The Expression of Myeloproliferative Neoplasm-Associated Calreticulin Variants Depends on the Functionality of ER-Associated Degradation

BACKGROUND: Mutations in CALR observed in myeloproliferative neoplasms (MPN) were recently shown to be pathogenic via their interaction with MPL and the subsequent activation of the Janus Kinase - Signal Transducer and Activator of Transcription (JAK-STAT) pathway. However, little is known on the impact of those variant CALR proteins on endoplasmic reticulum (ER) homeostasis. METHODS: The impact of the expression of Wild Type (WT) or mutant CALR on ER homeostasis was assessed by quantifying the expression level of Unfolded Protein Response (UPR) target genes, splicing of X-box Binding Protein 1 (XBP1), and the expression level of endogenous lectins. Pharmacological and molecular (siRNA) screens were used to identify mechanisms involved in CALR mutant proteins degradation. Coimmunoprecipitations were performed to define more precisely actors involved in CALR proteins disposal. RESULTS: We showed that the expression of CALR mutants alters neither ER homeostasis nor the sensitivity of hematopoietic cells towards ER stress-induced apoptosis. In contrast, the expression of CALR variants is generally low because of a combination of secretion and protein degradation mechanisms mostly mediated through the ER-Associated Degradation (ERAD)-proteasome pathway. Moreover, we identified a specific ERAD network involved in the degradation of CALR variants. CONCLUSIONS: We propose that this ERAD network could be considered as a potential therapeutic target for selectively inhibiting CALR mutant-dependent proliferation associated with MPN, and therefore attenuate the associated pathogenic outcomes

STAT5-and hypoxia-dependent upregulation of AXL

Internal tandem duplication in Fms-like tyrosine kinase 3 (FLT3-ITD) is the most frequent mutation observed in acute myeloid leukemia (AML) and correlates with poor prognosis. FLT3 tyrosine kinase inhibitors are promising for targeted therapy. Here, we investigated mechanisms dampening the response to the FLT3 inhibitor quizartinib, which is specific to the hematopoietic niche. Using AML primary samples and cell lines, we demonstrate that convergent signals from the hematopoietic microenvironment drive FLT3-ITD cell resistance to quizartinib through the expression and activation of the tyrosine kinase receptor AXL. Indeed, cytokines sustained phosphorylation of the transcription factor STAT5 in quizartinib-treated cells, which enhanced AXL expression by direct binding of a conserved motif in its genomic sequence. Likewise, hypoxia, another well-known hematopoietic niche hallmark, also enhanced AXL expression. Finally, in a xenograft mouse model, inhibition of AXL significantly increased the response of FLT3-ITD cells to quizartinib exclusively within a bone marrow environment. These data highlight a new bypass mechanism specific to the hematopoietic niche that hampers the response to quizartinib through combined upregulation of AXL activity. Targeting this signaling offers the prospect of a new therapy to eradicate resistant FLT3-ITD leukemic cells hidden within their specific microenvironment, thereby preventing relapses from FLT3-ITD clones

LIF-Dependent Signaling: New Pieces in the Lego

LIF, a member of the IL6 family of cytokine, displays pleiotropic effects on various cell types and organs. Its critical role in stem cell models (e.g.: murine ES, human mesenchymal cells) and its essential non redundant function during the implantation process of embryos, in eutherian mammals, put this cytokine at the core of many studies aiming to understand its mechanisms of action, which could benefit to medical applications. In addition, its conservation upon evolution raised the challenging question concerning the function of LIF in species in which there is no implantation. We present the recent knowledge about the established and potential functions of LIF in different stem cell models, (embryonic, hematopoietic, mesenchymal, muscle, neural stem cells and iPSC). We will also discuss EVO-DEVO aspects of this multifaceted cytokine

