Publications by authors named "William Vainchenker"

228 Publications

Role of Rho-GTPases in megakaryopoiesis.

Small GTPases 2021 Feb 11:1-17. Epub 2021 Feb 11.

INSERM, UMR 1287, Gustave Roussy, Equipe Labellisée LNCC , Villejuif, France.

Megakaryocytes (MKs) are the bone marrow (BM) cells that generate blood platelets by a process that requires: i) polyploidization responsible for the increased MK size and ii) cytoplasmic organization leading to extension of long pseudopods, called proplatelets, through the endothelial barrier to allow platelet release into blood. Low level of localized RHOA activation prevents actomyosin accumulation at the cleavage furrow and participates in MK polyploidization. In the platelet production, RHOA and CDC42 play opposite, but complementary roles. RHOA inhibits both proplatelet formation and MK exit from BM, whereas CDC42 drives the development of the demarcation membranes and MK migration in BM. Moreover, the or MK specific knock-out in mice and the genetic alterations in their down-stream effectors in human induce a thrombocytopenia demonstrating their key roles in platelet production. A better knowledge of Rho-GTPase signalling is thus necessary to develop therapies for diseases associated with platelet production defects. AKT: Protein Kinase BARHGEF2: Rho/Rac Guanine Nucleotide Exchange Factor 2ARP2/3: Actin related protein 2/3BM: Bone marrowCDC42: Cell division control protein 42 homologCFU-MK: Colony-forming-unit megakaryocyteCIP4: Cdc42-interacting protein 4mDIA: DiaphanousDIAPH1; Protein diaphanous homolog 1ECT2: Epithelial Cell Transforming Sequence 2FLNA: Filamin AGAP: GTPase-activating proteins or GTPase-accelerating proteinsGDI: GDP Dissociation InhibitorGEF: Guanine nucleotide exchange factorHDAC: Histone deacetylaseLIMK: LIM KinaseMAL: Megakaryoblastic leukaemiaMARCKS: Myristoylated alanine-rich C-kinase substrateMKL: Megakaryoblastic leukaemiaMLC: Myosin light chainMRTF: Myocardin Related Transcription FactorOTT: One-Twenty Two ProteinPACSIN2: Protein Kinase C And Casein Kinase Substrate In Neurons 2PAK: P21-Activated KinasePDK: Pyruvate Dehydrogenase kinasePI3K: Phosphoinositide 3-kinasePKC: Protein kinase CPTPRJ: Protein tyrosine phosphatase receptor type JRAC: Ras-related C3 botulinum toxin substrate 1RBM15: RNA Binding Motif Protein 15RHO: Ras homologousROCK: Rho-associated protein kinaseSCAR: Suppressor of cAMP receptorSRF: Serum response factorSRC: SarcTAZ: Transcriptional coactivator with PDZ motifTUBB1: Tubulin β1VEGF: Vascular endothelial growth factorWAS: Wiskott Aldrich syndromeWASP: Wiskott Aldrich syndrome proteinWAVE: WASP-family verprolin-homologous proteinWIP: WASP-interacting proteinYAP: Yes-associated protein.
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http://dx.doi.org/10.1080/21541248.2021.1885134DOI Listing
February 2021

IFN: Jekyll and Hyde.

Blood 2021 Jan;137(3):291-293

INSERM; Paris-Saclay University; Gustave Roussy.

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http://dx.doi.org/10.1182/blood.2020008560DOI Listing
January 2021

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

J Exp Med 2021 Feb;218(2)

INSERM U944, Centre National de la Recherche Scientifique (CNRS) UMR7212, IRSL, Hôpital Saint-Louis, Paris, France.

Interferon α (IFNα) is used to treat JAK2V617F-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 JAK2V617F 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.
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http://dx.doi.org/10.1084/jem.20201268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7579737PMC
February 2021

Regulation of Platelet Production and Life Span: Role of Bcl-xL and Potential Implications for Human Platelet Diseases.

Int J Mol Sci 2020 Oct 14;21(20). Epub 2020 Oct 14.

