Régulation et implication de la phosphatase CDC25A en aval de l'oncogène JAK2V617F dans les néoplasies myéloprolifératives

Les néoplasies myéloprolifératives (NMP) sont des pathologies malignes caractérisées par une expansion clonale excessive de cellules de la lignée myéloïde. La mutation JAK2V617F, qui entraîne une activation constitutive de la tyrosine kinase JAK2, est impliquée dans trois NMP, la maladie de Vaquez, la thrombocytémie essentielle et la myélofibrose primitive. Nous nous sommes ici intéressés à l'implication et à la régulation de la phosphatase CDC25A en aval de la mutation, une phosphatase impliquée dans la transition G1/S du cycle cellulaire. La première partie de nos travaux ont permis de mettre en évidence une surexpression de CDC25A en aval de JAK2V617F et notamment dans des précurseurs myéloïdes de patients atteints de NMP. Notre étude suggère que cette surexpression est la conséquence d'une dérégulation traductionnelle de CDC25A impliquant le facteur d'initiation de la traduction cap-dépendante eIF-2a et le facteur de transcription STAT5. De plus, l'inhibition de CDC25A diminue les capacités clonogènes de progéniteurs possédant JAK2V617F alors qu'elle a un effet moindre sur celle des progéniteurs de donneurs sains. La deuxième partie de nos travaux a mis en évidence une expression précoce de CDC25A au cours de différenciations érythroïdes et granulo-monocytaires de progéniteurs sains réalisées ex vivo, et son absence au cours des différenciations terminales. L'ensemble de nos résultats suggère l'implication d'une dérégulation traductionnelle de CDC25A dans l'expansion hématopoïétique observée chez les patients possédant la mutation JAK2V617F et ouvre la perspective d'une fenêtre thérapeutique quant à son inhibition.Myeloproliferative neoplasms (MPN) are hematological malignancies characterized by an excessive clonal amplification of myeloïd lineage. The JAK2V617F oncogene, which induces a constitutive activation of the tyrosine kinase JAK2, is involved in three MPN: polycythemia vera, essential thrombocythemia and primitive myelofibrosis. We kept interest on the G1/S transition CDC25A phosphatase for several reasons: (i) JAK2V617F favors this cell cycle transition; (ii) CDC25A is overexpressed in several cancers and downstream other tyrosine kinase oncogenes; (iii) the presence of CDC25A currently tested in clinical trials for other malignant pathologies. In our study, we demonstrate that the CDC25A phosphatase is constitutively over-expressed downstream of JAK2V617F mutant in cell lines, JAK2V617F-positive myeloid precursors of MPN patients and in bone marrow and spleen of JAK2V617F-positive knock-in mice. This over-expression appears to be the consequence of a translational deregulation of CDC25A involving the cap-dependant translation initiation factor eIF-2alpha and the STAT5 transcription factor. Furthermore, our results show that CDC25A inhibition reduces the clonogenic and proliferative potential of JAK2V617F-expressing erythroid progenitors, while moderately affecting normal counterpart. In the second part of our study, we show that CDC25A is early expressed in ex vivo erythroid and granulo-monocytic differentiations and absent in terminal differentiations. These results suggest that CDC25A deregulation may be involved in hematopoietic cells expansion in JAK2V617F patients, making this protein an attracting potential therapeutic target

Altered Ca2+ Homeostasis in Red Blood Cells of Polycythemia Vera Patients Following Disturbed Organelle Sorting during Terminal Erythropoiesis

The authors thank Thierry Peyrard, Dominique Gien, Sirandou Tounkara, and Eliane Véra at Centre National de Référence pour les Groupes Sanguins for the management of blood samples. The authors thank Sandrine Genetet and Isabelle Mouro-Chanteloup at the Inserm UMR_S1134 unit for their assistance in experiments. The authors also thank Michaël Dussiot at the Institute Imagine for his assistance in imaging flow cytometry. We thank Johanna Bruce and Virginie Salnot at 3P5 Proteomics Platform for sample preparation and analysis, and François Guillonneau and Patrick Mayeux for their management and strategies. Funding: The work was supported by Institut National de la Santé et de la Recherche Médicale (Inserm); Institut National de la Transfusion Sanguine (INTS); the University of Paris; and grants from Laboratory of Excellence (Labex) GR-Ex, reference No. ANR-11-LABX-0051. The Labex GR- Ex is funded by the IdEx program “Investissements d’avenir” of the French National Research Agency, reference No. ANR-11-IDEX-0005-02 and ANR-18-IDEX-0001. R.B., M.G.R., and D.M.A. were funded by the European Union’s Horizon 2020 Research and Innovation Program under grant agreement No. 675115-RELEVANCE-H2020-MSCA-ITN-2015. R.B. also received financial support from Société Française d’Hématologie (SFH) and Club du Globule Rouge et du Fer (CGRF). R.B. is currently funded by the Innovate UK Research and Innovation Knowledge Transfer Partnership (KTP) between University of Aberdeen and Vertebrate Antibodies Ltd. (Partnership No. KTP12327). T.D. was supported by PhD grants from Université Paris Saclay MESR (Ministère Enseignement Supérieur et de la Recherche) and then FRM (Fondation recherche médicale). The Orbitrap Fusion mass spectrometer was acquired with funds from Fonds Europeen de Developpement Regional (FEDER) through the Operational Program for Competitiveness Factors and Employment 2007-2013 and from the Canceropole Ile de France.Peer reviewedPublisher PD

