RESUMEN
Inferring which and how biological pathways and gene sets change is a key question in many studies that utilize single-cell RNA sequencing. Typically, these questions are addressed by quantifying the enrichment of known gene sets in lists of genes derived from global analysis. Here we offer SiPSiC, a new method to infer pathway activity in every single cell. This allows more sensitive differential analysis and utilization of pathway scores to cluster cells and compute UMAP or other similar projections. We apply our method to COVID-19, lung adenocarcinoma and glioma data sets, and demonstrate its utility. SiPSiC analysis results are consistent with findings reported in previous studies in many cases, but SiPSiC also reveals the differential activity of novel pathways, enabling us to suggest new mechanisms underlying the pathophysiology of these diseases and demonstrating SiPSiC's high accuracy and sensitivity in detecting biological function and traits. In addition, we demonstrate how it can be used to better classify cells based on activity of biological pathways instead of single genes and its ability to overcome patient-specific artifacts.
Asunto(s)
COVID-19 , Neoplasias Pulmonares , Análisis de la Célula Individual , Humanos , Análisis de la Célula Individual/métodos , COVID-19/virología , COVID-19/genética , Análisis por Conglomerados , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , SARS-CoV-2/genética , Glioma/genética , Glioma/patología , Glioma/metabolismo , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/metabolismo , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ARN/métodosRESUMEN
Structural maintenance of chromosomes flexible hinge domain-containing 1 (SMCHD1) is a noncanonical SMC protein and an epigenetic regulator. Mutations in SMCHD1 cause facioscapulohumeral muscular dystrophy (FSHD), by overexpressing DUX4 in muscle cells. Here, we demonstrate that SMCHD1 is a key regulator of alternative splicing in various cell types. We show how SMCHD1 loss causes splicing alterations of DNMT3B, which can lead to hypomethylation and DUX4 overexpression. Analyzing RNA sequencing data from muscle biopsies of patients with FSHD and Smchd1 knocked out cells, we found mis-splicing of hundreds of genes upon SMCHD1 loss. We conducted a high-throughput screen of splicing factors, revealing the involvement of the splicing factor RBM5 in the mis-splicing of DNMT3B. Subsequent RNA immunoprecipitation experiments confirmed that SMCHD1 is required for RBM5 recruitment. Last, we show that mis-splicing of DNMT3B leads to hypomethylation of the D4Z4 region and to DUX4 overexpression. These results suggest that DNMT3B mis-splicing due to SMCHD1 loss plays a major role in FSHD pathogenesis.
Asunto(s)
Proteínas Cromosómicas no Histona , ADN (Citosina-5-)-Metiltransferasas , Metilación de ADN , ADN Metiltransferasa 3B , Proteínas de Homeodominio , Distrofia Muscular Facioescapulohumeral , Humanos , Empalme Alternativo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Distrofia Muscular Facioescapulohumeral/genética , Distrofia Muscular Facioescapulohumeral/metabolismo , Distrofia Muscular Facioescapulohumeral/patología , Empalme del ARN , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Epigenetic defects caused by hereditary or de novo mutations are implicated in various human diseases. It remains uncertain whether correcting the underlying mutation can reverse these defects in patient cells. Here we show by the analysis of myotonic dystrophy type 1 (DM1)-related locus that in mutant human embryonic stem cells (hESCs), DNA methylation and H3K9me3 enrichments are completely abolished by repeat excision (CTG2000 expansion), whereas in patient myoblasts (CTG2600 expansion), repeat deletion fails to do so. This distinction between undifferentiated and differentiated cells arises during cell differentiation, and can be reversed by reprogramming of gene-edited myoblasts. We demonstrate that abnormal methylation in DM1 is distinctively maintained in the undifferentiated state by the activity of the de novo DNMTs (DNMT3b in tandem with DNMT3a). Overall, the findings highlight a crucial difference in heterochromatin maintenance between undifferentiated (sequence-dependent) and differentiated (sequence-independent) cells, thus underscoring the role of differentiation as a locking mechanism for repressive epigenetic modifications at the DM1 locus.
