RESUMEN
The activities of RNA polymerase and the spliceosome are responsible for the heterogeneity in the abundance and isoform composition of mRNA in human cells. However, the dynamics of these megadalton enzymatic complexes working in concert on endogenous genes have not been described. Here, we establish a quasi-genome-scale platform for observing synthesis and processing kinetics of single nascent RNA molecules in real time. We find that all observed genes show transcriptional bursting. We also observe large kinetic variation in intron removal for single introns in single cells, which is inconsistent with deterministic splice site selection. Transcriptome-wide footprinting of the U2AF complex, nascent RNA profiling, long-read sequencing, and lariat sequencing further reveal widespread stochastic recursive splicing within introns. We propose and validate a unified theoretical model to explain the general features of transcription and pervasive stochastic splice site selection.
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Precursores del ARN/genética , Sitios de Empalme de ARN/fisiología , Transcripción Genética , Exones/genética , Humanos , Intrones/genética , Precursores del ARN/metabolismo , Sitios de Empalme de ARN/genética , Empalme del ARN/genética , Empalme del ARN/fisiología , ARN Mensajero/metabolismo , Empalmosomas/metabolismo , TranscriptomaRESUMEN
Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an autosomal dominant condition characterized by the development of cutaneous and uterine leiomyomas and risk for development of an aggressive form of papillary renal cell cancer. HLRCC is caused by germline inactivating pathogenic variants in the fumarate hydratase (FH) gene, which encodes the enzyme that catalyzes the interconversion of fumarate and L-malate. We utilized enzyme and protein mobility assays to evaluate the FH enzyme in a cohort of patients who showed clinical manifestations of HLRCC but were negative for known pathogenic FH gene variants. FH enzyme activity and protein levels were decreased by 50% or greater in three family members, despite normal FH mRNA expression levels as measured by quantitative PCR. Direct Nanopore RNA sequencing demonstrated 57 base pairs of retained intron sequence between exons 9 and 10 of polyadenylated FH mRNA in these patients, resulting in a truncated FH protein. Genomic sequencing revealed a heterozygous intronic alteration of the FH gene (chr1: 241498239 T/C) resulting in formation of a splice acceptor site near a polypyrimidine tract, and a uterine fibroid obtained from a patient showed loss of heterozygosity at this site. The same intronic FH variant was identified in an unrelated patient who also showed a clinical phenotype of HLRCC. These data demonstrate that careful clinical assessment as well as biochemical characterization of FH enzyme activity, protein expression, direct RNA sequencing, and genomic DNA sequencing of patient-derived cells can identify pathogenic variants outside of the protein coding regions of the FH gene.
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Carcinoma de Células Renales , Neoplasias Renales , Leiomiomatosis , Neoplasias Cutáneas , Neoplasias Uterinas , Femenino , Humanos , Carcinoma de Células Renales/genética , Leiomiomatosis/genética , Leiomiomatosis/patología , Fumarato Hidratasa/genética , Fumarato Hidratasa/análisis , Neoplasias Renales/genética , Neoplasias Uterinas/genética , Neoplasias Uterinas/patología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Mutación , ARN Mensajero/genéticaRESUMEN
OBJECTIVES: Inflammatory cytokines that signal through the Janus kinases-signal transducer and activator of transcription (JAK-STAT) pathway, especially interferons (IFNs), are implicated in Sjögren's disease (SjD). Although inhibition of JAKs is effective in other autoimmune diseases, a systematic investigation of IFN-JAK-STAT signalling and the effect of JAK inhibitor (JAKi) therapy in SjD-affected human tissues has not been fully investigated. METHODS: Human minor salivary glands (MSGs) and peripheral blood mononuclear cells (PBMCs) were investigated using bulk or single-cell (sc) RNA sequencing (RNAseq), immunofluorescence (IF) microscopy and flow cytometry. Ex vivo culture assays on PBMCs and primary salivary gland epithelial cell (pSGEC) lines were performed to model changes in target tissues before and after JAKi. RESULTS: RNAseq and IF showed activated JAK-STAT pathway in SjD MSGs. Elevated IFN-stimulated gene (ISGs) expression associated with clinical variables (eg, focus scores, anti-SSA positivity). scRNAseq of MSGs exhibited cell type-specific upregulation of JAK-STAT and ISGs; PBMCs showed similar trends, including markedly upregulated ISGs in monocytes. Ex vivo studies showed elevated basal pSTAT levels in SjD MSGs and PBMCs that were corrected with JAKi. SjD-derived pSGECs exhibited higher basal ISG expressions and exaggerated responses to IFN-ß, which were normalised by JAKi without cytotoxicity. CONCLUSIONS: SjD patients' tissues exhibit increased expression of ISGs and activation of the JAK-STAT pathway in a cell type-dependent manner. JAKi normalises this aberrant signalling at the tissue level and in PBMCs, suggesting a putative viable therapy for SjD, targeting both glandular and extraglandular symptoms. Predicated on these data, a phase Ib/IIa randomised controlled trial to treat SjD with tofacitinib was initiated.