The epigenetic regulator RINF (CXXC5) maintains SMAD7 expression in human immature erythroid cells and sustains red blood cells expansion

The gene CXXC5, encoding a Retinoid-Inducible Nuclear Factor (RINF), is located within a region at 5q31.2 commonly deleted in myelodysplastic syndrome (MDS) and adult acute myeloid leukemia (AML). RINF may act as an epigenetic regulator and has been proposed as a tumor suppressor in hematopoietic malignancies. However, functional studies in normal hematopoiesis are lacking, and its mechanism of action is unknow. Here, we evaluated the consequences of RINF silencing on cytokineinduced erythroid differentiation of human primary CD34+ progenitors. We found that RINF is expressed in immature erythroid cells and that RINF-knockdown accelerated erythropoietin-driven maturation, leading to a significant reduction (~45%) in the number of red blood cells (RBCs), without affecting cell viability. The phenotype induced by RINF-silencing was TGFβ-dependent and mediated by SMAD7, a TGFβ- signaling inhibitor. RINF upregulates SMAD7 expression by direct binding to its promoter and we found a close correlation between RINF and SMAD7 mRNA levels both in CD34+ cells isolated from bone marrow of healthy donors and MDS patients with del(5q). Importantly, RINF knockdown attenuated SMAD7 expression in primary cells and ectopic SMAD7 expression was sufficient to prevent the RINF knockdowndependent erythroid phenotype. Finally, RINF silencing affects 5’-hydroxymethylation of human erythroblasts, in agreement with its recently described role as a Tet2- anchoring platform in mouse. Altogether, our data bring insight into how the epigenetic factor RINF, as a transcriptional regulator of SMAD7, may fine-tune cell sensitivity to TGFβ superfamily cytokines and thus play an important role in both normal and pathological erythropoiesis

Absolute proteome quantification of highly purified populations of circulating reticulocytes and mature erythrocytes

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Proteomic analysis of neutrophils from COVID-19 patients

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Synergistic effect of sildenafil combined with controlled hypothermia to alleviate microglial activation after neonatal hypoxia–ischemia in rats

Abstract Background and purpose The only validated treatment to prevent brain damage associated with hypoxia–ischemia (HI) encephalopathy of the newborn is controlled hypothermia with limited benefits. Additional putative neuroprotective drug candidates include sildenafil citrate, a phosphodiesterase-type 5 inhibitor. The main objective of this preclinical study is to assess its ability to reduce HI-induced neuroinflammation, in particular through its potential effect on microglial activation. Methods HI was induced in P10 Sprague–Dawley rats by unilateral carotid permanent artery occlusion and hypoxia (HI) and treated by either hypothermia (HT) alone, Sildenafil (Sild) alone or combined treatment (SildHT). Lesion size and glial activation were analyzed by immunohistochemistry, qRT-PCR, and proteomic analyses performed at P13. Results None of the treatments was associated with a significant early reduction in lesion size 72h after HI, despite significant changes in tissue loss distribution. Significant reductions in both Iba1 + (within the ipsilateral hemisphere) and GFAP + cells (within the ipsilateral hippocampus) were observed in SildHT group, but not in the other treatment groups. In microglia-sorted cells, pro-inflammatory markers, i.e. Il1b, Il6, Nos2, and CD86 were significantly downregulated in SildHT treatment group only. These changes were restricted to the ipsilateral hemisphere, were not evidenced in sorted astrocytes, and were not sex dependent. Proteomic analyses in sorted microglia refined the pro-inflammatory effect of HI and confirmed a biologically relevant impact of SildHT on specific molecular pathways including genes related to neutrophilic functions. Conclusions Our findings suggest that Sildenafil combined with controlled hypothermia produces maximum effect in mitigating microglial activation induced by HI through complex proteomic regulation. The reduction of neuroinflammation induced by Sildenafil may represent an interesting therapeutic strategy for neonatal neuroprotection

The proteome of neutrophils in sickle cell disease reveals an unexpected activation of interferon alpha signaling pathway

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Proteomic Landscape of Neutrophils in Sickle Cell Anemia: An Unexpected Autoimmune Profile