Asunto(s)
Distrofia Miotónica , Humanos , Distrofia Miotónica/genética , Heterocromatina/genética , Diferenciación Celular/genética , Metilación de ADN , Epigénesis GenéticaRESUMEN
Senescent cells can influence the function of tissues in which they reside, and their propensity for disease. A portion of adult human pancreatic beta cells express the senescence marker p16, yet it is unclear whether they are in a senescent state, and how this affects insulin secretion. We analyzed single-cell transcriptome datasets of adult human beta cells, and found that p16-positive cells express senescence gene signatures, as well as elevated levels of beta-cell maturation genes, consistent with enhanced functionality. Senescent human beta-like cells in culture undergo chromatin reorganization that leads to activation of enhancers regulating functional maturation genes and acquisition of glucose-stimulated insulin secretion capacity. Strikingly, Interferon-stimulated genes are elevated in senescent human beta cells, but genes encoding senescence-associated secretory phenotype (SASP) cytokines are not. Senescent beta cells in culture and in human tissue show elevated levels of cytoplasmic DNA, contributing to their increased interferon responsiveness. Human beta-cell senescence thus involves chromatin-driven upregulation of a functional-maturation program, and increased responsiveness of interferon-stimulated genes, changes that could increase both insulin secretion and immune reactivity.
Asunto(s)
Senescencia Celular , Ensamble y Desensamble de Cromatina , Células Secretoras de Insulina , Interferones , Humanos , Células Secretoras de Insulina/metabolismo , Senescencia Celular/genética , Interferones/metabolismo , Interferones/genética , Secreción de Insulina , Insulina/metabolismo , Cromatina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Células Cultivadas , Fenotipo Secretor Asociado a la Senescencia/genética , Transcriptoma , Análisis de la Célula IndividualRESUMEN
Osteosarcoma is an aggressive bone tumor that primarily affects children and adolescents. This malignancy is highly aggressive, associated with poor clinical outcomes, and primarily metastasizes to the lungs. Due to its rarity and biological heterogeneity, limited studies on its molecular basis exist, hindering the development of effective therapies. The WW domain-containing oxidoreductase (WWOX) is frequently altered in human osteosarcoma. Combined deletion of Wwox and Trp53 using Osterix1-Cre transgenic mice has been shown to accelerate osteosarcoma development. In this study, we generated a traceable osteosarcoma mouse model harboring the deletion of Trp53 alone (single-knockout) or combined deletion of Wwox/Trp53 (double-knockout) and expressing a tdTomato reporter. By tracking Tomato expression at different time points, we detected the early presence of tdTomato-positive cells in the bone marrow mesenchymal stem cells of non-osteosarcoma-bearing mice (young BM). We found that double-knockout young BM cells, but not single-knockout young BM cells, exhibited tumorigenic traits both in vitro and in vivo. Molecular and cellular characterization of these double-knockout young BM cells revealed their resemblance to osteosarcoma tumor cells. Interestingly, one of the observed significant transcriptomic changes in double-knockout young BM cells was the upregulation of Myc and its target genes compared to single-knockout young BM cells. Intriguingly, Myc-chromatin immunoprecipitation sequencing revealed its increased enrichment on Myc targets, which were upregulated in double-knockout young BM cells. Restoration of WWOX in double-knockout young BM cells reduced Myc protein levels. As a prototype target, we demonstrated the upregulation of MCM7, a known Myc target, in double-knockout young BM relative to single-knockout young BM cells. Inhibition of MCM7 expression using simvastatin resulted in reduced proliferation and tumor cell growth of double-knockout young BM cells. Our findings reveal BM mesenchymal stem cells as a platform to study osteosarcoma and Myc and its targets as WWOX effectors and early molecular events during osteosarcomagenesis.