University of Bordeaux, INSERM U1034, Biology of Cardiovascular Diseases, 33600 Pessac, France.

Blood platelets have important roles in haemostasis, where they quickly stop bleeding in response to vascular damage. They have also recognised functions in thrombosis, immunity, antimicrobal defense, cancer growth and metastasis, tumour angiogenesis, lymphangiogenesis, inflammatory diseases, wound healing, liver regeneration and neurodegeneration. Their brief life span in circulation is strictly controlled by intrinsic apoptosis, where the prosurvival Bcl-2 family protein, Bcl-xL, has a major role. Blood platelets are produced by large polyploid precursor cells, megakaryocytes, residing mainly in the bone marrow. Together with Mcl-1, Bcl-xL regulates megakaryocyte survival. This review describes megakaryocyte maturation and survival, platelet production, platelet life span and diseases of abnormal platelet number with a focus on the role of Bcl-xL during these processes.
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http://dx.doi.org/10.3390/ijms21207591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589436PMC
October 2020

Calreticulin del52 and ins5 knock-in mice recapitulate different myeloproliferative phenotypes observed in patients with MPN.

Nat Commun 2020 09 28;11(1):4886. Epub 2020 Sep 28.

INSERM, UMR 1287, Gustave Roussy, Villejuif, France.

Somatic mutations in the calreticulin (CALR) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). CALR mutations, including the two most frequent 52 bp deletion (del52) and 5 bp insertion (ins5), induce a frameshift to the same alternative reading frame generating new C-terminal tails. In patients, del52 and ins5 induce two phenotypically distinct myeloproliferative neoplasms (MPNs). They are equally found in ET, but del52 is more frequent in PMF. We generated heterozygous and homozygous conditional inducible knock-in (KI) mice expressing a chimeric murine CALR del52 or ins5 with the human mutated C-terminal tail to investigate their pathogenic effects on hematopoiesis. Del52 induces greater phenotypic changes than ins5 including thrombocytosis, leukocytosis, splenomegaly, bone marrow hypocellularity, megakaryocytic lineage amplification, expansion and competitive advantage of the hematopoietic stem cell compartment. Homozygosity amplifies these features, suggesting a distinct contribution of homozygous clones to human MPNs. Moreover, homozygous del52 KI mice display features of a penetrant myelofibrosis-like disorder with extramedullary hematopoiesis linked to splenomegaly, megakaryocyte hyperplasia and the presence of reticulin fibers. Overall, modeling del52 and ins5 mutations in mice successfully recapitulates the differences in phenotypes observed in patients.
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http://dx.doi.org/10.1038/s41467-020-18691-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522233PMC
September 2020

Megakaryocytes tame erythropoiesis with TGFβ1.

Blood 2020 08;136(9):1016-1017

INSERM, Unité 1287; Université Paris-Saclay; Laboratoire d'Excellence GR-Ex.

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http://dx.doi.org/10.1182/blood.2020006906DOI Listing
August 2020

A p53-JAK-STAT connection involved in myeloproliferative neoplasm pathogenesis and progression to secondary acute myeloid leukemia.

Blood Rev 2020 07 3;42:100712. Epub 2020 Jun 3.

Ludwig Institute for Cancer Research Brussels, Brussels, Belgium; Université catholique de Louvain and de Duve Institute, Brussels, Belgium; WELBIO (Walloon Excellence in Life Sciences and Biotechnology), Brussels, Belgium. Electronic address:

Since the discovery of JAK2 V617F as a highly prevalent somatic acquired mutation in the majority of myeloproliferative neoplasms (MPNs), it has become clear that these diseases are driven by pathologic activation of JAK2 and eventually of STAT5 and other members of the STAT family. The concept was strengthened by the discovery of the other activating driver mutations in MPL (thrombopoietin receptor, TpoR) and in calreticulin gene, which all lead to persistent activation of wild type JAK2. Although with a rare frequency, MPNs can evolve to secondary acute myeloid leukemia (sAML), a condition that is resistant to treatment. Here we focus on the role of p53 in this transition. In sAML mutations in TP53 or amplification in genes coding for negative regulators of p53 are much more frequent than in de novo AML. We review studies that explore a signaling and biochemical interaction between activated STATs and p53 in MPNs and other cancers. With the development of advanced sequencing efforts, strong evidence has been presented for dominant negative effects of mutated p53 in leukemia. In other studies, gain of function effects have been described that might be cell type specific. A more profound understanding of the potential interaction between p53 and activated STATs is necessary in order to take full advantage of novel p53-targeted therapies.
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http://dx.doi.org/10.1016/j.blre.2020.100712DOI Listing
July 2020

Different impact of calreticulin mutations on human hematopoiesis in myeloproliferative neoplasms.

Oncogene 2020 07 22;39(31):5323-5337. Epub 2020 Jun 22.

INSERM, UMR1287, Villejuif, France.

Mutations of calreticulin (CALRm) define a subtype of myeloproliferative neoplasms (MPN). We studied the biological and genetic features of CALR-mutated essential thrombocythemia and myelofibrosis patients. In most cases, CALRm were found in granulocytes, monocytes, B and NK cells, but also in T cells. However, the type 1 CALRm spreads more easily than the type 2 CALRm in lymphoid cells. The CALRm were also associated with an early clonal dominance at the level of hematopoietic stem and progenitor cells (HSPC) with no significant increase during granulo/monocytic differentiation in most cases. Moreover, we found that half of type 2 CALRm patients harbors some homozygous progenitors. Those patients were associated with a higher clonal dominance during granulo/monocytic differentiation than patients with only heterozygous type 2 CALRm progenitors. When associated mutations were present, CALRm were the first genetic event suggesting that they are both the initiating and phenotypic event. In blood, type 1 CALRm led to a greater increased number of all types of progenitors compared with the type 2 CALRm. However, both types of CALRm induced an increase in megakaryocytic progenitors associated with a ruxolitinib-sensitive independent growth and with a mild constitutive signaling in megakaryocytes. At the transcriptional level, type 1 CALRm seems to deregulate more pathways than the type 2 CALRm in megakaryocytes. Altogether, our results show that CALRm modify both the HSPC and megakaryocyte biology with a stronger effect for type 1 than for type 2 CALRm.
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http://dx.doi.org/10.1038/s41388-020-1368-3DOI Listing
July 2020

Germline genetic factors in the pathogenesis of myeloproliferative neoplasms.

Blood Rev 2020 07 29;42:100710. Epub 2020 May 29.

INSERM, UMR1287, Laboratory of Excellence GR-Ex, Villejuif, France; Gustave Roussy, Villejuif, France; Université Paris XI, UMR1287, Gustave Roussy, Villejuif, France. Electronic address:

Myeloproliferative neoplasms (MPN) are clonal hematological malignancies that lead to overproduction of mature myeloid cells. They are due to acquired mutations in genes encoding for AK2, MPL and CALR that result in the activation of the cytokine receptor/JAK2 signaling pathway. In addition, it exists germline variants that can favor the initiation of the disease or may affect its phenotype. First, they can be common risk alleles, which correspond to frequent single nucleotide variants present in control population and that contribute to the development of either sporadic or familial MPN. Second, some variants predispose to the onset of MPN with a higher penetrance and lead to familial clustering of MPN. Finally, some extremely rare genetic variants can induce MPN-like hereditary disease. We will review these different subtypes of germline genetic variants and discuss how they impact the initiation and/or development of the MPN disease.
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http://dx.doi.org/10.1016/j.blre.2020.100710DOI Listing
July 2020

A new efficient tool for non-invasive diagnosis of fetomaternal platelet antigen incompatibility.

Br J Haematol 2020 09 7;190(5):787-798. Epub 2020 Apr 7.

