RESUMEN
BACKGROUND: Deleterious variation in the epidermal differentiation complex (EDC) on chromosome 1 is a well-known genetic determinant of atopic dermatitis (AD) and has been associated with risk of peanut allergy (PA) in population-based studies. OBJECTIVE: Our aim was to determine the effect of genetic variation in the EDC on AD trajectory and risk of PA in early life. METHODS: Genome sequencing was used to measure genetic variation in the EDC in the Learning Early about Peanut Allergy (LEAP) study participants. Association tests were done to identify gene- and variant-level predicted deleterious variation associated with AD severity by using the Scoring Atopic Dermatitis (SCORAD) tool (n = 559) at baseline and each follow-up visit, as well as PA and food allergy in peanut avoiders (n = 275). Predicted deleterious variants included missense variants that were frameshift insertions, frameshift deletions, stop-gain mutations, or stop-loss mutations. Associations between variant load, SCORAD score, and PA were tested by using linear and generalized linear regression models. RESULTS: The genes FLG, FLG2, HRNR, and TCHH1 harbored the most predicted deleterious variation (30, 6, 3, and 1 variant, respectively). FLG variants were associated with SCORAD score at all time points; 4 variants (R1798X, R501X, S126X, and S761fs) drove the association with SCORAD score at each time point, and higher variant load was associated with greater AD severity over time. There was an association between these variants and PA, which remained significant independent of baseline AD severity (odds ratio = 2.63 [95% CI = 1.11-6.01] [P = .02]). CONCLUSIONS: Variation in FLG predicted to be deleterious is associated with AD severity at baseline and longitudinally and has an association with PA independent of baseline severity.
Asunto(s)
Dermatitis Atópica , Hipersensibilidad al Cacahuete , Humanos , Hipersensibilidad al Cacahuete/genética , Dermatitis Atópica/genética , Mutación del Sistema de Lectura , Mutación , Arachis/genéticaRESUMEN
BACKGROUND: Genetic ancestry plays a role in asthma health disparities. OBJECTIVE: Our aim was to evaluate the impact of ancestry on and identify genetic variants associated with asthma, total serum IgE level, and lung function. METHODS: A total of 436 Peruvian children (aged 9-19 years) with asthma and 291 without asthma were genotyped by using the Illumina Multi-Ethnic Global Array. Genome-wide proportions of indigenous ancestry populations from continental America (NAT) and European ancestry from the Iberian populations in Spain (IBS) were estimated by using ADMIXTURE. We assessed the relationship between ancestry and the phenotypes and performed a genome-wide association study. RESULTS: The mean ancestry proportions were 84.7% NAT (case patients, 84.2%; controls, 85.4%) and 15.3% IBS (15.8%; 14.6%). With adjustment for asthma, NAT was associated with higher total serum IgE levels (P < .001) and IBS was associated with lower total serum IgE levels (P < .001). NAT was associated with higher FEV1 percent predicted values (P < .001), whereas IBS was associated with lower FEV1 values in the controls but not in the case patients. The HLA-DR/DQ region on chromosome 6 (Chr6) was strongly associated with total serum IgE (rs3135348; P = 3.438 × 10-10) and was independent of an association with the haplotype HLA-DQA1â¼HLA-DQB1:04.01â¼04.02 (P = 1.55 × 10-05). For lung function, we identified a locus (rs4410198; P = 5.536 × 10-11) mapping to Chr19, near a cluster of zinc finger interacting genes that colocalizes to the long noncoding RNA CTD-2537I9.5. This novel locus was replicated in an independent sample of pediatric case patients with asthma with similar admixture from Brazil (P = .005). CONCLUSION: This study confirms the role of HLA in atopy, and identifies a novel locus mapping to a long noncoding RNA for lung function that may be specific to children with NAT.
