RESUMO
Although neutrophils have been linked to the formation of the pre-metastatic niche, the mechanism of their migration to distant, uninvolved tissues has remained elusive. We report that bone marrow neutrophils from mice with early-stage cancer exhibited much more spontaneous migration than that of control neutrophils from tumor-free mice. These cells lacked immunosuppressive activity but had elevated rates of oxidative phosphorylation and glycolysis, and increased production of ATP, relative to that of control neutrophils. Their enhanced spontaneous migration was mediated by autocrine ATP signaling through purinergic receptors. In ectopic tumor models and late stages of cancer, bone marrow neutrophils demonstrated potent immunosuppressive activity. However, these cells had metabolic and migratory activity indistinguishable from that of control neutrophils. A similar pattern of migration was observed for neutrophils and polymorphonuclear myeloid-derived suppressor cells from patients with cancer. These results elucidate the dynamic changes that neutrophils undergo in cancer and demonstrate the mechanism of neutrophils' contribution to early tumor dissemination.
Assuntos
Quimiotaxia de Leucócito/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Idoso , Animais , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-IdadeRESUMO
Acute myeloid leukemia with KMT2A (MLL) rearrangements is characterized by specific patterns of gene expression and enhancer architecture, implying unique core transcriptional regulatory circuitry. Here, we identified the transcription factors MEF2D and IRF8 as selective transcriptional dependencies of KMT2A-rearranged AML, where MEF2D displays partially redundant functions with its paralog, MEF2C. Rapid transcription factor degradation followed by measurements of genome-wide transcription rates and superresolution microscopy revealed that MEF2D and IRF8 form a distinct core regulatory module with a narrow direct transcriptional program that includes activation of the key oncogenes MYC, HOXA9, and BCL2. Our study illustrates a mechanism of context-specific transcriptional addiction whereby a specific AML subclass depends on a highly specialized core regulatory module to directly enforce expression of common leukemia oncogenes.
Assuntos
Leucemia Mieloide Aguda , Proteína de Leucina Linfoide-Mieloide , Rearranjo Gênico , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Oncogenes/genéticaRESUMO
Disseminated cancer cells from primary tumours can seed in distal tissues, but may take several years to form overt metastases, a phenomenon that is termed tumour dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully characterize dormancy within melanoma. Here we show that the aged lung microenvironment facilitates a permissive niche for efficient outgrowth of dormant disseminated cancer cells-in contrast to the aged skin, in which age-related changes suppress melanoma growth but drive dissemination. These microenvironmental complexities can be explained by the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state1-3. It was previously shown that dermal fibroblasts promote phenotype switching in melanoma during ageing4-8. We now identify WNT5A as an activator of dormancy in melanoma disseminated cancer cells within the lung, which initially enables the efficient dissemination and seeding of melanoma cells in metastatic niches. Age-induced reprogramming of lung fibroblasts increases their secretion of the soluble WNT antagonist sFRP1, which inhibits WNT5A in melanoma cells and thereby enables efficient metastatic outgrowth. We also identify the tyrosine kinase receptors AXL and MER as promoting a dormancy-to-reactivation axis within melanoma cells. Overall, we find that age-induced changes in distal metastatic microenvironments promote the efficient reactivation of dormant melanoma cells in the lung.
Assuntos
Envelhecimento , Pulmão , Melanoma , Metástase Neoplásica , Células Estromais , Microambiente Tumoral , Idoso , Envelhecimento/patologia , Fibroblastos/patologia , Humanos , Pulmão/patologia , Melanoma/patologia , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Neoplasia Residual , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases , Pele/patologia , Células Estromais/patologia , Proteína Wnt-5a , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlRESUMO
Ferroptosis is a non-apoptotic form of regulated cell death that is triggered by the discoordination of regulatory redox mechanisms culminating in massive peroxidation of polyunsaturated phospholipids. Ferroptosis inducers have shown considerable effectiveness in killing tumour cells in vitro, yet there has been no obvious success in experimental animal models, with the notable exception of immunodeficient mice1,2. This suggests that the effect of ferroptosis on immune cells remains poorly understood. Pathologically activated neutrophils (PMNs), termed myeloid-derived suppressor cells (PMN-MDSCs), are major negative regulators of anti-tumour immunity3-5. Here we found that PMN-MDSCs in the tumour microenvironment spontaneously die by ferroptosis. Although decreasing the presence of PMN-MDSCs, ferroptosis induces the release of oxygenated lipids and limits the activity of human and mouse T cells. In immunocompetent mice, genetic and pharmacological inhibition of ferroptosis abrogates suppressive activity of PMN-MDSCs, reduces tumour progression and synergizes with immune checkpoint blockade to suppress the tumour growth. By contrast, induction of ferroptosis in immunocompetent mice promotes tumour growth. Thus, ferroptosis is a unique and targetable immunosuppressive mechanism of PMN-MDSCs in the tumour microenvironment that can be pharmacologically modulated to limit tumour progression.
