Targeting Runt-Related Transcription Factor 1 Prevents Pulmonary Fibrosis and Reduces Expression of Severe Acute Respiratory Syndrome Coronavirus 2 Host Mediators
2021; Elsevier BV; Volume: 191; Issue: 7 Linguagem: Inglês
10.1016/j.ajpath.2021.04.006
ISSN1525-2191
AutoresMichael O’Hare, Dhanesh Amarnani, Hannah Whitmore, Miranda An, Claudia Mariño, Leslie Ramos, Santiago Delgado‐Tirado, Xinyao Hu, Natalia Chmielewska, Anita Chandrahas, Antonia Fitzek, Fabian Heinrich, Stefan Steurer, Benjamin Ondruschka, Markus Glatzel, Susanne Krasemann, Diego Sepúlveda‐Falla, David Lagares, Julien Pedron, John H. Bushweller, Paul Liu, Joseph F. Arboleda‐Velásquez, Leo A. Kim,
Tópico(s)SARS-CoV-2 and COVID-19 Research
ResumoPulmonary fibrosis (PF) can arise from unknown causes, as in idiopathic PF, or as a consequence of infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current treatments for PF slow, but do not stop, disease progression. We report that treatment with a runt-related transcription factor 1 (RUNX1) inhibitor (Ro24-7429), previously found to be safe, although ineffective, as a Tat inhibitor in patients with HIV, robustly ameliorates lung fibrosis and inflammation in the bleomycin-induced PF mouse model. RUNX1 inhibition blunted fundamental mechanisms downstream pathologic mediators of fibrosis and inflammation, including transforming growth factor-β1 and tumor necrosis factor-α, in cultured lung epithelial cells, fibroblasts, and vascular endothelial cells, indicating pleiotropic effects. RUNX1 inhibition also reduced the expression of angiotensin-converting enzyme 2 and FES Upstream Region (FURIN), host proteins critical for SARS-CoV-2 infection, in mice and in vitro. A subset of human lungs with SARS-CoV-2 infection overexpress RUNX1. These data suggest that RUNX1 inhibition via repurposing of Ro24-7429 may be beneficial for PF and to battle SARS-CoV-2, by reducing expression of viral mediators and by preventing respiratory complications. Pulmonary fibrosis (PF) can arise from unknown causes, as in idiopathic PF, or as a consequence of infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current treatments for PF slow, but do not stop, disease progression. We report that treatment with a runt-related transcription factor 1 (RUNX1) inhibitor (Ro24-7429), previously found to be safe, although ineffective, as a Tat inhibitor in patients with HIV, robustly ameliorates lung fibrosis and inflammation in the bleomycin-induced PF mouse model. RUNX1 inhibition blunted fundamental mechanisms downstream pathologic mediators of fibrosis and inflammation, including transforming growth factor-β1 and tumor necrosis factor-α, in cultured lung epithelial cells, fibroblasts, and vascular endothelial cells, indicating pleiotropic effects. RUNX1 inhibition also reduced the expression of angiotensin-converting enzyme 2 and FES Upstream Region (FURIN), host proteins critical for SARS-CoV-2 infection, in mice and in vitro. A subset of human lungs with SARS-CoV-2 infection overexpress RUNX1. These data suggest that RUNX1 inhibition via repurposing of Ro24-7429 may be beneficial for PF and to battle SARS-CoV-2, by reducing expression of viral mediators and by preventing respiratory complications. Pulmonary fibrosis (PF) is a chronic and often fatal lung disease characterized by the accumulation of extracellular matrix and the destruction of the lung parenchyma.1Lederer D.J. Martinez F.J. 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Topical delivery of a small molecule RUNX1 transcription factor inhibitor for the treatment of proliferative vitreoretinopathy.Sci Rep. 2020; 10: 20554Crossref PubMed Scopus (14) Google Scholar These results suggest that RUNX1 activity is associated with both pathologic angiogenesis and fibrosis, key cellular processes found in response to SARS-CoV-2 infection. Thus, RUNX1 modulation may result in novel modalities of treatment for prevalent, nonneoplastic conditions.27Whitmore H.A.B. Amarnani D. O'Hare M. Delgado-Tirado S. Gonzalez-Buendia L. An M. Pedron J. Bushweller J.H. Arboleda-Velasquez J.F. Kim L.A. TNF-α signaling regulates RUNX1 function in endothelial cells.FASEB. 