Summary RNA modifications play critical roles in important biological processes. However, the functions of N6-methyladenosine (m6A) mRNA modification in cancer biology and cancer stem cells remain largely unknown. Here, we show that m6A mRNA modification is critical for glioblastoma stem cell (GSC) self-renewal and tumorigenesis. Knockdown of METTL3 or METTL14, key components of the RNA methyltransferase complex, dramatically promotes human GSC growth, self-renewal, and tumorigenesis. In contrast, overexpression of METTL3 or inhibition of the RNA demethylase FTO suppresses GSC growth and self-renewal. Moreover, inhibition of FTO suppresses tumor progression and prolongs lifespan of GSC-grafted mice substantially. m6A sequencing reveals that knockdown of METTL3 or METTL14 induced changes in mRNA m6A enrichment and altered mRNA expression of genes (e.g., ADAM19) with critical biological functions in GSCs. In summary, this study identifies the m6A mRNA methylation machinery as promising therapeutic targets for glioblastoma.
Public health measures have successfully identified and contained outbreaks of the severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), but concerns remain over the possibility of future recurrences. Finding a vaccine for this virus therefore remains a high priority. Here, we show that a DNA vaccine encoding the spike (S) glycoprotein of the SARS-CoV induces T cell and neutralizing antibody responses, as well as protective immunity, in a mouse model. Alternative forms of S were analysed by DNA immunization. These expression vectors induced robust immune responses mediated by CD4 and CD8 cells, as well as significant antibody titres, measured by enzyme-linked immunosorbent assay. Moreover, antibody responses in mice vaccinated with an expression vector encoding a form of S that includes its transmembrane domain elicited neutralizing antibodies. Viral replication was reduced by more than six orders of magnitude in the lungs of mice vaccinated with these S plasmid DNA expression vectors, and protection was mediated by a humoral but not a T-cell-dependent immune mechanism. Gene-based vaccination for the SARS-CoV elicits effective immune responses that generate protective immunity in an animal model.
The severe acute respiratory syndrome coronavirus (SARS-CoV) synthesizes several putative viral envelope proteins, including the spike (S), membrane (M), and small envelope (E) glycoproteins. Although these proteins likely are essential for viral replication, their specific roles in SARS-CoV entry have not been defined. In this report, we show that the SARS-CoV S glycoprotein mediates viral entry through pH-dependent endocytosis. Further, we define its cellular tropism and demonstrate that virus transmission occurs through cell-mediated transfer by dendritic cells. The S glycoprotein was used successfully to pseudotype replicationdefective retroviral and lentiviral vectors that readily infected Vero cells as well as primary pulmonary and renal epithelial cells from human, nonhuman primate, and, to a lesser extent, feline species. The tropism of this reporter virus was similar to that of wild-type, replication-competent SARS-CoV, and binding of purified S to susceptible target cells was demonstrated by flow cytometry. Although myeloid dendritic cells were able to interact with S and to bind virus, these cells could not be infected by SARS-CoV. However, these cells were able to transfer the virus to susceptible target cells through a synapse-like structure. Both cell-mediated infection and direct infection were inhibited by anti-S antisera, indicating that strategies directed toward this gene product are likely to confer a therapeutic benefit for antiviral drugs or the development of a SARS vaccine.
Graphical AbstractHighlights d Structure-guided design and optimization yield potent FTO inhibitors d mRNA m 6 A acts as the major effector of the inhibitor/FTO axis in AML cells d FTO inhibitor FB23-2 displays therapeutic effects in PDX AML models d Targeting epitranscriptomic RNA methylation holds potential to treat AML SUMMARY FTO, an mRNA N 6 -methyladenosine (m 6 A) demethylase, was reported to promote leukemogenesis. Using structure-based rational design, we have developed two promising FTO inhibitors, namely FB23 and FB23-2, which directly bind to FTO and selectively inhibit FTO's m 6 A demethylase activity. Mimicking FTO depletion, FB23-2 dramatically suppresses proliferation and promotes the differentiation/apoptosis of human acute myeloid leukemia (AML) cell line cells and primary blast AML cells in vitro. Moreover, FB23-2 significantly inhibits the progression of human AML cell lines and primary cells in xeno-transplanted mice. Collectively, our data suggest that FTO is a druggable target and that targeting FTO by small-molecule inhibitors holds potential to treat AML. SignificanceAs the most abundant internal mRNA modification, m 6 A impacts various biological processes. As a major m 6 A demethylase, FTO is overexpressed in certain subtypes of AMLs and promotes leukemogenesis. Thus, the development of effective inhibitors to target FTO's aberrant m 6 A demethylase activity is urgently needed for leukemia therapy. Here we report two selective FTO inhibitors that efficiently reverse/suppress FTO-mediated aberrant epitranscriptome in AML cells and significantly inhibit AML progression in vivo. Our studies provide the proof-of-concept evidence demonstrating that small-molecule inhibitors targeting oncogenic FTO represent a promising targeted therapeutic strategy for the effective treatment of AML. Moreover, given the overexpression of FTO in various cancers, our work may have a broad impact on cancer therapy by targeting the FTO-mediated epitranscriptome.
