RNA Activation-A Novel Approach to Therapeutically Upregulate Gene Transcription.

RNA activation (RNAa) is a mechanism whereby RNA oligos complementary to genomic sequences around the promoter region of genes increase the transcription output of their target gene. Small activating RNA (saRNA) mediate RNAa through interaction with protein co-factors to facilitate RNA polymerase II activity and nucleosome remodeling. As saRNA are small, versatile and safe, they represent a new class of therapeutics that can rescue the downregulation of critical genes in disease settings. This review highlights our current understanding of saRNA biology and describes various examples of how saRNA are successfully used to treat various oncological, neurological and monogenic diseases. MTL-CEBPA, a first-in-class compound that reverses CEBPA downregulation in oncogenic processes using CEBPA-51 saRNA has entered clinical trial for the treatment of hepatocellular carcinoma (HCC). Preclinical models demonstrate that MTL-CEBPA reverses the immunosuppressive effects of myeloid cells and allows for the synergistic enhancement of other anticancer drugs. Encouraging results led to the initiation of a clinical trial combining MTL-CEBPA with a PD-1 inhibitor for treatment of solid tumors.

Cetuximab, docetaxel, and cisplatin versus platinum, fluorouracil, and cetuximab as first-line treatment in patients with recurrent or metastatic head and neck squamous-cell carcinoma (GORTEC 2014-01 TPExtreme): a multicentre, open-label, randomised, phase 2 trial.

BACKGROUND: Results from a phase 2 trial of the TPEx chemotherapy regimen (docetaxel-platinum-cetuximab) showed promising results, with a median overall survival of 14·0 months in first-line recurrent or metastatic head and neck squamous-cell carcinoma (HNSCC). We therefore aimed to compare the efficacy and safety of the TPEx regimen with the standard of care EXTREME regimen (platinum-fluorouracil-cetuximab) in this setting. METHODS: This was a multicentre, open-label, randomised, phase 2 trial, done in 68 centres (cancer centres, university and general hospitals, and private clinics) in France, Spain, and Germany. Eligible patients were aged 18-70 years with histologically confirmed recurrent or metastatic HNSCC unsuitable for curative treatment; had at least one measurable lesion according to Response Evaluation Criteria in Solid Tumors version 1.1; and had an Eastern Cooperative Oncology Group (ECOG) performance status of 1 or less. Participants were randomly assigned (1:1) using the TenAlea website by investigators or delegated clinical research associates to the TPEx regimen or the EXTREME regimen, with minimisation by ECOG performance status, type of disease evolution, previous cetuximab treatment, and country. The TPEx regimen consisted of docetaxel 75 mg/m2 and cisplatin 75 mg/m2, both intravenously on day 1, and cetuximab on days 1, 8, and 15 (intravenously 400 mg/m2 on day 1 of cycle 1 and 250 mg/m2 weekly subsequently). Four cycles were repeated every 21 days with systematic granulocyte colony-stimulating factor (G-CSF) support at each cycle. In case of disease control after four cycles, intravenous cetuximab 500 mg/m2 was continued every 2 weeks as maintenance therapy until progression or unacceptable toxicity. The EXTREME regimen consisted of fluorouracil 4000 mg/m2 on day 1-4, cisplatin 100 mg/m2 on day 1, and cetuximab on days 1, 8, and 15 (400 mg/m2 on day 1 of cycle 1 and 250 mg/m2 weekly subsequently) all delivered intravenously. Six cycles were delivered every 21 days followed by weekly 250 mg/m2 cetuximab as maintenance therapy in case of disease control. G-CSF support was not mandatory per the protocol in the EXTREME regimen. The primary endpoint was overall survival in the intention-to-treat population; safety was analysed in all patients who received at least one dose of chemotherapy or cetuximab. Enrolment is closed and this is the final analysis. This study is registered at ClinicalTrials.gov, NCT02268695. FINDINGS: Between Oct 10, 2014, and Nov 29, 2017, 541 patients were enrolled and randomly assigned to the two treatment regimens (271 to TPEx, 270 to EXTREME). Two patients in the TPEx group had major deviations in consent forms and were not included in the final analysis. Median follow-up was 34·4 months (IQR 26·6-44·8) in the TPEx group and 30·2 months (25·5-45·3) in the EXTREME group. At data cutoff, 209 patients had died in the TPEx group and 218 had died in the EXTREME group. Overall survival did not differ significantly between the groups (median 14·5 months [95% CI 12·5-15·7] in the TPEx group and 13·4 months [12·2-15·4] in the EXTREME group; hazard ratio 0·89 [95% CI 0·74-1·08]; p=0·23). 214 (81%) of 263 patients in the TPEx group versus 246 (93%) of 265 patients in the EXTREME group had grade 3 or worse adverse events during chemotherapy (p<0·0001). In the TPEx group, 118 (45%) of 263 patients had at least one serious adverse event versus 143 (54%) of 265 patients in the EXTREME group. 16 patients in the TPEx group and 21 in the EXTREME group died in association with adverse events, including seven patients in each group who had fatal infections (including febrile neutropenia). Eight deaths in the TPEx group and 11 deaths in the EXTREME group were assessed as treatment related, most frequently sepsis or septic shock (four in each treatment group). INTERPRETATION: Although the trial did not meet its primary endpoint, with no significant improvement in overall survival with TPEx versus EXTREME, the TPEx regimen had a favourable safety profile. The TPEx regimen could provide an alternative to standard of care with the EXTREME regimen in the first-line treatment of patients with recurrent or metastatic HNSCC, especially for those who might not be good candidates for up-front pembrolizumab treatment. FUNDING: Merck Santé and Chugai Pharma.

