The team studies the role of Heat Shock Proteins (HSPs), also called stress proteins, in pathologies in which a dysregulation in their expression or function is involved (ex. cancer, fibrosis, myeloproliferative syndromes or immune disorders).
HSPs are highly conserved molecular chaperones induced by a variety of exogenous or intracellular stresses. They participate in the correct folding, activity, transport and stability of proteins. HSPs have intra- (cytoprotective) and extracellular (danger signal) functions. Traditionally, HSPs are classified by their molecular weight into 5 families: HSP110 (also called HSPH), HSP90 (HSPD), HSP70 (HSPA), HSP60 (HSPD/E) and the small HSPs (HSPB). Stress proteins allow the cells to survive otherwise lethal conditions.
Collaborations : A Duval (H Saint Antoine, Paris), C Lepage (CHU Dijon), O Casasnovas (CHU Dijon), AM Cuervo (A Einstein Institute, NY); E Appella (NCI MIH, USA). Funding : I-SITE-BFC. ANR-15-IDE-0003 (2017-2021); Label d’excellence LNCC (2019-2023). INCa PLBIO 2022-26. Prix Ligue contre le Cancer 2021.
Rational. We found a mutation in HSP110 in 10-15 % of colorectal and gastric cancers and this HSP110 inactivation was associated with an excellent prognosis i.e. answer to an oxaliplatin/5-fluorouracil-based therapy (multicenter study including more than 3000 patients. Dorard et al, Nature Med 2011; Collura et al, Gastroenterol 2014). We demonstrated that this might be explained by HSP110 effect in the nucleus that co-localizes with DNA damaged sites and by its immunosuppressive role in the tumor microenvironment (Duvrez et al, Oncogene 2021; Causse et al, Oncogene 2020; Uyanik et al, Nature Commun 2021).
Objectives. 1) To study the nuclear role of HSP110 in chaperoning DNA repair; 2) To decipher the role of extracellular HSP110 on immune suppressive cells; 3) To verify in colorectal cancer patients (PRODIGE cohorts. Synopsis available on FFCD), if circulating levels (free or within exosomes) of HSP110 can be measured, can predict HSP110 expression in the tumor and can be used as a marker of response to chemo- or immuno-therapy. 4) To carry out structural and chemical studies so at least one of our molecules, selected for their ability to specifically inhibit HSP110 (Gozzi et al, Cell Death & Diff 2020; Marcion et al, Int J Cancer 2021), could be used in patients (in combination with chemotherapies or immunotherapies). Detection of circulating levels of HSP110 may help to monitor the disease evolution and/or to select the patients that may benefit the most from our HSP110-targeted therapy.
Collaborations : J Goetz, (Inserm 1109, Strasbourg), C Coutant (CGFL, Dijon), F Aubin (CHU Besançon), F Ghiringhelli (CGFL), L Briand (INRA, Dijon), W Boireau (FEMTO, Besançon), J Matias (CHU San Carlos Madrid) and V Cottet (CIC 1432). Funding : INCa EVRALTIC (2020-2023); INCa PLBIO20-160 (2020-2023); INCa PLBIO21-107 (2021-2024) Prizes :Ruban Rose Avenir 2016, Estée Lauder 2016, Odyssea 2017, Fondation Silab Jean Paufique 2019, Grand Prix de Recherche Ruban Rose 2021 INCa
Rational. Cancer cells release exosomes with HSP70 in their membrane (HSP70-exosomes) whereas “normal” cells do not. We have proposed a methodology to capture exosomes from the blood released by cancer cells using an interference biolayer approach and a peptide aptamer called A8, as a ligand, (PCT/EP2015/063186). Using this patented technology to capture circulating exosomes in cancer patients (EXODIAG and EXOMEL cohorts), we have provided proof of principle that HSP70-exosomes (associated or not to PD-L1) can be measured from human fluids and notably for cancer detection in a metastatic set-up (gobbo et al, JNCI 2016; Chanteloup et al, JEV 2020; Cordonnier et al, JEV 2020). From a therapeutic point of view, we already demonstrated that HSP70-exosomes were able to activate myeloid-derived suppressive cells (MDSC) and have selected a peptide aptamer (A8) and a chemical molecule (AC1LINNC) which, by binding to this extracellular HSP70, blocked the ability of these tumor-derived exosomes to activate MDSC (Hadi et al, JCI Insight 2020).