2021; 35: e21155Crossref PubMed Scopus (12) Google Scholar Ro24-7429 and Ro5-3335 are small-molecule inhibitors of RUNX1 activity. Ro5-3335 has been widely used in multiple studies as a RUNX1 inhibitor and is commercially available.25Cunningham L. Finckbeiner S. Hyde R.K. Southall N. Marugan J. Yedavalli V.R.K. Dehdashti S.J. Reinhold W.C. Alemu L. Zhao L. Yeh J.R. Sood R. Pommier Y. Austin C.P. Jeang K.T. Zheng W. Liu P. Identification of benzodiazepine Ro5-3335 as an inhibitor of CBF leukemia through quantitative high throughput screen against RUNX1–CBFβ interaction.Proc Natl Acad Sci U S A. 2012; 109: 14592Crossref PubMed Scopus (91) Google Scholar Ro24-7429 is of immediate translational interest because it was originally developed and tested in a phase 2 clinical trial for its potential effect as a Tat antagonist in the treatment of AIDS in patients infected with HIV.30Hsu M.C. Dhingra U. Earley J.V. Holly M. Keith D. Nalin C.M. Richou A.R. Schutt A.D. Tam S.Y. Potash M.J. Inhibition of type 1 human immunodeficiency virus replication by a tat antagonist to which the virus remains sensitive after prolonged exposure in vitro.Proc Natl Acad Sci U S A. 1993; 90: 6395-6399Crossref PubMed Scopus (52) Google Scholar Ro24-7429 had an acceptable safety profile in clinical trials but was found to have no detectable antiviral activity.31Haubrich R.H. Flexner C. Lederman M.M. Hirsch M. Pettinelli C.P. Ginsberg R. Lietman P. Hamzeh F.M. Spector S.A. Richman D.D. The AIDS Clinical Trials Group 213 TeamA randomized trial of the activity and safety of Ro 24-7429 (Tat antagonist) versus nucleoside for human immunodeficiency virus infection.J Infect Dis. 1995; 172: 1246-1252Crossref PubMed Scopus (28) Google Scholar In this study, we aim to evaluate the role of RUNX1 in lung fibrosis and test the potential antifibrotic effects of Ro24-7429 using the bleomycin-induced model of lung injury.32Tashiro J. Rubio G.A. Limper A.H. Williams K. Elliot S.J. Ninou I. Aidinis V. Tzouvelekis A. Glassberg M.K. Exploring animal models that resemble idiopathic pulmonary fibrosis.Front Med. 2017; 4: 118Crossref PubMed Scopus (153) Google Scholar An increase in RUNX1 RNA was found among a set of genes overexpressed in lung tissue of patients infected with SARS-CoV-2, a screen of differentially expressed angiogenesis-associated genes.16Ackermann M. Verleden S.E. Kuehnel M. Haverich A. Welte T. Laenger F. Vanstapel A. Werlein C. Stark H. Tzankov A. Li W.W. Li V.W. Mentzer S.J. Jonigk D. Pulmonary vascular endothelialitis, thrombosis, and angiogenesis in Covid-19.N Engl J Med. 2020; 383: 120-128Crossref PubMed Scopus (3475) Google Scholar In addition, RUNX1 has recently been predicted to modulate FES Upstream Region (FURIN) and angiotensin-converting enzyme 2 (ACE2) expression based on genomic-guided molecular maps of upstream regulatory elements.33Glinsky G.V. Tripartite combination of candidate pandemic mitigation agents: vitamin D, quercetin, and estradiol manifest properties of medicinal agents for targeted mitigation of the COVID-19 pandemic defined by genomics-guided tracing of SARS-CoV-2 targets in human cells.Biomedicines. 