Institut National de la Transfusion Sanguine (INTS), Département d'Immunologie Plaquettaire, Paris, France.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is the consequence of platelet destruction by maternal alloantibodies against fetal human platelet antigens (HPA). This may result in intracranial haemorrhages (ICH) or even fetal death. Currently, fetal HPA genotyping is performed using invasive procedures. Here, we carried out a proof-of-concept study for non-invasive prenatal diagnosis of fetal platelet genotyping in four HPA systems (HPA-1, -3, -5 and-15) by droplet digital polymerase chain reaction (ddPCR) using cell-free DNA extracts from the plasma of 47 pregnant women with suspected, or history of, FNAIT. Results showed that 74% (35/47) of pregnant women presented incompatibility in at least one HPA system, and 38% (18/47) of cases presented HPA-1 incompatibility, including nine women with multiple incompatibilities. ICH occurred in one case of profound fetal thrombocytopenia with HPA-15 incompatibility, confirming the need for non-invasive prenatal genotyping in systems other than HPA-1. Fetal HPA genotypes predicted by ddPCR were confirmed in all FNAIT cases after amniocentesis or delivery. Fetal HPA genotyping on maternal plasma based on ddPCR is a fast, safe and reliable non-invasive method. This technique will be useful for the early identification of pregnancies at high risk of FNAIT requiring antenatal management to minimize the risk of fetal/neonatal haemorrhage.
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http://dx.doi.org/10.1111/bjh.16593DOI Listing
September 2020

TET2 haploinsufficiency alters reprogramming into induced pluripotent stem cells.

Stem Cell Res 2020 04 6;44:101755. Epub 2020 Mar 6.

INSERM, UMR 1170, Laboratory of Excellence GR-Ex, Villejuif, France; Université Paris XI, UMR 1170, Gustave Roussy, Villejuif, France; UMR U1170,Gustave Roussy, 114 rue Edouard Vaillant, Villejuif 94805, France; Laboratory of Excellence GR-Ex, Villejuif, France. Electronic address:

The discovery of the Ten-Eleven Translocation (TET) protein family was initiated by the identification of the MLL partner TET1, and of mutations in the TET2 gene in hematological malignancies including myeloproliferative neoplasms (MPN). TET1, 2 and 3 proteins hydroxylate 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) and further oxidize 5-hmC into 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Previous studies highlight the involvement of TET proteins in somatic cells reprogramming into induced pluripotent stem cells (iPSC), particularly Tet1 and 2 in mouse and TET1 in human. Here, we asked whether endogenous TET2 knockdown also displays this function. Using different shRNA against TET2, we provide evidence that TET2 strongly decreases the reprogramming of human hematopoietic progenitor cells into iPSC. Importantly, using 2 MPN patients, we observed that TET2 mutations affecting catalytic domain allowed iPSC generation. Instead, using another TET2 and TET3-mutated patient, we could only reprogram IPSC with TET3 mutation alone, suggesting that the type of TET2 mutation and/or the cooperation with TET3 mutations may alter the reprogramming activity. Altogether, this work highlights the importance of endogenous TET in the reprogramming process of human hematopoietic progenitors.
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http://dx.doi.org/10.1016/j.scr.2020.101755DOI Listing
April 2020

Immunosuppression by Mutated Calreticulin Released from Malignant Cells.

Mol Cell 2020 02 27;77(4):748-760.e9. Epub 2019 Nov 27.

Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France; Equipe 11 labellisée Ligue contre le Cancer, Centre de Recherche des Cordeliers, INSERM UMR 1138, Paris, France; Sorbonne Université, Paris, France; Université of Paris, Paris, France; Suzhou Institute for Systems Medicine, Chinese Academy of Sciences, Suzhou, China; Karolinska Institutet, Department of Women's and Children's Health, Stockholm, Sweden; Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France. Electronic address:

