GIGA - Annual report 2021

ANNUAL REPORT 2 2 1 0 R E S E A R C H

Editor Michel Georges Graphic design Aurélie Gouverneur Pictures ©Geoffrey Meuli - Photography & Images (p.2, 4, 7, 9, 10, 11, 13, 27, 33, 71, 89) Shutterstock ULiège GIGA

GIGA 2021 Annual Report 1

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GIGA 2021 Annual Report 3 «Completing an annual report is a good opportunity to reflect about GIGA’s state. The end of the pandemic appears in sight, which will - to some extend - equate to a new start. A good opportunity to re-articulate priorities. GIGA’s motto is “Striving for academic excellence in the biomedical sciences to foster groundbreaking medical innovation.” The “academic” emphasis stems fromGIGA’s dual mission: research and education. GIGA’s members share the ambition to conduct research that is internationally recognized for its excellence. An honest indicator of research performance is the number of ERC grants awarded to GIGA PIs. It presently stands at eight. Compared to other centers in French-speaking Belgium, this is excellent. Yet, we can increase the number of ERCs [and other grants promoting excellence such as FNRS andWelbio] even more by joining forces. Strength in science lies in cross-fertilization: the I of GIGA (interdisciplinary). GIGA counts more than 100 postdoctoral fellows and ~260 PhD students. It is their PI’s responsibility to ensure that the time spent at GIGA becomes a springboard for a rich and fulfilling life. Young scientists should want to join GIGA not only to become skilled researchers but also to acquire soft skills required by public and private organizations alike. It is the mission of GIGA’s doctoral school to support PIs as mentor. If we reach the primary goal of academic excellence, innovations benefitting society will – with appropriate support – naturally follow. In 2021 alone, GIGA scientists have filed five patents and started three companies. The tech transfer office of ULiège is undergoing profound restructuring to better assist PIs in translating acquired knowledge into medical solutions. GIGA will proactively participate in these efforts: the A of GIGA (applied). In an increasingly competitive and ruthless research environment, it is important to define for one-self some guiding principles, key values that are not to be compromised. GIGA proposes four: BALANCE one cannot be a good scientist for the long run, if at the expense of family, loved ones, or health. ACCOUNTABILITY the quality of the working environment is not a given, but rather a reflection of how much each one of us contributes to make it better. INTEGRITY it is better to publish negative results that have been obtained rigorously, than to publish results that you know or should know are not going to stand the test of time. KINDNESS have you, today, expressed your gratitude to at least one of the many people, scientists or not, that in various capacities make it possible for you to do your work? Along these lines, I express my sincere thanks to all members of GIGA’s technological platforms and support teams for their dedication in supporting our community. They are a major ingredient of GIGA’s success and attractiveness». Michel Georges GIGA Director April 22 I G G A

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GIGA 2021 Annual Report 5 CONTENTS GIGA Organization Contacts Key Figures Grants Awards Research Highlighted publications GIGA-Cancer GIGA-Cardiovascular GIGA-Consciousness GIGA-CRC in vivo Imaging GIGA-I3 GIGA-In Silico Medicine GIGA-Medical Genomics GIGA-MBD GIGA-Neurosciences GIGA-Stem Cells Platforms GIGA-Genomics GIGA-Cell Imaging & Flow Cytometry GIGA-CRC In vivo Imaging GIGA-Immunohistology GIGA-Viral vectors GIGA-Proteomics GIGA-Zebrafish Facility GIGA-Mouse Facilities Education GIGA-Doctoral School for Health Sciences GIGA-Forem Biotechnologies Training Center Innovation 06 08 10 14 16 18 20 44 46 48 50 52 54 56 58 60 62 64 66 68 72 74 76 78 80 82 84 86 88 90

6 GIGA 2021 Annual Report GIGA ORGANIZATION RESEARCH y GIGA-Cancer y GIGA-Cardiovascular y GIGA-Consciousness y GIGA-CRC In vivo Imaging y GIGA-I3 y GIGA-In silico Medicine y GIGA-Medical Genomics y GIGA-Molecular Biology of Diseases y GIGA-Neurosciences y GIGA-Stem Cells PLATFORMS y GIGA-Genomics y GIGA-Bioinformatics y GIGA-Cell Imaging y GIGA-Flow Cytometry y GIGA-CRC In vivo Imaging y GIGA-Immunohistology y GIGA-Viral Vectors y GIGA-Proteomics y GIGA-Animal Facilities EDUCATION y GIGA-Doctoral School For Health Sciences y Forem-GIGA Biotechnologies Training Center INNOVATION y Biotech Companies y Patents Close collaboration with

GIGA 2021 Annual Report 7 Michel Georges Director Brigitte Malgrange Vice-Director GIGA DIRECTION

8 GIGA 2021 Annual Report GIGA-CONTACTS DIRECTION Michel Georges michel.georges@uliege.be Brigitte Malgrange bmalgrange@uliege.be RESEARCH GIGA-Cancer Agnès Noël agnes.noel@uliege.be GIGA-Cardiovascular Patrizio Lancellotti plancellotti@uliege.be GIGA-Consciousness Steven Laureys steven.laureys@uliege.be GIGA-CRC in vivo Imaging Eric Salmon eric.salmon@uliege.be GIGA-I3 Catherine Sadzot csadzot@uliege.be GIGA-In Silico Medicine Thomas Desaive tdesaive@uliege.be GIGA-Medical Genomics Tom Druet tom.druet@uliege.be GIGA-MBD Franck Dequiedt fdequiedt@uliege.be GIGA-Neurosciences Julie Bakker jbakker@uliege.be GIGA-Stem Cells Laurent Nguyen lnguyen@uliege.be GIGA-Grants Marilou Ramos Pamplona marilou.ramospamplona@uliege.be PLATFORMS Platforms manager Carine Bebrone carine.bebrone@uliege.be GIGA-Genomics Wouter Coppieters wouter.coppieters@uliege.be GIGA-Cell Imaging & GIGA-Flow Cytometry Sandra Ormenese sandra.ormenese@uliege.be GIGA-In vivo Imaging Eric Salmon eric.salmon@uliege.be GIGA-Immunohistology Chantal Humblet chantal.humblet@uliege.be GIGA-Viral Vectors Emmanuel Di Valentin edivalentin@uliege.be GIGA-Proteomics Dominique Baiwir d.baiwir@uliege.be GIGA-Zebrafish Facilities Hélène Pendeville hpendeville@uliege.be GIGA-Mouse Facilities Pierre Drion transgenics.giga@uliege.be EDUCATION GIGA-Doctoral School for Health Sciences Marianna Bevova marianna.bevova@uliege.be FOREM-GIGA Biotechnologies Training Center Pierre Gillain biotech-liege@forem.be