Assuntos
Neoplasias , Humanos , Camundongos , Animais , Microambiente TumoralRESUMO
Metastatic melanoma is an aggressive disease, despite recent improvements in therapy. Eradicating all melanoma cells even in drug-sensitive tumors is unsuccessful in patients because a subset of cells can transition to a slow-cycling state, rendering them resistant to most targeted therapy. It is still unclear what pathways define these subpopulations and promote this resistant phenotype. In the current study, we show that Wnt5A, a non-canonical Wnt ligand that drives a metastatic, therapy-resistant phenotype, stabilizes the half-life of p53 and uses p53 to initiate a slow-cycling state following stress (DNA damage, targeted therapy, and aging). Inhibiting p53 blocks the slow-cycling phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. In vivo, this can be accomplished with a single dose of p53 inhibitor at the commencement of BRAF/MEK inhibitor therapy. These data suggest that taking the paradoxical approach of inhibiting rather than activating wild-type p53 may sensitize previously resistant metastatic melanoma cells to therapy.
Assuntos
Melanoma/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Wnt-5a/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , MAP Quinase Quinase Quinases/metabolismo , Melanoma/genética , Melanoma/patologia , Terapia de Alvo Molecular , Mutação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Microambiente Tumoral/efeitos dos fármacos , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Although commensal flora is involved in the regulation of immunity, the interplay between cytokine signaling and microbiota in atherosclerosis remains unknown. We found that interleukin (IL)-23 and its downstream target IL-22 restricted atherosclerosis by repressing pro-atherogenic microbiota. Inactivation of IL-23-IL-22 signaling led to deterioration of the intestinal barrier, dysbiosis, and expansion of pathogenic bacteria with distinct biosynthetic and metabolic properties, causing systemic increase in pro-atherogenic metabolites such as lipopolysaccharide (LPS) and trimethylamine N-oxide (TMAO). Augmented disease in the absence of the IL-23-IL-22 pathway was mediated in part by pro-atherogenic osteopontin, controlled by microbial metabolites. Microbiota transfer from IL-23-deficient mice accelerated atherosclerosis, whereas microbial depletion or IL-22 supplementation reduced inflammation and ameliorated disease. Our work uncovers the IL-23-IL-22 signaling as a regulator of atherosclerosis that restrains expansion of pro-atherogenic microbiota and argues for informed use of cytokine blockers to avoid cardiovascular side effects driven by microbiota and inflammation.
Assuntos
Aterosclerose/etiologia , Aterosclerose/metabolismo , Dieta , Microbioma Gastrointestinal , Homeostase , Interleucina-23/metabolismo , Interleucinas/metabolismo , Animais , Aterosclerose/patologia , Biomarcadores , Modelos Animais de Doenças , Progressão da Doença , Expressão Gênica , Imunofenotipagem , Interleucina-23/deficiência , Metabolismo dos Lipídeos , Camundongos , Camundongos Knockout , Osteopontina/genética , Osteopontina/metabolismo , Transdução de Sinais , Interleucina 22RESUMO
Isoprenoids are vital for all organisms, in which they maintain membrane stability and support core functions such as respiration1. IspH, an enzyme in the methyl erythritol phosphate pathway of isoprenoid synthesis, is essential for Gram-negative bacteria, mycobacteria and apicomplexans2,3. Its substrate, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), is not produced in metazoans, and in humans and other primates it activates cytotoxic Vγ9Vδ2 T cells at extremely low concentrations4-6. Here we describe a class of IspH inhibitors and refine their potency to nanomolar levels through structure-guided analogue design. After modification of these compounds into prodrugs for delivery into bacteria, we show that they kill clinical isolates of several multidrug-resistant bacteria-including those from the genera Acinetobacter, Pseudomonas, Klebsiella, Enterobacter, Vibrio, Shigella, Salmonella, Yersinia, Mycobacterium and Bacillus-yet are relatively non-toxic to mammalian cells. Proteomic analysis reveals that bacteria treated with these prodrugs resemble those after conditional IspH knockdown. Notably, these prodrugs also induce the expansion and activation of human Vγ9Vδ2 T cells in a humanized mouse model of bacterial infection. The prodrugs we describe here synergize the direct killing of bacteria with a simultaneous rapid immune response by cytotoxic γδ T cells, which may limit the increase of antibiotic-resistant bacterial populations.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/imunologia , Ativação Linfocitária/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Oxirredutases/antagonistas & inibidores , Linfócitos T Citotóxicos/efeitos dos fármacos , Animais , Resistência Microbiana a Medicamentos , Resistência a Múltiplos Medicamentos , Inibidores Enzimáticos/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Feminino , Meia-Vida , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Oxirredutases/deficiência , Oxirredutases/genética , Oxirredutases/metabolismo , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Especificidade por Substrato , Suínos/sangue , Linfócitos T Citotóxicos/imunologiaRESUMO