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Asma/genética , Genotipo , Inmunoglobulina E/metabolismo , Pueblos Indígenas , Pulmón/metabolismo , Adolescente , Américas , Asma/epidemiología , Niño , Estudios de Cohortes , Femenino , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Antígenos HLA-DQ/metabolismo , Humanos , Pulmón/inmunología , Masculino , Perú/epidemiología , Polimorfismo de Nucleótido Simple , ARN Largo no Codificante/genética , España , Adulto JovenRESUMEN
BACKGROUND: Eczema herpeticum (EH) is a rare complication of atopic dermatitis (AD) caused by disseminated herpes simplex virus (HSV) infection. The role of rare and/or deleterious genetic variants in disease etiology is largely unknown. This study aimed to identify genes that harbor damaging genetic variants associated with HSV infection in AD with a history of recurrent eczema herpeticum (ADEH+). METHODS: Whole genome sequencing (WGS) was performed on 49 recurrent ADEH+ (≥3 EH episodes), 491 AD without a history of eczema herpeticum (ADEH-) and 237 non-atopic control (NA) subjects. Variants were annotated, and a gene-based approach (SKAT-O) was used to identify genes harboring damaging genetic variants associated with ADEH+. Genes identified through WGS were studied for effects on HSV responses and keratinocyte differentiation. RESULTS: Eight genes were identified in the comparison of recurrent ADEH+to ADEH-and NA subjects: SIDT2, CLEC7A, GSTZ1, TPSG1, SP110, RBBP8NL, TRIM15, and FRMD3. Silencing SIDT2 and RBBP8NL in normal human primary keratinocytes (NHPKs) led to significantly increased HSV-1 replication. SIDT2-silenced NHPKs had decreased gene expression of IFNk and IL1b in response to HSV-1 infection. RBBP8NL-silenced NHPKs had decreased gene expression of IFNk, but increased IL1b. Additionally, silencing SIDT2 and RBBP8NL also inhibited gene expression of keratinocyte differentiation markers keratin 10 (KRT10) and loricrin (LOR). CONCLUSION: SIDT2 and RBBP8NL participate in keratinocyte's response to HSV-1 infection. SIDT2 and RBBP8NL also regulate expression of keratinocyte differentiation genes of KRT10 and LOR.
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Dermatitis Atópica , Herpesvirus Humano 1 , Erupción Variceliforme de Kaposi , Proteínas de Transporte de Nucleótidos , Dermatitis Atópica/genética , Glutatión Transferasa , Herpesvirus Humano 1/genética , Humanos , Erupción Variceliforme de Kaposi/genética , Mutación , Secuenciación Completa del GenomaRESUMEN
Lymphatic endothelial cells (LECs) of the lymph node (LN) parenchyma orchestrate leukocyte trafficking and peripheral T cell dynamics. T cell responses to immunotherapy largely rely on peripheral T cell recruitment in tumors. Yet, a systematic and molecular understanding of how LECs within the LNs control T cell dynamics under steady-state and tumor-bearing conditions is lacking. Intravital imaging combined with immune phenotyping shows that LEC-specific deletion of the essential autophagy gene Atg5 alters intranodal positioning of lymphocytes and accrues their persistence in the LNs by increasing the availability of the main egress signal sphingosine-1-phosphate. Single-cell RNA sequencing of tumor-draining LNs shows that loss of ATG5 remodels niche-specific LEC phenotypes involved in molecular pathways regulating lymphocyte trafficking and LEC-T cell interactions. Functionally, loss of LEC autophagy prevents recruitment of tumor-infiltrating T and natural killer cells and abrogates response to immunotherapy. Thus, an LEC-autophagy program boosts immune-checkpoint responses by guiding systemic T cell dynamics.
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Autofagia , Inhibidores de Puntos de Control Inmunológico , Ganglios Linfáticos , Esfingosina/análogos & derivados , Linfocitos T , Autofagia/efectos de los fármacos , Animales , Ganglios Linfáticos/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Ratones , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ratones Endogámicos C57BL , Proteína 5 Relacionada con la Autofagia/metabolismo , Proteína 5 Relacionada con la Autofagia/genética , Células Endoteliales/metabolismo , Esfingosina/farmacología , Esfingosina/metabolismo , Humanos , Lisofosfolípidos/metabolismo , Inmunoterapia/métodos , Movimiento CelularRESUMEN
An important challenge in the real-world management of patients with advanced clear-cell renal cell carcinoma (aRCC) is determining who might benefit from immune checkpoint blockade (ICB). Here we performed a comprehensive multiomics mapping of aRCC in the context of ICB treatment, involving discovery analyses in a real-world data cohort followed by validation in independent cohorts. We cross-connected bulk-tumor transcriptomes across >1,000 patients with validations at single-cell and spatial resolutions, revealing a patient-specific crosstalk between proinflammatory tumor-associated macrophages and (pre-)exhausted CD8+ T cells that was distinguished by a human leukocyte antigen repertoire with higher preference for tumoral neoantigens. A cross-omics machine learning pipeline helped derive a new tumor transcriptomic footprint of neoantigen-favoring human leukocyte antigen alleles. This machine learning signature correlated with positive outcome following ICB treatment in both real-world data and independent clinical cohorts. In experiments using the RENCA-tumor mouse model, CD40 agonism combined with PD1 blockade potentiated both proinflammatory tumor-associated macrophages and CD8+ T cells, thereby achieving maximal antitumor efficacy relative to other tested regimens. Thus, we present a new multiomics and spatial map of the immune-community architecture that drives ICB response in patients with aRCC.
