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

J Clin Invest

Arterial cardiovascular events are the leading cause of death in patients with JAK2V617F myeloproliferative neoplasms (MPN). However, their mechanisms are poorly understood. The high prevalence of myocardial infarction without significant coronary stenosis or atherosclerosis in patients with MPN suggests that vascular function is altered. Consequences of JAK2V617F mutation on vascular reactivity are unknown. We observe here increased responses to vasoconstrictors in arteries from Jak2V617F mice, resulting from disturbed endothelial nitric oxide pathway and increased endothelial oxidative stress. This response was reproduced in wild-type mice by circulating microvesicles isolated from patients carrying JAK2V617F and by erythrocyte-derived microvesicles from transgenic mice. Microvesicles of other cellular origins had no effect. This effect was observed ex vivo on isolated aortas, but also in vivo on femoral arteries. Proteomic analysis of microvesicles derived from JAK2V617F erythrocytes identified increased expression of myeloperoxidase as the likely mechanism accounting for microvesicles effect. Myeloperoxidase inhibition in microvesicles derived from JAK2V617F erythrocytes supressed their effect on oxidative stress. Antioxidants, such as simvastatin and N-acetyl-cysteine, improved arterial dysfunction in Jak2V617F mice. In conclusion, JAK2V617F MPN are characterized by exacerbated vasoconstrictor responses resulting from increased endothelial oxidative stress caused by circulating erythrocyte-derived microvesicles. Simvastatin appears as promising therapeutic strategy in this setting

Effects of eight neuropsychiatric copy number variants on human brain structure

Many copy number variants (CNVs) confer risk for the same range of neurodevelopmental symptoms and psychiatric conditions including autism and schizophrenia. Yet, to date neuroimaging studies have typically been carried out one mutation at a time, showing that CNVs have large effects on brain anatomy. Here, we aimed to characterize and quantify the distinct brain morphometry effects and latent dimensions across 8 neuropsychiatric CNVs. We analyzed T1-weighted MRI data from clinically and non-clinically ascertained CNV carriers (deletion/duplication) at the 1q21.1 (n = 39/28), 16p11.2 (n = 87/78), 22q11.2 (n = 75/30), and 15q11.2 (n = 72/76) loci as well as 1296 non-carriers (controls). Case-control contrasts of all examined genomic loci demonstrated effects on brain anatomy, with deletions and duplications showing mirror effects at the global and regional levels. Although CNVs mainly showed distinct brain patterns, principal component analysis (PCA) loaded subsets of CNVs on two latent brain dimensions, which explained 32 and 29% of the variance of the 8 Cohen’s d maps. The cingulate gyrus, insula, supplementary motor cortex, and cerebellum were identified by PCA and multi-view pattern learning as top regions contributing to latent dimension shared across subsets of CNVs. The large proportion of distinct CNV effects on brain morphology may explain the small neuroimaging effect sizes reported in polygenic psychiatric conditions. Nevertheless, latent gene brain morphology dimensions will help subgroup the rapidly expanding landscape of neuropsychiatric variants and dissect the heterogeneity of idiopathic conditions

Mécanisme d’action de l’interféron dans le traitement des néoplasies myéloprolifératives JAK2V617F

