Franco-Brazilian Collaboration in Hematology International Research Network on Hematology (IRNH)

Franco-Brazilian collaboration in Hematology International Research Network on Hematology (IRNH)

New emerging collaboration The genesis of a project: from the late 90s

Analysis of the 677C→T mutation of the Methylenetetrahydrofolate Reductase Gene in Different Ethnic Groups R. F. Franco, A. G. Araújo, J. F. Guerreiro, J. Elion, M. A. Zago Thromb Haemost. 1998 Jan;79(1):119-21.

The phylogeography of mitochondrial DNA haplogroup L3g in Africa and the Atlantic slave trade. Bortoloni MC, Da Silva WA Jr, Zago MA, Elion J, Krishnamoorthy R, Goncalves VF, Pena SD Am. J. Hum. Genet. 75:523–524, 2004 from population genetics… ….to cellular and molecular hematology

Cellular and Molecular Effects of Hydroxycarbamide in Sickle Cell Patients Silva-Pinto AC, de Cássia Viu Carrara R, V.B. Palma P, Zago MA, Elion J, Covas DT Current Pharmacogenomics and Personalized Medicine, 2014, 12, 114-122

15 publications 1998-2015 The genesis of a project (II): from the early 2000s clinical hematology

Host defense and inflammatory gene polymorphisms are associated with outcomes after HLA-identical sibling bone marrow transplantation Vanderson Rocha, Rendrik F. Franco, Raphael Porcher, Henrique Bittencourt, Wilson A. Silva Jr, Aurelien Latouche, Agnes Devergie, Helene Esperou, Patricia Ribaud, Gerard Socie, Marco Antonio Zago, and Eliane Gluckman Blood. 2002 Dec 1;100(12):3908-18

Age-adjusted recipient pretransplantation telomere length and treatment-related mortality after hematopoietic stem cell transplantation.

Peffault de Latour R, Calado RT, Busson M, Abrams J, Adoui N, Robin M, Larghero J, Dhedin N, Xhaard A, Clave E, Charron D, Toubert A, Loiseau P, Socié G, Young NS. Blood. 2012;120(16):3353-9

7 publications 2002-2015 The genesis of a project (III): 2013

USP-Paris-Diderot Symposium on Hematology and Immunology

July 2015

2016-2019

Initial purpose

The purpose of the “International research network on Hematology” (INRH) is to facilitate collaborations around the following topic: Basic and translational research on non-malignant hematological diseases: - sickle cell anemia - aplastic anemia - autoimmune diseases Actions

- integrate activities from different fields: hematology, biochemistry, genetics, genomics, cell biology, immunology, cell therapy, clinical research and allied health sciences - search for funding - exchange of researchers and students, - organization of training courses and scientific meetings - development of training tools Coordination and Partners

French scientific coordinators Eliane Gluckman Laboratoire Eurocord EA4412 IUH Paris Diderot - USPC Jacques Elion UMR_S 1134 Inserm Paris Diderot - USPC

Brazilian scientific coordinators Rodrigo T. Calado Faculdade de Medicina de Ribeirão Preto FMRP-USP Belinda P. Simões Faculdade de Medicina de Ribeirão Preto FMRP-USP Partners

UMR_S 1134 Inserm Paris Diderot Caroline Le Van Kim, Wassim El Nemer, Jacques Elion UMR_S1134I UH Inserm Paris Diderot Antoine Toubert EA4412 IUH Paris Diderot Eliane Gluckman, Régis Peffault de la Tour

UMR_S1163 Imagine Inserm Paris Descartes Olivier Hermine, Marina Cavazzana

Hemocentro FMRP CTC Rodrigo Calado, Belinda Simoes, Dimas Covas, Simone Kashima-Haddad Network themes

Three major themes: - sickle cell anemia - aplastic anemia - autoimmune diseases

Present SCD geographic distribution

Healthy carriers ≈ 5% of the world population 300.000 newborns/year (80% in low income countries) Present SCD geographic distribution

clearly SCD is a global issue SCD incidence Global 1/2,700 Parisian area 1/1,050

SCD incidence 1/300 Sickle Cell Disease 10/15,000 patients in Mainland France 2,500 patients in ovesea territories