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Inhibidores de las Cinasas Janus , Quinasas Janus , Leucocitos Mononucleares , Factores de Transcripción STAT , Glándulas Salivales Menores , Transducción de Señal , Síndrome de Sjögren , Humanos , Síndrome de Sjögren/tratamiento farmacológico , Síndrome de Sjögren/inmunología , Inhibidores de las Cinasas Janus/farmacología , Inhibidores de las Cinasas Janus/uso terapéutico , Transducción de Señal/efectos de los fármacos , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Glándulas Salivales Menores/inmunología , Femenino , Interferones , Piperidinas/farmacología , Piperidinas/uso terapéutico , Persona de Mediana Edad , Masculino , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Adulto , Inflamación , Pirroles/farmacología , Pirroles/uso terapéutico , Células Epiteliales/efectos de los fármacosRESUMEN
Von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary tumour susceptibility disease caused by germline pathogenic variation of the VHL tumour suppressor gene. Affected individuals are at risk of developing multiple malignant and benign tumours in a number of organs.In this report, a male patient in his 20s who presented to the Urologic Oncology Branch at the National Cancer Institute with a clinical diagnosis of VHL was found to have multiple cerebellar haemangioblastomas, bilateral epididymal cysts, multiple pancreatic cysts, and multiple, bilateral renal tumours and cysts. The patient had no family history of VHL and was negative for germline VHL mutation by standard genetic testing. Further genetic analysis demonstrated a germline balanced translocation between chromosomes 1 and 3, t(1;3)(p36.3;p25) with a breakpoint on chromosome 3 within the second intron of the VHL gene. This created a pathogenic germline alteration in VHL by a novel mechanism that was not detectable by standard genetic testing.Karyotype analysis is not commonly performed in existing genetic screening protocols for patients with VHL. Based on this case, protocols should be updated to include karyotype analysis in patients who are clinically diagnosed with VHL but demonstrate no detectable mutation by existing genetic testing.
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Mutación de Línea Germinal , Translocación Genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Enfermedad de von Hippel-Lindau/genética , Neoplasias Cerebelosas/etiología , Análisis Mutacional de ADN , Hemangioblastoma/etiología , Humanos , Neoplasias Renales/etiología , Masculino , Secuenciación del Exoma , Enfermedad de von Hippel-Lindau/complicacionesRESUMEN
BACKGROUND: We conducted a phase II study of the combination of pembrolizumab with capecitabine and oxaliplatin (CAPOX) in patients with advanced biliary tract carcinoma (BTC) to assess response rate and clinical efficacy. Exploratory objectives included correlative studies of immune marker expression, tumor evolution, and immune infiltration in response to treatment. PATIENTS AND METHODS: Adult patients with histologically confirmed BTC were enrolled and received oxaliplatin and pembrolizumab on day 1 of cycles 1-6. Capecitabine was administered orally twice daily as intermittent treatment, with the first dose on day 1 and the last dose on day 14 of cycles 1-6. Starting on cycle 7, pembrolizumab monotherapy was continued until disease progression. The primary endpoint was progression-free survival (PFS). Secondary endpoints were safety, tolerability, feasibility, and response rate. Immunohistochemistry (IHC) for PD-L1 and immune infiltrates was analyzed in paired tumor biopsies, as well as bulk transcriptome and exome profiling for five patients and single-cell RNA sequencing for one partial responder. RESULTS: Eleven patients enrolled, three of whom had received no prior systemic therapy. Treatment was well tolerated, and the most common treatment-related grade 3 or 4 adverse events were lymphocytopenia, anemia, and decreased platelet count. Three patients (27.3%) achieved a partial response, and six (54%) had stable disease. The disease control rate was 81.8%. The median PFS was 4.1 months with a 6-month PFS rate of 45.5%. Molecular profiling suggests qualitative differences in immune infiltration and clonal evolution based on response. CONCLUSION: Capecitabine and oxaliplatin in combination with pembrolizumab is tolerable and a potentially effective treatment for refractory advanced BTC. This study highlights a design framework for the precise characterization of individual BTC tumors. TRIAL REGISTRATION: This study was registered in ClinicalTrials.gov (NCT03111732).
