RESUMEN
Sorafenib represents the current standard of care for patients with advanced-stage hepatocellular carcinoma (HCC). However, acquired drug resistance occurs frequently during therapy and is accompanied by rapid tumor regrowth after sorafenib therapy termination. To identify the mechanism of this therapy-limiting growth resumption, we established robust sorafenib resistance HCC cell models that exhibited mitochondrial dysfunction and chemotherapeutic crossresistance. We found a rapid relapse of tumor cell proliferation after sorafenib withdrawal, which was caused by renewal of mitochondrial structures alongside a metabolic switch toward high electron transport system (ETS) activity. The translation-inhibiting antibiotic tigecycline impaired the biogenesis of mitochondrial DNA-encoded ETS subunits and limited the electron acceptor turnover required for glutamine oxidation. Thereby, tigecycline prevented the tumor relapse in vitro and in murine xenografts in vivo. These results offer a promising second-line therapeutic approach for advanced-stage HCC patients with progressive disease undergoing sorafenib therapy or treatment interruption due to severe adverse events.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Hepáticas/tratamiento farmacológico , Sorafenib/farmacología , Tigeciclina/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Femenino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Ratones SCID , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Recurrencia Local de Neoplasia/prevención & control , Inhibidores de la Síntesis de la Proteína/farmacologíaRESUMEN
Cytoplasmic double-stranded RNA is sensed by RIG-I-like receptors (RLRs), leading to induction of type I interferons (IFN-Is), proinflammatory cytokines, and apoptosis. Here, we elucidate signaling mechanisms that lead to cytokine secretion and cell death induction upon stimulation with the bona fide RIG-I ligand 5'-triphosphate RNA (3p-RNA) in tumor cells. We show that both outcomes are mediated by dsRNA-receptor families with RLR being essential for cytokine production and IFN-I-mediated priming of effector pathways but not for apoptosis. Affinity purification followed by mass spectrometry and subsequent functional analysis revealed that 3p-RNA bound and activated oligoadenylate synthetase 1 and RNase L. RNase L-deficient cells were profoundly impaired in their ability to undergo apoptosis. Mechanistically, the concerted action of translational arrest triggered by RNase L and up-regulation of NOXA was needed to deplete the antiapoptotic MCL-1 to cause intrinsic apoptosis. Thus, 3p-RNA-induced apoptosis is a two-step process consisting of RIG-I-dependent priming and an RNase L-dependent effector phase.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/inmunología , Endorribonucleasas/inmunología , Neoplasias/inmunología , Receptores de Ácido Retinoico/inmunología , 2',5'-Oligoadenilato Sintetasa/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Apoptosis , Línea Celular Tumoral , Técnicas de Cocultivo , Proteína 58 DEAD Box/genética , Endorribonucleasas/genética , Humanos , Helicasa Inducida por Interferón IFIH1/genética , Ligandos , Ratones , Receptores Inmunológicos/genéticaRESUMEN
Replication competent vesicular stomatitis virus (VSV) is the basis of a vaccine against Ebola and VSV strains are developed as oncolytic viruses. Both functions depend on the ability of VSV to induce adequate amounts of interferon-α/ß. It is therefore important to understand how VSV triggers interferon responses. VSV activates innate immunity via retinoic acid-inducible gene I (RIG-I), a sensor for viral RNA. Our results show that VSV needs to replicate for a robust interferon response. Analysis of RIG-I-associated RNA identified a copy-back defective-interfering (DI) genome and full-length viral genomes as main trigger of RIG-I. VSV stocks depleted of DI genomes lost most of their interferon-stimulating activity. The remaining full-length genome and leader-N-read-through sequences, however, still triggered RIG-I. Awareness for DI genomes as trigger of innate immune responses will help to standardize DI genome content and to purposefully deplete or use DI genomes as natural adjuvants in VSV-based therapeutics.