Asunto(s)
Neoplasias Óseas , Osteosarcoma , Proteínas Proto-Oncogénicas c-myc , Oxidorreductasa que Contiene Dominios WW , Animales , Humanos , Ratones , Neoplasias Óseas/genética , Osteosarcoma/genética , Proteínas Supresoras de Tumor/genética , Regulación hacia Arriba/genética , Oxidorreductasa que Contiene Dominios WW/genética , Oxidorreductasa que Contiene Dominios WW/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Despite extensive studies on the effects of SARS-CoV-2 infection, there is still a lack of understanding of the downstream epigenetic and regulatory alterations in infected cells. In this study, we investigated changes in enhancer acetylation in epithelial lung cells infected with SARS-CoV-2 and their influence on transcriptional regulation and pathway activity. To achieve this, we integrated and reanalyzed data of enhancer acetylation, ex-vivo infection and single cell RNA-seq data from human patients. Our findings revealed coordinated changes in enhancers and transcriptional networks. We found that infected cells lose the WT1 transcription factor and demonstrate disruption of WT1-bound enhancers and of their associated target genes. Downstream targets of WT1 are involved in the regulation of the Wnt signaling and the mitogen-activated protein kinase cascade, which indeed exhibit increased activation levels. These findings may provide a potential explanation for the development of pulmonary fibrosis, a lethal complication of COVID-19. Moreover, we revealed over-acetylated enhancers associated with upregulated genes involved in cell adhesion, which could contribute to cell-cell infection of SARS-CoV-2. Furthermore, we demonstrated that enhancers may play a role in the activation of pro-inflammatory cytokines and contribute to excessive inflammation in the lungs, a typical complication of COVID-19. Overall, our analysis provided novel insights into the cell-autonomous dysregulation of enhancer regulation caused by SARS-CoV-2 infection, a step on the path to a deeper molecular understanding of the disease.
Asunto(s)
COVID-19 , Humanos , COVID-19/genética , SARS-CoV-2/genética , Regulación de la Expresión Génica , Inflamación , PulmónRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) protein 1 (TAL1) is a central transcription factor in hematopoiesis. The timing and level of TAL1 expression orchestrate the differentiation to specialized blood cells and its overexpression is a common cause of T-ALL. Here, we studied the 2 protein isoforms of TAL1, short and long, which are generated by the use of alternative promoters as well as by alternative splicing. We analyzed the expression of each isoform by deleting an enhancer or insulator, or by opening chromatin at the enhancer location. Our results show that each enhancer promotes expression from a specific TAL1 promoter. Expression from a specific promoter gives rise to a unique 5' UTR with differential regulation of translation. Moreover, our study suggests that the enhancers regulate TAL1 exon 3 alternative splicing by inducing changes in the chromatin at the splice site, which we demonstrate is mediated by KMT2B. Furthermore, our results indicate that TAL1-short binds more strongly to TAL1 E-protein partners and functions as a stronger transcription factor than TAL1-long. Specifically TAL1-short has a unique transcription signature promoting apoptosis. Finally, when we expressed both isoforms in mice bone marrow, we found that while overexpression of both isoforms prevents lymphoid differentiation, expression of TAL1-short alone leads to hematopoietic stem cell exhaustion. Furthermore, we found that TAL1-short promoted erythropoiesis and reduced cell survival in the CML cell line K562. While TAL1 and its partners are considered promising therapeutic targets in the treatment of T-ALL, our results show that TAL1-short could act as a tumor suppressor and suggest that altering TAL1 isoform's ratio could be a preferred therapeutic approach.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animales , Ratones , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cromatina , Hematopoyesis/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteína 1 de la Leucemia Linfocítica T Aguda/genética , Factores de Transcripción/metabolismoRESUMEN
Salivary adenoid cystic carcinoma (ACC) is a rare, biologically unique biphasic tumor that consists of malignant myoepithelial and luminal cells. MYB and Notch signaling have been implicated in ACC pathophysiology, but in vivo descriptions of these two programs in human tumors and investigation into their active coordination remain incomplete. We utilize single-cell RNA sequencing to profile human head and neck ACC, including a comparison of primary ACC with a matched local recurrence. We define expression heterogeneity in these rare tumors, uncovering diversity in myoepithelial and luminal cell expression. We find differential expression of Notch ligands DLL1, JAG1, and JAG2 in myoepithelial cells, suggesting a paracrine interaction that may support oncogenic Notch signaling. We validate this selective expression in three published cohorts of patients with ACC. Our data provide a potential explanation for the biphasic nature of low- and intermediate-grade ACC and may help direct new therapeutic strategies against these tumors.