GIGA 2021 Annual Report 9 ADMINISTRATION Finances/Accounting Sandrina Evrard, Manager sandrina.evrard@uliege.be Team Donatienne Boxus Marie Castronovo Marc Jacquemotte Sandrine L’Heureux Axelle Lio Eva Mashyaka Teresa Orsini Infrastructure Isabelle Danese, Manager idanese@uliege.be Team Audrey Hoffmann Gaëlle Massart Sihem Moudjed Fabienne Simart Welcome desk Gaëlle Massart gaelle.massart@uliege.be Communication Aurélie Gouverneur aurelie.gouverneur@uliege.be

10 GIGA 2021 Annual Report GIGA-KEY FIGURES 685 PEOPLE 215 Scientists with PhD 266 PhD Candidates 58 Clinicians 81 Technicians 43 Platforms staff 23 Administratives 56% WOMEN 44% MEN 27%NONBELGIANPEOPLE 42 NATIONALITIES KEY FIGURES

GIGA 2021 Annual Report 11 449 PUBLICATIONS 42 PHD THESES DEFENDED 490 ACTIVE RESEARCH CONVENTIONS 112 NEWRESEARCH CONVENTIONS COLLABORATIONS WITH 52 COUNTRIES 52 123 150 124 IF>10 IF 5-10 IF 3-5 IF<3

12 GIGA 2021 Annual Report FUNDINGS TOTAL 28 692 326€ Wallonie-Bruxelles Federation 22% Walloon Region 16% Europe 15% Contract research 12% ULiège 11% FEDER/ERDF 9% Federal 7% Other 5% Donation 4% Wallonie-Bruxelles Federation Wallon Region Europe Contract research ULiège FEDER/ERDF Federal Other Donation 6 364 758 € 4 579 191 € 4 242 645 € 3 586 258 € 3 085 543 € 2 558 613 € 1 902 861 € 1 332 510 € 1 000 947 €

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14 GIGA 2021 Annual Report GRANTS H2020 Ontology-driven and artificial intelligence-based repeated dose toxicity testing of chemicals for next generation risk assessment ONTOX Horizon2020 - Health, Demographic Change and Wellbeing Liesbet Geris In Silico World : Lowering barriers to ubiquitous adoption of In Silico Trials Horizon2020 - Health, Demographic Change and Wellbeing Liesbet Geris Towards improvement of ruminant breeding through genomic and epigenomic approaches (RumiGen) Horizon2020 - Food Security, Sustainable Agriculture and Forestry, Marine, Maritime and Inland Water Resea Carole Charlier JPI SCAIFIELD Spinocerebellar ataxias: Advanced imaging with ultra highfield MRI JPI - Joint Programme - Neurodegenerative Disease Research (JPND) Pierre Maquet EOS (2021 CALL) Understanding and tackling the spatio-temporal changes of the metastatic lymph node epicenter Patrizia Agostinis (KU Leuven) Gabriele Bergers (KU Leuven) Frédéric Kridelka (U Liège) Sophie Lucas (Université catholique de Louvain) David Lyden (Weill Cornell Medicine Gale and Ira Druckier Institute for Children’s Health (United States)) NOËL Agnès Noël (ULiège) Colinda Scheele (KU Leuven) Taking IBD Genes from GWAS to Function to Drug Target Rudi Beyaert (Universiteit Gent) Laure Dumoutier (Université catholique de Louvain) Denis Franchimont (Université libre de Bruxelles) Michel Georges (ULiège) Claude Libert (Universiteit Gent) Souad Rahmouni (Université de Liège) Ramnik Xavier (Massachusetts General Hospital (United States)) Host-microbe interaction in intestinal homeostasis: unravelling the mechanisms involved in the onset of multiple inflammation-related diseases Laure Bindels (Université catholique de Louvain) Patrice Cani(Université catholique de Louvain) Alain Chariot (ULiège) Edouard Louis (ULiège) Kathy McCoy (University of Calgary (Canada)) Jeroen Raes (KU Leuven) Tom Van de Wiele (Universiteit Gent) Geert Van Loo (Universiteit Gent) Lars Vereecke (Universiteit Gent) Severine Vermeire (KU Leuven) Endoplasmic reticulum and mitochondria: common drivers of human β cell and neuronal fate Miriam Cnop (Université libre de Bruxelles) Patrick Gilon (Université catholique de Louvain) Jean-Christophe Jonas (Université catholique de Louvain) Lynette Lim (KU Leuven) Laurent Nguyen (ULiège) Vincent Pasque (KU Leuven) Pierre Vanderhaeghen (KU Leuven) An integrated approach to unravel eosinophil function in chronic eosinophilic disorders Guy Brusselle (Universiteit Gent) Fabrice Bureau (ULiège) Christophe Desmet (ULiège) Philippe Gevaert (Universiteit Gent) Hamida Hammad (Universiteit Gent) Bart Lambrecht (Universiteit Gent) Renaud Louis (ULiège) Florence Roufosse (Université libre de Bruxelles) Savvas Savvides (Universiteit Gent) Eva Van Braeckel (Universiteit Gent) Tricking tumor immunosuppression: RNA epigenetics as novel target to improve cancer immunotherapy Pierre Close (ULiège) François Fuks (Université libre de Bruxelles) Massimiliano Mazzone (KU Leuven) Jo Van Ginderachter (Vrije Universiteit Brussel) Cell Death Regulation and Role in Infection and Inflammatory Diseases Koen Augustyns (Universiteit Antwerpen) Mathieu Bertrand (Universiteit Gent) Wouter Coppieters (ULiège) Emmanuel Dejardin (ULiège) Raffaella Gozzelino (NOVA University of Lisbon (Portugal)) Mathias Heikenwälder (German

