RESUMO
BACKGROUND AND AIMS: Genomic alterations that encourage stem cell activity and hinder proper maturation are central to the development of colorectal cancer (CRC). Key molecular mediators that promote these malignant properties require further elucidation to galvanize translational advances. We therefore aimed to characterize a key factor that blocks intestinal differentiation, define its transcriptional and epigenetic program, and provide preclinical evidence for therapeutic targeting in CRC. METHODS: Intestinal tissue from transgenic mice and patients were analyzed by means of histopathology and immunostaining. Human CRC cells and neoplastic murine organoids were genetically manipulated for functional studies. Gene expression profiling was obtained through RNA sequencing. Histone modifications and transcription factor binding were determined with the use of chromatin immunoprecipitation sequencing. RESULTS: We demonstrate that SRY-box transcription factor 9 (SOX9) promotes CRC by activating a stem cell-like program that hinders intestinal differentiation. Intestinal adenomas and colorectal adenocarcinomas from mouse models and patients demonstrate ectopic and elevated expression of SOX9. Functional experiments indicate a requirement for SOX9 in human CRC cell lines and engineered neoplastic organoids. Disrupting SOX9 activity impairs primary CRC tumor growth by inducing intestinal differentiation. By binding to genome wide enhancers, SOX9 directly activates genes associated with Paneth and stem cell activity, including prominin 1 (PROM1). SOX9 up-regulates PROM1 via a Wnt-responsive intronic enhancer. A pentaspan transmembrane protein, PROM1 uses its first intracellular domain to support stem cell signaling, at least in part through SOX9, reinforcing a PROM1-SOX9 positive feedback loop. CONCLUSIONS: These studies establish SOX9 as a central regulator of an enhancer-driven stem cell-like program and carry important implications for developing therapeutics directed at overcoming differentiation defects in CRC.
Assuntos
Diferenciação Celular , Neoplasias Colorretais/metabolismo , Elementos Facilitadores Genéticos , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição SOX9/metabolismo , Antígeno AC133/genética , Antígeno AC133/metabolismo , Animais , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Genes APC , Células HT29 , Humanos , Camundongos Transgênicos , Células-Tronco Neoplásicas/patologia , Fatores de Transcrição SOX9/genética , Carga Tumoral , Células Tumorais Cultivadas , Via de Sinalização WntRESUMO
OBJECTIVE: Oesophageal squamous cell carcinoma (OSCC), like other squamous carcinomas, harbour highly recurrent cell cycle pathway alterations, especially hyperactivation of the CCND1/CDK4/6 axis, raising the potential for use of existing CDK4/6 inhibitors in these cancers. Although CDK4/6 inhibition has shown striking success when combined with endocrine therapy in oestrogen receptor positive breast cancer, CDK4/6 inhibitor palbociclib monotherapy has not revealed evidence of efficacy to date in OSCC clinical studies. Herein, we sought to elucidate the identification of key dependencies in OSCC as a foundation for the selection of targets whose blockade could be combined with CDK4/6 inhibition. DESIGN: We combined large-scale genomic dependency and pharmaceutical screening datasets with preclinical cell line models, to identified potential combination therapies in squamous cell cancer. RESULTS: We identified sensitivity to inhibitors to the ERBB family of receptor kinases, results clearly extending beyond the previously described minority of tumours with EGFR amplification/dependence, specifically finding a subset of OSCCs with dual dependence on ERBB3 and ERBB2. Subsequently. we demonstrated marked efficacy of combined pan-ERBB and CDK4/6 inhibition in vitro and in vivo. Furthermore, we demonstrated that squamous lineage transcription factor KLF5 facilitated activation of ERBBs in OSCC. CONCLUSION: These results provide clear rationale for development of combined ERBB and CDK4/6 inhibition in these cancers and raises the potential for KLF5 expression as a candidate biomarker to guide the use of these agents. These data suggested that by combining existing Food and Drug Administration (FDA)-approved agents, we have the capacity to improve therapy for OSCC and other squamous cancer.