Asunto(s)
Carcinoma Adenoide Quístico , Humanos , Carcinoma Adenoide Quístico/genética , Oncogenes , Carcinogénesis , Secuenciación del Exoma , Análisis de Secuencia de ARNRESUMEN
We describe a protocol for H3K27ac ChIP paired-end sequencing and computational analysis of rearrangements. Our approach can be used to simultaneously map enhancers and their activity and to identify structural variations at enhancers. Since changes in enhancer activity and new enhancer translocations both play a major role in tumor initiation, progression, and response to therapy, this approach holds promise to uncover some of the mechanisms behind these processes.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Elementos de Facilitación Genéticos , Reordenamiento Génico , Histonas/genética , Histonas/metabolismoRESUMEN
Enhancer demethylation in leukemia has been shown to lead to overexpression of genes which promote cancer characteristics. The vascular endothelial growth factor A (VEGFA) enhancer, located 157 Kb downstream of its promoter, is demethylated in chronic myeloid leukemia (CML). VEGFA has several alternative splicing isoforms with different roles in cancer progression. Since transcription and splicing are coupled, we wondered whether VEGFA enhancer activity can also regulate the gene's alternative splicing to contribute to the pathology of CML. Our results show that mutating the VEGFA +157 enhancer promotes exclusion of exons 6a and 7 and activating the enhancer by tethering a chromatin activator has the opposite effect. In line with these results, CML patients present with high expression of +157 eRNA and inclusion of VEGFA exons 6a and 7. In addition, our results show that the positive regulator of RNAPII transcription elongation, CCNT2, binds VEGFA's promoter and enhancer, and its silencing promotes exclusion of exons 6a and 7 as it slows down RNAPII elongation rate. Thus our results suggest that VEGFA's +157 enhancer regulates its alternative splicing by increasing RNAPII elongation rate via CCNT2. Our work demonstrates for the first time a connection between an endogenous enhancer and alternative splicing regulation of its target gene.
RESUMEN
Lineage plasticity and stemness have been invoked as causes of therapy resistance in cancer, because these flexible states allow cancer cells to dedifferentiate and alter their dependencies. We investigated such resistance mechanisms in relapsed/refractory early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL) carrying activating NOTCH1 mutations via full-length single-cell RNA sequencing (scRNA-seq) of malignant and microenvironmental cells. We identified 2 highly distinct stem-like states that critically differed with regard to cell cycle and oncogenic signaling. Fast-cycling stem-like leukemia cells demonstrated Notch activation and were effectively eliminated in patients by Notch inhibition, whereas slow-cycling stem-like cells were Notch independent and rather relied on PI3K signaling, likely explaining the poor efficacy of Notch inhibition in this disease. Remarkably, we found that both stem-like states could differentiate into a more mature leukemia state with prominent immunomodulatory functions, including high expression of the LGALS9 checkpoint molecule. These cells promoted an immunosuppressive leukemia ecosystem with clonal accumulation of dysfunctional CD8+ T cells that expressed HAVCR2, the cognate receptor for LGALS9. Our study identified complex interactions between signaling programs, cellular plasticity, and immune programs that characterize ETP-ALL, illustrating the multidimensionality of tumor heterogeneity. In this scenario, combination therapies targeting diverse oncogenic states and the immune ecosystem seem most promising to successfully eliminate tumor cells that escape treatment through coexisting transcriptional programs.