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Proteína 58 DEAD Box/metabolismo , Genoma Viral , Mutación , Receptores Inmunológicos/metabolismo , Estomatitis Vesicular/metabolismo , Estomatitis Vesicular/virología , Virus de la Estomatitis Vesicular Indiana/fisiología , Replicación Viral , Animales , Línea Celular , Genoma Viral/genética , Genoma Viral/inmunología , Interacciones Huésped-Patógeno , Humanos , Inmunomodulación , ARN Viral/genética , ARN Viral/inmunologíaRESUMEN
Analysis of autoinflammatory and immunodeficiency disorders elucidates human immunity and fosters the development of targeted therapies. Oligoadenylate synthetase 1 is a type I interferon-induced, intracellular double-stranded RNA (dsRNA) sensor that generates 2'-5'-oligoadenylate to activate ribonuclease L (RNase L) as a means of antiviral defense. We identified four de novo heterozygous OAS1 gain-of-function variants in six patients with a polymorphic autoinflammatory immunodeficiency characterized by recurrent fever, dermatitis, inflammatory bowel disease, pulmonary alveolar proteinosis, and hypogammaglobulinemia. To establish causality, we applied genetic, molecular dynamics simulation, biochemical, and cellular functional analyses in heterologous, autologous, and inducible pluripotent stem cell-derived macrophages and/or monocytes and B cells. We found that upon interferon-induced expression, OAS1 variant proteins displayed dsRNA-independent activity, which resulted in RNase L-mediated RNA cleavage, transcriptomic alteration, translational arrest, and dysfunction and apoptosis of monocytes, macrophages, and B cells. RNase L inhibition with curcumin modulated and allogeneic hematopoietic cell transplantation cured the disorder. Together, these data suggest that human OAS1 is a regulator of interferon-induced hyperinflammatory monocyte, macrophage, and B cell pathophysiology.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/genética , Enfermedades Autoinflamatorias Hereditarias/genética , Enfermedades de Inmunodeficiencia Primaria/genética , 2',5'-Oligoadenilato Sintetasa/inmunología , 2',5'-Oligoadenilato Sintetasa/aislamiento & purificación , 2',5'-Oligoadenilato Sintetasa/metabolismo , Linfocitos B/inmunología , Células Cultivadas , Análisis Mutacional de ADN , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Pruebas de Enzimas , Mutación con Ganancia de Función/inmunología , Técnicas de Inactivación de Genes , Trasplante de Células Madre Hematopoyéticas , Enfermedades Autoinflamatorias Hereditarias/diagnóstico , Enfermedades Autoinflamatorias Hereditarias/inmunología , Enfermedades Autoinflamatorias Hereditarias/terapia , Heterocigoto , Humanos , Lactante , Recién Nacido , Interferón Tipo I/metabolismo , Macrófagos/inmunología , Simulación de Dinámica Molecular , Monocitos/inmunología , Cultivo Primario de Células , Enfermedades de Inmunodeficiencia Primaria/diagnóstico , Enfermedades de Inmunodeficiencia Primaria/inmunología , Enfermedades de Inmunodeficiencia Primaria/terapia , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
An exacerbated and unbalanced immune response may account for the severity of COVID-19, the disease caused by the novel severe acute respiratory syndrome (SARS) coronavirus 2, SARS-CoV-2. In this Viewpoint, we summarize recent evidence for the role of neutrophils in the pathogenesis of COVID-19 and propose CXCR2 inhibition as a promising treatment option to block neutrophil recruitment and activation.
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Betacoronavirus , Infecciones por Coronavirus/epidemiología , Pandemias , Neumonía Viral , COVID-19 , Humanos , Inflamación , SARS-CoV-2RESUMEN
The Tousled-like kinases 1 and 2 (TLK1/2) control histone deposition through the ASF1 histone chaperone and influence cell cycle progression and genome maintenance, yet the mechanisms underlying TLK-mediated genome stability remain uncertain. Here, we show that TLK loss results in severe chromatin decompaction and altered genome accessibility, particularly affecting heterochromatic regions. Failure to maintain heterochromatin increases spurious transcription of repetitive elements and induces features of alternative lengthening of telomeres (ALT). TLK depletion culminates in a cGAS-STING-TBK1-mediated innate immune response that is independent of replication-stress signaling and attenuated by the depletion of factors required to produce extra-telomeric DNA. Analysis of human cancers reveals that chromosomal instability correlates with high TLK2 and low STING levels in many cohorts. Based on these findings, we propose that high TLK levels contribute to immune evasion in chromosomally unstable and ALT+ cancers.