Assuntos
Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Humanos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Although juvenile hormone (JH) is known to prevent insect larval metamorphosis and stimulate adult reproduction, the molecular mechanisms of JH action in insect reproduction remain largely unknown. Earlier, we reported that the JH-receptor complex, composed of methoprene-tolerant and steroid receptor co-activator, acts on mini-chromosome maintenance (Mcm) genes Mcm4 and Mcm7 to promote DNA replication and polyploidy for the massive vitellogenin (Vg) synthesis required for egg production in the migratory locust (Guo, W., Wu, Z., Song, J., Jiang, F., Wang, Z., Deng, S., Walker, V. K., and Zhou, S. (2014) PLoS Genet. 10, e1004702). In this study we have investigated the involvement of cell-division-cycle 6 (Cdc6) in JH-dependent vitellogenesis and oogenesis, as Cdc6 is essential for the formation of prereplication complex. We demonstrate here that Cdc6 is expressed in response to JH and methoprene-tolerant, and Cdc6 transcription is directly regulated by the JH-receptor complex. Knockdown of Cdc6 inhibits polyploidization of fat body and follicle cells, resulting in the substantial reduction of Vg expression in the fat body as well as severely impaired oocyte maturation and ovarian growth. Our data indicate the involvement of Cdc6 in JH pathway and a pivotal role of Cdc6 in JH-mediated polyploidization, vitellogenesis, and oogenesis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Hormônios Juvenis/metabolismo , Ativação Transcricional , Vitelogênese , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiologia , Corpo Adiposo/metabolismo , Feminino , Gafanhotos/genética , Gafanhotos/metabolismo , Gafanhotos/fisiologia , Dados de Sequência Molecular , Folículo Ovariano/metabolismo , Poliploidia , Vitelinas/genética , Vitelinas/metabolismoRESUMO
Elevated CO2 and temperature strongly affect crop production, but understanding of the crop response to combined CO2 and temperature increases under field conditions is still limited while data are scarce. We grew wheat (Triticum aestivum L.) and rice (Oryza sativa L.) under two levels of CO2 (ambient and enriched up to 500 µmol mol(-1) ) and two levels of canopy temperature (ambient and increased by 1.5-2.0 °C) in free-air CO2 enrichment (FACE) systems and carried out a detailed growth and yield component analysis during two growing seasons for both crops. An increase in CO2 resulted in higher grain yield, whereas an increase in temperature reduced grain yield, in both crops. An increase in CO2 was unable to compensate for the negative impact of an increase in temperature on biomass and yield of wheat and rice. Yields of wheat and rice were decreased by 10-12% and 17-35%, respectively, under the combination of elevated CO2 and temperature. The number of filled grains per unit area was the most important yield component accounting for the effects of elevated CO2 and temperature in wheat and rice. Our data showed complex treatment effects on the interplay between preheading duration, nitrogen uptake, tillering, leaf area index, and radiation-use efficiency, and thus on yield components and yield. Nitrogen uptake before heading was crucial in minimizing yield loss due to climate change in both crops. For rice, however, a breeding strategy to increase grain number per m(2) and % filled grains (or to reduce spikelet sterility) at high temperature is also required to prevent yield reduction under conditions of global change.
Assuntos
Dióxido de Carbono , Oryza/crescimento & desenvolvimento , Triticum/crescimento & desenvolvimento , Biomassa , Mudança Climática , Nitrogênio/metabolismo , Oryza/metabolismo , Fotossíntese , Componentes Aéreos da Planta/crescimento & desenvolvimento , Componentes Aéreos da Planta/metabolismo , Temperatura , Triticum/metabolismoRESUMO
Recent advances in single-cell epigenomic techniques have created a growing demand for scATAC-seq analysis. One key analysis task is to determine cell type identity based on the epigenetic data. We introduce scATAnno, a python package designed to automatically annotate scATAC-seq data using large-scale scATAC-seq reference atlases. This workflow generates the reference atlases from publicly available datasets enabling accurate cell type annotation by integrating query data with reference atlases, without the use of scRNA-seq data. To enhance annotation accuracy, we have incorporated KNN-based and weighted distance-based uncertainty scores to effectively detect cell populations within the query data that are distinct from all cell types in the reference data. We compare and benchmark scATAnno against 7 other published approaches for cell annotation and show superior performance in multiple data sets and metrics. We showcase the utility of scATAnno across multiple datasets, including peripheral blood mononuclear cell (PBMC), Triple Negative Breast Cancer (TNBC), and basal cell carcinoma (BCC), and demonstrate that scATAnno accurately annotates cell types across conditions. Overall, scATAnno is a useful tool for scATAC-seq reference building and cell type annotation in scATAC-seq data and can aid in the interpretation of new scATAC-seq datasets in complex biological systems.