2020; 8: 129Google Scholar These data indicate that RUNX1 may play an important role during SARS-CoV-2 infection, and through the modulation of RUNX1 it may be possible to repress key host mediators to viral entry. We herein examined a potential direct link between RUNX1 function and the expression of proteins ACE2 and FURIN, two proteins with critical roles in SARS-CoV-2 infection of the lung. TNF-α and TGF-β1 were purchased from PeproTech (Rocky Hill, NJ). RUNX1 inhibitor Ro5-3335 was purchased from Millipore-Sigma (Burlington, MA). We contracted the synthesis of Ro24-7429 as fee for service from MedKoo Biosciences (Morrisville, NC), which confirmed the correct structure by 1H nuclear magnetic resonance and mass spectrometry, and purity >99% by high-performance liquid chromatography (data not shown). The remaining Ro24-7429 was received as a kind gift from Paul Liu (National Institutes of Health, National Human Genome Research Institute, Bethesda, MD). The core-binding factor-β–RUNX1 protein-protein interaction inhibitor, AI-14-91, was synthesized as described previously19Rai D.K. Sharma P. Kumar R. Post covid 19 pulmonary fibrosis- is it reversible?.Indian J Tuberc. 2020; ([Epub ahead of print] doi:)10.1016/j.ijtb.2020.11.003Crossref Scopus (73) Google Scholar and was received as a kind gift from the Bushweller Lab (Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA). Animal procedures were approved by the Institutional Animal Care and Use Committee of Massachusetts Eye and Ear, and performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. C57BL/6J male and female mice, aged 6 to 8 weeks, were purchased from Jackson Laboratory (Bar Harbor, ME). For all procedures, mice were anesthetized by i.p. injection of ketamine/xylazine mixture (100/50 mg/kg). Bleomycin sulfate (Sigma-Aldrich, St. Louis, MO) was dissolved in sterile 0.9% saline and administered as a single dose of 0.05 units in a total volume of 50 μL in saline solution per animal intratracheally. Control animals received 0.05 mL saline alone. A preventative regimen was chosen, and each animal received either Ro24-7429 drug, 70 mg/kg, or vehicle every other day for 7 days before the induction of the model and continued until the end of the experiment for 1 or 2 weeks. All animals received intratracheal instillations of bleomycin on day 0, as previously described.34Kremer S. Breuer R. Lossos I.S. Berkman N. Christensen T.G. Connor M.W. Breuer R. Effect of immunomodulators on bleomycin-induced lung injury.Respiration. 1999; 66: 455-462Crossref PubMed Scopus (27) Google Scholar, 35Laxer U. Lossos I.S. Gillis S. Or R. Christensen T.G. Goldstein R.H. Breuer R. The effect of enoxaparin on bleomycin-induced lung injury in mice.Exp Lung Res. 1999; 25: 531-541Crossref PubMed Scopus (22) Google Scholar, 36Berkman N. Kremer S. Or R. Lossos I.S. Christensen T.G. Goldstein R.H. Breuer R. Human recombinant interferon-alpha2a and interferon-alphaA/D have different effects on bleomycin-induced lung injury.Respiration. 2001; 68: 169-177Crossref PubMed Scopus (18) Google Scholar, 37Edler C. Schröder A.S. Apfelbacher M. Fitzek A. Heinemann A. Heinrich F. Klein A. Langenwalder F. Lutgehetmann M. Meissner K. Puschel K. Schadler J. Steurer S. Mushumba H. Sperhake J.-P. Dying with SARS-CoV-2 infection—an autopsy study of the first consecutive 80 cases in Hamburg, Germany.Int J Legal Med. 2020; 134: 1275-1284Crossref PubMed Scopus (303) Google Scholar A separate experiment was performed with similar drug-vehicle treatments and intratracheal saline instillation for controls. The surgeon performing intratracheal instillations was masked to the identity of the treatment groups. Mice were sacrificed at day 7 or day 14 after bleomycin instillations, and mouse lungs were perfused with phosphate-buffered saline to inflate the lungs before fixation. Lung tissue sections were fixed in 10% neutral-buffered formalin (Sigma-Aldrich; HT501128-4L) for 24 hours for histologic analysis. Fixed lungs were paraffin embedded and sectioned (5 μm thick), stained with hematoxylin and eosin to examine gross morphology and Masson trichrome stain to visualize collagen deposition, and examined by microscopy. Lung fibrosis was