CD4(+) follicular helper T cells (T(FH) cells) are essential for germinal center (GC) responses and long-lived antibody responses. Here we report that naive CD4(+) T cells deficient in the transcription factor Foxp1 'preferentially' differentiated into T(FH) cells, which resulted in substantially enhanced GC and antibody responses. We found that Foxp1 used both constitutive Foxp1A and Foxp1D induced by stimulation of the T cell antigen receptor (TCR) to inhibit the generation of T(FH) cells. Mechanistically, Foxp1 directly and negatively regulated interleukin 21 (IL-21); Foxp1 also dampened expression of the costimulatory molecule ICOS and its downstream signaling at early stages of T cell activation, which rendered Foxp1-deficient CD4(+) T cells partially resistant to blockade of the ICOS ligand (ICOSL) during T(FH) cell development. Our findings demonstrate that Foxp1 is a critical negative regulator of T(FH) cell differentiation.
Assuntos
Diferenciação Celular , Fatores de Transcrição Forkhead/fisiologia , Proteínas Repressoras/fisiologia , Linfócitos T Auxiliares-Indutores/citologia , Animais , Linfócitos T CD4-Positivos/imunologia , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Interleucinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/fisiologiaRESUMO
Missense mutations that inactivate p53 occur commonly in cancer, and germline mutations in TP53 cause Li Fraumeni syndrome, which is associated with early-onset cancer. In addition, there are over two hundred germline missense variants of p53 that remain uncharacterized. In some cases, these germline variants have been shown to encode lesser-functioning, or hypomorphic, p53 protein, and these alleles are associated with increased cancer risk in humans and mouse models. However, most hypomorphic p53 variants remain un- or mis-classified in clinical genetics databases. There thus exists a significant need to better understand the behavior of p53 hypomorphs and to develop a functional assay that can distinguish hypomorphs from wild-type p53 or benign variants. We report the surprising finding that two different African-centric genetic hypomorphs of p53 that occur in distinct functional domains of the protein share common activities. Specifically, the Pro47Ser variant, located in the transactivation domain, and the Tyr107His variant, located in the DNA binding domain, both share increased propensity to misfold into a conformation specific for mutant, misfolded p53. Additionally, cells and tissues containing these hypomorphic variants show increased NF-κB activity. We identify a common gene expression signature from unstressed lymphocyte cell lines that is shared between multiple germline hypomorphic variants of TP53, and which successfully distinguishes wild-type p53 and a benign variant from lesser-functioning hypomorphic p53 variants. Our findings will allow us to better understand the contribution of p53 hypomorphs to disease risk and should help better inform cancer risk in the carriers of p53 variants.
Assuntos
Síndrome de Li-Fraumeni , Proteína Supressora de Tumor p53 , Animais , Camundongos , Humanos , Proteína Supressora de Tumor p53/metabolismo , Predisposição Genética para Doença , Síndrome de Li-Fraumeni/genética , Genes p53 , Heterozigoto , Mutação em Linhagem GerminativaRESUMO
Mutant forms of p53 protein often possess protumorigenic functions, conferring increased survival and migration to tumor cells via their "gain-of-function" activity. Whether and how a common polymorphism in TP53 at amino acid 72 (Pro72Arg; referred to here as P72 and R72) impacts this gain of function has not been determined. We show that mutant p53 enhances migration and metastasis of tumors through the ability to bind and regulate PGC-1α and that this regulation is markedly impacted by the codon 72 polymorphism. Tumor cells with the R72 variant of mutant p53 show increased PGC-1α function along with greatly increased mitochondrial function and metastatic capability. Breast cancers containing mutant p53 and the R72 variant show poorer prognosis compared with P72. The combined results reveal PGC-1α as a novel "gain-of-function" partner of mutant p53 and indicate that the codon 72 polymorphism influences the impact of mutant p53 on metabolism and metastasis.