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Linfocitos T CD8-positivos , Carcinoma de Células Renales , Antígenos HLA , Inmunoterapia , Neoplasias Renales , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/terapia , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Humanos , Neoplasias Renales/inmunología , Neoplasias Renales/terapia , Neoplasias Renales/genética , Neoplasias Renales/patología , Animales , Inmunoterapia/métodos , Linfocitos T CD8-positivos/inmunología , Ratones , Antígenos HLA/inmunología , Antígenos HLA/genética , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/farmacología , Aprendizaje Automático , Antígenos CD40/inmunología , Antígenos CD40/genética , Macrófagos Asociados a Tumores/inmunología , Transcriptoma , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , FemeninoRESUMEN
BACKGROUND: Uterine leiomyosarcomas (uLMS) are aggressive tumours with poor prognosis and limited treatment options. Although immune checkpoint blockade (ICB) has proven effective in some 'challenging-to-treat' cancers, clinical trials showed that uLMS do not respond to ICB. Emerging evidence suggests that aberrant PI3K/mTOR signalling can drive resistance to ICB. We therefore explored the relevance of the PI3K/mTOR pathway for ICB treatment in uLMS and explored pharmacological inhibition of this pathway to sensitise these tumours to ICB. METHODS: We performed an integrated multiomics analysis based on TCGA data to explore the correlation between PI3K/mTOR dysregulation and immune infiltration in 101 LMS. We assessed response to PI3K/mTOR inhibitors in immunodeficient and humanized uLMS patient-derived xenografts (PDXs) by evaluating tumour microenvironment modulation using multiplex immunofluorescence. We explored response to single-agent and a combination of PI3K/mTOR inhibitors with PD-1 blockade in humanized uLMS PDXs. We mapped intratumoural dynamics using single-cell RNA/TCR sequencing of serially collected biopsies. RESULTS: PI3K/mTOR over-activation (pS6high) associated with lymphocyte depletion and wound healing immune landscapes in (u)LMS, suggesting it contributes to immune evasion. In contrast, PI3K/mTOR inhibition induced profound tumour microenvironment remodelling in an ICB-resistant humanized uLMS PDX model, fostering adaptive anti-tumour immune responses. Indeed, PI3K/mTOR inhibition induced macrophage repolarisation towards an anti-tumourigenic phenotype and increased antigen presentation on dendritic and tumour cells, but also promoted infiltration of PD-1+ T cells displaying an exhausted phenotype. When combined with anti-PD-1, PI3K/mTOR inhibition led to partial or complete tumour responses, whereas no response to single-agent anti-PD-1 was observed. Combination therapy reinvigorated exhausted T cells and induced clonal hyper-expansion of a cytotoxic CD8+ T-cell population supported by a CD4+ Th1 niche. CONCLUSIONS: Our findings indicate that aberrant PI3K/mTOR pathway activation contributes to immune escape in uLMS and provides a rationale for combining PI3K/mTOR inhibition with ICB for the treatment of this patient population.