GIGA 2021 Annual Report 15 Cancer Research Center (Germany)) Denis Lafontaine (Université libre de Bruxelles) Mohamed Lamkanfi (Universiteit Gent) Jonathan Maelfait (Universiteit Gent) Michel Moutschen (ULiège) Tom Vanden Berghe (Universiteit Antwerpen) Peter Vandenabeele (Universiteit Gent) PIT – Biowin THERAMIR Development of a unique platform of specific exosomes: Proof of concept for the solid tumor treatment Ingrid Struman MONITOSED A system for monitoring patient’s level of non-pharmacological sedation Steven Laureys WIN2WAL CHIPOMICS Development of a microfluidic demonstrator allowing to carry out all the steps of preanalytical preparation of the sample for a proteomic analysis of single cells Gabriel Mazzucchelli Laurent Nguyen GEPI4REE Specific rare earth ion microflotation system for the development of a process for the recovery and purification of Neodymium from electronic scrap Patricia Lassaux PDR (FNRS) NOCINEUTRASTHMA Study of the pulmonary nociceptor-neutrophil axis in environmentally mediated regulation of allergic asthma. Thomas Marichal INPP5K in B cell receptor signaling Stéphane Schurmans CXCL12 and translational control in GBM Bernard Rogister ADENOMinside New approaches to study the pathophysiology of adenomyosis Michelle Nisolle Brain stimulations in post-coma patients Steven Laureys Télévie Metabolism and therapy resistance in CRC Akeila Bellahcène GARP in tumor microenvironment Agnès Noël Cholangiocarcinoma tumorigenesis Alain Colige tRNA editing in colon cancer Pierre Close Eosinophils inhibit chemotherapy Luc Willems Lymph node premetastatic niche Agnès Noël PDAC characterization by sc-RNAseq Marianne Voz Inflammatory bowel disease intestinal organoids as a model for colitis-associated carcinogenesis characterization Edouard Louis TRAIN Targeting Resistant AdenocarcINoma Didier Cataldo Characterization of the tRNA epitrancriptome of lung cancer stem cells Francesca Rapino Mechanistic insights of estrogen receptor signaling by continuous or pulsed estrogenic treatments paving the way to breast cancer prevention Christel Pequeux FET oncogenic fusions and mRNA decay: towards a novel paradigm Franck Dequiedt DDT improves MPM immunotherapy Luc Willems Targeting glioblastoma propagating cells Bernard Rogister Gs/cAMP in pituitary tumors Pit-GAP Julien Hanson

16 GIGA 2021 Annual Report AWARDS/NEWS February 21 Arnaud Blomme obtains a Skłodowska-Curie Individual Fellowship for his TransMetTOR project Arnaud Blomme is a post-doctoral researcher at the Cancer Signaling Laboratory (GIGA Stem Cells). This grant will allow him to continue his research on the role(s) of mTORC2 in mRNA translation and cellular metabolism, in the context of lung cancer development. March 21 Solène Dauby, Emilie Lommers and Pierre Maquet, laureates of a Charcot Foundation Prize This award will allow them to better understand the disease mechanisms in multiple sclerosis. The aim of their research is to use new, extremely high-performance imaging tools (7 tesla MRI, PET CT scanner) to better observe brain structures, gain a more detailed understanding of the changes induced by the disease and thus improve the therapeutic management of multiple sclerosis patients. April 21 Pierre Foidart gets a BAEF fellowship for a post-doctoral stay at the Dana-Farber Cancer Institute Pierre Foidart, who completed his doctorate in the Laboratory of Tumour and Developmental Biology (GIGA - Cancer) at ULiège, has been awarded a BAEF (Belgian American Education Foundation) grant. This one-year grant will allow him to continue his research at the prestigious Dana-Farber Cancer Institute (Harvard Medical School) on triple-negative breast cancer.

GIGA 2021 Annual Report 17 May 21 A cross-border research center to better understand the microbiome Scientists from the Universities of Liège, Aachen, Maastricht and Venlo (UM campus) have just set up the Euregional Microbiome Center, a high-level research centre to better understand the viruses, bacteria, fungi and other microbes that live in our bodies. At the ULiège level the project is led by the BIO3 research group (GIGA/ Montefiore Institute - Kristel Van Steen), which works at the intersection of biostatistics, biomedicine and bioinformatics. March 21 Creation of CMD-Coat, a new spin-off from research conducted at the GIGA The company CMD-COAT S.A. was born from a collaboration between research centers of the University of Liege and the Cardiology Department of the University Hospital of Liege (CHU of Liege). It is the result of research by the team of Cécile Oury, FNRS research director, and Patrizio Lancellotti (director of the GIGA-Cardiovascular Research Unit) conducted in collaboration with the «Centre d’Étude et de Recherche sur les Macromolécules» (Department of Chemistry, ULiège). Thanks to this collaboration and to an ERC-Consolidator grant to Patrizio Lancellotti, the research team has developed an innovative bio- and hemo-compatible coating, which can be loaded with drugs, called Coatigel. May 21 Emilie Lommers, laureate of the Claire Fauconnier & Guy Sallets Fund Emilie Lommers, researcher at the GIGA-CRC in Vivo Imaging at the University of Liège and neurologist at the Liège University Hospital, is the laureate of the Claire Fauconnier and Guy Sallets Fund awarded by the King Baudouin Foundation. The prize of €50,000 will enable her to develop a medical imaging protocol to better reveal the various aspects of multiple sclerosis (MS) at the cerebral level.