Assuntos
Genes p53 , Mutação , Neoplasias/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Movimento Celular , Feminino , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Masculino , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/genética , Neoplasias/patologia , Fosforilação Oxidativa , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/antagonistas & inibidoresRESUMO
The SAGA coactivator complex is essential for eukaryotic transcription and comprises four distinct modules, one of which contains the ubiquitin hydrolase USP22. In yeast, the USP22 ortholog deubiquitylates H2B, resulting in Pol II Ser2 phosphorylation and subsequent transcriptional elongation. In contrast to this H2B-associated role in transcription, we report here that human USP22 contributes to the early stages of stimulus-responsive transcription, where USP22 is required for pre-initiation complex (PIC) stability. Specifically, USP22 maintains long-range enhancer-promoter contacts and controls loading of Mediator tail and general transcription factors (GTFs) onto promoters, with Mediator core recruitment being USP22-independent. In addition, we identify Mediator tail subunits MED16 and MED24 and the Pol II subunit RBP1 as potential non-histone substrates of USP22. Overall, these findings define a role for human SAGA within the earliest steps of transcription.
Assuntos
Ubiquitina Tiolesterase/genética , Apoptose , Estresse do Retículo Endoplasmático/genética , Células HCT116 , Humanos , Complexo Mediador/genética , Regiões Promotoras Genéticas , RNA Polimerase II , Transcrição GênicaRESUMO
Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are pathologically activated neutrophils that are crucial for the regulation of immune responses in cancer. These cells contribute to the failure of cancer therapies and are associated with poor clinical outcomes. Despite recent advances in the understanding of PMN-MDSC biology, the mechanisms responsible for the pathological activation of neutrophils are not well defined, and this limits the selective targeting of these cells. Here we report that mouse and human PMN-MDSCs exclusively upregulate fatty acid transport protein 2 (FATP2). Overexpression of FATP2 in PMN-MDSCs was controlled by granulocyte-macrophage colony-stimulating factor, through the activation of the STAT5 transcription factor. Deletion of FATP2 abrogated the suppressive activity of PMN-MDSCs. The main mechanism of FATP2-mediated suppressive activity involved the uptake of arachidonic acid and the synthesis of prostaglandin E2. The selective pharmacological inhibition of FATP2 abrogated the activity of PMN-MDSCs and substantially delayed tumour progression. In combination with checkpoint inhibitors, FATP2 inhibition blocked tumour progression in mice. Thus, FATP2 mediates the acquisition of immunosuppressive activity by PMN-MDSCs and represents a target to inhibit the functions of PMN-MDSCs selectively and to improve the efficiency of cancer therapy.