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Neoplasias de los Conductos Biliares , Sistema Biliar , Carcinoma , Neoplasias Gastrointestinales , Adulto , Anticuerpos Monoclonales Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Neoplasias de los Conductos Biliares/tratamiento farmacológico , Capecitabina/efectos adversos , Carcinoma/tratamiento farmacológico , Neoplasias Gastrointestinales/tratamiento farmacológico , Humanos , OxaliplatinoRESUMEN
BACKGROUND & AIMS: Many genetic and environmental factors, including family history, dietary fat, and inflammation, increase risk for colon cancer development. Peroxisome proliferator-activated receptor alpha (PPARα) is a nuclear receptor that regulates systemic lipid homeostasis. We explored the role of intestinal PPARα in colon carcinogenesis. METHODS: Colon cancer was induced in mice with intestine-specific disruption of Ppara (PparaΔIE), Pparafl/fl (control), and mice with disruption of Ppara that express human PPARA (human PPARA transgenic mice), by administration of azoxymethane with or without dextran sulfate sodium (DSS). Colons were collected from mice and analyzed by immunoblots, quantitative polymerase chain reaction, and histopathology. Liquid chromatography coupled with mass spectrometry-based metabolomic analyses were performed on urine and colons. We used molecular biology and biochemical approaches to study mechanisms in mouse colons, primary intestinal epithelial cells, and colon cancer cell lines. Gene expression data and clinical features of patients with colorectal tumors were obtained from Oncomine, and human colorectal-tumor specimens and adjacent normal tissues were collected and analyzed by immunohistochemistry. RESULTS: Levels of Ppara messenger RNA were reduced in colon tumors from mice. PparaΔIE mice developed more and larger colon tumors than control mice following administration of azoxymethane, with or without DSS. Metabolomic analyses revealed increases in methylation-related metabolites in urine and colons from PparaΔIE mice, compared with control mice, following administration of azoxymethane, with or without DSS. Levels of DNA methyltransferase 1 (DNMT1) and protein arginine methyltransferase 6 (PRMT6) were increased in colon tumors from PparaΔIE mice, compared with colon tumors from control mice. Depletion of PPARα reduced the expression of retinoblastoma protein, resulting in increased expression of DNMT1 and PRMT6. DNMT1 and PRMT6 decreased expression of the tumor suppressor genes Cdkn1a (P21) and Cdkn1b (p27) via DNA methylation and histone H3R2 dimethylation-mediated repression of transcription, respectively. Fenofibrate protected human PPARA transgenic mice from azoxymethane and DSS-induced colon cancer. Human colon adenocarcinoma specimens had lower levels of PPARA and retinoblastoma protein and higher levels of DNMT1 and PRMT6 than normal colon tissues. CONCLUSIONS: Loss of PPARα from the intestine promotes colon carcinogenesis by increasing DNMT1-mediated methylation of P21 and PRMT6-mediated methylation of p27 in mice. Human colorectal tumors have lower levels of PPARA messenger RNA and protein than nontumor tissues. Agents that activate PPARα might be developed for chemoprevention or treatment of colon cancer.
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Adenocarcinoma/prevención & control , Colon/enzimología , Neoplasias del Colon/prevención & control , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN , Proteínas Nucleares/metabolismo , PPAR alfa/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Adenocarcinoma/enzimología , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Anticarcinógenos/farmacología , Estudios de Casos y Controles , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Colon/patología , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , ADN (Citosina-5-)-Metiltransferasa 1/genética , Metilación de ADN/efectos de los fármacos , Bases de Datos Genéticas , Modelos Animales de Enfermedad , Fenofibrato/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/genética , PPAR alfa/agonistas , PPAR alfa/deficiencia , PPAR alfa/genética , Proteína-Arginina N-Metiltransferasas/genética , Transducción de SeñalRESUMEN
Recent work indicates that salivary glands are able to constitutively recruit CD8+ T cells and retain them as tissue-resident memory T cells, independently of local infection, inflammation, or Ag. To understand the mechanisms supporting T cell recruitment to the salivary gland, we compared T cell migration to the salivary gland in mice that were infected or not with murine CMV (MCMV), a herpesvirus that infects the salivary gland and promotes the accumulation of salivary gland tissue-resident memory T cells. We found that acute MCMV infection increased rapid T cell recruitment to the salivary gland but that equal numbers of activated CD8+ T cells eventually accumulated in infected and uninfected glands. T cell recruitment to uninfected salivary glands depended on chemokines and the integrin α4 Several chemokines were expressed in the salivary glands of infected and uninfected mice, and many of these could promote the migration of MCMV-specific T cells in vitro. MCMV infection increased the expression of chemokines that interact with the receptors CXCR3 and CCR5, but neither receptor was needed for T cell recruitment to the salivary gland during MCMV infection. Unexpectedly, however, the chemokine receptor CXCR3 was critical for T cell accumulation in uninfected salivary glands. Together, these data suggest that CXCR3 and the integrin α4 mediate T cell recruitment to uninfected salivary glands but that redundant mechanisms mediate T cell recruitment after MCMV infection.
