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International audience; In low-risk myelodysplastic syndrome (LR-MDS), erythropoietin (EPO) is widely used for the treatment of chronic anemia. However, initial response to EPO has time-limited effects. Luspatercept reduces red blood cell transfusion dependence in LR-MDS patients. Here, we investigated the molecular action of luspatercept (RAP-536) in an in vitro model of erythroid differentiation of MDS, and also in a in vivo PDX murine model with primary samples of MDS patients carrying or not SF3B1 mutation. In our in vitro model, RAP-536 promotes erythroid proliferation by increasing the number of cycling cells without any impact on apoptosis rates. RAP-536 promoted late erythroid precursor maturation while decreasing intracellular reactive oxygen species level. RNA sequencing of erythroid progenitors obtained under RAP-536 treatment showed an enrichment of genes implicated in positive regulation of response to oxidative stress and erythroid differentiation. In our PDX model, RAP-536 induces a higher hemoglobin level. RAP-536 did not modify variant allele frequencies in vitro and did not have any effect against leukemic burden in our PDX model. These results suggest that RAP-536 promotes in vivo and in vitro erythroid cell differentiation by decreasing ROS level without any remarkable impact on iron homeostasis and on mutated allele burden.

International audience; Physiopathology of myelodysplastic syndrome (MDS) remains poorly understood and the role of the microenvironment is increasingly highlighted. Recent studies in mouse models demonstrate that abnormalities of mesenchymal stromal cells (MSC) contribute to the physiopathology of MDS. In particular, genetic deletion of dicer1, a gene encoding a type III RNase essential for the genesis of miRNA, in murine MSC-derived osteoprogenitors led to a pathological microenvironment generating myelodysplastic features in hematopoietic progenitors and ultimately leading to acute myeloid leukemia [1]. In human, there is an increased susceptibility to senescence of the MDS mesenchymal stem cells and defects in the support properties of the growth of hematopoietic stem cells (HSC) [2]. These observations establish a causal relationship between deregulation of the hematopoietic niche and MDS pathogenesis. However, so far only few studies have addressed the mechanisms by microenvironmental MSC and HSC exchange signals that may interfere with miRNA processing, specifically in the human MDS microenvironment.

Chronic myelomonocytic leukemia (CMML) is a myeloid hematological malignancy with overlapping features of myelodysplastic syndromes (MDSs) and myeloproliferative neoplasms (MPNs). The knowledge of the role of the tumor microenvironment (TME), particularly mesenchymal stromal cells (MSCs), in MDS pathogenesis is increasing. Generally, cancer is associated with a procoagulant state participating in tumor development. Monocytes release procoagulant, tissue factor (TF)–bearing microparticles. We hypothesized that MSCs and clonal monocytes release procoagulant extracellular vesicles (EVs) within the CMML TME, inducing a procoagulant state that could modify hematopoietic stem cell (HSC) homeostasis. We isolated and cultured MSCs and monocytes from CMML patients and MSCs from healthy donors (HDs). Their medium EVs and small EVs (sEVs) were collected after iterative ultracentrifugations and characterized by nanoparticle tracking analysis. Their impact on hemostasis was studied with a thrombin generation assay and fibrinography. CMML or HD HSCs were exposed to sEVs from either CMML or HD MSCs. CMML MSC sEVs increased HD HSC procoagulant activity, suggesting a transfer of TF from the CMML TME to HD HSCs. The presence of TF on sEVs was shown by electron microscopy and western blot. Moreover, CMML monocyte EVs conferred a procoagulant activity to HD MSCs, which was reversed by an anti-TF antibody, suggesting the presence of TF on the EVs. Our findings revealed a procoagulant “climate” within the CMML environment related to TF-bearing sEVs secreted by CMML MSCs and monocytes.

Introduction: Anemia is the most frequent cytopenia in myelodysplastic syndromes (MDS) and patients will require red blood cell transfusion. Most low-risk MDS patients with anemia are currently treated with erythropoietin stimulating agents (ESA). This treatment gives 45-60% of erythroid response but responders will lose their erythroid response after a median time of 20-24 months (Park, Blood, 2008). Recently, luspatercept (RAP-536), a trap ligand of transforming growth factor-β superfamily, including activins and growth differentiation factors (GDFs), has shown positive effects on erythroid response in MDS patients especially in SF3B1 mutated patients (Platzbecker, Lancet Oncology, 2017). The objective of our work was to evaluate the effect of RAP-536 on human engraftment in PDX mice models of MDS. Methods: We performed patient derived xenograft (PDX) NOD SCID gamma (NSG) murine model by intrabone injections of mesenchymal stromal cells (MSC) and CD34+ hematopoietic stem progenitor cells (HSPC) from 3 MDS patients with SF3B1 mutations and 3 MDS patients without SF3B1 mutations but carrying other recurrent mutations (ASXL1, TET2, SRSF2, CBL, RUNX1, STAG2) seen in MDS (Table A). Three months after xenograft, we have realized a bone marrow aspiration for monitoring human engraftment using flow cytometry to check that there was a human engraftment. Then, the mice were treated for 2 months with 30mg/kg/week of RAP-536. At the end of the procedure, serum, blood and bone marrow were collected. Results: As GDF11 is a target of RAP-536, we have measured GDF11 level in serum in order to confirm the expected effects of RAP-536. We observed decreased levels of GDF11 in RAP-536 treated mice versus mock treated (p Conclusion: RAP-536 improves hemoglobin and WBC levels and seems to enhance the engraftment of human myeloid hematopoiesis in this PDX murine model of MDS. Molecular analyses are ongoing to clarify the relative proportions of normal and pathological human hematopoiesis before and after RAP-536. Figure Disclosures Park: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: RAP-536

Contexte Les Concentres de Globules Rouges (CGR) transfuses contiennent tres majoritairement des globules rouges, mais aussi des proteines et surtout des vesicules extracellulaires (EV). La liberation d’EV est un mecanisme de communication intercellulaire interessant car les EV vehiculent divers signaux : acides nucleiques, microARN, molecules membranaires, phosphatidylserines, facteur tissulaire, recepteurs/ligands, enzymes, facteurs de transcription, etc. qui peuvent influencer les cellules cibles. Bien que depourvu de systeme endosomal, les globules rouges en fin de vie liberent des EV. Les patients atteints de syndromes myelodysplasiques souffrent majoritairement d’anemie et recevront au cours de leur maladie des transfusions de culots globulaires. Dans ce travail, nous analysons ex vivo les interactions des EV de CGR sur l’hematopoiese des SMD dans son environnement medullaire. Resultats et conclusion Nous avons purifie les EV de CGR par ultracentrifugations differentielles et les avons caracterisees par Nanoparticle Tracking Analysis (Malvern NanoSight). Les EV sont plus nombreuses dans les poches pres de la date de peremption et sont plus petites. En microscopie confocale, nous avons mis en evidence que les EV marquees par un fluorochrome lipophile penetrent dans les CSH et les cellules souches mesenchymateuses (MSC) obtenues de moelle osseuse de patients atteints de SMD. Nous analysons maintenant le profil des microARN contenus dans ces EV et l’impact potentiel des EV sur l’hematopoiese en contexte pathologique SMD dans des modeles de coculture a long terme.