Réunion & Mayote Islands population 1 million

France: a country with overseas territories Integrated Red Blood Cell Biology

Paris

UMR_S1134

Guadeloupe

CAribbean network of REsearchers on Pointe-à-Pitre

Sickle cell disease and Thalassemia

Réunion Sickle Cell Disease in Brazil

MA 1:1400

PE 1:1400 BA 1:650 GO 1:1400 Number of births: 3 millions (2012) MG 1:1400 MS 1:8300 ES 1:1800 SP 1:4000 New SCD cases: 2.5 - 3,000 RJ 1:1200 PR e Mean incidence : 1/1000 SC 1:13000 RS 1:13500 HbS: a single mutation but a polysystemic disease

p.Val6Glu → HbS

free Hb NO deoxyHbS twopolymerisation trigerring scavenging factors

Haemolysis

Abnormal adhesion to endothelial cells

Vasoocclusion Acute complications - painful crises - dactilytis

Steinberg M.H., 1999 - acute chest syndrome

Inserm U1134U1134 HbS: a single mutation but a polysystemic disease

p.Val6Glu → HbS Polysystemic chronic complications free Hb NO deoxyHbS twopolymerisation trigerring scavenging Nonhemorrhagic Pulmonary factors Stroke Hypertension

HaemolysisHaemolysis Renal Cholelithiasis Failure Abnormal adhesion to endothelial cells

Priapism Osteonecrosis VasoocclusionVasoocclusion Acute complications Leg (VOC) Bacteremia- painful crises Ulceration - dactilytis

Steinberg M.H., 1999 - acute chest syndrome - splenic sequestration Inserm U1134U1134 Vasoocclusion: a complex mechanism a cascade of interactions between RBC, neutrophils, platelets and activated endothelium - inflammation

intravital microscopy (mouse) RBC (blue), neutrophils (red) adapted from Frenette et al, 2016 The IRNH Work program on SCD I Basic and translational research Jacques Elion and Dimas Covas WP1: Search for modifying genes WP2: Adenosine signaling and pathophysiology of priapism WP3: Innovative approaches for novel biomarkers and anti- adhesive therapies II Clinical research Eliane Gluckman and Belinda P. Simões WP1: Outcome of hematopoietic stem cell transplantation for SCD WP2: Elaboration of transplant protocols with alternative donors WP3: Endothelial damage in SCD WP4: HLA and other genetic polymorphisms in sickle cell disease WP5: Immunological reconstitution after HSCT The IRNH Work program on SCD I Basic and translational research Jacques Elion and Dimas Covas WP1: Search for modifying genes WP2: Adenosine signaling and pathophysiology of priapism WP3: Innovative approaches for novel biomarkers and anti- adhesive therapies II Clinical research Eliane Gluckman and Belinda P. Simões WP1: Outcome of hematopoietic stem cell transplantation for SCD WP2: Elaboration of transplant protocols with alternative donors WP3: Endothelial damage in SCD WP4: HLA and other genetic polymorphisms in sickle cell disease WP5: Immunological reconstitution after HSCT Combined variants

• Cell-to-Cell Signalling and Interaction; • Hematological System Development and Function; • Immune Cell Trafficking. • Cardiac Arrythmia, Cardiovascular Disease; • Cell Morphology.

Histamine Biosynhesis

IL-10 Signaling

Toll-like Receptor Signaling

Arsenate Detoxification I (Glutaredoxin)