Retinoic Acid Inducible Gene I and Protein Kinase R, but Not Stress Granules, Mediate the Proinflammatory Response to Yellow Fever Virus.

Yellow fever virus (YFV) is an RNA virus primarily targeting the liver. Severe YF cases are responsible for hemorrhagic fever, plausibly precipitated by excessive proinflammatory cytokine response. Pathogen recognition receptors (PRRs), such as the cytoplasmic retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), and the viral RNA sensor protein kinase R (PKR), are known to initiate a proinflammatory response upon recognition of viral genomes. Here, we sought to reveal the main determinants responsible for the acute cytokine expression occurring in human hepatocytes following YFV infection. Using a RIG-I-defective human hepatoma cell line, we found that RIG-I largely contributes to cytokine secretion upon YFV infection. In infected RIG-I-proficient hepatoma cells, RIG-I was localized in stress granules. These granules are large aggregates of stalled translation preinitiation complexes known to concentrate RLRs and PKR and are so far recognized as hubs orchestrating RNA virus sensing. Stable knockdown of PKR in hepatoma cells revealed that PKR contributes to both stress granule formation and cytokine induction upon YFV infection. However, stress granule disruption did not affect the cytokine response to YFV infection, as assessed by small interfering RNA (siRNA)-knockdown-mediated inhibition of stress granule assembly. Finally, no viral RNA was detected in stress granules using a fluorescence in situ hybridization approach coupled with immunofluorescence. Our findings suggest that both RIG-I and PKR mediate proinflammatory cytokine induction in YFV-infected hepatocytes, in a stress granule-independent manner. Therefore, by showing the uncoupling of the cytokine response from the stress granule formation, our model challenges the current view in which stress granules are required for the mounting of the acute antiviral response.IMPORTANCE Yellow fever is a mosquito-borne acute hemorrhagic disease caused by yellow fever virus (YFV). The mechanisms responsible for its pathogenesis remain largely unknown, although increased inflammation has been linked to worsened outcome. YFV targets the liver, where it primarily infects hepatocytes. We found that two RNA-sensing proteins, RIG-I and PKR, participate in the induction of proinflammatory mediators in human hepatocytes infected with YFV. We show that YFV infection promotes the formation of cytoplasmic structures, termed stress granules, in a PKR- but not RIG-I-dependent manner. While stress granules were previously postulated to be essential platforms for immune activation, we found that they are not required for the production of proinflammatory mediators upon YFV infection. Collectively, our work uncovered molecular events triggered by the replication of YFV, which could prove instrumental in clarifying the pathogenesis of the disease, with possible repercussions for disease management.

The Polyphenol-Rich Extract from Psiloxylon mauritianum, an Endemic Medicinal Plant from Reunion Island, Inhibits the Early Stages of Dengue and Zika Virus Infection.