GIGA-RESEARCH

20 GIGA 2021 Annual Report Loss of tRNA-modifying enzyme Elp3 activates a p53-dependent antitumor checkpoint in hematopoiesis Rosu A, El Hachem N, Rapino F, Rouault-Pierre K, Jorssen J, Somja J, Ramery E, Thiry M, Nguyen L, Jacquemyn M, Daelemans D, Adams CM, Bonnet D, Chariot A, Close P, Bureau F, Desmet CJ. J Exp Med. 2021 Mar 1;218(3):e20200662. doi: 10.1084/jem.20200662. HIGHLIGHTED PUBLICATIONS

GIGA 2021 Annual Report 21 The expression of the proteins that make up our bodies is very tightly regulated at many levels. In particular, the abundance of our various proteins is regulated via the transcription of the genes coding for their messenger RNA (mRNA) and then via the regulation of the half-life of the proteins formed. The expression of many proteins is also regulated in a more subtle, but no less biologically important way, by controlling the translation of mRNAs via various molecular mechanisms known as «translational» regulation. It has in this regard recently been shown that many mRNAs are the target of chemical modifications catalysed by dedicated enzymes, which consequently regulate their translation. An abundant literature is beginning to describe the role of what is currently called the mRNA epitranscriptome (i.e. mRNA modifications) in normal and pathological biological processes. However, mRNAs are not the most modified RNAs in our cells. The «award» of the modifications undoubtedly goes to another family of RNAs essential for protein production: transfer RNAs (tRNAs). tRNAs allow the translation of proteins by «pairing» the codons of mRNAs with their corresponding amino acid within the ribosomes. tRNAs can be subjected to almost a hundred distinct modifications, catalysed by a machinery involving hundreds of proteins. The biological role of this vast «tRNA epitranscriptome» is only just beginning to be studied in mammals, but it is already clear that the tRNA epitranscriptome is very important in central biological processes such as embryogenesis, brain development or even cancer, as shown by various publications notably by Alain Chariot, Pierre Close, Brigitte Malgrange and Laurent Nguyen of GIGA. Two 2021 publications by Christophe Desmet’s team (GIGA-I3) now contribute to extending the scope of action of the tRNA epitranscriptome to the fields of immunity and haematopoiesis. In this publication, the authors invalidated the tRNA modifying enzyme Elp3 in mouse haematopoietic stem cells (HSC). HSCs are responsible for the production of our blood cells and a large part of our immune cells throughout our lives. The loss of Elp3 leads a majority of HSC daughter cells to commit suicide when they need to differentiate, preventing them from giving birth to mature blood and immune cells. Thus, mice deficient in Elp3 in their HSCs develop severe anemia reminiscent of certain human blood diseases. The reason for the suicide of the transgenic cells in the process of differentiation seems to be linked to the perception of a threat of cancerisation. Indeed, the suicide of cells not expressing Elp3 is entirely controlled by the tumour suppressor p53. Moreover, mice deficient for Elp3 in their HSCs spontaneously develop lymphomas/leukemias with mutations in p53. In support of the transferability of these findings to human haematopoiesis, mutations affecting another enzyme acting in the same signalling pathway as Elp3 have very recently been identified as a probable cause of clonal haematopoiesis in humans, a pre-leukemic process. Whether in T lymphocytes or HSCs, it seems that the loss of the changes catalysed by Elp3 is perceived by cells as a major metabolic problem, leading to the activation of a central regulator of metabolic stress called Activating Transcription Factor-4 through non-canonical mechanisms. These results thus suggest that while the tRNA epitranscriptome plays a direct role in mRNA translation, it is also integrated into cellular signalling pathways, and thereby plays important regulatory roles in immunity and haematopoiesis.

22 GIGA 2021 Annual Report Extracellular HMGB1 blockade inhibits tumor growth through profoundly remodeling immune microenvironment and enhances checkpoint inhibitor-based immunotherapy Hubert P, Roncarati P, Demoulin S, Pilard C, Ancion M, Reynders C, Lerho T, Bruyere D, Lebeau A, Radermecker C, Meunier M, Nokin MJ, Hendrick E, Peulen O, Delvenne P, Herfs M. J Immunother Cancer. 2021 Mar;9(3):e001966. doi: 10.1136/jitc-2020-001966. HIGHLIGHTED PUBLICATIONS

GIGA 2021 Annual Report 23 A study conducted by researchers from the Laboratory of Experimental Pathology (Michaël Herfs, GIGA-Cancer) demonstrated that, contrary to what was assumed, HMGB1 does not act as an «alarmin» within the tumor microenvironment but rather as a highly immunosuppressive molecule. First discovered in the 1970s for its implication in the mechanisms of transcription and chromatin remodeling, HMGB1 became, over the years, more and more interesting for immunologists. Indeed, active or passive secretions of this protein were observed in various contexts (inflammation, infection, cancer) and the “alarmin” function of HMGB1 was demonstrated. As such, along with ATP and Calreticulin, HMGB1 is still frequently used as a marker for immunogenic cell death. This study highlights the “dark side” of HMGB1 during carcinogenesis. Actually, in this highly oxidizing microenvironment, HMGB1 does not act as a stimulator of the immune system but rather as an immunosuppressive molecule. Associated with a poor prognosis, its extracellular presence is correlated with higher densities of myeloid-derived suppressor cells (granulocytic and monocytic), regulatory T lymphocytes and M2 macrophages. Using several inhibitors, the researchers showed that HMGB1 blockade significantly reduced cancer progression through improving anti-tumor immunity, including by the activation of antigen-presenting cells (DCs and pDCs), the switch from M2 to M1 macrophages and increased apoptosis of cancer cells. To conclude this study, the synergistic effect of anti-HMGB1/anti-PD-1 combination as well as the antagonist functions of oxidized and reduced forms of HMGB1 on DCs/pDCs activation were shown. Used in traditional Japanese medicine for patients displaying a chronic infection/inflammation, inhibitors targeting HMGB1 may prove to be allies of choice for improving the current low response rate of immune checkpoint inhibitors (anti-PD-1/PD -L1) in the context of most cancers.