PPAR Signaling

Creatine-phosphate Biosynthesis

IL-6 Signaling

Aryl Hydrocarbon Receptor Signaling

Tec Kinase Signaling

NF-kB Signaling

Differential Regulation of Cytokine Production in Intestinal Ephitelial Cells by IL-17A and IL-17F The IRNH Work program on SCD I Basic and translational research Jacques Elion and Dimas Covas WP1: Search for modifying genes WP2: Adenosine signaling and pathophysiology of priapism WP3: Innovative approaches for novel biomarkers and anti- adhesive therapies II Clinical research Eliane Gluckman and Belinda P. Simões WP1: Outcome of hematopoietic stem cell transplantation for SCD WP2: Elaboration of transplant protocols with alternative donors WP3: Endothelial damage in SCD WP4: HLA and other genetic polymorphisms in sickle cell disease WP5: Immunological reconstitution after HSCT The IRNH Work program on SCD I Basic and translational research Jacques Elion and Dimas Covas WP1: Search for modifying genes WP2: Adenosine signaling and pathophysiology of priapism WP3: Innovative approaches for novel biomarkers and anti- adhesive therapies II Clinical research Sickle cell disease: an international Elianesurvey of Gluckman results of HLA-identical and Belinda sibling P. hematopoietic Simões WP1:stem cell Outcome transplantation. of hematopoietic stem cell transplantation for SCD Gluckman E1,2, Cappelli B2, Bernaudin F3, Labopin M4, Volt F1,2, Carreras J5, Pinto Simões B6, Ferster A7, Dupont S8, de la WP2:Fuente J9 ,ElaborationDalle JH10, Zecca M11 of, Walters transplant MC12, Krishnamurti protocols L13, Bhatia withM14, Leung alternative K15, Yanik G16, Kurtzberg donors J17, Dhedin N18, Kuentz M3, Michel G19, Apperley J20, Lutz P21, Neven B22, Bertrand Y23, Vannier JP24, Ayas M25, Cavazzana WP3:M26,27,28, Matthes-Martin Endothelial S29, Rochadamage V1,30,31, Elayoubi in SCD H1,2, Kenzey C1,2, Bader P32, Locatelli F33,34, Ruggeri A1,2,35, Eapen M5; Eurocord, the Pediatric Working Party of the European Society for Blood and Marrow Transplantation, and the WP4:Center for HLA International and Blood other and Marrowgenetic Transplant polymorphisms Research. in sickle cell disease WP5:Blood. 2017 Immunological Mar 16;129(11):1548-1556. reconstitution after HSCT Actions

- integrate activities from different fields: hematology, biochemistry, genetics, genomics, cell biology, immunology, cell therapy, clinical research and allied health sciences - search for funding - exchange of researchers and students 20 short term exchanges of researchers 2 visiting professorships to Brasil 6 PhD thesis codirected or cotutelle 1 Frenc postdoc in Brasil Actions

- integrate activities from different fields: hematology, biochemistry, genetics, genomics, cell biology, immunology, cell therapy, clinical research and allied health sciences - search for funding - exchange of researchers and students, - organization of training courses and scientific meetings USP-USPC IRNH Inaugural Meeting, São Paulo/Ribeirão Preto, 2015 USPC-USP Hematology/Bioinformatics Meeting, Paris, 2016 Projects

Based on Laboratory of excellence GR-Ex

Université de Paris COMPREHENSIVE PLATFORM

French Laboratory of Excellence on the Red Cell (GR-Ex) COMPREHENSIVE PLATFORM

French Laboratory of Excellence on the Red Cell (GR-Ex)

International Research Network on Hematology (IRNH) COMPREHENSIVE PLATFORM

French Laboratory of Excellence Coordinator: Olivier Hermine on the Red Cell (GR-Ex)Co-cordinator: Caroline Ke Van Kim

GENOMIC PLATFORM

The Imagine genomic platform is equipped with

• Two high throughput next generation sequencers HiSeq2500, Illumina • One next generation sequencer Ion Torrent PGM (Personal Genome Machine), Life Technologies • One next generation sequencer MiSeq, Illumina • A GeneChip microarray plateform, Affymetrix

Other equipment

• DNA shearing: Covaris S2, Covaris E220, Hydroshear, GeneMachines • Capillary electrophoresis: Bioanalyseur 2100 et Tape Station 2200 Agilent Technologies • Nucleic acids measurement: Nanodrop ND-1000 spectrophotometer (Thermo Scientific), QuBit • fluorimeter (Invitrogen), real time PCR StepOnePlus (Life Technologies) GENE EXPRESSION PLATFORM

Eric Soler  part of the epigenetic/gene regulation network in France A GENE REGULATORY Identification and functional characterization of novel NETWORK transcription factor complexes

- Proteomics analysis of Transcription factor complexes (IP-MS) - Genome-wide analysis of transcription factor binding sites (ChIP-Seq) - Functional characterization of enhancer elements PROTEOMICS GENOMICS (CRISPR/CAS9) (Soler et al. Genes Dev 2010; Stadhouders et al. Nat Comm. (in revision)) B Dynamics of genome organization and impact on gene FACS expression Sequential analyses on sorted cell populations II III - Transcriptome (RNA-Seq) - Dynamics of Transcription factor occupancy