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Leiomiosarcoma , Microambiente Tumoral , Neoplasias Uterinas , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Leiomiosarcoma/tratamiento farmacológico , Humanos , Femenino , Neoplasias Uterinas/tratamiento farmacológico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inhibidores mTOR/farmacología , Inhibidores mTOR/uso terapéutico , Animales , Ratones , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3/uso terapéuticoRESUMEN
Asthma has striking disparities across ancestral groups, but the molecular underpinning of these differences is poorly understood and minimally studied. A goal of the Consortium on Asthma among African-ancestry Populations in the Americas (CAAPA) is to understand multi-omic signatures of asthma focusing on populations of African ancestry. RNASeq and DNA methylation data are generated from nasal epithelium including cases (current asthma, N = 253) and controls (never-asthma, N = 283) from 7 different geographic sites to identify differentially expressed genes (DEGs) and gene networks. We identify 389 DEGs; the top DEG, FN1, was downregulated in cases (q = 3.26 × 10-9) and encodes fibronectin which plays a role in wound healing. The top three gene expression modules implicate networks related to immune response (CEACAM5; p = 9.62 × 10-16 and CPA3; p = 2.39 × 10-14) and wound healing (FN1; p = 7.63 × 10-9). Multi-omic analysis identifies FKBP5, a co-chaperone of glucocorticoid receptor signaling known to be involved in drug response in asthma, where the association between nasal epithelium gene expression is likely regulated by methylation and is associated with increased use of inhaled corticosteroids. This work reveals molecular dysregulation on three axes - increased Th2 inflammation, decreased capacity for wound healing, and impaired drug response - that may play a critical role in asthma within the African Diaspora.
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Asma , Población Negra , Metilación de ADN , Mucosa Nasal , Proteínas de Unión a Tacrolimus , Humanos , Asma/genética , Asma/metabolismo , Mucosa Nasal/metabolismo , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismo , Femenino , Masculino , Población Negra/genética , Adulto , Redes Reguladoras de Genes , Fibronectinas/metabolismo , Fibronectinas/genética , Estudios de Casos y Controles , Regulación de la Expresión Génica , Persona de Mediana Edad , MultiómicaRESUMEN
Glioblastoma (GBM) remains the most malignant primary brain tumor, with a median survival rarely exceeding 2 years. Tumor heterogeneity and an immunosuppressive microenvironment are key factors contributing to the poor response rates of current therapeutic approaches. GBM-associated macrophages (GAMs) often exhibit immunosuppressive features that promote tumor progression. However, their dynamic interactions with GBM tumor cells remain poorly understood. Here, we used patient-derived GBM stem cell cultures and combined single-cell RNA sequencing of GAM-GBM co-cultures and real-time in vivo monitoring of GAM-GBM interactions in orthotopic zebrafish xenograft models to provide insight into the cellular, molecular, and spatial heterogeneity. Our analyses revealed substantial heterogeneity across GBM patients in GBM-induced GAM polarization and the ability to attract and activate GAMs-features that correlated with patient survival. Differential gene expression analysis, immunohistochemistry on original tumor samples, and knock-out experiments in zebrafish subsequently identified LGALS1 as a primary regulator of immunosuppression. Overall, our work highlights that GAM-GBM interactions can be studied in a clinically relevant way using co-cultures and avatar models, while offering new opportunities to identify promising immune-modulating targets.
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Neoplasias Encefálicas , Glioblastoma , Animales , Humanos , Glioblastoma/patología , Pez Cebra , Galectina 1/genética , Galectina 1/metabolismo , Galectina 1/uso terapéutico , Línea Celular Tumoral , Macrófagos/metabolismo , Neoplasias Encefálicas/patología , Microambiente Tumoral/genéticaRESUMEN
Tumor endothelial cells (TECs) actively repress inflammatory responses and maintain an immune-excluded tumor phenotype. However, the molecular mechanisms that sustain TEC-mediated immunosuppression remain largely elusive. Here, we show that autophagy ablation in TECs boosts antitumor immunity by supporting infiltration and effector function of T-cells, thereby restricting melanoma growth. In melanoma-bearing mice, loss of TEC autophagy leads to the transcriptional expression of an immunostimulatory/inflammatory TEC phenotype driven by heightened NF-kB and STING signaling. In line, single-cell transcriptomic datasets from melanoma patients disclose an enriched InflammatoryHigh /AutophagyLow TEC phenotype in correlation with clinical responses to immunotherapy, and responders exhibit an increased presence of inflamed vessels interfacing with infiltrating CD8+ T-cells. Mechanistically, STING-dependent immunity in TECs is not critical for the immunomodulatory effects of autophagy ablation, since NF-kB-driven inflammation remains functional in STING/ATG5 double knockout TECs. Hence, our study identifies autophagy as a principal tumor vascular anti-inflammatory mechanism dampening melanoma antitumor immunity.