measured using quantitative histology using the Ashcroft method of analysis. All measurements were performed by two independent graders (N.C., A.C.) in a blinded manner. Images were acquired with the Nikon Eclipse E800 microscope (Minato City, Tokyo, Japan) with an Olympus DP70 camera (OM Digital Solutions, Hachioji, Tokyo, Japan). Adjacent 2× images of the lung were stitched together using Adobe Photoshop CS6 (San Jose, CA). Paraffin-embedded sections were processed for immunofluorescence using the following antibodies: anti-RUNX1 (1:100; LS-B13948; Lifespan Biosciences, Seattle, WA), anti–α-smooth muscle actin (SMA) antibody (1:200; A-2547; Millipore-Sigma), anti-ionized calcium binding adaptor molecule 1 (Iba1) antibody (1:100; ab5076; Abcam, Cambridge, UK), anti-Ly6g antibody (1:100; ab25377; Abcam), and Isolectin GS-IB4 Alexa Fluoro 594 Conjugate (1:250; l21413; Invitrogen, Carlsbad, CA). For heat-induced antigen retrieval, the slides were boiled in 10 mmol/L sodium citrate buffer (pH 6.0) and then maintained at a subboiling temperature (95°C to 100°C) for 20 minutes and subsequently cooled on the bench top for 30 minutes. Slides were washed with distilled water, permeabilized with 0.5% Triton X-100 in phosphate-buffered saline for 5 minutes, and blocked (10% goat serum in phosphate-buffered saline) for 1 hour at room temperature. The primary antibody was prepared in antibody dilution buffer (5% goat serum), and samples were incubated overnight with the antibody solution at 4°C. Sections were washed with phosphate-buffered saline and incubated with goat anti-rabbit Alexa Fluor 594 secondary antibody (1:500; A-11012; Invitrogen) for 2 hours at room temperature. Slides were mounted and visualized using Prolong Gold Antifade Reagent with DAPI (P36935; Invitrogen). Images were obtained using an EVOS FL automated stage live cell imaging system (Life Technologies, Cambridge, MA). Mouse lungs were perfused with saline and fixed with half strength Karnovsky fixative (2% formaldehyde + 2.5% glutaraldehyde, in 0.1 mol/L sodium cacodylate buffer, pH 7.4) for 24 hours under refrigeration. After fixation, samples were trimmed into 1-mm thick segments, rinsed with 0.1 mol/L sodium cacodylate buffer, post-fixed with 2% osmium tetroxide in 0.1 mol/L sodium cacodylate buffer for 1.5 hours, en bloc stained with 2% gadolinium triacetate in 0.05 mol/L sodium maleate buffer, pH 6, for 30 minutes, then dehydrated with graded ethyl alcohol solutions, transitioned with propylene oxide, and infiltrated in tEPON-812 epoxy resin (Tousimis, Rockville, MD) utilizing an automated EMS Lynx 2 EM tissue processor (Electron Microscopy Sciences, Hatfield, PA). The processed samples were oriented into tEPON-812 epoxy resin inside flat molds and polymerized using a 60°C oven. Semithin and ultrathin sections were obtained using a Leica UC7 ultramicrotome (Leica Microsystems, Buffalo Grove, IL) and diamond knives (Diatome, Hatfield, PA). Semithin sections were cut at 1 μm thickness through different lobes stained with 1% toluidine blue in 1% sodium tetraborate aqueous solution for assessment by light microscopy. Ultrathin sections on grids were stained with aqueous 2.5% gadolinium triacetate and modified Sato lead citrate. Grids were imaged using an FEI Tecnai G2 Spirit transmission electron microscope (FEI, Hillsboro, OR) at 80 kV interfaced with an AMT XR41 digital charge-coupled device camera (Advanced Microscopy Techniques, Woburn, MA) for digital TIFF file image acquisition. Transmission electron microscopy digital images were captured at 2000 × 2000 pixels at 16-bit resolution. Human alveolar epithelial cells (A549) were a generous gift from Dr. David Lagares (Massachusetts General Hospital, Boston, MA). Cells were maintained in low-glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mmol/L