Mutations affecting exon 9 of the CALR gene lead to the generation of a C-terminally modified calreticulin (CALR) protein that lacks the KDEL endoplasmic reticulum (ER) retention signal and consequently mislocalizes outside of the ER where it activates the thrombopoietin receptor in a cell-autonomous fashion, thus driving myeloproliferative diseases. Here, we used the retention using selective hooks (RUSH) assay to monitor the trafficking of CALR. We found that exon-9-mutated CALR was released from cells in response to the biotin-mediated detachment from its ER-localized hook, in vitro and in vivo. Cellular CALR release was confirmed in suitable mouse models bearing exon-9-mutated hematopoietic systems or tumors. Extracellular CALR mediated immunomodulatory effects and inhibited the phagocytosis of dying cancer cells by dendritic cells (DC), thereby suppressing antineoplastic immune responses elicited by chemotherapeutic agents or by PD-1 blockade. Altogether, our results demonstrate paracrine immunosuppressive effects for exon-9-mutated CALR.
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http://dx.doi.org/10.1016/j.molcel.2019.11.004DOI Listing
February 2020

Description of a knock-in mouse model of JAK2V617F MPN emerging from a minority of mutated hematopoietic stem cells.

Blood 2019 12;134(26):2383-2387

Biology of Cardiovascular Diseases, University of Bordeaux, INSERM, UMR1034, Pessac, France.

The major weakness of most knock-in JAK2V617F mouse models is the presence of the JAK2 mutation in all rather than in a few hematopoietic stem cells (HSC), such as in human "early-stage" myeloproliferative neoplasms (MPN). Understanding the mechanisms of disease initiation is critical as underscored by the incidence of clonal hematopoiesis of indeterminate potential associated with JAK2V617F. Currently, such studies require competitive transplantation. Here, we report a mouse model obtained by crossing JAK2V617F/WT knock-in mice with PF4iCre transgenic mice. As expected, PF4iCre;JAK2V617F/WT mice developed an early thrombocytosis resulting from the expression of JAK2V617F in the megakaryocytes. However, these mice then developed a polycythemia vera-like phenotype at 10 weeks of age. Using mT/mG reporter mice, we demonstrated that Cre recombination was present in all hematopoietic compartments, including in a low number of HSC. The frequency of mutated cells increased along hematopoietic differentiation mimicking the clonal expansion observed in essential thrombocythemia and polycythemia vera patients. This model thus mimics the HSC compartment observed in early-stage MPN, with a small number of JAK2V617F HSC competing with a majority of JAK2WT HSC. PF4iCre;JAK2V617F/WT mice are a promising tool to investigate the mechanisms that regulate clonal dominance and progression to myelofibrosis.
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http://dx.doi.org/10.1182/blood.2019001163DOI Listing
December 2019

Ontogenic Changes in Hematopoietic Hierarchy Determine Pediatric Specificity and Disease Phenotype in Fusion Oncogene-Driven Myeloid Leukemia.

Cancer Discov 2019 12 29;9(12):1736-1753. Epub 2019 Oct 29.

INSERM U1170, Gustave Roussy, Villejuif, France.

Fusion oncogenes are prevalent in several pediatric cancers, yet little is known about the specific associations between age and phenotype. We observed that fusion oncogenes, such as , are associated with acute megakaryoblastic or other myeloid leukemia subtypes in an age-dependent manner. Analysis of a novel inducible transgenic mouse model showed that expression in fetal hematopoietic stem cells induced rapid megakaryoblastic leukemia whereas expression in adult bone marrow hematopoietic stem cells resulted in a shift toward myeloid transformation with a strikingly delayed leukemogenic potential. Chromatin accessibility and single-cell transcriptome analyses indicate ontogeny-dependent intrinsic and -induced differences in the activities of key transcription factors, including ERG, SPI1, GATA1, and CEBPA. Importantly, switching off the fusion oncogene restored terminal differentiation of the leukemic blasts. Together, these data show that aggressiveness and phenotypes in pediatric acute myeloid leukemia result from an ontogeny-related differential susceptibility to transformation by fusion oncogenes. SIGNIFICANCE: This work demonstrates that the clinical phenotype of pediatric acute myeloid leukemia is determined by ontogeny-dependent susceptibility for transformation by oncogenic fusion genes. The phenotype is maintained by potentially reversible alteration of key transcription factors, indicating that targeting of the fusions may overcome the differentiation blockage and revert the leukemic state...
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http://dx.doi.org/10.1158/2159-8290.CD-18-1463DOI Listing
December 2019

Megakaryocyte polyploidization: role in platelet production.