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Inmunidad Innata , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Homeostasis del Telómero , Línea Celular Tumoral , Heterocromatina/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Neoplasias/enzimología , Neoplasias/inmunología , Nucleotidiltransferasas/metabolismo , Recombinación Genética/genética , Telómero/metabolismoRESUMEN
Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic immune receptor sensing viral RNA. It triggers the release of type I interferons (IFN) and proinflammatory cytokines inducing an adaptive cellular immune response. We investigated the therapeutic potential of systemic RIG-I activation by short 5'-triphosphate-modified RNA (ppp-RNA) for the treatment of acute myeloid leukemia (AML) in the syngeneic murine C1498 AML tumor model. ppp-RNA treatment significantly reduced tumor burden, delayed disease onset and led to complete remission including immunological memory formation in a substantial proportion of animals. Therapy-induced tumor rejection was dependent on CD4+ and CD8+ T cells, but not on NK or B cells, and relied on intact IFN and mitochondrial antiviral signaling protein (MAVS) signaling in the host. Interestingly, ppp-RNA treatment induced programmed death ligand 1 (PD-L1) expression on AML cells and established therapeutic sensitivity to anti-PD-1 checkpoint blockade in vivo. In immune-reconstituted humanized mice, ppp-RNA treatment reduced the number of patient-derived xenografted (PDX) AML cells in blood and bone marrow while concomitantly enhancing CD3+ T cell counts in the respective tissues. Due to its ability to establish a state of full remission and immunological memory, our findings show that ppp-RNA treatment is a promising strategy for the immunotherapy of AML.
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Anticuerpos Neutralizantes/farmacología , Proteína 58 DEAD Box/inmunología , Inmunoterapia/métodos , Leucemia Mieloide Aguda/terapia , ARN Bicatenario/farmacología , Receptores Virales/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Proteína 58 DEAD Box/genética , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica , Xenoinjertos , Humanos , Memoria Inmunológica/efectos de los fármacos , Interferones/genética , Interferones/inmunología , Isoinjertos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/mortalidad , Ratones , Receptores Virales/agonistas , Receptores Virales/genética , Inducción de Remisión , Transducción de Señal , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
BACKGROUND: Complete signal transducer and activator of transcription 1 (STAT1) deficiency causes a rare primary immunodeficiency that is characterized by defective IFN-dependent gene expression leading to life-threatening viral and mycobacterial infections early in life. OBJECTIVE: To characterize a novel STAT1 loss-of-function variant leading to pathological infection susceptibility and hyperinflammation. METHODS: Clinical, immunologic, and genetic characterization of a patient with severe infections and hemophagocytic lymphohistiocytosis-like hyperinflammation was investigated. RESULTS: We reported a child of consanguineous parents who presented with multiple severe viral infections that ultimately triggered hemophagocytic lymphohistiocytosis and liver failure. Despite intensified therapy with antivirals and cytomegalovirus-specific donor cells, the child died after hematopoietic stem cell transplantation because of cytomegalovirus reactivation with acute respiratory distress syndrome. Exome sequencing revealed a homozygous STAT1 variant (p.Val339ProfsTer18), leading to loss of STAT1 protein expression. Upon type I and type II IFN stimulation, immune and nonimmune cells showed defective upregulation of IFN-stimulated genes and increased susceptibility to viral infection in vitro. Increased viral infection rates were paralleled by hyperinflammatory ex vivo cytokine responses with increased production of TNF, IL-6, and IL-18. CONCLUSIONS: Complete STAT1 deficiency is a devastating disorder characterized by severe viral infections and ensuing hyperinflammatory responses. Early diagnosis can be made by exome sequencing and variant validation by functional testing of STAT1-dependent programmed cell death 1 ligand 1 surface expression on monocytes. Furthermore, high awareness for hyperinflammatory complications and potential targeted treatment strategies such as IL-18 binding protein could be considered. Hematopoietic stem cell transplantation is the only definitive treatment strategy but remains challenging.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Síndromes de Inmunodeficiencia , Linfohistiocitosis Hemofagocítica , Virosis , Niño , Citomegalovirus , Humanos , Síndromes de Inmunodeficiencia/diagnóstico , Síndromes de Inmunodeficiencia/genética , Linfohistiocitosis Hemofagocítica/diagnóstico , Linfohistiocitosis Hemofagocítica/genética , Factor de Transcripción STAT1/genéticaRESUMEN
Following publication of the original article [1], the authors have reported that Fig. 2 and Additional file 1: Figure S1, S2 partially show red scripts.