l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified 5% CO2 atmosphere. Confluent cultures of cells were pretreated with Ro24-7429 for 24 hours, followed by stimulation with 5 ng/mL of TGF-β1. Human lung fibroblasts (HLFs) were also a kind gift from Dr. David Lagares. Cells were maintained as above before treatment with Ro24-7429 and nintedanib for 72 hours. Human microvascular endothelial cells (lung) (HMEC-Ls; CC-2527) purchased at Passage 1 from Lonza (Basel, Switzerland) were incubated at 37°C with 5% CO2. HMEC-Ls were plated at Passage 2-6 using endothelial growth media (EGM-2; Lonza) supplemented with EGMTM-2 MV Microvascular Endothelial Cell Growth Medium-2 BulletKit (CC-2527). Cells were treated at Passage 3-7 in endothelial basal media (EBM-2; Lonza) supplemented with 5% fetal bovine serum, 1% gentamicin/amphotericin, and selected stimulants. Human pulmonary alveolar epithelial cells were purchased from ScienCell (Carlsbad, CA) and cultured in alveolar epithelial cell medium (ScienCell) supplemented with 2% fetal bovine serum, epithelial cell growth supplement, 100 U/mL penicillin G, and 100 μg/mL streptomycin. The cells were cultured and maintained in 6-well plates for experimental purposes. Angiogenesis-associated genes in COVID-19 deceased patients were previously reported.16Ackermann M. Verleden S.E. Kuehnel M. Haverich A. Welte T. Laenger F. Vanstapel A. Werlein C. Stark H. Tzankov A. Li W.W. Li V.W. Mentzer S.J. Jonigk D. Pulmonary vascular endothelialitis, thrombosis, and angiogenesis in Covid-19.N Engl J Med. 2020; 383: 120-128Crossref PubMed Scopus (3475) Google Scholar Analysis of this gene set was performed using oPOSSUM version 3.0 (http://opossum.cisreg.ca, last accessed June 1, 2021), human single site analysis tool with either RUNX1 or activator protein 1 (AP-1) as the JASPAR CORE transcription factor binding site (TFBS) profile. Conservation cutoff was 0.4; matrix score threshold was 85%; and upstream/downstream sequence was 5000. Target gene hits shared between AP-1 and RUNX1 were manually compared, whereas genes associated with the c-Jun N-terminal kinase (JNK) pathway were manually matched with JNK signaling pathway genes reported by AmiGO 2 (http://amigo.geneontology.org/amigo, last accessed April 13, 2021). All autopsies of SARS-CoV-2 infected deceased were performed at the Institute of Legal Medicine, University Medical-Center of Hamburg-Eppendorf, as described in previous works,37Edler C. Schröder A.S. Apfelbacher M. Fitzek A. Heinemann A. Heinrich F. Klein A. Langenwalder F. Lutgehetmann M. Meissner K. Puschel K. Schadler J. Steurer S. Mushumba H. Sperhake J.-P. Dying with SARS-CoV-2 infection—an autopsy study of the first consecutive 80 cases in Hamburg, Germany.Int J Legal Med. 2020; 134: 1275-1284Crossref PubMed Scopus (303) Google Scholar between March and September 2020 in the dissection room, with institutional review board approval from the independent ethics committee of the Hamburg University (protocol PV7311). Seven COVID-19 patients and one case with negative PCR virus test were selected. Clinical data, including pre-existing medical conditions, medical course before death, and antemortem diagnostic findings, were assessed (Supplemental Table S1). Lung tissue samples were formalin fixed and paraffin embedded. Samples were immunohistochemically stained using a Ventana Benchmark XT Autostainer (Ventana, Tucson, AZ). RUNX1 staining was performed in accordance with the manufacturer's recommendations, using a RUNX1 antibody (HPA004176; rabbit polyclonal; Sigma Aldrich, Hamburg, Germany; dilution 1:200). For detection of specific binding, the Ultra View Universal 3,3′-Diaminobenzidine Detection Kit (Ventana, Roche, Basel, Switzerland) was used, which contains secondary antibodies, 3,3′-diam
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