24 GIGA 2021 Annual Report Anti-IL5 mepolizumab minimally influences residual blood eosinophils in severe asthma Van Hulst G, Jorssen J, Jacobs N, Henket M, Louis R, Schleich F, Bureau F, Desmet CJ. Eur Respir J. 2022 Mar 17;59(3):2100935. doi:10.1183/13993003.00935-2021. HIGHLIGHTED PUBLICATIONS

GIGA 2021 Annual Report 25 Asthma is a respiratory disease characterized by recurrent episodes of coughing and respiratory oppression, which are usually treated with anti-inflammatory or bronchodilator treatments. Slightly more than half of asthma patients have so-called «eosinophilic asthma». This type of asthma is characterized by an increase in blood eosi-nophils and their infiltration into the lung. In eosi-nophilic asthma, numbers of eosinophils are associated with the severity of the disease, including the rate of exacerbations, which are periods of worsening disease, and with the risk of irreversible airway obstruction. Conventional treatments fail to control asthma in about 5 to 10% of patients with severe asthma. Biological treatments targeting eosinophils have therefore been developed to control severe eosinophilic asthma that reduce exacerbations and loss of lung function. Anti-eosinophilic treatments are based on monoclonal antibodies that exploit the dependence of eosinophils on a specific cytokine: interleukin-5 (IL5), which is described in the literature as an essential growth factor of eosinophils. One such treatment, benralizumab, destroys eosinophils and their progenitors by binding to the IL5 receptor on their surface. Patients treated with benralizumab then usually become totally eosinopenic, i.e. without eosinophils. Two other treatments, reslizumab and mepolizumab, neutralize IL5. Patients treated with these two molecules become partially eosinopenic, but retain a population of residual eosinophils representing about half the number of eosinophils in the general population. Therefore, the teams of Christophe Desmet and Fabrice Bureau’s team (Cellular and Molecular Immunology Laboratory, associated with the Pneumology Laboratory - GIGA-I3) asked the following questions. What happens to these residual eosinophils? Does the suppression of IL5 during eosinophil development also change their biological activities in addition to reducing their number? Could this have long-term consequences given the various immune and homeostatic roles experimentally attributed to eosinophils, including in metabolism, antimicrobial and anticancer defence? The question of whether residual eosinophils from patients treated with mepolizumab or reslizumab have any particular biological activity is therefore important in predicting the potential long-term effects of IL5 neutralization. In this study published in the prestigious European Respiratory Journal, GIGA researchers used a comparative transcriptomics approach in mice and humans to determine whether residual eosinophils growing under IL5 restriction conditions in vivo exhibit alterations in their gene expression program. They compared blood eosinophils from patients with severe eosinophilic allergic asthma treated with mepolizumab to those from healthy controls and control asthma patients receiving another biologic therapy, the anti-immunoglobulin type E biological omalizumab. After performing similar comparisons on bone marrow eosinophils from mice genetically deficient or not for IL5, they observed that reduced IL5 availability does not elicit any detectable response in gene expression of residual eosinophils in humans or mice. They then stimulated human and mouse residual eosinophils with a potent eosinophil activator, IL33, and detected only a minimal effect of IL5 deficiency on their response to activation. From a basic perspective, this work suggests that the main effect of IL5 on eosinophilopoiesis in the steady state is to control the number of peripheral eosinophils rather than to influence their differentiation and subsequent activity. From a clinical perspective, these results suggest that treatment with IL5 neutralizing antibodies such as mepolizumab or reslizumab spares a pool of residual circulating eosinophils that largely resemble those of «healthy» individuals. This work was funded by a research project and an EOS from the FRS-FNRS.

26 GIGA 2021 Annual Report Wobble tRNA modification and hydrophilic amino acid patterns dictate protein fate Rapino F, Zhou Z, Roncero Sanchez AM, Joiret M, Seca C, El Hachem N, Valenti G, Latini S, Shostak K, Geris L, Li P, Huang G, Mazzucchelli G, Baiwir D, Desmet CJ, Chariot A, Georges M, Close P. Nat Commun. 2021 Apr 15;12(1):2170. doi: 10.1038/s41467-021-22254-5. HIGHLIGHTED PUBLICATIONS

GIGA 2021 Annual Report 27 In this article, Francesca Rapino and Pierre Close, from the Cancer Signaling Laboratory (GIGA-Stem Cells), discover how the regulation of transfer RNAs and modification of their anticodon impact the dynamics of protein synthesis and regulate protein folding and stability. The study of the genetic mutations responsible for the formation of cancers has led to the development of new drugs that specifically target cancer cells and which significantly improve the prognosis of patients. However, tumor development also involves a series of non-genetic mechanisms that allow cancer cells to adapt and escape therapy. The regulation of protein synthesis appears to be a central mechanism allowing the adaptation and reprogramming of cancer cells during tumor development. Recent work by Dr Pierre Close’s team has demonstrated the importance of the regulation of transfer RNAs (tRNAs) in the growth, invasion and therapeutic resistance of cancers, by regulating protein synthesis. In this study, the team set out to investigate the mechanisms underlying the reprogramming of protein synthesis through the regulation of tRNAs. The synthesis of new proteins is a highly regulated process that is responsible for the expression of all the proteins present in a cell, in a given context. During protein synthesis, messenger RNAs (mRNAs) are translated into proteins, via adaptor molecules, tRNAs which, within the ribosomes, ensure recognition of the codon sequence of mRNAs and add the corresponding amino acid to the nascent peptide chain. In order to ensure optimal translation and protein synthesis under the best conditions, the tRNAs are modified at their anticodon. In particular, the modifications at their wobble base make it possible to ensure perfect fidelity in the translation of mRNAs by tRNAs. A lack of fidelity during translation results in problems in protein folding, which leads to their subsequent aggregation and degradation. In this study, the role of the modification of the tRNAs on the uridines located at the wobble base of the anticodon (U34-tRNA), which is catalyzed by an enzymatic cascade enclosing among others the enzymes ELONGATOR and CTU1 / CTU2 was studied in more details. The results have shed light on the mechanisms that link the regulation of translation by tRNA modification to protein stability and expression. Surprisingly, it has been discovered that a large majority of genes are insensitive to U34-tRNA modifications. On the other hand, for a certain number of genes, these modifications are essential during their translation, otherwise, the proteins produced are unstable and are degraded. It appears that the presence of specific sequences of hydrophilic amino acids, when combined with a slowdown in translation, precipitates proteins towards aggregation and leads to their degradation. These results provide key insights into the impact of the regulation of translation by tRNA modification on protein stability and function. This work makes it possible to envision a more systematic prediction of the identity of proteins whose expression depends on the regulation of the numerous modifications of tRNAs. Conceptually, this will allow a more precise identification of the different cellular vulnerabilities towards the regulation of mRNA translation and tRNA modifications, based on the set of proteins expressed by the cells.