Assuntos
Proteínas de Transporte de Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Neutrófilos/metabolismo , Idoso , Animais , Ácido Araquidônico/metabolismo , Dinoprostona/metabolismo , Proteínas de Transporte de Ácido Graxo/antagonistas & inibidores , Feminino , Humanos , Metabolismo dos Lipídeos , Lipídeos , Masculino , Camundongos , Pessoa de Meia-Idade , Neutrófilos/patologia , Fator de Transcrição STAT5/metabolismoRESUMO
Epstein-Barr virus (EBV) establishes lifelong asymptomatic infection by replication of its chromatinized episomes with the host genome. EBV exhibits different latency-associated transcriptional repertoires, each with distinct three-dimensional structures. CTCF, Cohesin and PARP1 are involved in maintaining viral latency and establishing episome architecture. Epstein-Barr virus-associated gastric cancer (EBVaGC) represents 1.3-30.9% of all gastric cancers globally. EBV-positive gastric cancers exhibit an intermediate viral transcription profile known as 'Latency II', expressing specific viral genes and noncoding RNAs. In this study, we investigated the impact of PARP1 inhibition on CTCF/Cohesin binding in Type II latency. We observed destabilization of the binding of both factors, leading to a disrupted three-dimensional architecture of the episomes and an altered viral gene expression. Despite sharing the same CTCF binding profile, Type I, II and III latencies exhibit different 3D structures that correlate with variations in viral gene expression. Additionally, our analysis of H3K27ac-enriched interactions revealed differences between Type II latency episomes and a link to cellular transformation through docking of the EBV genome at specific sites of the Human genome, thus promoting oncogene expression. Overall, this work provides insights into the role of PARP1 in maintaining active latency and novel mechanisms of EBV-induced cellular transformation.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Neoplasias Gástricas , Humanos , Infecções por Vírus Epstein-Barr/virologia , Expressão Gênica , Genoma Viral , Herpesvirus Humano 4/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/virologia , Latência Viral/genética , Regulação Viral da Expressão GênicaRESUMO
Cancer metabolism, including in mitochondria, is a disease hallmark and therapeutic target, but its regulation is poorly understood. Here, we show that many human tumors have heterogeneous and often reduced levels of Mic60, or Mitofilin, an essential scaffold of mitochondrial structure. Despite a catastrophic collapse of mitochondrial integrity, loss of bioenergetics, and oxidative damage, tumors with Mic60 depletion slow down cell proliferation, evade cell death, and activate a nuclear gene expression program of innate immunity and cytokine/chemokine signaling. In turn, this induces epithelial-mesenchymal transition (EMT), activates tumor cell movements through exaggerated mitochondrial dynamics, and promotes metastatic dissemination in vivo. In a small-molecule drug screen, compensatory activation of stress response (GCN2) and survival (Akt) signaling maintains the viability of Mic60-low tumors and provides a selective therapeutic vulnerability. These data demonstrate that acutely damaged, "ghost" mitochondria drive tumor progression and expose an actionable therapeutic target in metastasis-prone cancers.
Assuntos
Mitocôndrias/fisiologia , Metástase Neoplásica/fisiopatologia , Neoplasias/genética , Morte Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Transição Epitelial-Mesenquimal , Humanos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Proteínas Mitocondriais/metabolismo , Proteínas Musculares/metabolismo , Invasividade Neoplásica/genética , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Processos Neoplásicos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio , Transdução de SinaisRESUMO
Immunotherapy has revolutionized cancer treatment, but many cancers are not impacted by currently available immunotherapeutic strategies. Here, we investigated inflammatory signaling pathways in neuroblastoma, a classically "cold" pediatric cancer. By testing the functional response of a panel of 20 diverse neuroblastoma cell lines to three different inflammatory stimuli, we found that all cell lines have intact interferon signaling, and all but one lack functional cytosolic DNA sensing via cGAS-STING. However, double-stranded RNA (dsRNA) sensing via Toll-like receptor 3 (TLR3) was heterogeneous, as was signaling through other dsRNA sensors and TLRs more broadly. Seven cell lines showed robust response to dsRNA, six of which are in the mesenchymal epigenetic state, while all unresponsive cell lines are in the adrenergic state. Genetically switching adrenergic cell lines toward the mesenchymal state fully restored responsiveness. In responsive cells, dsRNA sensing results in the secretion of proinflammatory cytokines, enrichment of inflammatory transcriptomic signatures, and increased tumor killing by T cells in vitro. Using single-cell RNA sequencing data, we show that human neuroblastoma cells with stronger mesenchymal signatures have a higher basal inflammatory state, demonstrating intratumoral heterogeneity in inflammatory signaling that has significant implications for immunotherapeutic strategies in this aggressive childhood cancer.