RESUMO
Small cell lung cancer (SCLC) exists broadly in four molecular subtypes: ASCL1, NEUROD1, POU2F3 and Inflammatory. Initially, SCLC subtypes were thought to be mutually exclusive, but recent evidence shows intra-tumoural subtype heterogeneity and plasticity between subtypes. Here, using a CRISPR-based autochthonous SCLC genetically engineered mouse model to study the consequences of KDM6A/UTX inactivation, we show that KDM6A inactivation induced plasticity from ASCL1 to NEUROD1 resulting in SCLC tumours that express both ASCL1 and NEUROD1. Mechanistically, KDM6A normally maintains an active chromatin state that favours the ASCL1 subtype with its loss decreasing H3K4me1 and increasing H3K27me3 at enhancers of neuroendocrine genes leading to a cell state that is primed for ASCL1-to-NEUROD1 subtype switching. This work identifies KDM6A as an epigenetic regulator that controls ASCL1 to NEUROD1 subtype plasticity and provides an autochthonous SCLC genetically engineered mouse model to model ASCL1 and NEUROD1 subtype heterogeneity and plasticity, which is found in 35-40% of human SCLCs.
Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Animais , Camundongos , Carcinoma de Pequenas Células do Pulmão/genética , Histona Desmetilases/genética , Cromatina , Epigenômica , Neoplasias Pulmonares/genéticaRESUMO
Immunotherapies have yet to demonstrate significant efficacy in the treatment of hormone receptor-positive (HR+) breast cancer. Given that endocrine therapy (ET) is the primary approach for treating HR+ breast cancer, we investigated the effects of ET on the tumor immune microenvironment (TME) in HR+ breast cancer. Spatial proteomics of primary HR+ breast cancer samples obtained at baseline and after ET from patients enrolled in a neoadjuvant clinical trial (NCT02764541) indicated that ET upregulated ß2-microglobulin and influenced the TME in a manner that promotes enhanced immunogenicity. To gain a deeper understanding of the underlying mechanisms, the intrinsic effects of ET on cancer cells were explored, which revealed that ET plays a crucial role in facilitating the chromatin binding of RelA, a key component of the NF-κB complex. Consequently, heightened NF-κB signaling enhanced the response to interferon-gamma, leading to the upregulation of ß2-microglobulin and other antigen presentation-related genes. Further, modulation of NF-κB signaling using a SMAC mimetic in conjunction with ET augmented T-cell migration and enhanced MHC-I-specific T-cell-mediated cytotoxicity. Remarkably, the combination of ET and SMAC mimetics, which also blocks prosurvival effects of NF-κB signaling through the degradation of inhibitors of apoptosis proteins, elicited tumor regression through cell autonomous mechanisms, providing additional support for their combined use in HR+ breast cancer. SIGNIFICANCE: Adding SMAC mimetics to endocrine therapy enhances tumor regression in a cell autonomous manner while increasing tumor immunogenicity, indicating that this combination could be an effective treatment for HR+ patients with breast cancer.
Assuntos
Neoplasias da Mama , NF-kappa B , Humanos , Feminino , NF-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias da Mama/patologia , Apresentação de Antígeno , Proteínas Reguladoras de Apoptose , Apoptose , Linhagem Celular Tumoral , Proteínas Mitocondriais/metabolismo , Microambiente TumoralRESUMO
The transcriptional co-activator YAP1 oncogene is the downstream effector of the Hippo pathway, which regulates tissue homeostasis, organ size, regeneration, and tumorigenesis. Multiple cancers are dependent on sustained expression of YAP1 for cell proliferation, survival, and tumorigenesis, but the molecular basis of this oncogene dependency is not well understood. To identify genes that can functionally substitute for YAP1, we performed a genome-scale genetic rescue screen in YAP1-dependent colon cancer cells expressing an inducible YAP1-specific shRNA. We found that the transcription factor PRDM14 rescued cell proliferation and tumorigenesis upon YAP1 suppression in YAP1-dependent cells, xenografts, and colon cancer organoids. YAP1 and PRDM14 individually activated the transcription of calmodulin 2 (CALM2) and a glucose transporter SLC2A1 upon YAP1 suppression, and CALM2 or SLC2A1 expression was required for the rescue of YAP1 suppression. Together, these findings implicate PRDM14-mediated transcriptional upregulation of CALM2 and SLC2A1 as key components of oncogenic YAP1 signaling and dependency.
