Interleukin-18 produced by bone marrow- derived stromal cells supports T-cell acute leukaemia progression

International audienceDevelopment of novel therapies is critical for T-cell acute leukae-mia (T-ALL). Here, we investigated the effect of inhibiting the MAPK/MEK/ERK pathway on T-ALL cell growth. Unexpectedly, MEK inhibitors (MEKi) enhanced growth of 70% of human T-ALL cell samples cultured on stromal cells independently of NOTCH activa-tion and maintained their ability to propagate in vivo. Similar results were obtained when T-ALL cells were cultured with ERK1/ 2-knockdown stromal cells or with conditioned medium from MEKi-treated stromal cells. Microarray analysis identified interleu-kin 18 (IL-18) as transcriptionally up-regulated in MEKi-treated MS5 cells. Recombinant IL-18 promoted T-ALL growth in vitro, whereas the loss of function of IL-18 receptor in T-ALL blast cells decreased blast proliferation in vitro and in NSG mice. The NFKB pathway that is downstream to IL-18R was activated by IL-18 in blast cells. IL-18 circulating levels were increased in T-ALL-xeno-grafted mice and also in T-ALL patients in comparison with controls. This study uncovers a novel role of the pro-inflammatory cytokine IL-18 and outlines the microenvironment involvement in human T-ALL development

The Cricket Paralysis Virus Suppressor Inhibits microRNA Silencing Mediated by the Drosophila Argonaute-2 Protein

International audienceSmall RNAs are potent regulators of gene expression. They also act in defense pathways against invading nucleic acids such as transposable elements or viruses. To counteract these defenses, viruses have evolved viral suppressors of RNA silencing (VSRs). Plant viruses encoded VSRs interfere with siRNAs or miRNAs by targeting common mediators of these two pathways. In contrast, VSRs identified in insect viruses to date only interfere with the siRNA pathway whose effector Argonaute protein is Argonaute-2 (Ago-2). Although a majority of Drosophila miRNAs exerts their silencing activity through their loading into the Argonaute-1 protein, recent studies highlighted that a fraction of miRNAs can be loaded into Ago-2, thus acting as siRNAs. In light of these recent findings, we reexamined the role of insect VSRs on Ago-2-mediated miRNA silencing in Drosophila melanogaster. Using specific reporter systems in cultured Schneider-2 cells and transgenic flies, we showed here that the Cricket Paralysis virus VSR CrPV1-A but not the Flock House virus B2 VSR abolishes silencing by miRNAs loaded into the Ago-2 protein. Thus, our results provide the first evidence that insect VSR have the potential to directly interfere with the miRNA silencing pathway

CrPV-1A interferes with miRNA silencing mediated by perfect or imperfect target sites in S2 cells.

<p>Western blot analysis of S2 cells co-transfected with pMTmRFP-IM or pMTmRFP-PM, pAct-miG-2 and CrPV-1A-HA or no VSR expression constructs. α-tubulin is shown as loading control. One representative experiment out of four is shown.</p

CrPV-1A suppresses the Ago-2-dependent miRNA silencing in S2 cells.

<p>(A) Schematic representation of the reporter system for miRNA silencing. Pre-miRNAs inserted in an <i>Rpl17</i> intron and the mRFP gene are expressed from the same unspliced transcript under the control of the <i>ubiquitin</i> promoter. The miR-5-miR-6.1-mRFP construct (left), expresses miR-5 and miR-6.1 without complementarity to the GFP and was used as a control. The miG-1-miR-6.1–mRFP construct (right) expresses miR-6.1 as well as miG-1 which targets the GFP mRNA expressed from pUbi-GFP with perfect complementarity. (B) Western blot analysis of Drosophila S2 cell lysates, co-transfected with miRNA expression constructs described in (A), the pUbi-GFP sensor plasmid, and the C-terminal HA-tagged CrPV-1A or B2 expression vectors. Control plasmid with non-cognate miR-5 and miR-6 was used as a control for miRNA target specificity and α-tubulin is the loading control. One representative experiment out of five is shown.</p

Silencing of mRFP reporters bearing four imperfect or perfect miG-2 binding sites in Drosophila S2 cells.

<p>(A) Schematic representation of miG-2 silencing sensors. The copper inducible <i>metallothionein</i> promoter drive the expression of the mRFP transcript bearing perfectly matched (pMTmRFP-PM) or imperfectly matched (pMTmRFP-IM) miG-2 target sites. Expression of miG-2 is provided in <i>trans</i> by the pAct-miG-2 construct. (B) Western blot analysis of Drosophila S2 cells co-transfected with pAct-miG-2, pMTmRFP-IM or pMTmRFP-PM and the indicated dsRNAs targeting Ago-2 (A2), Ago-1 (A1) or the control luciferase dsRNA (L). α-tubulin is used as loading control. One representative experiment out of four is shown.</p

CrPV-1A but not B2 suppresses Ago-2 mediated miRNA silencing in adult flies.

<p>(A) Structure of the automiW transgene used to induce <i>white</i> silencing in adult eyes. The GMR promoter drives transgene expression of the miW miRNAs together with the white gene in differentiated eye. The two miW-1 and miW-2 will repress expression of the white gene resulting in white colored eyes. (B) Analysis of the automiW silencing in adult fly eyes. Silencing of the <i>white</i> maker gene of automiW was triggered in the presence of a GMR-Gal4 transgene and analyzed upon GFP, Drosha or Ago-2 RNAi knockdown by inverted repeats transgenes (IR). Fly genotypes and transgenes dosages are indicated above panels. (C) Analysis of automiW silencing in adult female eyes in the presence of one copy of the GMR-gal4 driver and one copy of the indicated VSR transgene (lane 1 to 3). A UAS-GFP transgene was used a control (lane 4). Note also that in the absence of the automiW transgene in heterozygous combinations of GMR-GAL4 with a UAS-VSR or UAS-GFP (lane 5 to 8), the mini-white markers of the two transgenes produced equivalent strong red eye pigmentation. Upper panel: eye pigmentation content was measured by pigment dosage for flies carrying the indicated transgenes. Histogram shows the mean values and error bars indicated standard deviation for three experiments. Bottom panels: one representative eye image is shown for each genotype.</p