Assuntos
Epigênese Genética/genética , Inflamação/genética , Neuroblastoma/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Citocinas/genética , Humanos , Fatores Imunológicos/genética , Imunoterapia/métodos , Masculino , Camundongos , Camundongos SCID , Nucleotidiltransferases/genética , RNA de Cadeia Dupla/genética , Transdução de Sinais/genética , Receptor 3 Toll-Like/genética , Transcriptoma/genéticaRESUMO
The Epstein Barr virus (EBV) infects almost 95% of the population worldwide. While typically asymptomatic, EBV latent infection is associated with several malignancies of epithelial and lymphoid origin in immunocompromised individuals. In latently infected cells, the EBV genome persists as a chromatinized episome that expresses a limited set of viral genes in different patterns, referred to as latency types, which coincide with varying stages of infection and various malignancies. We have previously demonstrated that latency types correlate with differences in the composition and structure of the EBV episome. Several cellular factors, including the nuclear lamina, regulate chromatin composition and architecture. While the interaction of the viral genome with the nuclear lamina has been studied in the context of EBV lytic reactivation, the role of the nuclear lamina in controlling EBV latency has not been investigated. Here, we report that the nuclear lamina is an essential epigenetic regulator of the EBV episome. We observed that in B cells, EBV infection affects the composition of the nuclear lamina by inducing the expression of lamin A/C, but only in EBV+ cells expressing the Type III latency program. Using ChIP-Seq, we determined that lamin B1 and lamin A/C bind the EBV genome, and their binding correlates with deposition of the histone repressive mark H3K9me2. By RNA-Seq, we observed that knock-out of lamin A/C in B cells alters EBV gene expression. Our data indicate that the interaction between lamins and the EBV episome contributes to the epigenetic control of viral gene expression during latency, suggesting a restrictive function of the nuclear lamina as part of the host response against viral DNA entry into the nucleus.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Infecções por Vírus Epstein-Barr/genética , Expressão Gênica , Regulação Viral da Expressão Gênica , Genoma Viral , Herpesvirus Humano 4/genética , Humanos , Lamina Tipo A/genética , Lâmina Nuclear/genética , Latência Viral/genéticaRESUMO
Tumours evade immune control by creating hostile microenvironments that perturb T cell metabolism and effector function1-4. However, it remains unclear how intra-tumoral T cells integrate and interpret metabolic stress signals. Here we report that ovarian cancer-an aggressive malignancy that is refractory to standard treatments and current immunotherapies5-8-induces endoplasmic reticulum stress and activates the IRE1α-XBP1 arm of the unfolded protein response9,10 in T cells to control their mitochondrial respiration and anti-tumour function. In T cells isolated from specimens collected from patients with ovarian cancer, upregulation of XBP1 was associated with decreased infiltration of T cells into tumours and with reduced IFNG mRNA expression. Malignant ascites fluid obtained from patients with ovarian cancer inhibited glucose uptake and caused N-linked protein glycosylation defects in T cells, which triggered IRE1α-XBP1 activation that suppressed mitochondrial activity and IFNγ production. Mechanistically, induction of XBP1 regulated the abundance of glutamine carriers and thus limited the influx of glutamine that is necessary to sustain mitochondrial respiration in T cells under glucose-deprived conditions. Restoring N-linked protein glycosylation, abrogating IRE1α-XBP1 activation or enforcing expression of glutamine transporters enhanced mitochondrial respiration in human T cells exposed to ovarian cancer ascites. XBP1-deficient T cells in the metastatic ovarian cancer milieu exhibited global transcriptional reprogramming and improved effector capacity. Accordingly, mice that bear ovarian cancer and lack XBP1 selectively in T cells demonstrate superior anti-tumour immunity, delayed malignant progression and increased overall survival. Controlling endoplasmic reticulum stress or targeting IRE1α-XBP1 signalling may help to restore the metabolic fitness and anti-tumour capacity of T cells in cancer hosts.