Assuntos
Carcinogênese/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Calmodulina/genética , Calmodulina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Neoplasias do Colo/genética , Proteínas de Ligação a DNA/genética , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Transportador de Glucose Tipo 1/genética , Humanos , Camundongos , Camundongos Nus , Organoides , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Ativação Transcricional , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Sinalização YAP/genética , Proteínas de Sinalização YAP/fisiologiaRESUMO
Polycomb repressive epigenetic complexes are recurrently dysregulated in cancer. Unlike polycomb repressive complex 2 (PRC2), the role of PRC1 in oncogenesis and therapy resistance is not well-defined. Here, we demonstrate that highly recurrent mutations of the PRC1 subunits BCOR and BCORL1 in leukemia disrupt assembly of a noncanonical PRC1.1 complex, thereby selectively unlinking the RING-PCGF enzymatic core from the chromatin-targeting auxiliary subcomplex. As a result, BCOR-mutated PRC1.1 is localized to chromatin but lacks repressive activity, leading to epigenetic reprogramming and transcriptional activation at target loci. We define a set of functional targets that drive aberrant oncogenic signaling programs in PRC1.1-mutated cells and primary patient samples. Activation of these PRC1.1 targets in BCOR-mutated cells confers acquired resistance to treatment while sensitizing to targeted kinase inhibition. Our study thus reveals a novel epigenetic mechanism that explains PRC1.1 tumor-suppressive activity and identifies a therapeutic strategy in PRC1.1-mutated cancer. SIGNIFICANCE: We demonstrate that BCOR and BCORL1 mutations in leukemia unlink PRC1.1 repressive function from target genes, resulting in epigenetic reprogramming and activation of aberrant cell signaling programs that mediate treatment resistance. Our study provides mechanistic insights into the pathogenesis of PRC1.1-mutated leukemia that inform novel therapeutic approaches. This article is highlighted in the In This Issue feature, p. 85.
Assuntos
Carcinogênese , Leucemia , Carcinogênese/genética , Proteínas de Ciclo Celular/genética , Cromatina , Epigênese Genética/genética , Humanos , Leucemia/genética , Mutação/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Transdução de Sinais/genéticaRESUMO
Most invasive lobular breast cancers (ILC) are of the luminal A subtype and are strongly hormone receptor-positive. Yet, ILC is relatively resistant to tamoxifen and associated with inferior long-term outcomes compared with invasive ductal cancers (IDC). In this study, we sought to gain mechanistic insights into these clinical findings that are not explained by the genetic landscape of ILC and to identify strategies to improve patient outcomes. A comprehensive analysis of the epigenome of ILC in preclinical models and clinical samples showed that, compared with IDC, ILC harbored a distinct chromatin state linked to gained recruitment of FOXA1, a lineage-defining pioneer transcription factor. This resulted in an ILC-unique FOXA1-estrogen receptor (ER) axis that promoted the transcription of genes associated with tumor progression and poor outcomes. The ILC-unique FOXA1-ER axis led to retained ER chromatin binding after tamoxifen treatment, which facilitated tamoxifen resistance while remaining strongly dependent on ER signaling. Mechanistically, gained FOXA1 binding was associated with the autoinduction of FOXA1 in ILC through an ILC-unique FOXA1 binding site. Targeted silencing of this regulatory site resulted in the disruption of the feed-forward loop and growth inhibition in ILC. In summary, ILC is characterized by a unique chromatin state and FOXA1-ER axis that is associated with tumor progression, offering a novel mechanism of tamoxifen resistance. These results underscore the importance of conducting clinical trials dedicated to patients with ILC in order to optimize treatments in this breast cancer subtype. SIGNIFICANCE: A unique FOXA1-ER axis in invasive lobular breast cancer promotes disease progression and tamoxifen resistance, highlighting a potential therapeutic avenue for clinical investigations dedicated to this disease. See related commentary by Blawski and Toska, p. 3668.