Asunto(s)
Carcinogénesis , Galectinas/metabolismo , Regulación Leucémica de la Expresión Génica , Evasión Inmune , Células Madre Neoplásicas/patología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Análisis de la Célula Individual/métodos , Adolescente , Adulto , Anciano , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Niño , Preescolar , Resistencia a Antineoplásicos , Femenino , Estudios de Seguimiento , Galectinas/genética , Receptor 2 Celular del Virus de la Hepatitis A/genética , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Humanos , Lactante , Masculino , Persona de Mediana Edad , Mutación , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Pronóstico , RNA-Seq/métodos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto JovenRESUMEN
Widespread changes to DNA methylation and chromatin are well documented in cancer, but the fate of higher-order chromosomal structure remains obscure. Here we integrated topological maps for colon tumors and normal colons with epigenetic, transcriptional, and imaging data to characterize alterations to chromatin loops, topologically associated domains, and large-scale compartments. We found that spatial partitioning of the open and closed genome compartments is profoundly compromised in tumors. This reorganization is accompanied by compartment-specific hypomethylation and chromatin changes. Additionally, we identify a compartment at the interface between the canonical A and B compartments that is reorganized in tumors. Remarkably, similar shifts were evident in non-malignant cells that have accumulated excess divisions. Our analyses suggest that these topological changes repress stemness and invasion programs while inducing anti-tumor immunity genes and may therefore restrain malignant progression. Our findings call into question the conventional view that tumor-associated epigenomic alterations are primarily oncogenic.
Asunto(s)
Cromatina/metabolismo , Cromosomas/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Metilación de ADN , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica/genética , División Celular , Senescencia Celular/genética , Secuenciación de Inmunoprecipitación de Cromatina , Cromosomas/genética , Estudios de Cohortes , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Biología Computacional , Metilación de ADN/genética , Epigenómica , Células HCT116 , Humanos , Hibridación Fluorescente in Situ , Microscopía Electrónica de Transmisión , Simulación de Dinámica Molecular , RNA-Seq , Análisis Espacial , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Epigenetic regulators, when genomically altered, may become driver oncogenes that mediate otherwise unexplained pro-oncogenic changes lacking a clear genetic stimulus, such as activation of the WNT/ß-catenin pathway in melanoma. This study identifies previously unrecognized recurrent activating mutations in the G9a histone methyltransferase gene, as well as G9a genomic copy gains in approximately 26% of human melanomas, which collectively drive tumor growth and an immunologically sterile microenvironment beyond melanoma. Furthermore, the WNT pathway is identified as a key tumorigenic target of G9a gain-of-function, via suppression of the WNT antagonist DKK1. Importantly, genetic or pharmacologic suppression of mutated or amplified G9a using multiple in vitro and in vivo models demonstrates that G9a is a druggable target for therapeutic intervention in melanoma and other cancers harboring G9a genomic aberrations. SIGNIFICANCE: Oncogenic G9a abnormalities drive tumorigenesis and the "cold" immune microenvironment by activating WNT signaling through DKK1 repression. These results reveal a key druggable mechanism for tumor development and identify strategies to restore "hot" tumor immune microenvironments.This article is highlighted in the In This Issue feature, p. 890.
