RESUMEN
Many oncogenic transcription factors (TFs) are considered to be undruggable because of their reliance on large protein-protein and protein-DNA interfaces. TFs such as hypoxia-inducible factors (HIFs) and X-box-binding protein 1 (XBP1) are induced by hypoxia and other stressors in solid tumors and bind to unfolded protein response element (UPRE) and hypoxia-induced response element (HRE) motifs to control oncogenic gene programs. Here, we report a strategy to create synthetic transcriptional repressors (STRs) that mimic the basic leucine zipper domain of XBP1 and recognize UPRE and HRE motifs. A lead molecule, STR22, binds UPRE and HRE DNA sequences with high fidelity and competes with both TFs in cells. Under hypoxia, STR22 globally suppresses HIF1α binding to HRE-containing promoters and enhancers, inhibits hypoxia-induced gene expression and blocks protumorigenic phenotypes in triple-negative breast cancer (TNBC) cells. In vivo, intratumoral and systemic STR22 treatment inhibited hypoxia-dependent gene expression, primary tumor growth and metastasis of TNBC tumors. These data validate a novel strategy to target the tumor hypoxia response through coordinated inhibition of TF-DNA binding.
RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed over 6 million individuals worldwide and continues to spread in countries where vaccines are not yet widely available or its citizens are hesitant to become vaccinated. Therefore, it is critical to unravel the molecular mechanisms that allow SARS-CoV-2 and other coronaviruses to infect and overtake the host machinery of human cells. Coronavirus replication triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), a key host cell pathway widely believed to be essential for viral replication. We examined the master UPR sensor IRE1α kinase/RNase and its downstream transcription factor effector XBP1s, which is processed through an IRE1α-mediated mRNA splicing event, in human lung-derived cells infected with betacoronaviruses. We found that human respiratory coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome coronavirus (MERS-CoV), and murine coronavirus (MHV) all induce ER stress and strongly trigger the kinase and RNase activities of IRE1α as well as XBP1 splicing. In contrast, SARS-CoV-2 only partially activates IRE1α through autophosphorylation, but its RNase activity fails to splice XBP1. Moreover, while IRE1α was dispensable for replication in human cells for all coronaviruses tested, it was required for maximal expression of genes associated with several key cellular functions, including the interferon signaling pathway, during SARS-CoV-2 infection. Our data suggest that SARS-CoV-2 actively inhibits the RNase of autophosphorylated IRE1α, perhaps as a strategy to eliminate detection by the host immune system. IMPORTANCE SARS-CoV-2 is the third lethal respiratory coronavirus, after MERS-CoV and SARS-CoV, to emerge this century, causing millions of deaths worldwide. Other common coronaviruses such as HCoV-OC43 cause less severe respiratory disease. Thus, it is imperative to understand the similarities and differences among these viruses in how each interacts with host cells. We focused here on the inositol-requiring enzyme 1α (IRE1α) pathway, part of the host unfolded protein response to virus-induced stress. We found that while MERS-CoV and HCoV-OC43 fully activate the IRE1α kinase and RNase activities, SARS-CoV-2 only partially activates IRE1α, promoting its kinase activity but not RNase activity. Based on IRE1α-dependent gene expression changes during infection, we propose that SARS-CoV-2 prevents IRE1α RNase activation as a strategy to limit detection by the host immune system.
Asunto(s)
COVID-19 , Coronavirus del Síndrome Respiratorio de Oriente Medio , Animales , Ratones , Humanos , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Estrés del Retículo Endoplásmico/genética , SARS-CoV-2/genética , Inositol , Proteínas Serina-Treonina Quinasas/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/metabolismo , Ribonucleasas/genética , Factores de Transcripción , ARN Mensajero , Pulmón/metabolismo , Interferones , Proteína 1 de Unión a la X-Box/genéticaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed over 6 million individuals worldwide and continues to spread in countries where vaccines are not yet widely available, or its citizens are hesitant to become vaccinated. Therefore, it is critical to unravel the molecular mechanisms that allow SARS-CoV-2 and other coronaviruses to infect and overtake the host machinery of human cells. Coronavirus replication triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), a key host cell pathway widely believed essential for viral replication. We examined the master UPR sensor IRE1α kinase/RNase and its downstream transcription factor effector XBP1s, which is processed through an IRE1α-mediated mRNA splicing event, in human lung-derived cells infected with betacoronaviruses. We found human respiratory coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome coronavirus (MERS-CoV), and murine coronavirus (MHV) all induce ER stress and strongly trigger the kinase and RNase activities of IRE1α as well as XBP1 splicing. In contrast, SARS-CoV-2 only partially activates IRE1α through autophosphorylation, but its RNase activity fails to splice XBP1. Moreover, while IRE1α was dispensable for replication in human cells for all coronaviruses tested, it was required for maximal expression of genes associated with several key cellular functions, including the interferon signaling pathway, during SARS-CoV-2 infection. Our data suggest that SARS-CoV-2 actively inhibits the RNase of autophosphorylated IRE1α, perhaps as a strategy to eliminate detection by the host immune system. IMPORTANCE: SARS-CoV-2 is the third lethal respiratory coronavirus after MERS-CoV and SARS-CoV to emerge this century, causing millions of deaths world-wide. Other common coronaviruses such as HCoV-OC43 cause less severe respiratory disease. Thus, it is imperative to understand the similarities and differences among these viruses in how each interacts with host cells. We focused here on the inositol-requiring enzyme 1α (IRE1α) pathway, part of the host unfolded protein response to virus-induced stress. We found that while MERS-CoV and HCoV-OC43 fully activate the IRE1α kinase and RNase activities, SARS-CoV-2 only partially activates IRE1α, promoting its kinase activity but not RNase activity. Based on IRE1α-dependent gene expression changes during infection, we propose that SARS-CoV-2 prevents IRE1α RNase activation as a strategy to limit detection by the host immune system.