Assuntos
Neoplasias da Mama , Carcinoma Ductal de Mama , Carcinoma Lobular , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/tratamento farmacológico , Carcinoma Lobular/genética , Carcinoma Lobular/metabolismo , Cromatina/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Prognóstico , Receptores de Estrogênio/metabolismo , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêuticoRESUMO
c-MYC (MYC) is a major driver of prostate cancer tumorigenesis and progression. Although MYC is overexpressed in both early and metastatic disease and associated with poor survival, its impact on prostate transcriptional reprogramming remains elusive. We demonstrate that MYC overexpression significantly diminishes the androgen receptor (AR) transcriptional program (the set of genes directly targeted by the AR protein) in luminal prostate cells without altering AR expression. Analyses of clinical specimens reveal that concurrent low AR and high MYC transcriptional programs accelerate prostate cancer progression toward a metastatic, castration-resistant disease. Data integration of single-cell transcriptomics together with ChIP-seq uncover an increase in RNA polymerase II (Pol II) promoter-proximal pausing at AR-dependent genes following MYC overexpression without an accompanying deactivation of AR-bound enhancers. Altogether, our findings suggest that MYC overexpression antagonizes the canonical AR transcriptional program and contributes to prostate tumor initiation and progression by disrupting transcriptional pause release at AR-regulated genes.
Assuntos
Neoplasias da Próstata , Receptores Androgênicos , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Genes myc , Humanos , Masculino , Próstata/patologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-myc , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismoRESUMO
Chromatin immunoprecipitation sequencing (ChIP-seq) and the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) have become essential technologies to effectively measure protein-DNA interactions and chromatin accessibility. However, there is a need for a scalable and reproducible pipeline that incorporates proper normalization between samples, correction of copy number variations, and integration of new downstream analysis tools. Here we present Containerized Bioinformatics workflow for Reproducible ChIP/ATAC-seq Analysis (CoBRA), a modularized computational workflow which quantifies ChIP-seq and ATAC-seq peak regions and performs unsupervised and supervised analyses. CoBRA provides a comprehensive state-of-the-art ChIP-seq and ATAC-seq analysis pipeline that can be used by scientists with limited computational experience. This enables researchers to gain rapid insight into protein-DNA interactions and chromatin accessibility through sample clustering, differential peak calling, motif enrichment, comparison of sites to a reference database, and pathway analysis. CoBRA is publicly available online at https://bitbucket.org/cfce/cobra.
Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Biologia Computacional , Cromatina/genética , Variações do Número de Cópias de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA , Fluxo de TrabalhoRESUMO
How cancer cells adapt to evade the therapeutic effects of drugs targeting oncogenic drivers is poorly understood. Here we report an epigenetic mechanism leading to the adaptive resistance of triple-negative breast cancer (TNBC) to fibroblast growth factor receptor (FGFR) inhibitors. Prolonged FGFR inhibition suppresses the function of BRG1-dependent chromatin remodelling, leading to an epigenetic state that derepresses YAP-associated enhancers. These chromatin changes induce the expression of several amino acid transporters, resulting in increased intracellular levels of specific amino acids that reactivate mTORC1. Consistent with this mechanism, addition of mTORC1 or YAP inhibitors to FGFR blockade synergistically attenuated the growth of TNBC patient-derived xenograft models. Collectively, these findings reveal a feedback loop involving an epigenetic state transition and metabolic reprogramming that leads to adaptive therapeutic resistance and provides potential therapeutic strategies to overcome this mechanism of resistance.