Objectives. 1) to develop a platform for cancer follow-up from blood and urine samples enabling the enumeration of (i) HSP70-exosomes probed by high-affinity ligands (HSP70 peptide aptamer) and of (ii) identify other markers in these tumor-derived exosomes (lipids, mi-RNAs, proteins) that can be associated to HSP70. 2) In our preclinical models, we will test our inhibitors of HSP70 combined to other immunotherapeutic therapies: PD-L1/PD-1 antibodies. 3) HSP70-exosomes as diagnosis markers that are of huge interest in many diseases. With the French network of gynecology and obstetrics INSERM clinical research unit, we will measure HSP70-exosomes in the urine of pregnant women with preeclampsia (before and one year after delivery) to determine whether these stress-exosomes can be used as markers of the high-risk cardiovascular profile observed in preeclamptic women and often associated with cardiovascular adverse events.
Collaborations : The team also belong to the Reference Constitutive Center for Rare Pulmonary Disease, Univeristy Hospital Dijon). M Kolb (Hamilton, Canada), B Crestani (Inserm U700 Bichat, Paris), V Cottin (HCL- OrphaLung, Lyon) Andreas Guenther (Giessen, Allemagne) P Rocchi (Inserm 1068, Institute P Calmettes, Marseilles), P Barthélémy (INSERM U1212) ; M Königshoff (Pittsburgh, USA). Funding : European Contract, HEALTH-F2-eurIPFnet; ANR MESO –IPF (2012-2015), ANR SHOT-IPF (2016-2020); ANR-I-SITE-BFC (2019-2022); European Joint Programme on Rare Diseases; RARE-ILD; European FP7 eurILDFreg; ANR- AAPG-2021-Smart Progress (2021-2025).
Rational. Fibroproliferative diseases can affect all tissues and organ systems and are responsible for 45 % of deaths around the world. Among them, progressive pulmonary fibrosis with different disease including idiopathic pulmonary fibrosis (IPF) is a major clinical issue. Despite the apparent heterogeneity among progressive fibrosing diseases, a common denominator is the important role of the pro-fibrotic mediator Transforming Growth Factor (TGF)-β1. Except for pirfenidone and nintedanib that delay clinical worsening, no other pharmacologic treatment is currently available. It becomes urgent to propose new and efficient therapeutic targets/drugs for fibrosis. We have previously demonstrated that HSP27 (HSPB1) and alpha-B-crystallin (HSPB5) were overexpressed in IPF patients’ lungs and demonstrated in experimental models that they favored fibrosis by stabilizing key proteins of the TGF-β1 pro-fibrotic signaling pathway (Smad4, Snail) (Burgy et al, Science Transl Med 2016; Bonniaud et al, Eur Resp J 2018; Boutanquoi et al Eur Resp J 2020). Translational research is allowed by the links with the Reference Constitutive Center for Rare Pulmonary Disease, CHU- Dijon.
Objectives. 1) to study HSPB1/Snail and HSPB5/Smad4 associations to identify druggable domains. 2) To screen specific HSP inhibitors (chemical molecules, antisense oligonucleotides, ASO) able to block the TGF β fibrotic pathway. 3) To test them in our preclinical models, alone or in combination with pirfenidone and nintedanib. 4) To decipher the role of the extracellular HSPs in the early inflammatory phase that precedes the fibrotic process. 5) To explore the possibility of using circulating HSPs (free or within exosomes) as early markers of fibrosis and/or to predict response to therapies. 6) To Develop of companion biomarkers for anti-HSPB1, -HSPB5 using our ASO (aim 2) radiolabelled with 68-Gallium, for which the team has already an expertise (with the Inserm Transfert). It should be noted that our molecules aimed to interfere with the fibrotic process may represent pharmacological leads for treating not only pulmonary fibrosis but also renal fibrosis (with K El Karoui, Mondor Hospital, Paris) and myelofibrosis (see below).B Impact of HSP27 in myeloproliferative neoplasms and myelofibrosis
Collaborations : Olivier Hermine (Inserm 1163, Institut Imagine, Paris), I Plo (INSERM UMR U1170, IGR, Villejuif); Funding : ANR PRTS (2016-2021), Association “Laurette Fugain” and Labex GR-Ex.
Rational. We demonstrated that HSP27 was a new player in myeloproliferative neoplasms (MPN) through its effect facilitating the assembly JAK2/STAT5. Using an HSP27 specific ASO in 2 animal models of myelofibrosis, we observed a decrease in myelofibrosis progression, associated with a marked decrease in the platelet count as well as in megakaryocytes (Sevin et al, Nature Commun 2018.