Assuntos
Endorribonucleases/metabolismo , Mitocôndrias/metabolismo , Neoplasias Ovarianas/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Proteína 1 de Ligação a X-Box/metabolismo , Sistemas de Transporte de Aminoácidos Básicos , Animais , Ascite/metabolismo , Respiração Celular , Progressão da Doença , Estresse do Retículo Endoplasmático , Feminino , Regulação Neoplásica da Expressão Gênica , Glucose/metabolismo , Glutamina/metabolismo , Glicosilação , Humanos , Interferon gama/biossíntese , Interferon gama/genética , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias Ovarianas/patologia , Transdução de Sinais , Taxa de Sobrevida , Linfócitos T/metabolismo , Evasão Tumoral/imunologia , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-Box/biossíntese , Proteína 1 de Ligação a X-Box/deficiênciaRESUMO
Long non-coding (lnc) RNAs can regulate gene expression and protein functions. However, the proportion of lncRNAs with biological activities among the thousands expressed in mammalian cells is controversial. We studied Lockd (lncRNA downstream of Cdkn1b), a 434-nt polyadenylated lncRNA originating 4 kb 3' to the Cdkn1b gene. Deletion of the 25-kb Lockd locus reduced Cdkn1b transcription by approximately 70% in an erythroid cell line. In contrast, homozygous insertion of a polyadenylation cassette 80 bp downstream of the Lockd transcription start site reduced the entire lncRNA transcript level by >90% with no effect on Cdkn1b transcription. The Lockd promoter contains a DNase-hypersensitive site, binds numerous transcription factors, and physically associates with the Cdkn1b promoter in chromosomal conformation capture studies. Therefore, the Lockd gene positively regulates Cdkn1b transcription through an enhancer-like cis element, whereas the lncRNA itself is dispensable, which may be the case for other lncRNAs.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/genética , Elementos Facilitadores Genéticos , RNA Longo não Codificante/genética , Animais , Linhagem Celular , Regulação da Expressão Gênica , Camundongos , Poli A/metabolismo , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Despite effective combination antiretroviral therapy (cART), people living with HIV (PLWH) continue to harbor replication-competent and transcriptionally active virus in infected cells, which in turn can lead to ongoing viral antigen production, chronic inflammation, and increased risk of age-related comorbidities. To identify new agents that may inhibit postintegration HIV beyond cART, we screened a library of 512 pure compounds derived from natural products and identified (-)-hopeaphenol as an inhibitor of HIV postintegration transcription at low to submicromolar concentrations without cytotoxicity. Using a combination of global RNA sequencing, plasmid-based reporter assays, and enzyme activity studies, we document that hopeaphenol inhibits protein kinase C (PKC)- and downstream NF-κB-dependent HIV transcription as well as a subset of PKC-dependent T-cell activation markers, including interleukin-2 (IL-2) cytokine and CD25 and HLA-DRB1 RNA production. In contrast, it does not substantially inhibit the early PKC-mediated T-cell activation marker CD69 production of IL-6 or NF-κB signaling induced by tumor necrosis factor alpha (TNF-α). We further show that hopeaphenol can inhibit cyclin-dependent kinase 9 (CDK9) enzymatic activity required for HIV transcription. Finally, it inhibits HIV replication in peripheral blood mononuclear cells (PBMCs) infected in vitro and dampens viral reactivation in CD4+ cells from PLWH. Our study identifies hopeaphenol as a novel inhibitor that targets a subset of PKC-mediated T-cell activation pathways in addition to CDK9 to block HIV expression. Hopeaphenol-based therapies could complement current antiretroviral therapy otherwise not targeting cell-associated HIV RNA and residual antigen production in PLWH.
Assuntos
Infecções por HIV , Estilbenos , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Proteína Quinase C/genética , Quinase 9 Dependente de Ciclina/metabolismo , Leucócitos Mononucleares/metabolismo , Replicação Viral , Latência Viral , Estilbenos/farmacologia , Infecções por HIV/metabolismo , RNARESUMO
Viruses suppress immune recognition through diverse mechanisms. Epstein-Barr Virus (EBV) establishes latent infection in memory B-lymphocytes and B-cell malignancies where it impacts B-cell immune function. We show here that EBV primary infection of naïve B-cells results in a robust down-regulation of HLA genes. We found that the viral encoded transcriptional regulatory factor EBNA2 bound to multiple regulatory regions in the HLA locus. Conditional expression of EBNA2 correlated with the down regulation of HLA class II transcription. EBNA2 down-regulation of HLA transcription was found to be dependent on CIITA, the major transcriptional activator of HLA class II gene transcription. We identified a major EBNA2 binding site downstream of the CIITA gene and upstream of DEXI, a dexamethasone inducible gene that is oriented head-to-head with CIITA gene transcripts. CRISPR/Cas9 deletion of the EBNA2 site upstream of DEXI attenuated CIITA transcriptional repression. EBNA2 caused an increase in DEXI transcription and a graded change in histone modifications with activation mark H3K27ac near the DEXI locus, and a loss of activation marks at the CIITA locus. A prominent CTCF binding site between CIITA and DEXI enhancers was mutated and further diminished the effects of EBNA2 on CIITA. Analysis of HiC data indicate that DEXI and CIITA enhancers are situated in different chromosome topological associated domains (TADs). These findings suggest that EBNA2 down regulates HLA-II genes through the down regulation of CIITA, and that this down regulation is an indirect consequence of EBNA2 enhancer formation at a neighboring TAD. We propose that enhancer competition between these neighboring chromosome domains represents a novel mechanism for gene regulation demonstrated by EBNA2.