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Linfocitos T CD8-positivos/inmunología , Infecciones por Herpesviridae/inmunología , Integrina alfa4/genética , Muromegalovirus/inmunología , Receptores CXCR3/genética , Glándulas Salivales/inmunología , Animales , Movimiento Celular/inmunología , Células Cultivadas , Quimiocinas/metabolismo , Infecciones por Herpesviridae/virología , Memoria Inmunológica/inmunología , Interferón gamma/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CCR5/genética , Glándulas Salivales/virologíaRESUMEN
OBJECTIVES: Little is known about the molecular details regarding the contribution of different cell types of the salivary gland to the altered gene expression profile seen in Sjögren's syndrome (SS). METHODS: Using laser microdissection, tissue samples enriched in acini, ducts and inflammatory foci in subjects with and without SS were isolated for RNA-seq analysis. Gene expression profiles were analysed and selected enriched genes were further examined using real time PCR and by immunofluorescence. RESULTS: RNA-seq analysis of salivary biopsies from subjects with and without SS revealed marked differences in gene expression occurring in the ductal and infiltrating cells compared to acinar cells. Up-regulated genes in the SS ductal cells included C4A complement and the SLC26A9 ion channel. The inflammatory infiltrate showed the most dramatic differences in gene expression and contained up-regulated genes associated with T-cells, natural killer, dendritic and basophils/mast cells. qPCR with total salivary gland mRNA confirmed the differential mRNA expression of several genes (MMP9, FOL1HB, CCL21, CCR7), thereby validating the approach. Additional immunofluorescence studies demonstrated high expression and co-localisation of CCL21 chemokine and CCR7 chemokine receptor within the SS infiltrates. CONCLUSIONS: Major gene expression changes in the salivary gland of SS were detected in the ductal and inflammatory cells and not in the acinar cells. Two chemokines involved in immune cell trafficking to secondary lymphoid tissue, CCR7 and CCL21, showed markedly increased expression and may contribute to the recruitment of diverse immune cells to the salivary glands, causing inflammation and loss of secretory function.
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Perfilación de la Expresión Génica , Captura por Microdisección con Láser , ARN Mensajero/genética , Glándulas Salivales/química , Análisis de Secuencia de ARN , Síndrome de Sjögren/genética , Estudios de Casos y Controles , Quimiocina CCL21/análisis , Quimiocina CCL21/genética , Femenino , Técnica del Anticuerpo Fluorescente , Estudios de Asociación Genética , Marcadores Genéticos , Humanos , Masculino , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores CCR7/análisis , Receptores CCR7/genética , Glándulas Salivales/inmunología , Glándulas Salivales/patología , Síndrome de Sjögren/diagnóstico , Síndrome de Sjögren/inmunología , TranscriptomaRESUMEN
Reconstitution of CMV-specific immunity after transplant remains a primary clinical objective to prevent CMV disease, and adoptive immunotherapy of CMV-specific T cells can be an effective therapeutic approach. Because of viral persistence, most CMV-specific CD8(+) T cells become terminally differentiated effector phenotype CD8(+) T cells (TEFF). A minor subset retains a memory-like phenotype (memory phenotype CD8(+) T cells [TM]), but it is unknown whether these cells retain memory function or persist over time. Interestingly, recent studies suggest that CMV-specific CD8(+) T cells with different phenotypes have different abilities to reconstitute sustained immunity after transfer. The immunology of human CMV infections is reflected in the murine CMV (MCMV) model. We found that human CMV- and MCMV-specific T cells displayed shared genetic programs, validating the MCMV model for studies of CMV-specific T cells in vivo. The MCMV-specific TM population was stable over time and retained a proliferative capacity that was vastly superior to TEFF. Strikingly, after transfer, TM established sustained and diverse T cell populations even after multiple challenges. Although both TEFF and TM could protect Rag(-/-) mice, only TM persisted after transfer into immune replete, latently infected recipients and responded if recipient immunity was lost. Interestingly, transferred TM did not expand until recipient immunity was lost, supporting that competition limits the Ag stimulation of TM. Ultimately, these data show that CMV-specific TM retain memory function during MCMV infection and can re-establish CMV immunity when necessary. Thus, TM may be a critical component for consistent, long-term adoptive immunotherapy success.