Asunto(s)
Carcinogénesis/genética , Mutación con Ganancia de Función/genética , Antígenos de Histocompatibilidad/genética , N-Metiltransferasa de Histona-Lisina/genética , Oncogenes/genética , Línea Celular Tumoral , Humanos , MutaciónRESUMEN
Epigenetic aberrations are widespread in cancer, yet the underlying mechanisms and causality remain poorly understood1-3. A subset of gastrointestinal stromal tumours (GISTs) lack canonical kinase mutations but instead have succinate dehydrogenase (SDH) deficiency and global DNA hyper-methylation4,5. Here, we associate this hyper-methylation with changes in genome topology that activate oncogenic programs. To investigate epigenetic alterations systematically, we mapped DNA methylation, CTCF insulators, enhancers, and chromosome topology in KIT-mutant, PDGFRA-mutant and SDH-deficient GISTs. Although these respective subtypes shared similar enhancer landscapes, we identified hundreds of putative insulators where DNA methylation replaced CTCF binding in SDH-deficient GISTs. We focused on a disrupted insulator that normally partitions a core GIST super-enhancer from the FGF4 oncogene. Recurrent loss of this insulator alters locus topology in SDH-deficient GISTs, allowing aberrant physical interaction between enhancer and oncogene. CRISPR-mediated excision of the corresponding CTCF motifs in an SDH-intact GIST model disrupted the boundary between enhancer and oncogene, and strongly upregulated FGF4 expression. We also identified a second recurrent insulator loss event near the KIT oncogene, which is also highly expressed across SDH-deficient GISTs. Finally, we established a patient-derived xenograft (PDX) from an SDH-deficient GIST that faithfully maintains the epigenetics of the parental tumour, including hypermethylation and insulator defects. This PDX model is highly sensitive to FGF receptor (FGFR) inhibition, and more so to combined FGFR and KIT inhibition, validating the functional significance of the underlying epigenetic lesions. Our study reveals how epigenetic alterations can drive oncogenic programs in the absence of canonical kinase mutations, with implications for mechanistic targeting of aberrant pathways in cancers.
Asunto(s)
Carcinogénesis/genética , Aberraciones Cromosómicas , Tumores del Estroma Gastrointestinal/genética , Tumores del Estroma Gastrointestinal/patología , Oncogenes/genética , Succinato Deshidrogenasa/deficiencia , Animales , Sistemas CRISPR-Cas/genética , Metilación de ADN , Elementos de Facilitación Genéticos/genética , Epigénesis Genética , Factor 4 de Crecimiento de Fibroblastos/genética , Tumores del Estroma Gastrointestinal/enzimología , Humanos , Ratones , Mutación , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Succinato Deshidrogenasa/genéticaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Evolved resistance to tyrosine kinase inhibitor (TKI)-targeted therapies remains a major clinical challenge. In epidermal growth factor receptor (EGFR) mutant non-small-cell lung cancer (NSCLC), failure of EGFR TKIs can result from both genetic and epigenetic mechanisms of acquired drug resistance. Widespread reports of histologic and gene expression changes consistent with an epithelial-to-mesenchymal transition (EMT) have been associated with initially surviving drug-tolerant persister cells, which can seed bona fide genetic mechanisms of resistance to EGFR TKIs. While therapeutic approaches targeting fully resistant cells, such as those harboring an EGFRT790M mutation, have been developed, a clinical strategy for preventing the emergence of persister cells remains elusive. Using mesenchymal cell lines derived from biopsies of patients who progressed on EGFR TKI as surrogates for persister populations, we performed whole-genome CRISPR screening and identified fibroblast growth factor receptor 1 (FGFR1) as the top target promoting survival of mesenchymal EGFR mutant cancers. Although numerous previous reports of FGFR signaling contributing to EGFR TKI resistance in vitro exist, the data have not yet been sufficiently compelling to instigate a clinical trial testing this hypothesis, nor has the role of FGFR in promoting the survival of persister cells been elucidated. In this study, we find that combining EGFR and FGFR inhibitors inhibited the survival and expansion of EGFR mutant drug-tolerant cells over long time periods, preventing the development of fully resistant cancers in multiple vitro models and in vivo. These results suggest that dual EGFR and FGFR blockade may be a promising clinical strategy for both preventing and overcoming EMT-associated acquired drug resistance and provide motivation for the clinical study of combined EGFR and FGFR inhibition in EGFR-mutated NSCLCs.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Resistencia a Antineoplásicos/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias Pulmonares , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal/genética , Receptores ErbB/genética , Receptores ErbB/fisiología , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Terapia Molecular Dirigida , Mutación , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/farmacología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Most pancreatic neuroendocrine tumors (PNETs) do not produce excess hormones and are therefore considered 'non-functional'1-3. As clinical behaviors vary widely and distant metastases are eventually lethal2,4, biological classifications might guide treatment. Using enhancer maps to infer gene regulatory programs, we find that non-functional PNETs fall into two major subtypes, with epigenomes and transcriptomes that partially resemble islet α- and ß-cells. Transcription factors ARX and PDX1 specify these normal cells, respectively5,6, and 84% of 142 non-functional PNETs expressed one or the other factor, occasionally both. Among 103 cases, distant relapses occurred almost exclusively in patients with ARX+PDX1- tumors and, within this subtype, in cases with alternative lengthening of telomeres. These markedly different outcomes belied similar clinical presentations and histology and, in one cohort, occurred irrespective of MEN1 mutation. This robust molecular stratification provides insight into cell lineage correlates of non-functional PNETs, accurately predicts disease course and can inform postoperative clinical decisions.