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Melanoma , Humanos , Ratones , Animales , Melanoma/patología , Células Endoteliales/metabolismo , Linfocitos T CD8-positivos , FN-kappa B/metabolismo , Autofagia , Inmunoterapia , Microambiente TumoralRESUMEN
We investigated the interplay between genetics and oral peanut protein exposure in the determination of the immunological response to peanut using the targeted intervention in the LEAP clinical trial. We identified an association between peanut-specific IgG4 and HLA-DQA1*01:02 that was only observed in the presence of sustained oral peanut protein exposure. The association between IgG4 and HLA-DQA1*01:02 was driven by IgG4 specific for the Ara h 2 component. Once peanut consumption ceased, the association between IgG4-specific Ara h 2 and HLA-DQA1*01:02 was attenuated. The association was validated by observing expanded IgG4-specific epitopes in people who carried HLA-DQA1*01:02. Notably, we confirmed the previously reported associations with HLA-DQA1*01:02 and peanut allergy risk in the absence of oral peanut protein exposure. Interaction between HLA and presence or absence of exposure to peanut in an allergen- and epitope-specific manner implicates a mechanism of antigen recognition that is fundamental to driving immune responses related to allergy risk or protection.
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Albuminas 2S de Plantas/inmunología , Alelos , Formación de Anticuerpos , Antígenos de Plantas/inmunología , Arachis , Cadenas alfa de HLA-DQ , Inmunoglobulina G/inmunología , Hipersensibilidad al Cacahuete , Formación de Anticuerpos/genética , Formación de Anticuerpos/inmunología , Niño , Femenino , Cadenas alfa de HLA-DQ/genética , Cadenas alfa de HLA-DQ/inmunología , Humanos , Masculino , Hipersensibilidad al Cacahuete/genética , Hipersensibilidad al Cacahuete/inmunologíaRESUMEN
Rationale: Previous studies identified an interaction between HLA and oral peanut exposure. HLA-DQA1*01:02 had a protective role with the induction of Ara h 2 epitope-specific IgG4 associated with peanut consumption during the LEAP clinical trial for prevention of peanut allergy, while it was a risk allele for peanut allergy in the peanut avoidance group. We have now evaluated this gene-environment interaction in two subsequent peanut oral immunotherapy (OIT) trials - IMPACT and POISED - to better understand the potential for the HLA-DQA1*01:02 allele as an indicator of higher likelihood of desensitization, sustained unresponsiveness, and peanut allergy remission. Methods: We determined HLA-DQA1*01:02 carrier status using genome sequencing from POISED (N=118, age: 7-55yr) and IMPACT (N=126, age: 12-<48mo). We tested for association with remission, sustained unresponsiveness (SU), and desensitization in the OIT groups, as well as peanut component specific IgG4 (psIgG4) using generalized linear models and adjusting for relevant covariates and ancestry. Results: While not quite statistically significant, a higher proportion of HLA-DQA1*01:02 carriers receiving OIT in IMPACT were desensitized (93%) compared to non-carriers (78%); odds ratio (OR)=5.74 (p=0.06). In this sample we also observed that a higher proportion of carriers achieved remission (35%) compared to non-carriers (22%); OR=1.26 (p=0.80). In POISED, carriers more frequently attained continued desensitization (80% versus 61% among non-carriers; OR=1.28, p=0.86) and achieved SU (52% versus 31%; OR=2.32, p=0.19). psIgG4 associations with HLA-DQA1*01:02 in the OIT arm of IMPACT which included younger study subjects recapitulated patterns noted in LEAP, but no associations of note were observed in the older POISED study subjects. Conclusions: Findings across three clinical trials show a pattern of a gene environment interaction between HLA and oral peanut exposure. Age, and prior sensitization contribute additional determinants of outcomes, consistent with a mechanism of restricted antigen recognition fundamental to driving protective immune responses to OIT.