28 GIGA 2021 Annual Report Alternative glycosylation controls endoplasmic reticulum dynamics and tubular extension in mammalian cells Kerselidou D, Dohai BS, Nelson DR, Daakour S, De Cock N, Hassoun ZAO, Kim DK, Olivet J, El Assal DC, Jaiswal A, Alzahmi A, Saha D, Pain C, Matthijssens F, Lemaitre P, Herfs M, Chapuis J, Ghesquiere B, Vertommen D, Kriechbaumer V, Knoops K, Lopez-Iglesias C, van Zandvoort M, Lambert JC, Hanson J, Desmet C, Thiry M, Lauersen KJ, Vidal M, Van Vlierberghe P, Dequiedt F, Salehi-Ashtiani K, Twizere JC. Sci Adv. 2021 May 7;7(19):eabe8349. doi: 10.1126/sciadv.abe8349. HIGHLIGHTED PUBLICATIONS

GIGA 2021 Annual Report 29 The team of Jean-Claude Twizere (Laboratory of Viral Interactomes, GIGA-MBD), in collaboration with 5 other GIGA laboratories, as well as several other Belgian and international teams, published a study in Science Advances that may have a major impact in the understanding of the internal dynamics in an animal cell. The Endoplasmic Reticulum (ER) is the largest organelle of eucaryotic cells. This organelle structurally organized into a vast membranous network, was first observed in the 1950s by Porter and Palade using electron microscopy. Constitutive proteins of the ER tubular network were identified in 2010 (Shibata et al. Cell 143, 774-788) and 2017 (Powers et al. Nature 543, 257-260). However, as the ER adapts its architecture according to the cellular state, the control of this dynamic rearrangement remained unclear. In this study, Despoina Kerselidou, PhD student in Jean-Claude Twizere’s lab, showed that exostosin-1 (EXT1) glycosyltransferase, an enzyme involved in the polymerization of glycans destined to the cellular membrane, is also a key regulator of ER morphology and dynamics. Using genetic engineering techniques, electron and super-resolution microscopy, and a series of complex -omics analyses, these teams showed that knock-out or -down of EXT1 affects ER morphology and induces ER membrane network extension. These phenotypic changes originate from reprogramming of N-glycosylation reactions and significantly increase cellular metabolic activities. This study could have implications both inmedicine, with the understanding of various diseases such as numerous cancers and Alzheimer’s diseases in which ER dynamics are affected. Reprogramming of N-glycosylation in the ER could also impact the production of recombinant proteins, therapeutic viral vectors and vaccines in mammalian cells.

30 GIGA 2021 Annual Report ERG transcription factors have a splicing regulatory function involving RBFOX2 that is altered in the EWS-FLI1 oncogenic fusion Saulnier O, Guedri-Idjouadiene K, Aynaud MM, Chakraborty A, Bruyr J, Pineau J, O’Grady T, Mirabeau O, Grossetête S, Galvan B, Claes M, Al Oula Hassoun Z, Sadacca B, Laud K, Zaïdi S, Surdez D, Baulande S, Rambout X, Tirode F, Dutertre M, Delattre O, Dequiedt F. Nucleic Acids Res. 2021 May 21;49(9):5038-5056. doi: 10.1093/nar/gkab305. HIGHLIGHTED PUBLICATIONS

GIGA 2021 Annual Report 31 Ewing sarcoma is a bone and soft tissue cancer that predominantly affects children and young adults. Current treatments are limited to intensive chemotherapy that can be brutal and have multiple short-term and long-term side effects. Ewing sarcomas are typified by a chromosomal translocation (i.e. an exchange of DNA regions between two chromosomes) leading to a fusion between two genes: EWS and FLI1. The resulting EWS-FLI1 chimeric protein carries a DNA-binding region from FLI1 that brings the potent gene-activating region of EWS to inappropriate regions of the genome. According to the current model, the consequence of the presence of EWS-FLI1 is a complete deregulation of the gene expression landscape in Ewing sarcoma cells contributing to tumor growth and disease progression. To date, deregulation of gene expression has been exclusively studied at the quantitative level, with hundreds of mRNAs, the key intermediate in the process of gene expression being up or down-regulated by EWS-FLI1, leading to abnormal production of the corresponding proteins and deregulated cellular functions. In a collaborative study with Institut Curie in Paris, the Laboratory of Gene expression and Cancers (GIGA-MBD) took a new look at how EWSFLI1 might modify the gene expression landscape in Ewing sarcoma cells. The researchers hypothesized that EWS-FLI1 might also perturb gene expression programs at the qualitative levels, by affecting mRNA splicing. Splicing of mRNA is a fundamental step in gene expression that allows the cells to express multiple variants (they are called isoforms) of proteins, from a single gene. In their study, the collaborative French and Belgian teams have shown that beyond their role in transcription, EWS-FLI1 has a direct role in alternative splicing of pre-mRNA. Via its splicing function, EWS-FLI1 imposes the expression of specific protein isoforms by Ewing sarcoma cells that affects their adhesion and migration properties, both of which are key in tumor progression and metastasis. This study not only adds significant new insights into the pathological functions of EWS-FLI1 but also suggest new therapeutic avenues for Ewing sarcoma. Specifically targeting this new function of EWS-FLI1 in splicing gives hopes for the future, for more effective and personalized treatments for Ewing sarcoma young patients.

32 GIGA 2021 Annual Report Tumor resistance to ferroptosis driven by Stearoyl-CoA Desaturase-1 (SCD1) in cancer cells and Fatty Acid Biding Protein-4 (FABP4) in tumor microenvironment promote tumor recurrence Luis G, Godfroid A, Nishiumi S, Cimino J, Blacher S, Maquoi E, Wery C, Collignon A, Longuespée R, Montero-Ruiz L, Dassoul I, Maloujahmoum N, Pottier C, Mazzucchelli G, Depauw E, Bellahcène A, Yoshida M, Noel A, Sounni NE. Redox Biol. 2021 Jul;43:102006. doi: 10.1016/j.redox.2021.102006. HIGHLIGHTED PUBLICATIONS