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Linfocitos T CD8-positivos/inmunología , Infecciones por Herpesviridae/inmunología , Memoria Inmunológica/inmunología , Subgrupos de Linfocitos T/inmunología , Traslado Adoptivo , Animales , Modelos Animales de Enfermedad , Humanos , Inmunoterapia Adoptiva/métodos , Ratones , Ratones Transgénicos , Muromegalovirus/inmunología , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
MicroRNAs (miRs) seem to mediate renal fibrosis in several renal diseases, with some miRs having profibrotic effects and others having opposing effects. Although differential expression of certain miRs has been described in lupus nephritis, it is unknown whether miRs contribute to fibrosis or could serve as biomarkers of specific histologic manifestations of lupus nephritis. Here, we compared miR expression in kidney biopsies from patients with lupus nephritis and identified miR-150 as the most differentially expressed miR in kidneys with high chronicity (chronicity index [CI] ≥ 4); miR-150 positively correlated with chronicity scores and the expression of profibrotic proteins. Overexpression of miR-150 significantly reduced expression of the antifibrotic protein suppressor of cytokine signaling 1 (SOCS1) and upregulated profibrotic proteins in both proximal tubular and mesangial cells. Directly targeting SOCS1 with a small interfering RNA produced similar results. Furthermore, TGF-ß1 induced miR-150 expression, decreased SOCS1, and increased profibrotic proteins in proximal tubular cells and podocytes; a miR-150 inhibitor reversed these changes, suggesting that the profibrotic effects of TGF-ß1 are, at least in part, mediated by miR-150. Consistent with these in vitro observations, biopsies with high miR-150 and high CI exhibited substantial expression of TGF-ß1, reduced SOCS1, and an increase in profibrotic proteins. In summary, miR-150 is a promising quantitative renal biomarker of kidney injury in lupus nephritis. Our results suggest that miR-150 promotes renal fibrosis by increasing profibrotic molecules through downregulation of SOCS1.
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Fibrosis/metabolismo , Riñón/metabolismo , Nefritis Lúpica/metabolismo , MicroARNs/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Biomarcadores , Biopsia , Regulación hacia Abajo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Riñón/patología , Análisis por Micromatrices , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Proteína 1 Supresora de la Señalización de CitocinasRESUMEN
Immune checkpoint blockade (ICB) targeting the programmed cell death protein 1 (PD-1) and its ligand 1 (PD-L1) fails to provide clinical benefit for most cancer patients due to primary or acquired resistance. Drivers of ICB resistance include tumor antigen processing/presentation machinery (APM) and IFNγ signaling mutations. Thus, there is an unmet clinical need to develop alternative therapies for these patients. To this end, we have developed a CRISPR/Cas9 approach to generate murine tumor models refractory to PD-1/-L1 inhibition due to APM/IFNγ signaling mutations. Guide RNAs were employed to delete B2m, Jak1, or Psmb9 genes in ICB-responsive EMT6 murine tumor cells. B2m was deleted in ICB-responsive MC38 murine colon cancer cells. We report a detailed development and validation workflow including whole exome and Sanger sequencing, western blotting, and flow cytometry to assess target gene deletion. Tumor response to ICB and immune effects of gene deletion were assessed in syngeneic mice. This workflow can help accelerate the discovery and development of alternative therapies and a deeper understanding of the immune consequences of tumor mutations, with potential clinical implications.
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Presentación de Antígeno , Receptor de Muerte Celular Programada 1 , Animales , Ratones , Antígeno B7-H1 , Línea Celular Tumoral , Sistemas CRISPR-Cas/genética , Receptor de Muerte Celular Programada 1/genética , ARN Guía de Sistemas CRISPR-Cas , Transducción de SeñalRESUMEN
Small-cell lung cancer (SCLC) is the most fatal form of lung cancer. Intratumoral heterogeneity, marked by neuroendocrine (NE) and non-neuroendocrine (non-NE) cell states, defines SCLC, but the cell-extrinsic drivers of SCLC plasticity are poorly understood. To map the landscape of SCLC tumor microenvironment (TME), we apply spatially resolved transcriptomics and quantitative mass spectrometry-based proteomics to metastatic SCLC tumors obtained via rapid autopsy. The phenotype and overall composition of non-malignant cells in the TME exhibit substantial variability, closely mirroring the tumor phenotype, suggesting TME-driven reprogramming of NE cell states. We identify cancer-associated fibroblasts (CAFs) as a crucial element of SCLC TME heterogeneity, contributing to immune exclusion, and predicting exceptionally poor prognosis. Our work provides a comprehensive map of SCLC tumor and TME ecosystems, emphasizing their pivotal role in SCLC's adaptable nature, opening possibilities for reprogramming the TME-tumor communications that shape SCLC tumor states.
