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Poster Session 4: Disease & Therapeutics 21:00 - 22:00 Wednesday, 27Th May, 2020 Poster

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Poster Session 4: Disease & Therapeutics 21:00 - 22:00 Wednesday, 27Th May, 2020 Poster Poster Session 4: Disease & Therapeutics 21:00 - 22:00 Wednesday, 27th May, 2020 Poster 219 Non-canonical immune response to inhibition of DNA methylation via stabilization of dsRNAs from endogenous retroviruses Yongsuk Ku1, Joo-Hwan Park2, Ryeongeun Cho1, Yongki Lee1, Sung-Soo Yoon2, Junshik Hong2, Yoosik Kim1 1Korea Advanced Institute of Science and Technology, Daejeon, Korea, Republic of. 2Seoul National University College of Medicine, Seoul, Korea, Republic of Abstract 5-Aza-2'-deocyxycitidine, also known as decitabine, is a DNA hypomethylating agent (HMA) used to treat acute myeloid leukemia (AML) and pre-leukemic disorder myelodysplastic syndrome (MDS). Recent studies have shown that decitabine induces apoptosis in cancer cells by activating transcription of endogenous retroviral elements (ERVs) through demethylation of their promoters. When transcribed, ERV RNAs can adopt double- stranded secondary structure and act as an activation cue for melanoma differentiation-associated protein 5 (MDA5), a member of the innate immune response proteins that recognize viral double-stranded RNAs (dsRNAs). Considering that many dsRNA binding proteins recognize the secondary structure of dsRNAs rather than their specific sequences, we investigated whether other dsRNA binding proteins can regulate decitabine- mediated cell death. Using image-based RNAi screening system, we analyzed the effect of downregulating 10 dsRNA binding proteins individually and in pairs on cellular response to decitabine. We found that STAUFEN (STAU) knockdown decreased the interferon signature and rescued decitabine-mediated cell death. Our subsequent analysis revealed that STAU directly binds to ERV RNAs and stabilizes the RNAs together with a long noncoding RNA. Analysis of a clinical patient cohort further showed that MDS and AML patients with low STAU expressions exhibited poor response to HMA. Collectively, our study reveals that decitabine-mediated cell death is a consequence of complex interactions among different dsRNA binding proteins for access to their commonly binding dsRNAs. Presenting author email [email protected] Topic category RNA & Cellular Immunity 711 Antiviral immunity in Drosophila: Identification of new protein partners of Dicer-2 Claire Rousseau1, Laurianne Kuhn2, Philippe Hamman2, Carine Meignin1 1M3i UPR9022, Université de Strasbourg, CNRS, Strasbourg, France. 2Plateforme Protéomique Strasbourg- Esplanade, Strasbourg, France Abstract Despite the massive impact of viral infection on health, it is still unclear how the innate immune system is activated. Work over the past ten years has revealed that viral nucleic acids, and in particular RNA, are sensed by cytosolic receptors. However, the exact mechanism by which non-self RNA (i.e. viral RNA) is discriminated from self RNA (i.e. cellular RNA) remains elusive. In Drosophila melanogaster there is only one known viral RNA sensor, Dicer-2, which is one of the main components of the antiviral RNA interference (RNAi) pathway. This pathway plays a major role in the control of viral infection in insects, as shown by the production of virus-derived siRNAs (small interfering RNA) in infected flies, and the increased susceptibility to viral infection of drosophila mutants for dicer-2, R2D2 and AGO2 genes. Dicer-2 is able to recognise viral dsRNA and synthesise siRNA duplexes, which will then be loaded onto the RNAi-induced Silencing Complex (RISC) in order to induce the cleavage and subsequent degradation of the complementary viral RNAs. Furthermore, Dicer-2 has been shown in vitro to recognise the extremities of the dsRNA molecules. However, how can Dicer-2 recognise viral RNAs, as they often have protected termini? As we know that the different cofactors of Dicer-2 can modulate its specificity for a given substrate, we wanted to define the partners of Dicer-2 in the context of viral infection. These partners could help the recognition of protected viral RNAs by Dicer-2. My PhD project focuses on the identification of Dicer-2 partners recruited during viral infection in vivo. I have performed a mass spectrometry experiment using three Dicer-2 versions: wild-type, ATP hydrolysis and RNAseIII mutants, in the course of viral infection by the Drosophila C Virus (DCV). The mass spectrometry data was analysed using three complementary approaches to identify: (1) Dicer-2 specific interactants; (2) the impact of the mutations on the partners of Dicer-2; and (3) proteins that interact with Dicer-2 specifically during the infection by DCV. I am currently performing an RNAi screen on the top candidates in order to test their impact on viral load. Presenting author