CD71 I (ChIP-Seq) - Chromatin structure (3C-Seq) (Soler et al. Methods 2011; Stadhouders et al. Ter119 Nat Protoc. 2013) PROTEOMIC PLATFORM

Deep proteomic analysis of erythroid differentiation

Partner 6 : Emilie-Fleur Gautier, Sarah Ducamp, Catherine Lacombe, Frédérique Verdier, Yael Zermati and Patrick Mayeux Partner 13 : Marie Catherine Giarratana and Luc Douay Paris Descartes University proteomic platform (3P5): Marjorie Leduc, Virginie Salnot and François Guillonneau

1. Evolution of the erythroblast proteome along erythroid differentiation

Optimization of erythroid culture methods to improve the synchronisation of terminal differentiation steps

Proteomic analyses - Relative expression of 2730 proteins mesured by ITRAQ labelling - Absolute expression of 6300 proteins mesured by label-free analysis (as copy number per cell) - Evolution of ribosome biogenesis mesured by pulsed Silac and related to the induction of terminal differentiation PROTEOMIC PLATFORM

Deep proteomic analysis of erythroid differentiation

1. Evolution of the erythroblast proteome along erythroid differentiation

ErythropoiesisOptimization of erythroid culture in SCDmethods toand improve mechanisms the synchronisation of terminalof the differentiation switch steps of globins expression Wassim El Nemer, Sara El Hoss Institut national de la santé et de la recherche médicale

Proteomic analyses -Alexander, Relative expression Jonathan, of 2730 proteins Simone mesured Kashima, by ITRAQ Prof labelling Dimas - Absolute expression of 6300 proteins mesured by label-free analysis (as copy number per cell) - Evolution of ribosome biogenesis mesured by pulsed Silac and related to the induction of terminal differentiation Proteomics Conventional NewBorn ScreeningFAPESP-PIPE methods project

Isoelectric focusing (IEF) CapillaryDr Patrickelectrophoresis Ducoroy, France Confirmatory tests

Hbs separated according to charge- IEF - HPLC - Capillary electrophoresis HPLC Dra Sandra Rodriguez Pereira Faça, Hbs separatedIncubadora SUPERA according (USP – Ribeirão to charge Preto) /polarity)

70-80.000 tests/year

Innovative high throughput NBS methods Mass spectrometry (MALDI-TOF) Globin chains separated according to mass 2-300.000 tests/year METABOLOMIC PLATFORM

Partner 11: Lydie Oliveira, funded by GR-Ex, Christophe Junot and Paul-Henri Roméo Two laboratories: LEMM (CEA-Saclay) and LRTS (CEA-Fontenay-aux-Roses) Fingerprinting Data processing Multivariate statistical analysis by LC-ESI HRMS Samples 100 EXPOSED Metabolite 90 4000 Collection 80 3000

70 CONTROLS extraction 2000 Biomarker 60 of blood 1000 50 0 identification (Boiling RBCs in 40 t[2]O samples Abundance Relative -1000 30 -2000 water) 20

10 -3000

0 -4000 0 20 40 60 80 100 120 140 Time (min)

VARIABLES (RT-MASSES) -4000 -3000 -2000 -1000 0 1000 2000 3000 4000 t[1]P

Use of 3 chromatographic High Resolution Mass Spectrometry methods to achieve an (HRMS): more detected metabolites

1% Amino Acids optimal metabolome coverage and to improved identifications 2% 4% Hydroxy Acids

6% Monocarboxylic Acids

3% Dicarboxylic Acids 33% RP-C8 / RP-C18 3% Tricarboxylic Acids Aromatic Acids Resolution Mass accuracy 5% Keto Acids

Carbohydrates C10H15O4 Alcohols and Polyols

Databases 15% Polyamines

Pyridines and derivatives

PentaFluoroPhenylPropyl 9% Nucleosides, Nucleotides, 2% and Analogues Carnitines and derivatives (PFPP) 6% 8% 2% Ketones 1% Others

205 metabolites detected, identified or annotated in ZICpHILIC RBC extracts FLOW CELL IMAGING PLATFORM

Partner 1 : Michaël Dussiot, Olivier Hermine Imagine Institute : Olivier Pellé, Meriem Garfa-Traore, Nicolas Goudin SFR Necker: Corinne Cordier, Jérôme Megret Imaging Flow Cytometry (Imagestream System) The ImageStreamX Mark II (AMNIS) fully ISX MKII Excitation Lasers integrates the features of classical flow cytometry 375nm 70mw Laser 561nm 200mw Laser and fluorescence microscopy combined with (DAPI, Hoechst, Pacific Blue…) (PE, Spectrum Orange…) extended software for digital image analysis.