GIGA 2021 Annual Report 33 Nor Eddine Sounni’s group (LBTD, GIGA-Cancer) has provided new insight on mechanisms driven by lipid transport and desaturation in protecting cancer cells from ferroptosis and promoting tumor recurrence, a serious impediment in cancer treatment. The group has previously reported evidence of tumor lipid addiction during recurrence after treatment and metastatic dissemination. Here, in collaboration with Yoshida’s laboratory from Kobe University in Japan and Bellahcène’s group at the GIGA-Cancer, they applied lipidomics to in vivo tumor models of hypoxia/reoxygenation and revealed a particular enrichment of recurrent tumors in mono-unsaturated fatty acids (MUFA). They demonstrated that lipid desaturation by Steroyl-CoA-Desaturase-1 (SCD1) and lipid transport by fatty-acid-binding-protein-4 (FABP4) contribute to such tumor recurrence. SCD1 and FABP4 were found highly overexpressed in residual tumors from mouse and human cancers. Mechanistically, they revealed a cooperation between cancer cells producing SCD1 upon therapy-derived metabolic stress and tumor endothelial cells releasing FABP4 that increases lipid uptake and lipid droplets in cancer cells. SCD1 switches the balance of MUFA/PUFA (poly-unsaturated fatty acids) in favor of MUFA. PUFA are the main substrates for lipid oxidation that induces ferroptosis, a redox- active iron-induced cell death. Notably, FABP4 and SCD1 inhibition sensitized cancer cells to ferroptosis. Overall, this work reveals that SCD1 and FABP4 constitute vulnerabilities that can be targeted to suppress tumor recurrence.

34 GIGA 2021 Annual Report Preservation of brain activity in unresponsive patients identifies MCS Star Thibaut A, Panda R, Annen J, Sanz LRD, Naccache L, Martial C, Chatelle C, Aubinet C, Bonin EAC, Barra A, Briand MM, Cecconi B, Wannez S, Stender J, Laureys S, Gosseries O. Ann Neurol. 2021 Jul;90(1):89-100. doi: 10.1002/ana.26095 HIGHLIGHTED PUBLICATIONS

GIGA 2021 Annual Report 35 A study conducted by the Coma Science Group (GIGA-Consciousness) provides new evidence that up to two third of patients who are diagnosed as being unresponsive at the bedside (i.e., in an unresponsive wakefulness syndrome, previously called the vegetative state) retain brain activity compatible with the diagnosis of minimally conscious state. Dr. Aurore Thibaut commented: “The notion of cognitive motor dissociation in patients with disorders of consciousness is more and more accepted in the scientific and clinical communities. Such patients present higher brain activity (measured with neuroimaging tools), compared to what they can express when assessed at the bedside. Such dissociation can occur due to many factors such as severe motor impairments. In the present study, we aimed to look at the patients’ brain function at rest (i.e., no tasks and no stimuli were applied) and investigate, in a blind way, the residual brain activity and grey matter atrophy of unresponsive patients compared to minimally conscious patients.” The researchers included 135 brain-injured patients with disorders of consciousness. Forty-eight were diagnosed in unresponsive wakefulness syndrome (e.g., no signs of consciousness) and 87 as minimally conscious (e.g., ability to perceive external stimuli such as pain and/or answer simple command). These patients were diagnosed using a standardized and validate behavioral scale, the Coma Recovery Scale-Revised, on multiple occasions to ensure a stable and reliable clinical diagnosis. When looking at the residual brain metabolism of these 48 unresponsive patients, three researchers who were blinded of their diagnosis, categorized 32 of them (67%) as being minimally conscious. Later on, these 32 patients with a mismatch between clinical and neuroimaging diagnosis were classified as being in minimally conscious state “star” (i.e., MCS* - * indicating that the diagnosis is made with neuroimaging). When looking at the different subgroups of patients, researchers found a better prognosis for patients in MCS* compared to those in unresponsive wakefulness syndrome. In addition, these patients presented higher brain activity as measured with electroencephalography, as well as less brain atrophy in specific brain regions. “These results suggest that patients in MCS* represent a distinct entity that differ from the unresponsive wakefulness syndrome in term of clinical outcome and brain activity patterns” says Dr. Olivia Gosseries. The authors conclude that patients who are unresponsive at the bedside but who present substantial brain activity (i.e., MCS*) is a frequent phenomenon that is associated with a better outcome and brain activity compatible with the minimally conscious state. In this scenario, they recommend that complementary exams should be performed to all unresponsive patients before taking medical decisions.

36 GIGA 2021 Annual Report Multi- and transgenerational outcomes of an exposure to a mixture of endocrine-disrupting chemicals (EDCs) on puberty and maternal behavior in the female rat López-Rodríguez D, Aylwin CF, Delli V, Sevrin E, Campanile M, Martin M, Franssen D, Gérard A, Blacher S, Tirelli E, Noël A, Lomniczi A, Parent AS. Environ Health Perspect. 2021 Aug;129(8):87003. doi: 10.1289/EHP8795. HIGHLIGHTED PUBLICATIONS

GIGA 2021 Annual Report 37 Currently, we are facing a major environmental, societal and health issue due to the increasing presence of environmental pollutants from the agri-food (e.g. pesticides and fungicides), petrochemical (e.g. bisphenols and phthalates), pharmaceutical (paracetamol) and cosmetic (sunscreens) industries. We are all exposed to dozens of substances on a daily basis. Among these pollutants, several hundred are identified as endocrine disruptors (EDs). These substances are capable of altering hormonal homeostasis and increasing the risk of numerous disorders. They are the main research topic of Anne-Simone Parent’s team at Neuroendocrinology Laboratory (GIGA-Neuroscience). The developing organism and in particular the brain is especially sensitive to EDs. Exposure to EDs during development can lead to alterations in the neural circuits that control sexual development and reproductive function in adulthood. Moreover, recent data suggest that these alterations may persist over several generations. In Anne-Simone Parent’s laboratory, a study published in Environmental Health Perspectives examined the effects of a mixture of 13 EDCs to which we are commonly exposed at low doses on sexual development and maternal behavior over three generations of female rats. The mixture containing plastics, pesticides, fungicides, sunscreens and paracetamol was administered before gestation until the end of lactation. The study shows that developmental exposure to a mixture of PEs leads to altered puberty, ovulatory cycle and ovarian folliculogenesis two and three generations after exposure. As Anne-Simone Parent points out, «these results call into question the current methods of evaluating the danger of endocrine disruptors since their effects are not seen directly after exposure during pregnancy, but in the second and third generations». These effects are therefore particularly worrying since the third generation has never been directly exposed to EPs. The study shows that the transmission of these effects on reproduction could be explained by alterations of germ cells which would then lead to an epigenetic reprogramming of the hypothalamic control of reproduction. In addition, the team identified alterations in maternal behavior caused by abnormalities in hypothalamic dopaminergic signaling. This altered maternal behavior is transmitted from generation to generation through learning. Overall, this study highlights the brain mechanisms explaining the effects of a mixture of EDs on reproduction across several generations of animals. These results raise concerns about the potential consequences of EDs on our future generations.