Two human demethylases, the fat mass and obesity-associated (FTO) enzyme and ALKBH5, oxidatively demethylate abundant N6-methyladenosine (m6A) residues in mRNA. Achieving a method for selective inhibition of FTO over ALKBH5 remains a challenge, however. Here, we have identified meclofenamic acid (MA) as a highly selective inhibitor of FTO. MA is a non-steroidal, anti-inflammatory drug that mechanistic studies indicate competes with FTO binding for the m6A-containing nucleic acid. The structure of FTO/MA has revealed much about the inhibitory function of FTO. Our newfound understanding, revealed herein, of the part of the nucleotide recognition lid (NRL) in FTO, for example, has helped elucidate the principles behind the selectivity of FTO over ALKBH5. Treatment of HeLa cells with the ethyl ester form of MA (MA2) has led to elevated levels of m6A modification in mRNA. Our collective results highlight the development of functional probes of the FTO enzyme that will (i) enable future biological studies and (ii) pave the way for the rational design of potent and specific inhibitors of FTO for use in medicine.
Highlights d Development of two potent FTO inhibitors with IC 50 values in the low nanomolar range d KD of FTO or pharmacological inhibition of FTO suppresses LSC/LIC self-renewal d Targeting FTO suppresses immune checkpoint gene expression and immune evasion d Targeting FTO by potent inhibitors holds therapeutic promise against various cancers
Ebola virus encodes seven viral structural and regulatory proteins that support its high rates of replication, but little is known about nucleocapsid assembly of this virus in infected cells. We report here that three viral proteins are necessary and sufficient for formation of Ebola virus particles and that intracellular posttranslational modification regulates this process. Expression of the nucleoprotein (NP) and virion-associated proteins VP35 and VP24 led to spontaneous assembly of nucleocapsids in transfected 293T cells by transmission electron microscopy. A specific biochemical interaction of these three proteins was demonstrated, and, interestingly, O-glycosylation and sialation of NP were demonstrated and necessary for their association. This distinct mechanism of regulation for filovirus assembly suggests new approaches for viral therapies and vaccines for Ebola and related viruses.
Study Type – Aetiology (case control) Level of Evidence 3b OBJECTIVE To investigate the progression of urodynamic changes, as well as histological and biochemical outcomes over a prolonged period of partial bladder outlet obstruction (pBOO) in an animal model with physiologically relevant pBOO. MATERIALS AND METHODS Healthy, adult, female Fischer rats underwent surgical creation of a pBOO for either 2, 4, 8, or 13 weeks and were compared with sham‐operated rats. Urodynamic measurements were used to compare bladder volumes and pressure. Tissue was grossly analysed with light microscopy and bladder weights and thicknesses were compared. Reverse transcription‐polymerase chain reaction for collagen, transforming growth factor β (TGF‐β), connective tissue growth factor (CTGF), hypoxia inducible factor 1α (HIF‐1α), and platelet‐derived growth factor (PDGF‐A) was performed on all samples, as well as immunohistochemistry (IHC) for α‐smooth muscle actin (α‐SMA). Finally, mass spectrometry was used to quantify the collagen content of the bladders as a measure of fibrosis. RESULTS After induction of pBOO, all rats remained healthy. Initial urodynamics showed an increase in capacity while maintaining normal pressures, but then deteriorated into small capacity, high‐pressure bladders. Haematoxylin and eosin (H&E) staining showed an initial inflammatory response, and this was confirmed with significantly increased mRNA levels of TGF‐β, CTGF, HIF‐1α, and PDGF. The progression to smooth muscle hypertrophy was evident on H&E and confirmed with increased bladder mass and thickness. IHC for α‐SMA showed a progressive increase associated with the elevated bladder pressures. Masson’s trichrome and mass spectrometry showed a progressive increase in collagen to 13 weeks. CONCLUSION With this model, we have effectively replicated the clinical scenario, with significant pathophysiological changes occurring insidiously in otherwise healthy rats. We believe that our observed changes represent distinct phases of bladder decompensation; with an initial inflammatory response to the stress of the pBOO, smooth muscle hypertrophy to overcome the increased urethral resistance, and eventual decompensation to fibrosis. The time course of the inflammatory markers implies the need for early intervention to prevent this cascade. Novel strategies targeting these observed physiological responses could lead to improved preventative strategies, with respect to biochemical pathways and the time course of their initiation.
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