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Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Microambiente Tumoral , Humanos , Carcinoma Pulmonar de Células Pequeñas/patología , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Tumores Neuroendocrinos/patología , Tumores Neuroendocrinos/genética , Tumores Neuroendocrinos/metabolismo , Células Neuroendocrinas/patología , Células Neuroendocrinas/metabolismo , Femenino , Masculino , PronósticoRESUMEN
Sequencing of genes, such as BRCA1 and BRCA2, is recommended for individuals with a personal or family history of early onset and/or bilateral breast and/or ovarian cancer or a history of male breast cancer. Such sequencing efforts have resulted in the identification of more than 17,000 BRCA2 variants. The functional significance of most variants remains unknown; consequently, they are called variants of uncertain clinical significance (VUSs). We have previously developed mouse embryonic stem cell (mESC)-based assays for functional classification of BRCA2 variants. We now developed a next-generation sequencing (NGS)-based approach for functional evaluation of BRCA2 variants using pools of mESCs expressing 10-25 BRCA2 variants from a given exon. We use this approach for functional evaluation of 223 variants listed in ClinVar. Our functional classification of BRCA2 variants is concordant with the classification reported in ClinVar or those reported by other orthogonal assays.
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Genes BRCA2 , Neoplasias Ováricas , Humanos , Femenino , Masculino , Animales , Ratones , Células Madre Embrionarias de Ratones , Neoplasias Ováricas/genética , Proteína BRCA2/genéticaRESUMEN
Objectives: Inflammatory cytokines that signal through the JAK- STAT pathway, especially interferons (IFNs), are implicated in Sjögren's Disease (SjD). Although inhibition of JAKs is effective in other autoimmune diseases, a systematic investigation of IFN-JAK-STAT signaling and effect of JAK inhibitor (JAKi) therapy in SjD-affected human tissues has not been reported. Methods: Human minor salivary glands (MSGs) and peripheral blood mononuclear cells (PBMCs) were investigated using bulk or single cell (sc) RNA sequencing (RNAseq), immunofluorescence microscopy (IF), and flow cytometry. Ex vivo culture assays on PBMCs and primary salivary gland epithelial cell (pSGEC) lines were performed to model changes in target tissues before and after JAKi. Results: RNAseq and IF showed activated JAK-STAT pathway in SjD MSGs. Elevated IFN-stimulated gene (ISGs) expression associated with clinical variables (e.g., focus scores, anti-SSA positivity). scRNAseq of MSGs exhibited cell-type specific upregulation of JAK-STAT and ISGs; PBMCs showed similar trends, including markedly upregulated ISGs in monocytes. Ex vivo studies showed elevated basal pSTAT levels in SjD MSGs and PBMCs that were corrected with JAKi. SjD-derived pSGECs exhibited higher basal ISG expressions and exaggerated responses to IFNß, which were normalized by JAKi without cytotoxicity. Conclusions: SjD patients' tissues exhibit increased expression of ISGs and activation of the JAK-STAT pathway in a cell type-dependent manner. JAKi normalizes this aberrant signaling at the tissue level and in PBMCs, suggesting a putative viable therapy for SjD, targeting both glandular and extraglandular symptoms. Predicated on these data, a Phase Ib/IIa randomized controlled trial to treat SjD with tofacitinib was initiated.