Assuntos
Antineoplásicos/farmacologia , Proteínas Cromossômicas não Histona/metabolismo , Resistencia a Medicamentos Antineoplásicos , Compostos de Fenilureia/farmacologia , Pirimidinas/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteínas de Sinalização YAP/metabolismo , Aminoácidos/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Terapia de Alvo Molecular , Complexos Multiproteicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Sinalização YAP/antagonistas & inibidores , Proteínas de Sinalização YAP/genéticaRESUMO
Esophageal squamous cell carcinomas (ESCCs) harbor recurrent chromosome 3q amplifications that target the transcription factor SOX2. Beyond its role as an oncogene in ESCC, SOX2 acts in development of the squamous esophagus and maintenance of adult esophageal precursor cells. To compare Sox2 activity in normal and malignant tissue, we developed engineered murine esophageal organoids spanning normal esophagus to Sox2-induced squamous cell carcinoma and mapped Sox2 binding and the epigenetic and transcriptional landscape with evolution from normal to cancer. While oncogenic Sox2 largely maintains actions observed in normal tissue, Sox2 overexpression with p53 and p16 inactivation promotes chromatin remodeling and evolution of the Sox2 cistrome. With Klf5, oncogenic Sox2 acquires new binding sites and enhances activity of oncogenes such as Stat3. Moreover, oncogenic Sox2 activates endogenous retroviruses, inducing expression of double-stranded RNA and dependence on the RNA editing enzyme ADAR1. These data reveal SOX2 functions in ESCC, defining targetable vulnerabilities.
Assuntos
Adenosina Desaminase/metabolismo , Epigenoma , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Carcinogênese/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Retrovirus Endógenos/genética , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Interferons/metabolismo , Íntrons/genética , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Organoides/patologia , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , Fatores de Transcrição SOXB1/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Immune checkpoint blockade (ICB) therapy revolutionized cancer treatment, but many patients with impaired MHC-I expression remain refractory. Here, we combined FACS-based genome-wide CRISPR screens with a data-mining approach to identify drugs that can upregulate MHC-I without inducing PD-L1. CRISPR screening identified TRAF3, a suppressor of the NFκB pathway, as a negative regulator of MHC-I but not PD-L1. The Traf3-knockout gene expression signature is associated with better survival in ICB-naïve patients with cancer and better ICB response. We then screened for drugs with similar transcriptional effects as this signature and identified Second Mitochondria-derived Activator of Caspase (SMAC) mimetics. We experimentally validated that the SMAC mimetic birinapant upregulates MHC-I, sensitizes cancer cells to T cell-dependent killing, and adds to ICB efficacy. Our findings provide preclinical rationale for treating tumors expressing low MHC-I expression with SMAC mimetics to enhance sensitivity to immunotherapy. The approach used in this study can be generalized to identify other drugs that enhance immunotherapy efficacy. SIGNIFICANCE: MHC-I loss or downregulation in cancer cells is a major mechanism of resistance to T cell-based immunotherapies. Our study reveals that birinapant may be used for patients with low baseline MHC-I to enhance ICB response. This represents promising immunotherapy opportunities given the biosafety profile of birinapant from multiple clinical trials.This article is highlighted in the In This Issue feature, p. 1307.
Assuntos
Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias/tratamento farmacológico , Antígeno B7-H1/metabolismo , Mineração de Dados , Perfilação da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Imunoterapia , Microambiente Tumoral/efeitos dos fármacosRESUMO
Neuroendocrine carcinomas (NEC) are tumors expressing markers of neuronal differentiation that can arise at different anatomic sites but have strong histological and clinical similarities. Here we report the chromatin landscapes of a range of human NECs and show convergence to the activation of a common epigenetic program. With a particular focus on treatment emergent neuroendocrine prostate cancer (NEPC), we analyze cell lines, patient-derived xenograft (PDX) models and human clinical samples to show the existence of two distinct NEPC subtypes based on the expression of the neuronal transcription factors ASCL1 and NEUROD1. While in cell lines and PDX models these subtypes are mutually exclusive, single-cell analysis of human clinical samples exhibits a more complex tumor structure with subtypes coexisting as separate sub-populations within the same tumor. These tumor sub-populations differ genetically and epigenetically contributing to intra- and inter-tumoral heterogeneity in human metastases. Overall, our results provide a deeper understanding of the shared clinicopathological characteristics shown by NECs. Furthermore, the intratumoral heterogeneity of human NEPCs suggests the requirement of simultaneous targeting of coexisting tumor populations as a therapeutic strategy.