PLoS Pathog

Cytomegalovirus (CMV) is a leading infectious cause of morbidity in immune-compromised patients. γδ T cells have been involved in the response to CMV but their role in protection has not been firmly established and their dependency on other lymphocytes has not been addressed. Using C57BL/6 αβ and/or γδ T cell-deficient mice, we here show that γδ T cells are as competent as αβ T cells to protect mice from CMV-induced death. γδ T cell-mediated protection involved control of viral load and prevented organ damage. γδ T cell recovery by bone marrow transplant or adoptive transfer experiments rescued CD3ε-/- mice from CMV-induced death confirming the protective antiviral role of γδ T cells. As observed in humans, different γδ T cell subsets were induced upon CMV challenge, which differentiated into effector memory cells. This response was observed in the liver and lungs and implicated both CD27+ and CD27- γδ T cells. NK cells were the largely preponderant producers of IFNγ and cytotoxic granules throughout the infection, suggesting that the protective role of γδ T cells did not principally rely on either of these two functions. Finally, γδ T cells were strikingly sufficient to fully protect Rag-/-γc-/- mice from death, demonstrating that they can act in the absence of B and NK cells. Altogether our results uncover an autonomous protective antiviral function of γδ T cells, and open new perspectives for the characterization of a non classical mode of action which should foster the design of new γδ T cell based therapies, especially useful in αβ T cell compromised patients

Rôle des basses concentrations en oxygène dans la préservation et la quiescence des cellules souches hématopoïétiques

L'hématopoièse siège dans la moelle osseuse et assure la production continue des cellules sanguines à partir des cellules souches hématopoiétiques (CSH). Les CSH sont caractérisées par leur capacité de greffe hématopoiétique à long terme, et leur maintien à vie dépend de 2 équilibres fonctionnels majeurs : autorenouvellement/différentiation et quiescence/prolifération. La différenciation hématopoiétique suit schématiquement le gradient en oxygène (O2) médullaire, les CSH se trouvant à proximité de l'endoste dans des zones peu oxygénées, et les progéniteurs engagés dans des zones médullaires juxta-vasculaires plus oxygénées (4-5 % d'O2). Ces concentrations d'O2 sont primordiales dans le contrôle et la préservation de l'homéostasie hématopoiétique. Nous avons montré que la culture à 0,9 % d'O2 maintient des progéniteurs primitifs et des CSH capables de reconstitution hématopoiétique, alors que la culture à 20 % d'O2 entraîne leur disparition. Cette disparition est indépendante d'une boucle de VEGF autocrine ou paracrine, et non modifiée par l'addition de VEGF165 exogène. Certains de nos clients inattendus suggèrent que des molécules mimétiques de "l'hypoxie", présentes dans certains milieux, permettent le maintien voire l'expansion des CSH en culture à 20 % d'O2. D'autre part, à très basses concentrations d'oxygène (0,1 % d'O2), les cellules hématopoiétiques primitives entrent en quiescence. Nous avons montré sur une lignée hématopoiétique primitive (FDCP-mix) que cette quiescence est associée à une hypophosphorylation de la protéine du rétinoblastome (pRb) et une augmentation de p27, sans induction d'apoptose.Haematopoiesis occurs in the bone marrow and allows the permanent and controlled production of peripheral blood cells from haematopoietic stem cell (HSC). HSC are characterized by their long term hematopoietic reconstitution capacity, and their maintenance depends on 2 major functional equilibriums : self-renewal vs differentiation, and quiescence vs proliferation. Haematopoietic differentiation follows the oxygen (O2) gradient in the bone marrow : most primitive cells reside in low oxygenated endosteal areas, while differentiated haematopoietic cells are located in more oxygenated (4-5 % O2) juxta-vascular areas. These oxygen tensions are central in control and preservation of hematopoietic homeostasis. We confirm that culture at 0,9 % O2 maintains primitive progenitors and haematopoietic stem cell capable of hematopoietic reconstitution, while culture at 20 % O2 makes them disappear. Hypoxia-related HSC preservation is independent of a paracrine or autocrine VEGF loop, and is not modified by VEGF165 addition in the culture, whatever the O2 concentration. Furthermore, some of our unexpected results suggest that some media contain hypoxia-mimicking molecules that could maintain and even expend HSC in 20 % O2 culture. In addition, very low oxygen tension (0,1 % O2) induces primitive haematopoietic cell quiescence. We showed that in an haematopoietic primitive cell line (FDCP-mix), this quiescence is associated with retinoblastoma (Rb) protein hypophosphorylation and increased p27 expression, without apoptosis induction.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Rôle des variations des concentrations d'O2 sur la prolifération, le cycle cellulaire et la fonctionnalité des cellules souches hématopoïétiques

