Scientific background Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the first cellular immunotherapy developed to cure hematologic malignancies. It is based on the anti-tumor allo-immune response (graft versus tumor effect) induced by the donor immune system also transferred during the transplant process. Despite its efficiency, hematologic malignancies relapse accounts for half of deceases and to date, no biomarker allow to predict whose patient will relapse after allogeneic HSCT and to identify these patients early before relapse. Traditional statistical methods used for biomarker identifications are limited, mostly by their parametric nature, and could benefit from advanced machine learning and optimization techniques to select relevant variables and link them to the relapsing process. This unmet medical need is of critical importance to improve prognosis of patients who are currently treated for a hematologic cancer with allo-HSCT and to adapt their treatment before relapse. Hypothesis Here, we assume that integration of clinical data with immune and metabolic variables could provide metadata for a mathematical model to predict relapse occurrence. Aims To characterize circulating immune subsets and metabolome in the donor and to compare them at 3 months and one year after transplantation in patients with or without relapse To build a calibrated stochastic simulator for the relapsing process, accounting for post-transplant events and integrating clinical data with immune and metabolic variables. Methodology This project will rely on a multicentric cohort of 369 patients who received an alloHSCT. We will use mass cytometry and mass spectrometry to decipher circulating immune subsets and metabolites associated with relapse and other post-transplantation events. We will then create a simulator that model the dynamics of post-transplant events to identify relevant biomarkers using advanced optimization techniques and to generate a tool to predict relapse after alloHSCT. Validation in animal model will finally help to identify relevant new therapeutic targets. Expected results and impact This project will use data from an already constituted large cohort of patients to develop a machine learning tool for clinicians to estimate the probability of relapse based on various clinical and immune-metabolic data.

MITOMORT is a basic science project focusing on the role of an endogenous interferon induced gene (ISG) encoding a small protein that targets the mitochondria leading to cell death. The title is a play on mitochondrion and the French word for death, mort. APOBEC3A is an ISG gene encoding a cytidine deaminase that is able to edit C residues in chromosomal DNA. The attack rate can be so high that it causes extensive double stranded breaks and apoptosis. This is referred to as hypermutation. Lower levels of mutation, hypomutation, occur and are associated with oncogenesis. The Molecular Retrovirology Unit at the Pasteur Institute was the first to show that the APOBEC3A enzyme could attack chromosomal DNA. We noted that the initiation codons (AUG) of the two APOBEC3A isoforms used the “adequate” context according to the terminology of Marylin Kozak. Accordingly, we can expect that only 30-40% of ribosomes will settle on these sites, the remainder will continue to scan the mRNA. We asked the question, where will they settle? The next AUG downstream is in an “adequate” context while the following AUG is in a “strong” context. It turns out that initiation at these two sites produce two small proteins isoforms termes A3Ap3 and A3Ap4 (10.5 kDa and 8.6 kDa) that are in the same reading frame but overlapping that of APOBEC3A. They encode transmembrane spanning proteins that target the mitochondrion resulting in apoptosis. We apparently have a unique situation where two pro-apoptotic proteins, APOBEC3A targeting the archive, the genome, and A3Ap3/A3Ap4 targeting the powerhouse, the mitochondrion, are encoded by a single gene – to date called APOBEC3A. Research of A3Ap3/A3Ap4 apparently links apoptosis to the network of stress sensors that constitutes the interferon signalling pathway. It provides a link between the live cell and the death signal. Low levels of APOBEC3A will provide ongoing hypomutation and a weakening the mitochondrial network through sub-lethal doses of A3Ap3/A3Ap4. To compensate this the cell might slowly switch to ATP production via glycolysis as opposed to oxidative phosphorylation, or the Warburg effect. The project seeks to understand the mechanism and biology of this small endogenous pro-apoptotic protein - the singular is used because, so far, the two isoforms appear to have exactly the same function. The MITOMORT team combines the technology to resolve many facets associated with A3Ap3/A3Ap4, notably electron microscopy, confocal imagery and video. As intracellular obligate parasites have to protect them from premature apoptosis it is possible that nature has already developed an antagonist. To explore this hypothesis the consortium includes a laboratory with considerable experience in finding protein interactors, screening of a library of viral orfs as well microbial anti-apoptotic proteins. It is likely that A3Ap3/A3Ap4 is regulated leading to a fine balance between life and death. This would extend considerably the subject and provide new leads. The two Pasteur labs have collaborated in the past while the lab in Tours is already collaborating with the MRU on the mitochondrial localization of A3Ap3/A3Ap4. The lab at Maison-Alfort is an obvious collaborating lab and is well known to the MRU even though they have never collaborated directly before. While MITOMORT is a basic research project, as it concerns apoptosis mediated by an endogenous ISG, we feel that the findings will appeal to a wide audience of cell biologists. It provides a link between inflammation and cell death and so there is the possibility of it shedding some light on some autoimmune diseases like systemic lupus erythematosus. As to patents and the like, it is a little premature to make any predictions.