405nm 120mw Laser 642nm 150mw Laser (DAPI, Hoechst, Pacific Blue…) (AF647, Cy5, APC, APCCy7…) ImageStreamX MKII Imaging Flow Cytometer • Brightfield, darkfield, and several fluorescent 488nm 200 or 400mw 785nm 70mw Laser images at high speed (FITC, PE, PI, ECD, PerCP…) (SSC in channel 6 or 12) IDEAS® Statistical Image Analysis Software • Quantitative cellular image analysis and population statistics Erythro-phagocytosis Results Applications

*** • Translocation 0,2X *** *** • Co-localization RBC *** *** 40 * • Cell classification 0,5X RBC 30 • Cell cycle

20 • Apoptosis

1X 10 Spot Count_Peak Spot RBC (M03, Channel 3, Bright, 23) Bright, 3, Channel (M03, 0

X X X C .5 1 0.2 0 o 60 r X ro o c 1 c cr 1X RBC Ma ro Ma Ma c 60°C Ma RED CELL PHYSIOLOGY PLATFORMS

Wassim El Nemer, Yves Colin Aronovicz, SID4Bio Caroline Le Van Kim

Dynamic Adhesion Platform (cellular interactions of blood cells) 600 Type 1 500 Type 2

400 2 Type 3 ECM purified proteins: 300 • Lamin

Lorrca Flow cytometry Stopped-flow (Membrane transport) A full panel of surface proteins

Marker 1 RED CELL PHYSIOLOGY PLATFORMS

Wassim El Nemer, Yves Colin Aronovicz, SID4Bio Caroline Le Van Kim Simone Kashima, Yann Lamarre

Dynamic Adhesion Platform (cellular interactions of blood cells) 600 Type 1 500 Type 2

400 2 Type 3 ECM purified proteins: 300 • Lamin

Lorrca Flow cytometry Stopped-flow (Membrane transport) A full panel of surface proteins

Marker 1 MICROFLUIDIC PLATFORM

Partner 14: Pierre Buffet and Partner 2: Yves Colin Aronovicz/Caroline Le Van Kim

Microfluidics to deconvolute the intensity and mechanisms of interactions between RBC and the human spleen

Application Screening approach for compounds blocking the transmission of malaria (Duez et al. Submitted)

Funding from the Gates Foundation and GSK MICROFLUIDIC PLATFORM

Partner 14: Pierre Buffet and Partner 2: Yves Colin Aronovicz/Caroline Le Van Kim

Microfluidics to deconvolute the intensity and mechanisms of interactions between RBC and the human spleen

Wassim El Nemer

Lizarralde Iragorri MA, El Hoss S, Brousse V, Lefevre SD, Dussiot M, Xu T, Ferreira AR, Lamarre Y, Silva Pinto AC, Kashima S, Lapouméroulie C, Covas DT, Le Van Kim C, Colin Y, Elion J, Français O, Le Pioufle B, El Nemer W. A microfluidic approach to study the effect of mechanicalApplication stress on erythrocytes in sickle cell disease. Screening approach for compounds Lab Chip. 2018 Sep 26;18(19):2975-2984. blocking the transmission of malaria (Duez et al. Submitted)

Funding from the Gates Foundation and GSK INTRAVITAL VIDEO MICROSCOPY PLATFORM

Partner 8 : Pierre-Louis Tharaux, Pierre Nivoit (funded by the GR-Ex) Intravital video microscopy to investigate the murine cremaster muscle microcirculation

Intravital microscopy MEMBRANE PROTEIN STRUCTURE STUDIES

Experimental + In silico approaches Structure modeling TM protein purification approaches