L'hématopoièse assure la production des cellules sanguines à partir des cellules souches hématopoiétiques (CSH) identifiables par l'expression de l'antigène CD34. L'hématopoièse est régulée par divers facteurs dont la concentration d'O2 qui varie dans la moelle osseuse de 0 à 4 % (hypoxie) permet l'expansion des cellules CD34+ avec une meilleure amplification des CSH qu'à 20 % d'O2. L'hypoxie importante (0,1 %) induit le maintien et le retour en quiescence des CSH après division. Dans les deux cas, les CSH après culture sont capables de xénogreffe chez les souris immunodéficientes. Ce maintien en quiescence est également observé sur la lignée hématopoiétique murine non tumorale (FDCP-Mix) en hypoxie ou avec un mimétique chimique, le chlorure de cobalt (CoC1 2). A l'inverse, le CoC1 2 induit l'apoptose de la lignée tumorale HeLa. Enfin, nous avons étudié les capacités d'expansion des CSH CD34+ de sang périphérique dans un milieu utilisé en pratique clinique.Hematopoiesis allows the permanent blood cell production from hematopoietic stem cells (HSC), which can be identified by their cell surface expression of the CD34 antigen. Hematopoiesis is regulated by various factors including O2 concentration ranging from 0 to 4 % in the bone marrow (hypoxia). We report here that culture of CD34+ cells at 3 % O2 allows their expansion with a better amplification of HSC. Severe hypoxia (0,1 %) favors the maintenance and return to quiescence of HSC after division. In both cases, cultured HSC engraft immunodeficient mice. The maintenance in quiescence is observed in the normal primitive hematopoietic cell line FDCP-Mix when cultured in hypoxia as well as with cobalt chloride (CoC1 2), a hypoxic mimetic. On the contrary, CoC1 2 induces HeLa cell apoptosis. Actually, we investigate peripheral blood HSC expansion in a medium used in clinic.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Etude de la production de M-CSF par les cellules stromales médullaires et les lymphocytes T

LIMOGES-BU Médecine pharmacie (870852108) / SudocPARIS-BIUM (751062103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Effets des médiateurs lipidiques au cours de l'hematopoiése humaine

LIMOGES-BU Médecine pharmacie (870852108) / SudocPARIS-BIUM (751062103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Régulation de l'hématopoïèse par les basses concentrations d'oxygène (rôles de l'antigène CD34 et du facteur de croissance VEGF165)

L'hématopoïèse, processus de production des cellules sanguines à partir des cellules souches, est notamment régulée par les concentrations d'O2 médullaires comprises entre 0 à 5 % (hypoxie). Nous avons établi des liens entre des facteurs intrinsèques à la cellule impliqués dans le processus de différenciation (antigène CD34) et leur sensibilité à l'hypoxie, et étudié les effets sur l'hématopoïèse de facteurs inductibles par l'hypoxie (Vascular Endothelial Growth Factor, VEGF). La culture de cellules hématopoiétiques CD34+ à 1 % d'O2 augmente ou stabilise l'expression du gène cd34 qui diminue à 20 % d'O2. Le maintien prolongé de cette expression associé à une expression membranaire durable de la protéine sont corrélés avec le statut immature des cellules. D'autre part, le VEGF165 permet la survie de cellules souches murines en culture liquide à 1 % d'O2. Ainsi l'hypoxie freine la différenciation des cellules souches par l'expression du gène cd34 et favorise leur survie par le VEGF165.Hematopoiesis, the process of mature blood production from stem cells, is in part regulated by bone marrow oxygen concentrations, which vary from 0 to 5 % (hypoxia). We studied in this work the relationships between cell intrinsic factors involved in the maturation process (CD34 antigen) and their sensitivity to hypoxia, and the effects of molecules inducible by hypoxia (Vascular Endothelial Growth Factor, VEGF) on hematopoiesis. We showed that cultures of CD34+ cells at 1 % O2 induce or stabilize the cd34 gene expression that decreases at 20 % O2. The prolonged maintenance of this expression associated with the long-lasting membrane expression of the protein were correlated with the primitiveness of cells. We also showed that VEGF165 led to the survival of murine stem cells cultured at 1 % O2. This work suggests that hypoxia slows down the differentiation of stem cells by inducing cd34 gene expression, and favours their survival through VEGF165.BORDEAUX2-BU Santé (330632101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Les cellules CD34+ du sang périphérique en condition d'homéostasie (Elution à partir de filtres de leucoréduction)