Meeting: 60th Annual Meeting of the American-Society-of-Hematology (ASH)Location: San Diego, CADate: DEC 01-04, 2018Sponsor: Amer Soc HematolInternational audienceAbstract Although sickle cell disease (SCD) is a red cell disorder, many cell types, including endothelial cells and polymorphonuclear neutrophils (PMNs), contribute to its pathophysiology. In particular, activated PMNs have been implicated to play an important role in the initiation and propagation of vaso-occlusive events in SCD. Activated PMNs engage in a complex process of abnormal interactions with activated endothelial cells, platelets and circulating erythrocytes contributing to endothelial injury and decreased blood flow. In the present study, global proteomic analysis was performed using label-free mass spectrometry of PMNs from 4 SCD patients (SS) in steady state and from 4 control subjects (AA). We identified a total of 4,534 proteins both in AA and SS PMNs with 3,080 of these proteins identified in at least three samples for each condition. 50 proteins were significantly over-expressed in SS PMNs compared to AA PMNs (ratio > 1.4). STRING employed to monitor potential interaction between the overexpressed proteins showed that the main interactive clusters consist of STAT1 and STAT2, OAS 1, 2 and 3, and many Interferon Signaling Proteins i.e. IFIT1, IFIT2, IFIT 3, ISG15, ISG20, GBP2, IFI35, MX1 and MX2, TLR8 proteins (Fig. 1). This finding implies a strong activation of the type I interferon (IFN) signaling pathway in the SS PMNs (between 10 and 100-fold increase in SS vs AA). In addition, 33 proteins showed significantly lower expression in SS PMNs compared to AA PMNs. Among these were L-selectin (CD62L) and IL-17 receptor A (IL17RA) (p = 0.01). These findings are consistent with previously described phenotypes of aged neutrophils and acute inflammatory responses in SCD. Similar proteomic analysis performed on PMNs from SS patients treated with hydroxycarbamide (HC, n=4) showed that 14 proteins had significantly lower expression compared to untreated-SS patients (ratio <0.7). Interestingly, HC restored a normal expression pattern for most of the interferon signaling proteins. Type I IFNs represent the major effector cytokines of the host immune response against viruses and other intracellular pathogens. These cytokines are produced via activation of STAT1 and of pattern recognition receptors, including the Toll-like receptor signaling network. To determine if type I IFN-α could be detected at the protein level in the plasma of SS patients, we used the novel digital-ELISA technology (SIMOA, Quanterix) developed by Wilson et al (J Lab Autom, 2016). Interestingly, we found an increased level of INFα in plasma from SS patients compared to AA (n=32) (p<0.001) and it is noteworthy that while 50% of SS patients have similar level of INFα compare to AA individuals the other 50% exhibit 10 to 1,000-fold increased levels (Fig. 2). In summary, our novel proteomic analysis documents a high level expression of interferon signaling proteins, STAT1 and TLR8 in the proteome of neutrophils from SS patients and strongly suggests autoimmune or auto-inflammatory phenomena at basal state in SCD. Our results provide strong support for an important role for the innate immune system in the pathophysiology of SCD. Future studies will help determine the relationship between the plasmatic level of IFN-α and clinical complications and will establish if interferon signaling proteins and IFN-α could represent new therapeutic targets in SCD. Disclosures Hermand-Tournamille: Imara: Research Funding. Le Va Kim:Imara: Research Funding. Koehl:Imara: Research Funding

Red blood cell proteomics reveal remnant protein biosynthesis and folding pathways in PIEZO1-related hereditary xerocytosis

International audienceHereditary xerocytosis is a dominant red cell membrane disorder characterized by an increased leak of potassium from the inside to outside the red blood cell membrane, associated with loss of water leading to red cell dehydration and chronic hemolysis. 90% of cases are related to heterozygous gain of function mutations in PIEZO1, encoding a mechanotransductor that translates a mechanical stimulus into a biological signaling. Data are still required to understand better PIEZO1-HX pathophysiology. Recent studies identified proteomics as an accurate and high-input tool to study erythroid progenitors and circulating red cell physiology. Here, we isolated red blood cells from 5 controls and 5 HX patients carrying an identified and pathogenic PIEZO1 mutation and performed a comparative deep proteomic analysis. A total of 603 proteins were identified among which 56 were differentially expressed (40 over expressed and 16 under expressed) between controls and HX with a homogenous expression profile within each group. We observed relevant modifications in the protein expression profile related to PIEZO1 mutations, identifying two main “knots”. The first contained both proteins of the chaperonin containing TCP1 complex involved in the assembly of unfolded proteins, and proteins involved in translation. The second contained proteins involved in ubiquitination. Deregulation of proteins involved in protein biosynthesis was also observed in in vitro -produced reticulocytes after Yoda1 exposure. Thus, our work identifies significant changes in the protein content of PIEZO1-HX erythrocytes, revealing a “PIEZO1 signature” and identifying potentially targetable pathways in this disease characterized by a heterogeneous clinical expression and contra-indication of splenectomy