Histone variants act through the replacement of conventional histones by dedicated chaperones. They confer novel structural properties to nucleosomes and change the chromatin landscape. The functional and physiological requirement of the replacement of conventional histones by histone variants during organ formation and post-natal life remains poorly described. The incorporation of the histone variant H3.3 into chromatin is DNA-synthesis independent and relies on two different chaperone complexes, HIRA and DAXX/ATRX, which have different genomic deposition domains. While most epigenetic studies are performed in vitro, we intend to study them in an in vivo context where cell behavior can be properly addressed and where consequences for tissue formation, growth, homeostasis and repair can be fully investigated. Skeletal muscle provides the possibility to address yet poorly explored biochemical, cell biology, and developmental aspects of chromatin biology during development and postnatal life. Based on published and preliminary data from the three partners involved in this project, we hypothesize that: (i) HIRA and DAXX play a key role in muscle stem cells identity and muscle fibers organization (ii) H3.3 contributes to genome stability and prevents premature aging in adult muscle fibers (iii) a third H3.3 chaperone exists, which allows H3.3 incorporation into chromatin in the absence of HIRA and DAXX. Therefore, the main objectives of this proposal are defined in three work packages as follows: WP1: Conserved and divergent functions of H3.3 and DAXX-ATRX/HIRA pathways in muscle progenitors: we have recently shown that in the absence of HIRA, the muscle stem cell pool is lost during muscle regeneration. In addition, conditional HIRA inactivation in muscle progenitors during development have reduced myoblast numbers and smaller muscle size. In this context, our investigations will be extended to DAXX and H3.3. Our preliminary results indicate that DAXX is regulates myogenic gene expression via its histone chaperone activity. WP2: Role of H3.3 and DAXX-ATRX/HIRA pathways in adult myofibers structure and function: H2A.Z inactivation in adult muscle causes accelerated aging due to accumulation of DNA damage consecutive defective DNA repair by non-homologous end joining (NHEJ). H3.3 is also required for NHEJ. We therefore predict that H3.3 inactivation in muscle fibers will cause DNA damage and premature aging. Many evidences indicate that H3.3 regulates gene expression. We will determine if similarly to H2A.Z, H3.3 function in muscle fibers is restricted to DNA repair or if it also regulates gene expression. Finally, the roles of H3.3 chaperones have not yet been investigated in post-mitotic muscle fibers. To address these points H3.3, HIRA and DAXX will be inactivated in muscle fibers. We have recently shown that muscle fibers contain several myonuclear domains with specific identity and function defined by nuclei-specific expression profiles. The epigenetic landscape and myonuclei identity will be evaluated by single nuclei RNA seq and ATAC seq in the KO muscles. WP3: characterization of a new H3.3 deposition pathway that can bypass DAXX-ATRX/HIRA: H3.3 Chip-seq in Hira KO and Daxx KO myoblasts show HIRA and DAXX independent H3.3 deposition at specific loci, suggesting the presence of a third chaperone. Like other chaperones, this new chaperone should be part of a large multiprotein complex. We will isolate this complex from myoblasts and identify its composition. The complex will then be reconstituted with recombinant proteins to analyze its deposition properties. We will also invalidate the expression of some of the important components of the new deposition complex in vivo and we will determine the presumably perturbed H3.3 distribution pattern and the resulting cell phenotype at molecular level. Taken collectively, the expected data should shed in depth light on the intimate mechanism of H3.3 deposition and H3.3 function.