RESUMEN
BACKGROUND: The tumor microenvironment (TME) combines features of regulatory cytokines and immune cell populations to evade the recognition by the immune system. Myeloid-derived suppressor cells (MDSC) comprise populations of immature myeloid cells in tumor-bearing hosts with a highly immunosuppressive capacity. We could previously identify RIG-I-like helicases (RLH) as targets for the immunotherapy of pancreatic cancer inducing immunogenic tumor cell death and type I interferons (IFN) as key mediators linking innate with adaptive immunity. METHODS: Mice with orthotopically implanted KrasG12D p53fl/R172H Ptf1a-Cre (KPC) pancreatic tumors were treated intravenously with the RLH ligand polyinosinic-polycytidylic acid (poly(I:C)), and the immune cell environment in tumor and spleen was characterized. A comprehensive analysis of the suppressive capacity as well as the whole transcriptomic profile of isolated MDSC subsets was performed. Antigen presentation capability of MDSC from mice with ovalbumin (OVA)-expressing tumors was investigated in T cell proliferation assays. The role of IFN in MDSC function was investigated in Ifnar1-/- mice. RESULTS: MDSC were strongly induced in orthotopic KPC-derived pancreatic cancer, and frequencies of MDSC subsets correlated with tumor weight and G-CSF serum levels, whereas other immune cell populations decreased. Administration of the RLH-ligand induced a IFN-driven immune response, with increased activation of T cells and dendritic cells (DC), and a reduced suppressive capacity of both polymorphonuclear (PMN)-MDSC and monocytic (M)-MDSC fractions. Whole transcriptomic analysis confirmed an IFN-driven gene signature of MDSC, a switch from a M2/G2- towards a M1/G1-polarized phenotype, and the induction of genes involved in the antigen presentation machinery. Nevertheless, MDSC failed to present tumor antigen to T cells. Interestingly, we found MDSC with reduced suppressive function in Ifnar1-deficient hosts; however, there was a common flaw in immune cell activation, which was reflected by defective immune cell activation and tumor control. CONCLUSIONS: We provide evidence that the treatment with immunostimulatory RNA reprograms the TME of pancreatic cancer by reducing the suppressive activity of MDSC, polarizing myeloid cells into a M1-like state and recruiting DC. We postulate that tumor cell-targeting combination strategies may benefit from RLH-based TME remodeling. In addition, we provide novel insights into the dual role of IFN signaling in MDSC's suppressive function and provide evidence that host-intrinsic IFN signaling may be critical for MDSC to gain suppressive function during tumor development.
RESUMEN
Checkpoint molecules such as programmed death 1 (PD-1) dampen excessive T cell activation to preserve immune homeostasis. PD-1-specific monoclonal antibodies have revolutionized cancer therapy, as they reverse tumour-induced T cell exhaustion and restore CTL activity. Based on this success, deciphering underlying mechanisms of PD-1-mediated immune functions has become an important field of immunological research. Initially described for T cells, there is emerging evidence of unconventional PD-1 expression by myeloid as well as tumor cells, yet, with cell-intrinsic functions in various animal tumor models. Here, we describe positive PD-1 antibody staining of various murine immune and tumour cells that is, unlike for T cells, not the PD-1 receptor and restricted to cells with low forward scatter characteristics. Based on flow cytometry and various approaches, including two established murine anti-PD-1 antibody clones, CRISPR/Cas9 genome editing and confocal imaging, we describe a staining pattern assigned to a nuclear antigen cross-reacting with anti-PD-1 monoclonal antibodies. Lack of PD-1 expression was further underlined by the analysis of PD-1 expression from B16-F10-derived 3D cultures and ex vivo tumours. Thus, our data provide multiple lines of evidence that PD-1 expression by non-T cells is unlikely to be the case and, taking recent data of PD-1 tumour cell-intrinsic functions into account, suggest that other antibody-mediated pathways might apply.