Não é possível exibir esta imagem no momento. Transfected cells

Mechanistic of solute / ion transports across the red cell membrane alcalinisation Proteoliposomes

Energy activatio n

Molecular Dynamics Catherine Etchebest Functional studies by stopped-flow fluorometry MEMBRANE PROTEIN STRUCTURE STUDIES

Experimental + In silico approaches Structure modeling TM protein purification approaches

Não é possível exibir esta imagem no momento. Transfected cells

Catherine Etchebest Mechanistic of solute / ion transports across Fernandothe red Barroso, cell membrane FCFRP alcalinisation Proteoliposomes

Energy activatio n

Molecular Dynamics

Functional studies by stopped-flow fluorometry SINGLE CELL ANALYSIS

genomics

single cell transcriptomics

(…proteomics)

Wilson Silva Jr Rodrigo Panepucci

Inserm Tokyo UMR1134 99

Cohort of 2,000 SCD Patients Projects on SCD Heterogeneity of Hb F expression Wassim El Nemer Simone Kashima, Dimas Covas Abnormal erythropoiesis Wassim El Nemer and Olivier Hermine Simone Kashima, Dimas Covas Fighting inflammation Olivier Hermine Catherine Etchebest Renato Monteiro Niels Camara Biomedical Science Institute USP Gene therapy Marina Cavazzana Vanderson Rocha, Renato Cunha Franco-Brazilian collaboration in Hematology International Research Network on Hematology (IRNH)

New emerging collaboration Collaborators at Hematology Center (Hemocentro)

Fernando Ferreira Costa Genomics Lab Hemocentro

Nicola Conran Vascular Inflammation Lab

Research Interests:

- Genetic and pathophysiological aspects of hemoglobinopathies (especially sickle cell disease) Vascular Inflammation Laboratory - Inflammatory mechanisms of hemolysis Current Collaborative Project : Use of an in vitro microfluidic assay to investigate a role for platelets in neutrophil recruitment in sickle cell anemia

Support: Process Fapesp 2014/009843 (Thematic Project)

Biologie Intégrée du Globule Rouge, UMR_S1134, Paris Caroline Le Van Kim, Pr. Bérengère Koehl, M.D., Ph.D. Mickaël Marin, B.Sc. (Microfluidics engineer) Yves Colin Aronovicz, Ph.D. Wassim Nemer, Ph.D. Jacques Elion, M.D., Ph.D.

Red cell research group, UNICAMP, Brazil Nicola Conran, Ph.D. Fernando Ferreira Costa, M.D., Ph.D. Hanan Chwieh, Ph.D. Student Flavia Costa Leonardo, Ph.D. Funded by Fapesp 2014/009843 (Thematic Project):

Visit by Hanan Chweih (PhD Student, UNICAMP) to INTS, Paris

Visit by Eng. Michael Marin (INTS, Paris) to Hemocentro, UNICAMP

Visit by Bérèngere Koehl (INTS, Paris) to Hemocentro, UNICAMP Future Grants?

Grant requested from FAPESP as part of a “Thematic Project” Grant Proposal

“Collaborative Project Proposal – Defining vaso-occlusion and approaches for its reversal”.

Principal Investigators: Nicola Conran Zorzetto/ Caroline Le Van Kim/ Bérengère Koehl (INTS, Paris)

Instituitions: Centro de Hematologia e Hemoterapia, UNICAMP / Biologie Intégrée du Globule Rouge, UMR_S 1134, INTS, Paris, France

Duration: 4 years (Post doctoral project)

Post doctoral fellow: To be recruited

Red Cell Disease research group, Hemocentro (Blood Center), UNICAMP, Brazil Nicola Conran, Ph.D.: Laboratory of Vascular Inflammation Fernando Ferreira Costa, M.D., Ph.D.: Laboratory of Genomics Hanan Chwieh, M.Sc., Ph.D. Laboratory of Vascular Inflammation Flávia Costa Leonardo, Ph.D.: Laboratory of Vascular Inflammation Carla Franco Penteado, Ph.D.: Laboratory of Genomics

Biologie Intégrée du Globule Rouge, UMR_S 1134, INTS, Paris, France Caroline Le Van Kim, Ph.D. Bérengère Koehl, M.D., Ph.D. Mickaël Marin, B.Sc. (Microfluidics engineer) Merci Obrigado