L obtention d un nombre élevé de cellules souches hématopoïétiques (CSH) représente un enjeu majeur pour le développement de protocoles de thérapies cellulaires d hémopathies ou de tumeurs solides. L expansion ex vivo de ces cellules met en jeu différents acteurs (cytokiniques, environnementaux) et notamment les basses concentrations d oxygène (O2), qui reflètent des conditions physiologiques retrouvées au sein de structures spécifiques de la moelle osseuse où résident les CSH et auxquelles notre équipe s intéresse depuis plusieurs années. Les effets bénéfiques de ces basses concentrations d O2 sur le maintien des CSH sont actuellement bien établis lors de courtes cultures de cellules de moelle osseuse, de sang placentaire ou mobilisées dans le sang. Nous avons cherché à confirmer et à étendre ces résultats à des cellules peu étudiées, les cellules souches de sang périphérique en situation d homéostasie (CSSP-H). Ces cellules représentent en effet une source possible de CSH à usage thérapeutique, du fait de leur disponibilité et de leur facilité d accès. Nos travaux ont permis d établir et d optimiser un protocole, rapide et simple, de purification de cellules CD34+ à partir de filtres de leucoréduction (LRF). La quantité et la pureté de ces cellules adaptées à la poursuite de nos travaux, ainsi que leur validation fonctionnelle, nous ont permis de les utiliser comme modèle pour l étude des effets de cultures de 7 jours très faiblement oxygénées (0,1% d O2). La détermination de combinaisons cytokiniques assurant le maintien et l expansion des cellules primitives a révélé un rôle bénéfique de l IL-3 et du SCF couplé à la TPO. Ces conditions de culture ont permis de révéler, comparativement à des cultures réalisées à 20% d O2, le rôle majeur des faibles concentrations d O2 dans le maintien de cellules quiescentes, indifférenciées, ne se divisant pas ou très peu et capables de reconstituer une hématopoïèse, suite à leur injection dans des souris NOG. Les mécanismes moléculaires et métaboliques intervenant dans ces processus restent, cependant, encore à établir.Obtaining a high number of hematopoietic stem cells (HSCs) is a major challenge for developing cell therapies for blood diseases. Ex vivo expansion of HSCs involves various factors (cytokines, environment), including low oxygen (O2) concentrations, that reflect the physiological conditions found in specific structures of the bone marrow where HSCs reside. Our team is interested with the study of these low O2 levels for several years and their beneficial effects are currently well established during short-term cultures of cells from bone marrow, cord blood or mobilized in the blood. We sought to confirm and extend these results to poorly studied cells: stem cells from steady state peripheral blood (SSPB). Indeed, these cells represent a possible HSCs source devoted to the therapeutic use, because of their availability and their easy access. Our work has led to the establishment and the optimisation of a procedure, rapid and easy to set up, for CD34+ cells purification from leukoreduction filters (LRFs). The cell quantities and purities, adapted to our further work, together with their functional validation, led us to use these cells as a model for 7-days in vitro cultures performed at very low O2 concentration (0.1%). Cytokine combination assays, allowing the maintenance and the expansion of primitive cells, have revealed a beneficial influence of IL-3 or SCF + TPO additions. These cultures have revealed, comparatively to those performed at 20% O2, a major role of the very low O2 concentrations in the maintenance of quiescent and undifferentiated cells, showing an un- or low-cycling status and able to reconstitute hematopoiesis, consecutively to their injection into NOG mice. However, the molecular and metabolic mechanisms involved in these processes remain unknown.BORDEAUX2-Bib. électronique (335229905) / SudocSudocFranceF