RESUMEN
The spread of SARS-CoV-2 and ongoing COVID-19 pandemic underscores the need for new treatments. Here we report that cannabidiol (CBD) inhibits infection of SARS-CoV-2 in cells and mice. CBD and its metabolite 7-OH-CBD, but not THC or other congeneric cannabinoids tested, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after viral entry, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD inhibits SARS-CoV-2 replication in part by up-regulating the host IRE1α RNase endoplasmic reticulum (ER) stress response and interferon signaling pathways. In matched groups of human patients from the National COVID Cohort Collaborative, CBD (100 mg/ml oral solution per medical records) had a significant negative association with positive SARS-CoV-2 tests. This study highlights CBD as a potential preventative agent for early-stage SARS-CoV-2 infection and merits future clinical trials. We caution against use of non-medical formulations including edibles, inhalants or topicals as a preventative or treatment therapy at the present time.
Asunto(s)
Antivirales/farmacología , Cannabidiol/farmacología , Interacciones Huésped-Patógeno/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , Células A549 , Animales , Antivirales/química , COVID-19/virología , Cannabidiol/química , Cannabidiol/metabolismo , Chlorocebus aethiops , Estrés del Retículo Endoplásmico/efectos de los fármacos , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Células Epiteliales/virología , Femenino , Regulación Viral de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno/fisiología , Humanos , Interferones/metabolismo , Ratones , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , SARS-CoV-2/fisiología , Células Vero , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
ABSTRACT: Coronavirus disease 2019 (COVID-19) was declared a pandemic by the World Health Organization on March 11, 2020. By mid-March, London had emerged as the epicenter in the United Kingdom, accounting for 45% of the COVID-19-related mortality. A cancer COVID-19-free "cold hub," through National Health Service partnership with independent institutions, was established to maintain a throughput of surgical oncology patients with an accessible triage system for oncologic specialties. The high population density, commuter volume, and rising COVID-19 incidence heightened the challenge of segregating a vulnerable population that was already at high risk for surgical morbidity and mortality.The aim of this review is to report the experience of developing a multicenter COVID-19-free cold hub with the aim of providing safe surgery for surgical oncology patients. We discuss the timeline, structure, and infection control policy and suggest practical points that may guide other health care systems.The surgical oncology hub treated 1542 patients between March 1 and July 1, 2020. There were no cases of COVID-19-related mortality in a 30-day follow-up. Key strategies for the restructuring of the cancer service included the following: (1) formation of an accessible referral pathway, (2) creation and structuring of cold hub hospitals, (3) development of protocols for infection control and preoperative testing, (4) rapid reorganization of services based on initial feedback, and (5) clear communication and leadership.It has been shown that a surgical oncology cold hub with an accessible referral system and an effective system of preoperative screening system can minimize COVID-19 transmission, morbidity, and mortality, in a region with heavy disease prevalence. This structure represents a safe, ethical, and viable system that can be replicated in other health care systems.
Asunto(s)
COVID-19 , Instituciones Oncológicas/organización & administración , Oncología Quirúrgica/organización & administración , Humanos , Reino Unido/epidemiologíaRESUMEN
Metastasis suppression by high-dose, multi-drug targeting is unsuccessful due to network heterogeneity and compensatory network activation. Here, we show that targeting driver network signaling capacity by limited inhibition of core pathways is a more effective anti-metastatic strategy. This principle underlies the action of a physiological metastasis suppressor, Raf Kinase Inhibitory Protein (RKIP), that moderately decreases stress-regulated MAP kinase network activity, reducing output to transcription factors such as pro-metastastic BACH1 and motility-related target genes. We developed a low-dose four-drug mimic that blocks metastatic colonization in mouse breast cancer models and increases survival. Experiments and network flow modeling show limited inhibition of multiple pathways is required to overcome variation in MAPK network topology and suppress signaling output across heterogeneous tumor cells. Restricting inhibition of individual kinases dissipates surplus signal, preventing threshold activation of compensatory kinase networks. This low-dose multi-drug approach to decrease signaling capacity of driver networks represents a transformative, clinically relevant strategy for anti-metastatic treatment.
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Redes y Vías Metabólicas/efectos de los fármacos , Metástasis de la Neoplasia/prevención & control , Proteínas de Unión a Fosfatidiletanolamina/genética , Transducción de Señal/efectos de los fármacos , Animales , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Movimiento Celular , Combinación de Medicamentos , Femenino , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos C57BL , Ratones DesnudosRESUMEN
The rapid spread of COVID-19 underscores the need for new treatments. Here we report that cannabidiol (CBD), a compound produced by the cannabis plant, inhibits SARS-CoV-2 infection. CBD and its metabolite, 7-OH-CBD, but not congeneric cannabinoids, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after cellular infection, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD induces interferon expression and up-regulates its antiviral signaling pathway. A cohort of human patients previously taking CBD had significantly lower SARS-CoV-2 infection incidence of up to an order of magnitude relative to matched pairs or the general population. This study highlights CBD, and its active metabolite, 7-OH-CBD, as potential preventative agents and therapeutic treatments for SARS-CoV-2 at early stages of infection.