RESUMEN
Primary liver cancer is a rising cause of cancer deaths in the US. Although immunotherapy with immune checkpoint inhibitors induces a potent response in a subset of patients, response rates vary among individuals. Predicting which patients will respond to immune checkpoint inhibitors is of great interest in the field. In a retrospective arm of the National Cancer Institute Cancers of the Liver: Accelerating Research of Immunotherapy by a Transdisciplinary Network (NCI-CLARITY) study, we use archived formalin-fixed, paraffin-embedded samples to profile the transcriptome and genomic alterations among 86 hepatocellular carcinoma and cholangiocarcinoma patients prior to and following immune checkpoint inhibitor treatment. Using supervised and unsupervised approaches, we identify stable molecular subtypes linked to overall survival and distinguished by two axes of aggressive tumor biology and microenvironmental features. Moreover, molecular responses to immune checkpoint inhibitor treatment differ between subtypes. Thus, patients with heterogeneous liver cancer may be stratified by molecular status indicative of treatment response to immune checkpoint inhibitors.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Estudios Retrospectivos , Inmunoterapia , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/terapia , GenómicaRESUMEN
Poly (ADP-ribose) polymerase inhibitors (PARPis) represent a major advance in ovarian cancer, now as a treatment and as a maintenance therapy in the upfront and recurrent settings. However, patients often develop resistance to PARPis, underlining the importance of dissecting resistance mechanisms. Here, we report different dosing/timing schemes of PARPi treatment in BRCA2-mutant PEO1 cells, resulting in the simultaneous development of distinct resistance mechanisms. PARPi-resistant variants PEO1/OlaJR, established by higher initial doses and short-term PARPi treatment, develops PARPi resistance by rapidly restoring functional BRCA2 and promoting drug efflux activity. In contrast, PEO1/OlaR, developed by lower initial doses with long-term PARPi exposure, shows no regained BRCA2 function but a mesenchymal-like phenotype with greater invasion ability, and exhibits activated ATR/CHK1 and suppressed EZH2/MUS81 signaling cascades to regain HR repair and fork stabilization, respectively. Our study suggests that PARPi resistance mechanisms can be governed by treatment strategies and have a molecular basis on BRCA2 functionality. Further, we define different mechanisms that may serve as useful biomarkers to assess subsequent treatment strategies in PARPi-resistant ovarian cancer.
Asunto(s)
Antineoplásicos , Neoplasias Ováricas , Humanos , Femenino , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Reparación del ADN por Recombinación , Resistencia a Antineoplásicos/genética , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Antineoplásicos/farmacología , Recombinación Homóloga , Proteína BRCA2/genéticaRESUMEN
BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (iCCA) accounts for a fraction of primary liver cancers but has a 5-year survival rate of only 10%. Immune checkpoint inhibitors are effective in treating many solid cancers, but immune checkpoint inhibitor monotherapy has no clear benefit in iCCA. Mitogen-activated kinase (MEK) inhibitors, such as trametinib, have shown promising results in preclinical studies for iCCA by inhibiting cell proliferation and modifying the tumor microenvironment. This study aimed to show the potential benefit of combining trametinib with anti-programmed cell death protein 1 (PD-1) therapy in different iCCA mouse models. METHODS: Here, we assessed the in vitro cytotoxicity of trametinib in mouse (SB1 and LD-1) and human (EGI-1) cholangiocarcinoma cell lines. We examined the efficacy of single-agent trametinib, anti-PD-1, and a combination of both in subcutaneous, orthotopic, and plasmid-induced iCCA mouse models. Flow cytometry analysis was used to elucidate changes in the tumor immune microenvironment upon treatment. Whole-exome sequencing (WES) was performed on the SB1 tumor cell line to correlate this preclinical model with iCCAs in patients. RESULTS: Trametinib reduced tumor cell growth of SB1, LD-1, and EGI-1 tumor cells in vitro. Trametinib treatment led to up-regulation of major histocompatibility complex (MHC-I) and programmed cell death ligand 1 (PD-L-1) (programmed cell death ligand 1) on tumor cells in vitro. The combination of trametinib and anti-PD-1 reduced tumor burden in several iCCA tumor models and improved survival in SB1 tumor-bearing mice compared with either agent alone. Immunoprofiling of tumor-bearing mice showed an increase of hepatic effector memory CD8+ and CD4+ T cells, as well as an increased degranulation of CD8+ T cells, indicating enhanced cytotoxicity. WES and somatic mutational analysis showed no mutations of KRAS, BRAF, and ERK in SB1 tumor cells, and showed a similar genetic signature of SB1 found in a cohort of patients with iCCA. CONCLUSIONS: Altogether, our study shows that trametinib improves the immunogenicity of tumor cells by up-regulating MHC-I surface expression. The combination with anti-PD-1 results in optimal treatment efficacy for iCCA. WES of SB1 cells suggests that KRAS wild-type iCCAs also respond to this combination therapy.