Platelets 2020 Aug 22;31(6):707-716. Epub 2019 Sep 22.

UMR 1170, Institut National de la Santé et de la Recherche Médicale, Univ. Paris-Sud, Université Paris-Saclay, Gustave Roussy Cancer Campus, Equipe Labellisée Ligue Nationale Contre le Cancer , Villejuif, France.

Mammal megakaryocytes (MK) undergo polyploidization during their differentiation. This process leads to a marked increase in the MK size and of their cytoplasm. Contrary to division by classical mitosis, ploidization allows an economical manner to produce platelets as they arise from the fragmentation of the MK cytoplasm. The platelet production correlates to the entire MK cytoplasm mass that depends both upon the number of MKs and their size. Polyploidization occurs by several rounds of DNA replication with at the end of each round an aborted mitosis at late phase of cytokinesis. As there is also a defect in karyokinesis, MKs are giant cells with a single polylobulated nucleus with a 2N ploidy. However, polyploidization does not increase platelet production because it requires a parallel development of MK organelles such as mitochondria, granules and the demarcation membrane system. MK polyploidization is regulated by extrinsic factors, more particularly by thrombopoietin (TPO), which during a platelet stress increases first polyploidization before enhancing the MK number and by transcription factors such as RUNX1, GATA1, and FLI1 that regulate MK differentiation explaining why polyploidization and cytoplasmic maturation are intermingled. MK polyploidization is ontogenically regulated and is markedly altered in malignant myeloid disorders such as acute megakaryoblastic leukemia and myeloproliferative disorders as well as in hereditary thrombocytopenia, more particularly those involving transcription factors or signaling pathways. In addition, MKs arising from progenitors have a much lower ploidy than leading to a low yield of platelet production . Thus, it is tempting to find approaches to increase MK polyploidization . However, these approaches require molecules that are able to simultaneously increase MK polyploidization and to induce terminal differentiation. Here, we will focus on the regulation by extrinsic and intrinsic factors of MK polyploidization during development and pathological conditions.
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http://dx.doi.org/10.1080/09537104.2019.1667497DOI Listing
August 2020

MCM8- and MCM9 Deficiencies Cause Lifelong Increased Hematopoietic DNA Damage Driving p53-Dependent Myeloid Tumors.

Cell Rep 2019 Sep;28(11):2851-2865.e4

Institute of Human Genetics, CNRS, DNA Replication and Genome Dynamics, 141, Rue de la Cardonille, 34396 Montpellier, France; Institute of Human Genetics, UMR 9002, CNRS-University of Montpellier, 141, Rue de la Cardonille, 34396 Montpellier, France. Electronic address:

Hematopoiesis is particularly sensitive to DNA damage. Myeloid tumor incidence increases in patients with DNA repair defects and after chemotherapy. It is not known why hematopoietic cells are highly vulnerable to DNA damage. Addressing this question is complicated by the paucity of mouse models of hematopoietic malignancies due to defective DNA repair. We show that DNA repair-deficient Mcm8- and Mcm9-knockout mice develop myeloid tumors, phenocopying prevalent myelodysplastic syndromes. We demonstrate that these tumors are preceded by a lifelong DNA damage burden in bone marrow and that they acquire proliferative capacity by suppressing signaling of the tumor suppressor and cell cycle controller RB, as often seen in patients. Finally, we found that absence of MCM9 and the tumor suppressor Tp53 switches tumorigenesis to lymphoid tumors without precedent myeloid malignancy. Our results demonstrate that MCM8/9 deficiency drives myeloid tumor development and establishes a DNA damage burdened mouse model for hematopoietic malignancies.
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http://dx.doi.org/10.1016/j.celrep.2019.07.095DOI Listing
September 2019

Knock-in of murine Calr del52 induces essential thrombocythemia with slow-rising dominance in mice and reveals key role of Calr exon 9 in cardiac development.