Assuntos
Carcinoma Neuroendócrino/genética , Neoplasias da Próstata/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Masculino , Fatores de Transcrição/genéticaRESUMO
Cytotoxic T cells play a key role in adaptive immunity by killing infected or cancerous cells. While the transcriptional control of CD8 T cell differentiation and effector function following T cell activation has been extensively studied, little is known about epigenetic regulation of these processes. Here we show that the histone deacetylase HDAC3 inhibits CD8 T cell cytotoxicity early during activation and is required for persistence of activated CD8 T cells following resolution of an acute infection. Mechanistically, HDAC3 inhibits gene programs associated with cytotoxicity and effector differentiation of CD8 T cells including genes encoding essential cytotoxicity proteins and key transcription factors. These data identify HDAC3 as an epigenetic regulator of the CD8 T cell cytotoxicity program.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epigênese Genética , Histona Desacetilases/metabolismo , Linfócitos T Citotóxicos , Acetilação/efeitos dos fármacos , Acrilamidas/farmacologia , Animais , Antígenos/metabolismo , Sequência de Bases , Linfócitos T CD8-Positivos/efeitos dos fármacos , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Epigênese Genética/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/deficiência , Histonas/metabolismo , Linfonodos/efeitos dos fármacos , Linfonodos/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Lisina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Fenilenodiaminas/farmacologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Linfócitos T Citotóxicos/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Fixed-tissue ChIP-seq for H3K27 acetylation (H3K27ac) profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27ac profiling. FiTAc-seq is a modified protocol that replaces the proteinase K digestion applied in FiT-seq with extended heating at 65 °C in a higher concentration of detergent and a minimized sonication step, to produce robust genome-wide H3K27ac maps from clinical samples. FiTAc-seq generates high-quality enhancer landscapes and super-enhancer (SE) annotation in numerous archived FFPE samples from distinct tumor types. This approach will be of great interest for both basic and clinical researchers. The entire protocol from FFPE blocks to sequence-ready library can be accomplished within 4 d.
Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Inclusão em Parafina , Fixação de Tecidos , Acetilação , Animais , Fígado/citologia , CamundongosRESUMO
We present Model-based AnalysEs of Transcriptome and RegulOme (MAESTRO), a comprehensive open-source computational workflow ( http://github.com/liulab-dfci/MAESTRO ) for the integrative analyses of single-cell RNA-seq (scRNA-seq) and ATAC-seq (scATAC-seq) data from multiple platforms. MAESTRO provides functions for pre-processing, alignment, quality control, expression and chromatin accessibility quantification, clustering, differential analysis, and annotation. By modeling gene regulatory potential from chromatin accessibilities at the single-cell level, MAESTRO outperforms the existing methods for integrating the cell clusters between scRNA-seq and scATAC-seq. Furthermore, MAESTRO supports automatic cell-type annotation using predefined cell type marker genes and identifies driver regulators from differential scRNA-seq genes and scATAC-seq peaks.
Assuntos
Regulação da Expressão Gênica , Modelos Genéticos , Análise de Célula Única , Software , Transcriptoma , Células da Medula Óssea/metabolismo , Estudos de Casos e Controles , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Análise de Sequência de RNA , Microambiente TumoralRESUMO
Chromatin organization is a highly orchestrated process that influences gene expression, in part by modulating access of regulatory factors to DNA and nucleosomes. Here, we report that the chromatin accessibility regulator HMGN1, a target of recurrent DNA copy gains in leukemia, controls myeloid differentiation. HMGN1 amplification is associated with increased accessibility, expression, and histone H3K27 acetylation of loci important for hematopoietic stem cells (HSCs) and leukemia, such as HoxA cluster genes. In vivo, HMGN1 overexpression is linked to decreased quiescence and increased HSC activity in bone marrow transplantation. HMGN1 overexpression also cooperates with the AML-ETO9a fusion oncoprotein to impair myeloid differentiation and enhance leukemia stem cell (LSC) activity. Inhibition of histone acetyltransferases CBP/p300 relieves the HMGN1-associated differentiation block. These data nominate factors that modulate chromatin accessibility as regulators of HSCs and LSCs, and suggest that targeting HMGN1 or its downstream effects on histone acetylation could be therapeutically active in AML.