The recent emergence and re-emergence of viral infections transmitted by vectors, such as the Zika virus (ZIKV) and Dengue virus (DENV), is a cause for international concern. These highly pathogenic arboviruses represent a serious health burden in tropical and subtropical areas of the world. Despite the high morbidity and mortality associated with these viral infections, antiviral therapies are missing. Medicinal plants have been widely used to treat various infectious diseases since millenaries. Several compounds extracted from plants exhibit potent effects against viruses in vitro, calling for further investigations regarding their efficacy as antiviral drugs. Here, we demonstrate that an extract from Psiloxylon mauritianum, an endemic medicinal plant from Reunion Island, inhibits the infection of ZIKV in vitro without exhibiting cytotoxic effects. The extract was active against different ZIKV African and Asian strains, including an epidemic one. Time-of-drug-addition assays revealed that the P. mauritianum extract interfered with the attachment of the viral particles to the host cells. Importantly, the P. mauritianum extract was also able to prevent the infection of human cells by four dengue virus serotypes. Due to its potency and ability to target ZIKV and DENV particles, P. mauritianum may be of value for identifying and characterizing antiviral compounds to fight medically-important flaviviruses.

Extract from Aphloia theiformis, an edible indigenous plant from Reunion Island, impairs Zika virus attachment to the host cell surface.

The mosquito-borne Zika virus (ZIKV) belongs to the flavivirus genus of the Flaviviridae family. Contemporary epidemic strains of ZIKV are associated with congenital malformations in infants, including microcephaly, as well as Guillain-Barré syndrome in adults. A risk of human-to-human transmission of ZIKV is also well documented. A worldwide research effort has been undertaken to identify safe and effective strategies to prevent or treat ZIKV infection. We show here that extract from Aphloia theiformis, an edible endemic plant from Indian Ocean islands, exerts a potent antiviral effect against ZIKV strains of African and Asian lineages, including epidemic strains. The antiviral effect of A. theiformis extract was extended to clinical isolates of dengue virus (DENV) of the four serotypes in human hepatocytes. A. theiformis inhibited virus entry in host cells by acting directly on viral particles, thus impairing their attachment to the cell surface. Electron microscopic observations revealed that organization of ZIKV particles was severely affected by A. theiformis. We propose a model of antiviral action for A. theiformis against flaviviruses that highlights the potential of medicinal plants as promising sources of naturally-derived antiviral compounds to prevent ZIKV and DENV infections.

Immature particles and capsid-free viral RNA produced by Yellow fever virus-infected cells stimulate plasmacytoid dendritic cells to secrete interferons.

Plasmacytoid dendritic cells (pDCs) are specialized in the production of interferons (IFNs) in response to viral infections. The Flaviviridae family comprises enveloped RNA viruses such as Hepatitis C virus (HCV) and Dengue virus (DENV). Cell-free flaviviridae virions poorly stimulate pDCs to produce IFN. By contrast, cells infected with HCV and DENV potently stimulate pDCs via short-range delivery of viral RNAs, which are either packaged within immature virions or secreted exosomes. We report that cells infected with Yellow fever virus (YFV), the prototypical flavivirus, stimulated pDCs to produce IFNs in a TLR7- and cell contact- dependent manner. Such stimulation was unaffected by the presence of YFV neutralizing antibodies. As reported for DENV, cells producing immature YFV particles were more potent at stimulating pDCs than cells releasing mature virions. Additionally, cells replicating a release-deficient YFV mutant or a YFV subgenomic RNA lacking structural protein-coding sequences participated in pDC stimulation. Thus, viral RNAs produced by YFV-infected cells reach pDCs via at least two mechanisms: within immature particles and as capsid-free RNAs. Our work highlights the ability of pDCs to respond to a variety of viral RNA-laden carriers generated from infected cells.

Zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells.

The cytopathic effects of Zika virus (ZIKV) are poorly characterized. Innate immunity controls ZIKV infection and disease in most infected patients through mechanisms that remain to be understood. Here, we studied the morphological cellular changes induced by ZIKV and addressed the role of interferon-induced transmembrane proteins (IFITM), a family of broad-spectrum antiviral factors, during viral replication. We report that ZIKV induces massive vacuolization followed by "implosive" cell death in human epithelial cells, primary skin fibroblasts and astrocytes, a phenomenon which is exacerbated when IFITM3 levels are low. It is reminiscent of paraptosis, a caspase-independent, non-apoptotic form of cell death associated with the formation of large cytoplasmic vacuoles. We further show that ZIKV-induced vacuoles are derived from the endoplasmic reticulum (ER) and dependent on the PI3K/Akt signaling axis. Inhibiting the Sec61 ER translocon in ZIKV-infected cells blocked vacuole formation and viral production. Our results provide mechanistic insight behind the ZIKV-induced cytopathic effect and indicate that IFITM3, by acting as a gatekeeper for incoming virus, restricts virus takeover of the ER and subsequent cell death.