Leukemia 2020 02 30;34(2):510-521. Epub 2019 Aug 30.

Ludwig Institute for Cancer Research, Brussels, Belgium.

Frameshifting mutations (-1/+2) of the calreticulin (CALR) gene are responsible for the development of essential thrombocythemia (ET) and primary myelofibrosis (PMF). The mutant CALR proteins activate the thrombopoietin receptor (TpoR) inducing cytokine-independent megakaryocyte progenitor proliferation. Here, we generated via CRISPR/Cas9 technology two knock-in mouse models that are heterozygous for a type-I murine Calr mutation. These mice exhibit an ET phenotype with elevated circulating platelets compared with wild-type controls, consistent with our previous results showing that murine CALR mutants activate TpoR. We also show that the mutant CALR proteins can be detected in plasma. The phenotype of Calr del52 is transplantable, and the Calr mutated hematopoietic cells have a slow-rising advantage over wild-type hematopoiesis. Importantly, a homozygous state of a type-1 Calr mutation is lethal at a late embryonic development stage, showing narrowed ventricular myocardium walls, similar to the murine Calr knockout phenotype, pointing to the C terminus of CALR as crucial for heart development.
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http://dx.doi.org/10.1038/s41375-019-0538-1DOI Listing
February 2020

Remodeling of Bone Marrow Hematopoietic Stem Cell Niches Promotes Myeloid Cell Expansion during Premature or Physiological Aging.

Cell Stem Cell 2019 09 11;25(3):407-418.e6. Epub 2019 Jul 11.

Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute and Department of Haematology, University of Cambridge, Cambridge CB2 0PT, UK; National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK; Centro Nacional de Investigaciones Cardiovasculares (CNIC), 28029 Madrid, Spain. Electronic address:

Hematopoietic stem cells (HSCs) residing in the bone marrow (BM) accumulate during aging but are functionally impaired. However, the role of HSC-intrinsic and -extrinsic aging mechanisms remains debated. Megakaryocytes promote quiescence of neighboring HSCs. Nonetheless, whether megakaryocyte-HSC interactions change during pathological/natural aging is unclear. Premature aging in Hutchinson-Gilford progeria syndrome recapitulates physiological aging features, but whether these arise from altered stem or niche cells is unknown. Here, we show that the BM microenvironment promotes myelopoiesis in premature/physiological aging. During physiological aging, HSC-supporting niches decrease near bone but expand further from bone. Increased BM noradrenergic innervation promotes β-adrenergic-receptor(AR)-interleukin-6-dependent megakaryopoiesis. Reduced β-AR-Nos1 activity correlates with decreased endosteal niches and megakaryocyte apposition to sinusoids. However, chronic treatment of progeroid mice with β-AR agonist decreases premature myeloid and HSC expansion and restores the proximal association of HSCs to megakaryocytes. Therefore, normal/premature aging of BM niches promotes myeloid expansion and can be improved by targeting the microenvironment.
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http://dx.doi.org/10.1016/j.stem.2019.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739444PMC
September 2019

The role of the thrombopoietin receptor MPL in myeloproliferative neoplasms: recent findings and potential therapeutic applications.

Expert Rev Hematol 2019 06 22;12(6):437-448. Epub 2019 May 22.

c Ludwig Institute for Cancer Research Brussels , Brussels , Belgium.