Objectives. 1) to explore the role of HSP27 in normal megakaryopoiesis, in Polycythemia Vera (PV) and Essential Thrombocythemia (ET); 2) To evaluate the impact of our HSP27 inhibitors (ASO and chemical compounds) in vivo in two mouse models (JAK2V617F and CALRdel52); 3) To study the interaction and interplay between HSP27 and CALR; 4) To measure HSP27 levels in PV and ET human samples including serum, CD34+circulating hematopoietic progenitor cells and bone marrow biopsies; 5) To evaluate the prognostic impact of HSP27 in MPN patients (cohorts CHU Dijon and IGR, Vilejuif).
Collaborations : Paola Ballerini (AP-HP, Paris), Pierre-Simon Bellaye (CGFL, Dijon), Jean-Paul Pais de Barros (UB). Funding : Ligue régionale contre le Cancer, Société Française de Lutte contre les Cancers et Leucémies de l'Enfant et de l'Adolescent (SFCE), Association pour la Recherche sur le Cancer (ARC), Hôpital Universitaire de Dijon (AAP Collection 2020).
Rational. HSP90 and HSP70 inhibitors have not yet been explored in patients with ALL. We found that a) HSP90 and HSP70 were highly expressed by ALL cells (Mshaik R, et al. Blood Cancer J. 2021, Georgievski A, et al. Cell Death Dis. 2022). ALL cells produce Extracellular Vesicles (EVs) which express HSP70 and which have an impact on the microenvironment in bone marrow (Georgievski A, et al. Cell Death Dis. 2022) and in particular on normal hematopoiesis (Hermetet et al. Nature Communication, 2019).
Objectives. Through the use of xenotransplantation of human cells isolated from patients with ALL (PDX models), we are developing preclinical studies to ; 1) analyze whether the use of HSP90 and HSP70 inhibitors is effective ; 2) their molecular mechanism of action ; 3) the role of EVs produced by ALL cells in the development of cancer ; 4) the purification of these EVs in patients’ blood in order to develop a sensitive tool for the detection of therapeutic relapses (coll. Paola Ballerini, AP-HP).B Extracellular HSP90, a marker and a target in hypoxia-related lung fibrosis
Collaborations : A Cochet (PU-PH CGFL). F Denat, C Bernhard and M Moreau (ICMUB Dijon). Funding : ANR HYMAGE-IPF.
Rational. We demonstrated that, in fibrotic conditions, lung epithelial cells secreted HSP90 which interacted with the low-density lipoprotein Receptor-related Protein-1 (LRP1) on surrounding fibroblasts to promote profibrotic signaling pathways (Bellaye et al, Eur Resp J, 2018; Tanguy et al, Int J Mol Sci 2021; Bonniaud et al, Eur Resp J 2018).
Objectives. To develop a theranostic agent based on Fab fragments of an anti-HSP90 antibody (MC5) radiolabelled with 111-Indium. This antibody (4C5) has been shown to target exclusively extracellular HSP90. The anti-HSP90 Fab will be bioconjugated with a chelator able to “trap” the radionuclide for SPECT imaging. The antifibrotic effect of this anti-HSP90 Fab will also be tested in vitro and in vivo in our pulmonary fibrosis models.C GRP94: Role in macrophage polarization. Interest as a therapeutic target
Collaborations : B Collin (Pharm’Image, CGFL Dijon); M Narce (UMR INSERM/uB/AGROSUP 1231 Dijon); W Zhou (Ningbo University,China). Funding : Label LNCC; ANR-10-EQPX-05-01.
Rational. Glucose-regulated protein 94 (GRP94) is an endoplasmic reticulum (ER) HSP90 protein that is constitutively expressed in all cell types yet increases during ER stress. GRP94 plays a major dual role in immune responses. It controls T cell functions by chaperoning GARP (Glycoprotein A repetition predominant), the docking receptor of TGF-β in Tregs, contributes to the immunosuppressive phenotype of M2 macrophages (Chaumonnot et al, CDDis 2021) and chaperones complement C3 (Joly et al, Oncogene 2017; Seignez et al, JCI 2017; Chaumonnot et al, Cell Death Dis 2021). In cancer, GRP94 inhibition with the inhibitor PU-WS13 induces a decrease in M2-like macrophages together which is associated to a decrease in tumor growth and an increase in CD8+ cells (Bouchard et al, Cells 2021).
Objectives. 1-To validate GRP94 as a therapeutic target in cancer. We will: a) decipher the role of GRP94 in M2 macrophages-mediated immunosuppression especially regarding TGFb; b) develop with Pharm’Image tools to optimize the in vivo detection of GRP94 and GARP by pharmaco-imaging (e.g. Positron Emission Tomography coupled with Resonance Imaging – PET-MRI); c) screen and evaluate GRP94 inhibitors for their capacity to inhibit its chaperone activity and reduce the immunosuppressive function of Tregs and M2 macrophages; d) vectorize the final selected inhibitor(s) using lipid nanocarriers to optimize the formulation and allow a more specific targeting. 2- In a mice model of drug addiction (with W Zhou) to study the role of GRP94 in microglial cells.