Interferon alpha is the only drug that treats patients with MPN in a durable way and gives 20% of Complete Molecular Remission (CMR). However, treatments are long (2 to 5 years) and failures are frequent. Moreover, the mechanism of action of IFNα is poorly understood which limits the improvement of treatment. Our hypothesis is the involvement of PML (promyelocytic leukemia), a tumor suppressor gene controlled at the transcriptional level by IFN. This "Interferon Stimulated Gene" (ISG) is the main organizer of the PML nuclear bodies (PML-NB) which recruit and modify partner proteins in a post-translational manner. In particular, they activate the P53 and Rb pathways with apoptosis, senescence and cell cycle arrest. The biogenesis of PML-NB is enhanced by the oxidative stress and action of arsenic trioxide (ATO). This was done with a mouse model mimicking human MPNs: Jak2V617F Knock-In (KI) mice. This preclinical study shows that ATO increases the efficacy of IFNα on the hematological and molecular response, the disappearance of disease-initiating cells (tested in secondary transplants) and the relapse rate of the disease after stopping the treatment. Treatment of mice transplanted with 50% Jak2V617F Pml GFP- 50% Jak2V617FPml ⁻⁄⁻ show that there is a selection of Pml ⁻⁄⁻ cells that are more resistant to the treatment and that PML is involved in these effects. Overall, these results highlight the involvement of PML and open the prospect of more efficient therapeutic trials in MPN by combining IFN and ATO in the treatment of patients.L’interféron alpha est le seul médicament qui traite de façon durable les patients atteints de NMP et donne 20% de rémissions moléculaires complètes (RMC). Cependant les traitements sont longs (2 à 5 ans) et les échecs fréquents. De plus, le mécanisme d'action de l'IFNα est mal connu, ce qui limite l'amélioration des traitements. Notre hypothèse de départ est l’implication de PML (promyelocytic leukemia), un gène suppresseur de tumeur contrôlé au niveau transcriptionnel par l’IFN. Ce gène, stimulé par l’interféron (ISG) est le principal organisateur des corps nucléaires PML (CN-PML) qui recrutent et modifient des protéines partenaires d'une façon post traductionnelle. Ils activent notamment les protéines p53 et Rb avec apoptose, sénescence et arrêt du cycle cellulaire. La biogenèse des CN-PML est augmentée par le stress oxydatif et l’action du trioxyde d’arsenic (ATO). Cela a été utilisé sur un modèle murin miman les NMP humaines : les souris Knock-In (KI) Jak2V617F. Cette étude préclinique montre que l’ATO augmente l'efficacité de l'IFNα sur la réponse hématologique et moléculaire, la disparition des cellules initiatrices de la maladie (testée en transplantations secondaires) et le taux de rechure de la maladie après l'arrêt du traitement. Le traitement des souris greffées 50% Jak2V617F Pml GFP-50% Jak2V617F Pml ⁻⁄⁻ montre qu'il y a une sélection des cellules Pml ⁻⁄⁻ qui sont résistantes au traitement. Cela montre que PML est impliquée dans ces effets. De plus, nous montrons qu'il s'agit d'une augmentation de sénescence, une des fonctions principales des CN-PML. Au total, ces résultats mettent en évidence l’implication de PML et ouvrent la perspective d’essais thérapeutiques plus performants dans les NMP

JAK2V617F myeloproliferative neoplasm eradication by a novel interferon/arsenic therapy involves PML

International audienceInterferon α (IFNα) is used to treat JAK2 V617F-driven myeloproliferative neoplasms (MPNs) but rarely clears the disease. We investigated the IFNα mechanism of action focusing on PML, an interferon target and key senescence gene whose targeting by arsenic trioxide (ATO) drives eradication of acute promyelocytic leukemia. ATO sharply potentiated IFNα-induced growth suppression of JAK2 V617F patient or mouse hematopoietic progenitors, which required PML and was associated with features of senescence. In a mouse MPN model, combining ATO with IFNα enhanced and accelerated responses, eradicating MPN in most mice by targeting disease-initiating cells. These results predict potent clinical efficacy of the IFNα+ATO combination in patients and identify PML as a major effector of therapy, even in malignancies with an intact PML gene.

SRSF2-P95H decreases JAK/STAT signaling in hematopoietic cells and delays myelofibrosis development in mice.

Heterozygous mutation targeting proline 95 in Serine/Arginine-rich Splicing Factor 2 (SRSF2) is associated with V617F mutation in Janus Activated Kinase 2 (JAK2) in some myeloproliferative neoplasms (MPNs), most commonly primary myelofibrosis. To explore the interaction of Srsf2 with Jak2, we generated Cre-inducible knock-in mice expressing these mutants under control of the stem cell leukemia (Scl) gene promoter. In transplantation experiments, Srsf2 unexpectedly delayed myelofibrosis induced by Jak2 and decreased TGFβ1 serum level. Srsf2 reduced the competitiveness of transplanted Jak2 hematopoietic stem cells while preventing their exhaustion. RNA sequencing of sorted megakaryocytes identified an increased number of splicing events when the two mutations were combined. Focusing on JAK/STAT pathway, Jak2 exon 14 skipping was promoted by Srsf2, an event detected in patients with JAK2 and SRSF2 co-mutation. The skipping event generates a truncated inactive JAK2 protein. Accordingly, Srsf2 delays myelofibrosis induced by the thrombopoietin receptor agonist Romiplostim in Jak2 wild-type animals. These results unveil JAK2 exon 14 skipping promotion as a strategy to reduce JAK/STAT signaling in pathological conditions