Bipolar disorder is a severe chronic psychiatric disorder affecting 1% of the population. Lithium is its gold standard treatment. Human MRI and preclinical studies suggest that it may increase neurogenesis, neuroprotection, myelination and modulate synaptic plasticity and neuroinflammation. However, many aspects of its mode of action remain unknown: which cellular effects are associated with the “MRI effects” of lithium in patients? Are its therapeutic cellular effects region specific or brain wide? We will conduct 2 parallel studies (in rats and humans) using similar (longitudinal) designs and methods ([11C]-UCB-J PET and MRI) plus histology and immunochemistry in the rats. We will assess synaptic plasticity, myelination, oligodendrocytes, neurogenesis and neuroinflammatory aspects associated with lithium in the same study. We will thus be able to draw inferences and inter species correspondences between PET/MR findings and immunohistological findings

Résumé: SARS-CoV-2 is a betacoronavirus that has recently emerged as a human pathogen in the city of Wuhan in China’s Hubei province. The disease caused by this newly identified virus has been named COVID-19 and symptoms include fever, severe respiratory illness and pneumonia. As of March 2020, the World Health Organization (WHO) has declared that SARS-CoV-2 is pandemic, and the number of confirmed cases is exponentially increasing. SARS-CoV-2 virus is closely related to SARS-CoV, which was responsible for the Severe Acute Respiratory Syndrome (SARS) in 2002. Similarly, to SARS-CoV, SARS-CoV-2 is of zoonotic origin and was demonstrated to cause life-threatening diseases in humans. So far, there is no vaccine or treatment for COVID-19. It is therefore critical to generate vaccines and drugs that will either prevent or treat COVID-19. At present, there are 8 candidate vaccines in clinical evaluation and more than 100 candidate vaccines in preclinical evaluation. Vaccine in clinical trials are represented by inactivated SARS-CoV-2 virus, non-replicating viral vectors, DNA and RNA vaccines (https://www.who.int). Surprisingly, no protein subunit vaccine are reported to be tested currently in clinical trial, as proposed in this project, which are generally safer and easier to produce. Moreover, all Human coronaviruses enter their host cells using the trimeric transmembrane spike (S) glycoprotein. The coronavirus’ S protein represents a major target for the human humoral immune response following SARS-CoV-2 infection. However, a large set of data from previous SARS-CoV-1 or CoV-2 infected individuals, or generated in preclinical models, pointed out the potential protective effect of cellular immunity. In this study, we propose to test the immunogenicity and the preventative effect of a combination of two vaccine platforms already in phase 1 to 3 clinical development; i.e the DNA-derived DREP platform and the anti-Dendritic cell (DC) targeting epitope-based vaccine. A series of DREP and DC-targeting constructs against SARS-CoV-2 are already available. A large set of data showed that these vaccines, either administered alone, or in a prime boost combination, elicited strong and durable T and B-cell immune responses against infectious agents. We develop here an original strategy aimed to induce a polyepitopic T and B cell responses. These vaccines are ready to be tested in two preclinical models, in humanized mice (mice reconstituted with a human immune system), allowing to study in depth human immune responses of different vaccine combinations, and in transgenic knock-in mice expressing the human CD40 and human ACE2, the receptor of SARS-CoV-2 to demonstrate the protective effect of these vaccines. The overreaching goal of this study is to identify within the 12-months time line of this project, vaccine (s) that will be moved forward to the clinical development.