Collaborations : C. Michiels, R. Maudinas (University Hospital of Dijon), T. Hadi (NYU Langone University), A. Schmitt (CGFL, Dijon), F. Neiers (CSGA, INRA, Dijon), S. Messaoudi (Laboratoire BioCIS UMR 8076, Paris), P. Couvreur (UMR CNRS 8612 Galien Institute), K. Pyrshev (University of Texas Health Science Center, Houston), Dr. JL Desseyn (Infinite – U1286 Inserm, Lille) .Funding : ANR JCJC, 2017-2021, AAP Plateforme 2021.
Rational. We have recently demonstrated that GroEL (prokaryotic HSP60) is released from cultures of Lactobacillus when grown in biofilms (to mimic the probiotic intestinal colonization) and induced, in monocytes, an anti-inflammatory response by blocking the release of TNF-alpha or IL-1-beta (Dias et al, J Gastroenterol 2021). We have also shown that the β3-adrenergic receptor is expressed and functional in human intestinal macrophages and Its stimulation leads to anti-inflammatory properties.
Objectives.a) to study the role of lactobacillus HSP60 (GroEL) and β3-AR on intestinal homeostasis, via the regulation of macrophages/lymphocytes using in vitro, ex vivo models (organotypic and 3D cultures performed with intestinal crypts isolated from colon resection of patients (Dr. B. Paquette, Digestive surgery, university hospital of Besançon) and in our mice models of acute or chronic inflammatory bowel diseases. Since our pharmacological agents (GroEL- and β3-agonists) are highly lipophilic, we intend to use our recently patented technology that uses lipoproteins as nanovectors (FR3051674-A1; WO2017207897-A1. Hadi et al, JCI Insight 2020).
Funding : Private company Hastim.
Rational. Autologous therapeutic vaccine in cancer will permit personalized and universal cancer treatment. To this goal, immunizing cancer patients with autologous tumor derived HSP-peptide complexes directly isolated from the tumor biopsy was a concept in personalized immunotherapy developed more than 20 years ago (Srivastava et al, Nat Immunol 2000). Although it showed some encouraging results in cancer patients, the results obtained were not conclusive. As we have learned the importance of the vectorization in vaccine strategies, this concept of personalized vaccination is being updated. Hastim, a pioneering French biotechnology company in research on immune stimulation vector has developed a support of hydroxyapatite that also allows an HSP-dependent concentration of tumor peptide antigens. These hydroxyapatite-based autologous therapeutic vaccines (APAVAC®) are already being used in clinical veterinary (dogs and horses with a B cell lymphoma) with outstanding results. With the final goal of testing this APAVAC vaccine in patients we need first to use preclinical cancer mice models to explore both its efficacy and mechanism of action.
Objectives. To characterize the immunotherapeutic effect of those hydroxyapatite-based vaccines using different rodent cancer experimental models including humanized murine model xenografted with patient-derived tumors. To identify universal biological markers to predict response to this personalized cancer treatment. To establish grounds to initiate a pilot phase 1 clinical trial study.
Rational. IAPs (inhibitors of apoptosis) constitute an evolutionary conserved family of proteins, discovered in baculovirus as potent inhibitors of apoptosis in infected insect cells (Gyrd-Hansen et al. Nat. Rev. 2010). Among IAPs, cIAP1 is an E3-ubiquitin ligase with oncogenic properties. Thanks to its E3-ubiquitine ligase activity, it has the capacity of modulate the stability, activity and the interaction network of its protein partners. It is thus able to control the duration and intensity of signaling pathways, allowing cells to adapt to changing environment and to cope with intracellular and extracellular stresses. We demonstrated that cIAP1 is expressed both in the nucleus and cytoplasm and we identified novel protein partners. We also demonstrated that alterations in cIAP1 expression in different tumor samples and demonstrated oncogenic properties in mice (Marivin et al. Oncogene 2014, Glorian et al. CDDi 2017, Allègre et al. PlosONE 2018, Tencer et al. Nat Struct Cell Biol in revision).
The Objective is to assess the oncogenic properties of cIAP1, determine its nuclear functions, analyze its role in regulating several signaling pathway (for example TGFβ signaling pathways), develop specific inhibitors and evaluate their efficacy in the treatment of tumor and fibrosis.