email [email protected] Topic category RNA & Cellular Immunity 764 A CRISPR screen for microRNAs that regulate self-renewal and reconstitution capacity of hematopoietic stem cells Tatenda Kadungure, Hong Zhang, PhD University of Massachusetts Medical School, Worcester, MA, USA Abstract Hematopoietic stem cells (HSCs) self-renew and differentiate to give rise to all blood cells in the body, but their function changes with age. Old HSCs have diminished self-renewal capacity and are skewed towards myeloid over lymphoid lineages during differentiation. The decline in HSC self-renewal and bias in differentiation are associated with decreased immunity and increased susceptibility to myeloid leukemias in the elderly. Understanding how HSC function is regulated will help identify potential targets for rejuvenation of aged stem cells. Deletion of Dicer in the mouse hematopoietic system results in loss of HSC self-renewal suggesting that microRNAs (miRNAs) play a critical role in regulating HSC self-renewal. We therefore hypothesize there is a set of miRNAs which regulate HSC function and deletion of these miRNAs will alter (enhance or reduce) self- renewal and reconstitution capacity of HSCs. We further hypothesize that targeting miRNAs which negatively regulate mouse HSC function can rejuvenate old HSCs and restore the differentiation balance between myeloid and lymphoid lineages. In the present study, we investigate how miRNAs regulate mouse HSC self-renewal and reconstitution capacity using a CRISPR/Cas9-based loss-of-function screen. We have done four independent screens using a library of 701 sgRNAs targeting 172 miRNAs and preliminary analysis of two of the screens identified 8 potential miRNA candidates. sgRNAs targeting these 8 miRNAs are enriched in CRISPR-edited HSCs after competitive serial transplantation suggesting that these miRNAs normally act as negative regulators of HSC self-renewal and reconstitution capacity. Moving forward, we will analyze two additional screens and validate the candidate miRNA targets individually to investigate their roles in regulating HSC self-renewal and rejuvenating old HSCs. Presenting author email [email protected] Topic category RNA & Cellular Immunity 783 Gluten consumption maintains viral induced type I interferon pathway through an increase of m6A levels Maialen Sebastian-delaCruz1, Ane Olazagoitia-Garmendia1, Luis Manuel Mendoza1, Maria Legarda2, Carlos Tutau2, Jose Ramon Bilbao1,3, Ainara Castellanos-Rubio1,4 1University of the Basque Country (UPV-EHU), BioCruces Health Research Institute, Leioa, Spain. 2Pediatric Gastroenterology Section, Cruces Hospital, Barakaldo, Spain. 3CIBERDEM, Madrid, Spain. 4IKERBASQUE, Basque Foundation for Science, Bilbao, Spain Abstract Celiac disease (CeD) is a complex autoimmune disorder in which gliadin from gluten is the known triggering agent, and enteroviral infections have been linked to the risk of developing the disease. The type I interferon (IFN-I) pathway, which is upregulated in CeD patients, is activated upon viral infections, being an important innate immune response mechanism. It has been described that m6A methylated RNAs are able to control the innate immune response to infections by targeting IFN-Is and have also been involved in the development of autoimmunity. We hypothesized that gluten consumption can reactivate the IFN-I pathway in patients that have been previously infected by enterovirus, and that this may be regulated by an m6A-dependent mechanism. To test this hypothesis, we used polyinosinic-polycytidylic acid (PIC) treatment as a viral mimic in combination with gluten stimulations in HCT15 intestinal cells. As expected, PIC treatment induced the IRF7-IFNB pathway, together with downregulation of ALKBH5 m6A eraser and an overall increase in m6A levels after 24h. We then combined PIC and gliadin stimulations to evaluate the effect of gluten on the m6A machinery and IFN-I pathway after a mimic viral infection. We observed a synergistic effect, since both ALKBH5 decrease and IRF7 induction were augmented in cells treated with both agents. METTL3 expression is also increased by gliadin stimulation after PIC treatment. In addition, silencing of ALKBH5 and METTL3 overexpression increased IRF7 expression, together with STAT1 and CXCL10, suggesting that the induction observed after viral mimic and gliadin stimulation is at least partially mediated by an m6A increase. Likewise, we observed that total m6A levels together with some m6A machinery genes are higher in biopsies from CeD patients. Besides, IRF7, STAT1 and CXCL10 are constitutively upregulated in celiac patients. In conclusion, our results suggest that in the context of viral infection, gliadin consumption may lead to an autoimmune response by the alteration of m6A machinery and the induction of immune activation pathways. Presenting author email [email protected]
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