Asunto(s)
Elementos de Facilitación Genéticos , Neoplasias Pancreáticas/genética , Linaje de la Célula , Proteínas de Homeodominio/análisis , Humanos , Mutación , Neoplasias Pancreáticas/química , Proteínas Proto-Oncogénicas/genética , Telómero , Transactivadores/análisis , Factores de Transcripción/análisisRESUMEN
Purpose: Epithelial-to-mesenchymal transition (EMT) confers resistance to a number of targeted therapies and chemotherapies. However, it has been unclear why EMT promotes resistance, thereby impairing progress to overcome it.Experimental Design: We have developed several models of EMT-mediated resistance to EGFR inhibitors (EGFRi) in EGFR-mutant lung cancers to evaluate a novel mechanism of EMT-mediated resistance.Results: We observed that mesenchymal EGFR-mutant lung cancers are resistant to EGFRi-induced apoptosis via insufficient expression of BIM, preventing cell death despite potent suppression of oncogenic signaling following EGFRi treatment. Mechanistically, we observed that the EMT transcription factor ZEB1 inhibits BIM expression by binding directly to the BIM promoter and repressing transcription. Derepression of BIM expression by depletion of ZEB1 or treatment with the BH3 mimetic ABT-263 to enhance "free" cellular BIM levels both led to resensitization of mesenchymal EGFR-mutant cancers to EGFRi. This relationship between EMT and loss of BIM is not restricted to EGFR-mutant lung cancers, as it was also observed in KRAS-mutant lung cancers and large datasets, including different cancer subtypes.Conclusions: Altogether, these data reveal a novel mechanistic link between EMT and resistance to lung cancer targeted therapies. Clin Cancer Res; 24(1); 197-208. ©2017 AACR.
Asunto(s)
Proteína 11 Similar a Bcl2/genética , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Terapia Molecular Dirigida , Compuestos de Anilina/farmacología , Animales , Apoptosis/genética , Ciclo Celular/genética , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Receptores ErbB/genética , Humanos , Ratones , Mutación , Regiones Promotoras Genéticas , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/genética , Sulfonamidas/farmacologíaRESUMEN
In prostate cancer, resistance to the antiandrogen enzalutamide (Enz) can occur through bypass of androgen receptor (AR) blockade by the glucocorticoid receptor (GR). In contrast to fixed genomic alterations, here we show that GR-mediated antiandrogen resistance is adaptive and reversible due to regulation of GR expression by a tissue-specific enhancer. GR expression is silenced in prostate cancer by a combination of AR binding and EZH2-mediated repression at the GR locus, but is restored in advanced prostate cancers upon reversion of both repressive signals. Remarkably, BET bromodomain inhibition resensitizes drug-resistant tumors to Enz by selectively impairing the GR signaling axis via this enhancer. In addition to revealing an underlying molecular mechanism of GR-driven drug resistance, these data suggest that inhibitors of broadly active chromatin-readers could have utility in nuanced clinical contexts of acquired drug resistance with a more favorable therapeutic index.