N change in the KRAS protein. Elevated levels of CRAF and phosphorylated AKT were also observed. In addition, combination treatment with vemurafenib and either a MAP/ERK kinase (MEK) inhibitor or an AKT inhibitor synergistically inhibited proliferation of resistant cells. These findings suggest that resistance to BRAF(V600E) inhibition could occur through several mechanisms, including elevated RAS-GTP levels and increased levels of AKT phosphorylation. Together, our data implicate reactivation of the RAS/RAF pathway by upstream signaling activation as a key mechanism of acquired resistance to vemurafenib, in support of clinical studies in which combination therapy with other targeted agents are being strategized to combat resistance.