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Arachis , Hipersensibilidad al Cacahuete , Adolescente , Adulto , Niño , Humanos , Persona de Mediana Edad , Adulto Joven , Inmunoglobulina G , Factores Inmunológicos , Inmunoterapia , Hipersensibilidad al Cacahuete/genética , Hipersensibilidad al Cacahuete/terapia , Ensayos Clínicos como AsuntoRESUMEN
Tumor heterogeneity and lack of knowledge about resistant cell states remain a barrier to targeted cancer therapies. Basal cell carcinomas (BCCs) depend on Hedgehog (Hh)/Gli signaling, but can develop mechanisms of Smoothened (SMO) inhibitor resistance. We previously identified a nuclear myocardin-related transcription factor (nMRTF) resistance pathway that amplifies noncanonical Gli1 activity, but characteristics and drivers of the nMRTF cell state remain unknown. Here, we use single cell RNA-sequencing of patient tumors to identify three prognostic surface markers (LYPD3, TACSTD2, and LY6D) which correlate with nMRTF and resistance to SMO inhibitors. The nMRTF cell state resembles transit-amplifying cells of the hair follicle matrix, with AP-1 and TGFß cooperativity driving nMRTF activation. JNK/AP-1 signaling commissions chromatin accessibility and Smad3 DNA binding leading to a transcriptional program of RhoGEFs that facilitate nMRTF activity. Importantly, small molecule AP-1 inhibitors selectively target LYPD3+/TACSTD2+/LY6D+ nMRTF human BCCs ex vivo, opening an avenue for improving combinatorial therapies.
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Carcinoma Basocelular/metabolismo , Proteínas Hedgehog/metabolismo , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Factor de Transcripción AP-1/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/metabolismo , ADN de Neoplasias/metabolismo , Resistencia a Antineoplásicos , Matriz Extracelular/metabolismo , Ontología de Genes , Factores de Intercambio de Guanina Nucleótido/metabolismo , Folículo Piloso/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Proteínas de Neoplasias/metabolismo , Unión Proteica , Proteína smad3/metabolismo , Transactivadores/metabolismo , Regulación hacia ArribaRESUMEN
Basal cell carcinomas (BCCs) rely on Hedgehog (HH) pathway growth signal amplification by the microtubule-based organelle, the primary cilium. Despite naive tumor responsiveness to Smoothened inhibitors (Smoi), resistance in advanced tumors remains common. Although the resistant BCCs usually maintain HH pathway activation, squamous cell carcinomas with Ras/MAPK pathway activation also arise, and the molecular basis of tumor type and pathway selection are still obscure. Here, we identify the primary cilium as a critical determinant controlling tumor pathway switching. Strikingly, Smoothened inhibitor-resistant BCCs have an increased mutational load in ciliome genes, resulting in reduced primary cilia and HH pathway activation compared with naive or Gorlin syndrome patient BCCs. Gene set enrichment analysis of resistant BCCs with a low HH pathway signature showed increased Ras/MAPK pathway activation. Tissue analysis confirmed an inverse relationship between primary cilia presence and Ras/MAPK activation, and primary cilia removal in BCCs potentiated Ras/MAPK pathway activation. Moreover, activating Ras in HH-responsive cell lines conferred resistance to both canonical (vismodegib) and noncanonical (atypical protein kinase C and MRTF inhibitors) HH pathway inhibitors and conferred sensitivity to MAPK inhibitors. Our results provide insights into BCC treatment and identify the primary cilium as an important lineage gatekeeper, preventing HH-to-Ras/MAPK pathway switching.
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Síndrome del Nevo Basocelular/metabolismo , Carcinoma Basocelular/metabolismo , Cilios/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Hedgehog/metabolismo , Neoplasias Cutáneas/metabolismo , Proteínas ras/metabolismo , Anilidas/uso terapéutico , Antineoplásicos/uso terapéutico , Síndrome del Nevo Basocelular/genética , Síndrome del Nevo Basocelular/patología , Carcinogénesis , Carcinoma Basocelular/genética , Carcinoma Basocelular/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Proteínas Hedgehog/antagonistas & inhibidores , Humanos , Mutación/genética , Piridinas/uso terapéutico , Transducción de Señal , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patologíaRESUMEN
Tissue development results from lineage-specific transcription factors (TFs) programming a dynamic chromatin landscape through progressive cell fate transitions. Here, we define epigenomic landscape during epidermal differentiation of human pluripotent stem cells (PSCs) and create inference networks that integrate gene expression, chromatin accessibility, and TF binding to define regulatory mechanisms during keratinocyte specification. We found two critical chromatin networks during surface ectoderm initiation and keratinocyte maturation, which are driven by TFAP2C and p63, respectively. Consistently, TFAP2C, but not p63, is sufficient to initiate surface ectoderm differentiation, and TFAP2C-initiated progenitor cells are capable of maturing into functional keratinocytes. Mechanistically, TFAP2C primes the surface ectoderm chromatin landscape and induces p63 expression and binding sites, thus allowing maturation factor p63 to positively autoregulate its own expression and close a subset of the TFAP2C-initiated surface ectoderm program. Our work provides a general framework to infer TF networks controlling chromatin transitions that will facilitate future regenerative medicine advances.