Axl Mediates ZIKA Virus Entry in Human Glial Cells and Modulates Innate Immune Responses.

ZIKA virus (ZIKV) is an emerging pathogen responsible for neurological disorders and congenital microcephaly. However, the molecular basis for ZIKV neurotropism remains poorly understood. Here, we show that Axl is expressed in human microglia and astrocytes in the developing brain and that it mediates ZIKV infection of glial cells. Axl-mediated ZIKV entry requires the Axl ligand Gas6, which bridges ZIKV particles to glial cells. Following binding, ZIKV is internalized through clathrin-mediated endocytosis and traffics to Rab5+ endosomes to establish productive infection. During entry, the ZIKV/Gas6 complex activates Axl kinase activity, which downmodulates interferon signaling and facilitates infection. ZIKV infection of human glial cells is inhibited by MYD1, an engineered Axl decoy receptor, and by the Axl kinase inhibitor R428. Our results highlight the dual role of Axl during ZIKV infection of glial cells: promoting viral entry and modulating innate immune responses. Therefore, inhibiting Axl function may represent a potential target for future antiviral therapies.

Mechanisms of HIV-1 Nucleocapsid Protein Inhibition by Lysyl-Peptidyl-Anthraquinone Conjugates.

The Nucleocapsid protein NCp7 (NC) is a nucleic acid chaperone responsible for essential steps of the HIV-1 life cycle and an attractive candidate for drug development. NC destabilizes nucleic acid structures and promotes the formation of annealed substrates for HIV-1 reverse transcription elongation. Short helical nucleic acid segments bordered by bulges and loops, such as the Trans-Activation Response element (TAR) of HIV-1 and its complementary sequence (cTAR), are nucleation elements for helix destabilization by NC and also preferred recognition sites for threading intercalators. Inspired by these observations, we have recently demonstrated that 2,6-disubstituted peptidyl-anthraquinone-conjugates inhibit the chaperone activities of recombinant NC in vitro, and that inhibition correlates with the stabilization of TAR and cTAR stem-loop structures. We describe here enhanced NC inhibitory activity by novel conjugates that exhibit longer peptidyl chains ending with a conserved N-terminal lysine. Their efficient inhibition of TAR/cTAR annealing mediated by NC originates from the combination of at least three different mechanisms, namely, their stabilizing effects on nucleic acids dynamics by threading intercalation, their ability to target TAR RNA substrate leading to a direct competition with the protein for the same binding sites on TAR, and, finally, their effective binding to the NC protein. Our results suggest that these molecules may represent the stepping-stone for the future development of NC-inhibitors capable of targeting the protein itself and its recognition site in RNA.

Viral entry route determines how human plasmacytoid dendritic cells produce type I interferons.

Although plasmacytoid dendritic cells (pDCs) represent a rare immune cell type, they are the most important source of type I interferons (IFNs) upon viral infection. Phagocytosed RNA viruses and RNA virus-infected cells are detected by pDCs with the endosomal pattern recognition receptor (PRR) toll-like receptor 7 (TLR7). We showed that replication of the yellow fever live vaccine YF-17D in human pDCs and pDC-like cell lines stimulated type I IFN production through RIG-I (retinoic acid-inducible gene I), a member of the RIG-I-like receptor (RLR) family of cytosolic PRRs. Thus, human pDCs sense replicative viral RNA. In contrast, direct contact between pDCs and YF-17D-infected cells stimulated a TLR7-dependent, viral replication-independent production of type I IFN. We also showed that the RLR pathway was dampened by the activities of interleukin-1 receptor-associated kinases 1 and 4 (IRAK1 and IRAK4), which are downstream effectors of the TLR7 pathway, suggesting that both kinases play opposing roles downstream of specific PRRs. Together, these data suggest that a virus can stimulate either TLR or RLR signaling in the same cell, depending on how its nucleic acid content is delivered.