38 GIGA 2021 Annual Report ATP-citrate lyase promotes axonal transport across species Even A, Morelli G, Turchetto S, Shilian M, Bail RL, Laguesse S, Krusy N, Brisker A, Brandis A, Inbar S, Chariot A, Saudou F, Dietrich P, Dragatsis I, Brone B, Broix L, Rigo JM, Weil M, Nguyen L. Nat Commun. 2021 Oct 7;12(1):5878. doi: 10.1038/s41467-021-25786-y. HIGHLIGHTED PUBLICATIONS

GIGA 2021 Annual Report 39 Laurent Nguyen’s laboratory (Laboratory for Molecular Regulation of Neurogenesis, GIGA-Stem Cells) has been studying the role of the Elongator complex in the nervous system for many years. Loss of Elongator’s activity has been associated with various neurological diseases, including familial dysautonomia. These researchers have just identified the mechanism by which Elongator controls axonal transport in projection neurons. Neurons possess a molecular network made up of microtubules that allow proteins and specialized structures (cargos) to be transported within the axon. This process, known as axonal transport, is necessary for the functioning and survival of neurons and is impaired in many neurodegenerative diseases. In order to better understand these diseases, it is important to determine the mechanisms underlying this process in neuronal axons. For optimal axonal transport, microtubules are acetylated by the enzyme ATAT1. This modification controls the recruitment of cargos and adapt their speed of transport along microtubules. Laurent Nguyen’s team previously showed that loss of Elongator complex in the brain leads to defect in migration and differentiation of cortical neurons, further characterized by a reduced acetylation of their microtubules (Cell 2009). More recently, these researchers discovered that motile vesicles that are transported along axonal microtubules in these neurons are enriched for ATAT1 enzymes, thereby regulating the acetylation of their microtubule tracks (Science Advances 2019). In the context of axonal transport, this work reveals a link between the activity of ATAT1 (acetylation of microtubules) and that of the Elongator complex, both of which are enriched on motile vesicles that move along axonal microtubules. Researchers from GIGA, in collaboration with Tel Aviv University, show that the absence of Elongator at motile vesicles reduces the stability of ACLY, the enzyme responsible for the synthesis of AcetylCoA, necessary for the acetylation of microtubules by ATAT1. This molecular pathway is important for axonal transport and is conserved across species, from flies to humans. This breakthrough in the mechanisms regulating axonal transport enables a better understanding of how its disruption can lead to neurodegenerative diseases, such as familial dysautonomia (resulting from the loss of Elongator activity), Huntington’s disease, and amyotrophic lateral sclerosis. On the long run, it would be conceivable to normalize axonal transport with pharmacological molecules that modify the activity of key enzymes regulating positively (Elongator, ATAT1, ACL1) or negatively (HADC6) the acetylation of microtubules and thus axonal transport.

40 GIGA 2021 Annual Report A non-internalised cd38-binding radiolabelled single-domain antibody fragment to monitor and treat multiple myeloma Duray E, Lejeune M, Baron F, Beguin Y, Devoogdt N, Krasniqi A, Lauwers Y, Zhao YJ, D’huyvetter M, Dumoulin M, Caers J. J Hematol Oncol. 2021 Nov 2;14(1):183. doi: 10.1186/s13045-021-01171-6. HIGHLIGHTED PUBLICATIONS

GIGA 2021 Annual Report 41 Antibody-based therapies targeting the CD38 enzyme are currently used as single agents as well as in combination regimens for multiple myeloma, a malignant disease of plasma cells, a population of B lymphocytes. In this study, researchers from the laboratory of Haematology (GIGA-I3) developed single-domain anti-CD38 antibodies (sdAbs) that can be used to track CD38-positive tumor cells and then used for targeted radionuclide therapy. The sdAbs are derived from camelid heavy chain antibodies and have shown to be promising theranostic agents due to their favorable pharmacological properties. At the beginning of this project, 4 anti-CD38 nanobodies were produced, purified and characterized at the Protein Engineering Center. A first phase of in vitro and in vivo characterization (radio-labeling with technetium-99m) allowed to identify nanobody 2F8 for which the properties sought in the context of diagnostic tools (specific accumulation within the tumor, very good tumor/background ratio as early as 1h post-injection) or therapeutic tools (absence of competition with daratumumab currently used on the market, absence of aspecific retention within non-targeted organs, ... ) were found. Then, the internalization of the receptor after treatment was evaluated. Indeed, internalization is observed when using the monoclonal antibody daratumumab (current therapeutic agent in the treatment of myeloma). In the case of Nb 2F8, a limited (20%) and constant internalization over time was demonstrated using a radio-labeling technique. These results were confirmed using a second method, flow cytometry (via the GIGA-Flow Cytometry platform). This low internalization opened the possibility, due to its novelty, to protect this tool by a European patent filed in 2020. After having verified the efficacy of this antibody in vitro, it was necessary to test its behavior in vivo when conjugated to a therapeutic radioisotope (beta or alpha emitter). In this step carried out in collaboration with the VUB, lutetium was chosen on the basis of its availability, its direct labelling after coupling with a chelator (as for indium) and its emission of β-particles (targeted radioimmunotherapy) and radiation (bio-distribution). In this first therapeutic experience, a significant tumor regression was observed. The efficacy of the anti-CD38 agent was then confirmed. During the different experiments (diagnosis and therapy), one of the rare side effects of this compound was the presence of a renal toxicity following an aspecific retention of the radio-labelled compound linked to the glomerular filtration. Some devices, such as the co-injection of gelofusine (a solution known to act on this renal retention), have been implemented during the last therapeutic experiments allowing to reduce considerably this side effect. New studies are currently underway to continue the project and further improve efficacy while reducing side effects.

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