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Linaje de la Célula , Cromatina/metabolismo , Epidermis/metabolismo , Redes Reguladoras de Genes , Factor de Transcripción AP-2/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Diferenciación Celular , Ectodermo/citología , Epigénesis Genética , Retroalimentación Fisiológica , Humanos , Queratinocitos/citología , Transcriptoma/genéticaRESUMEN
Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.
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Cromatina/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Humanos , Conformación MolecularRESUMEN
Hedgehog pathway-dependent cancers can escape Smoothened (SMO) inhibition through mutations in genes encoding canonical hedgehog pathway components; however, around 50% of drug-resistant basal cell carcinomas (BCCs) lack additional variants of these genes. Here we use multidimensional genomics analysis of human and mouse drug-resistant BCCs to identify a noncanonical hedgehog activation pathway driven by the transcription factor serum response factor (SRF). Active SRF along with its coactivator megakaryoblastic leukemia 1 (MKL1) binds DNA near hedgehog target genes and forms a previously unknown protein complex with the hedgehog transcription factor glioma-associated oncogene family zinc finger-1 (GLI1), causing amplification of GLI1 transcriptional activity. We show that cytoskeletal activation through Rho and the formin family member Diaphanous (mDia) is required for SRF-MKL-driven GLI1 activation and for tumor cell viability. Remarkably, nuclear MKL1 staining served as a biomarker in tumors from mice and human subjects to predict tumor responsiveness to MKL inhibitors, highlighting the therapeutic potential of targeting this pathway. Thus, our study illuminates, for the first time, cytoskeletal-activation-driven transcription as a personalized therapeutic target for combatting drug-resistant malignancies.
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Carcinoma Basocelular/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Factor de Respuesta Sérica/genética , Transactivadores/genética , Proteína con Dedos de Zinc GLI1/genética , Animales , Carcinoma Basocelular/genética , Carcinoma Basocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Proteínas Hedgehog , Humanos , Ratones , Complejos Multiproteicos/genética , Transducción de Señal , Activación TranscripcionalRESUMEN
Human embryonic stem cell (hESC) differentiation promises advances in regenerative medicine1-3, yet conversion of hESCs into transplantable cells or tissues remains poorly understood. Using our keratinocyte differentiation system, we employ a multi-dimensional genomics approach to interrogate the contributions of inductive morphogens retinoic acid and bone morphogenetic protein 4 (BMP4) and the epidermal master regulator p63 (encoded by TP63)4,5 during surface ectoderm commitment. In contrast to other master regulators6-9, p63 effects major transcriptional changes only after morphogens alter chromatin accessibility, establishing an epigenetic landscape for p63 to modify. p63 distally closes chromatin accessibility and promotes accumulation of H3K27me3 (trimethylated histone H3 lysine 27). Cohesin HiChIP10 visualizations of chromosome conformation show that p63 and the morphogens contribute to dynamic long-range chromatin interactions, as illustrated by TFAP2C regulation11. Our study demonstrates the unexpected dependency of p63 on morphogenetic signaling and provides novel insights into how a master regulator can specify diverse transcriptional programs based on the chromatin landscape induced by exposure to specific morphogens.
Asunto(s)
Proteína Morfogenética Ósea 4/farmacología , Diferenciación Celular , Ensamble y Desensamble de Cromatina , Queratinocitos/fisiología , Factores de Transcripción/fisiología , Tretinoina/farmacología , Proteínas Supresoras de Tumor/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Cromatina/efectos de los fármacos , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/genética , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/fisiología , Epidermis/efectos de los fármacos , Epidermis/fisiología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Queratinocitos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Royal jelly is the queen-maker for the honey bee Apis mellifera, and has cross-species effects on longevity, fertility, and regeneration in mammals. Despite this knowledge, how royal jelly or its components exert their myriad effects has remained poorly understood. Using mouse embryonic stem cells as a platform, here we report that through its major protein component Royalactin, royal jelly can maintain pluripotency by activating a ground-state pluripotency-like gene network. We further identify Regina, a mammalian structural analog of Royalactin that also induces a naive-like state in mouse embryonic stem cells. This reveals an important innate program for stem cell self-renewal with broad implications in understanding the molecular regulation of stem cell fate across species.