Asunto(s)
Neoplasias de los Conductos Biliares/tratamiento farmacológico , Colangiocarcinoma/tratamiento farmacológico , Inhibidores de Puntos de Control Inmunológico/administración & dosificación , Piridonas/administración & dosificación , Pirimidinonas/administración & dosificación , Animales , Neoplasias de los Conductos Biliares/metabolismo , Neoplasias de los Conductos Biliares/mortalidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colangiocarcinoma/metabolismo , Colangiocarcinoma/mortalidad , Sinergismo Farmacológico , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Ratones , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Piridonas/farmacología , Pirimidinonas/farmacología , Secuenciación del Exoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor necrosis happens commonly in advanced solid tumors. We reported that necroptosis plays a major role in tumor necrosis. Although several key necroptosis regulators including receptor interacting protein kinase 1 (RIPK1) have been identified, the regulation of tumor necroptosis during tumor development remains elusive. Here, we report that Z-DNA-binding protein 1 (ZBP1), not RIPK1, mediates tumor necroptosis during tumor development in preclinical cancer models. We found that ZBP1 expression is dramatically elevated in necrotic tumors. Importantly, ZBP1, not RIPK1, deletion blocks tumor necroptosis during tumor development and inhibits metastasis. We showed that glucose deprivation triggers ZBP1-depedent necroptosis in tumor cells. Glucose deprivation causes mitochondrial DNA (mtDNA) release to the cytoplasm and the binding of mtDNA to ZBP1 to activate MLKL in a BCL-2 family protein, NOXA-dependent manner. Therefore, our study reveals ZBP1 as the key regulator of tumor necroptosis and provides a potential drug target for controlling tumor metastasis.
Asunto(s)
Neoplasias de la Mama/genética , Necroptosis/genética , Proteínas de Unión al ARN/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Células HEK293 , Humanos , Células MCF-7 , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Proteínas de Unión al ARN/metabolismo , Tratamiento con ARN de Interferencia/métodos , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Understanding the mechanisms of the Warburg shift to aerobic glycolysis is critical to defining the metabolic basis of cancer. Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an aggressive cancer characterized by biallelic inactivation of the gene encoding the Krebs cycle enzyme fumarate hydratase, an early shift to aerobic glycolysis, and rapid metastasis. We observed impairment of the mitochondrial respiratory chain in tumors from patients with HLRCC. Biochemical and transcriptomic analyses revealed that respiratory chain dysfunction in the tumors was due to loss of expression of mitochondrial DNA (mtDNA)-encoded subunits of respiratory chain complexes, caused by a marked decrease in mtDNA content and increased mtDNA mutations. We demonstrated that accumulation of fumarate in HLRCC tumors inactivated the core factors responsible for replication and proofreading of mtDNA, leading to loss of respiratory chain components, thereby promoting the shift to aerobic glycolysis and disease progression in this prototypic model of glucose-dependent human cancer.
Asunto(s)
Carcinoma de Células Renales/genética , Ciclo del Ácido Cítrico , Daño del ADN , ADN Mitocondrial/metabolismo , Fumarato Hidratasa/genética , Neoplasias Renales/genética , Leiomiomatosis/enzimología , Síndromes Neoplásicos Hereditarios/enzimología , Neoplasias Cutáneas/enzimología , Neoplasias Uterinas/enzimología , Adulto , Anciano , Carcinoma de Células Renales/etiología , Carcinoma de Células Renales/metabolismo , Reparación del ADN , Replicación del ADN , Femenino , Fumarato Hidratasa/deficiencia , Perfilación de la Expresión Génica , Humanos , Neoplasias Renales/etiología , Neoplasias Renales/metabolismo , Leiomiomatosis/complicaciones , Masculino , Persona de Mediana Edad , Mitocondrias/genética , Mitocondrias/metabolismo , Mutación , Síndromes Neoplásicos Hereditarios/complicaciones , Neoplasias Cutáneas/complicaciones , Neoplasias Uterinas/complicaciones , Adulto JovenRESUMEN
Mongolia has the highest incidence of hepatocellular carcinoma (HCC) in the world, but its causative factors and underlying tumor biology remain unknown. Here, we describe molecular characteristics of HCC from 76 Mongolian patients by whole-exome and transcriptome sequencing. We present a comprehensive analysis of mutational signatures, driver genes, and molecular subtypes of Mongolian HCC compared to 373 HCC patients of different races and ethnicities and diverse etiologies. Mongolian HCC consists of prognostic molecular subtypes similar to those found in patients from other areas of Asia, Europe, and North America, as well as other unique subtypes, suggesting the presence of distinct etiologies linked to Mongolian patients. In addition to common driver mutations (TP53, CTNNB1) frequently found in pan-cancer analysis, Mongolian HCC exhibits unique drivers (most notably GTF2IRD2B, PNRC2, and SPTA1), the latter of which is associated with hepatitis D viral infection. These results suggest the existence of new molecular mechanisms at play in Mongolian hepatocarcinogenesis.