: Classical Myeloproliferative Neoplasms (MPNs) include three disorders: Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). MPNs are associated with constitutive activation of JAK2 leading to persistent cell signaling downstream of the dimeric myeloid cytokine receptors due to mutations in three genes encoding JAK2, calreticulin (CALR) and the thrombopoietin (TPO) receptor (MPL or TPOR). CALR and MPL mutants induce JAK2 activation that depends on MPL expression, thus explaining why they induce megakaryocyte pathologies including ET and PMF, but not PV. In contrast, JAK2 V617F drives all three diseases as it induces persistent signaling EPOR, G-CSFR (CSF3R) and MPL. : Here, we review how different pathogenic mutations of are translated into active receptors by inducing stable dimerization. We focus on the unique role of MPL on the hematopoietic stem cell (HSC), explaining why MPL is indispensable for the development of all MPNs. Last but not least, we describe how CALR mutants are pathogenic binding and activation of MPL. : Altogether, we believe that MPL is an important, but challenging, therapeutic target in MPNs that requires novel strategies to interrupt the specific conformational changes induced by each mutation or pathologic interaction without compromising the key functions of wild type MPL.
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http://dx.doi.org/10.1080/17474086.2019.1617129DOI Listing
June 2019

Multilayer intraclonal heterogeneity in chronic myelomonocytic leukemia.

Haematologica 2020 01 2;105(1):112-123. Epub 2019 May 2.

INSERM U1170, Gustave Roussy Cancer Center, Villejuif, France

The functional diversity of cells that compose myeloid malignancies, i.e., the respective roles of genetic and epigenetic heterogeneity in this diversity, remains poorly understood. This question is addressed in chronic myelomonocytic leukemia, a myeloid neoplasm in which clinical diversity contrasts with limited genetic heterogeneity. To generate induced pluripotent stem cell clones, we reprogrammed CD34 cells collected from a patient with a chronic myelomonocytic leukemia in which whole exome sequencing of peripheral blood monocyte DNA had identified 12 gene mutations, including a mutation in and two heterozygous mutations in in the founding clone and a secondary (G12D) mutation. CD34 cells from an age-matched healthy donor were also reprogrammed. We captured a part of the genetic heterogeneity observed in the patient, i.e. we analyzed five clones with two genetic backgrounds, without and with the (G12D) mutation. Hematopoietic differentiation of these clones recapitulated the main features of the patient's disease, including overproduction of granulomonocytes and dysmegakaryopoiesis. These analyses also disclosed significant discrepancies in the behavior of hematopoietic cells derived from induced pluripotent stem cell clones with similar genetic background, correlating with limited epigenetic changes. These analyses suggest that, beyond the coding mutations, several levels of intraclonal heterogeneity may participate in the yet unexplained clinical heterogeneity of the disease.
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http://dx.doi.org/10.3324/haematol.2018.208488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939510PMC
January 2020

Calreticulin mutants as oncogenic rogue chaperones for TpoR and traffic-defective pathogenic TpoR mutants.

Blood 2019 06 22;133(25):2669-2681. Epub 2019 Mar 22.

Ludwig Institute for Cancer Research Brussels, Brussels, Belgium.

Calreticulin (CALR) +1 frameshift mutations in exon 9 are prevalent in myeloproliferative neoplasms. Mutant CALRs possess a new C-terminal sequence rich in positively charged amino acids, leading to activation of the thrombopoietin receptor (TpoR/MPL). We show that the new sequence endows the mutant CALR with rogue chaperone activity, stabilizing a dimeric state and transporting TpoR and mutants thereof to the cell surface in states that would not pass quality control; this function is absolutely required for oncogenic transformation. Mutant CALRs determine traffic via the secretory pathway of partially immature TpoR, as they protect N117-linked glycans from further processing in the Golgi apparatus. A number of engineered or disease-associated TpoRs such as TpoR/MPL R102P, which causes congenital thrombocytopenia, are rescued for traffic and function by mutant CALRs, which can also overcome endoplasmic reticulum retention signals on TpoR. In addition to requiring -glycosylation of TpoR, mutant CALRs require a hydrophobic patch located in the extracellular domain of TpoR to induce TpoR thermal stability and initial intracellular activation, whereas full activation requires cell surface localization of TpoR. Thus, mutant CALRs are rogue chaperones for TpoR and traffic-defective TpoR mutants, a function required for the oncogenic effects.
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http://dx.doi.org/10.1182/blood-2018-09-874578DOI Listing
June 2019

Rare type 1-like and type 2-like calreticulin mutants induce similar myeloproliferative neoplasms as prevalent type 1 and 2 mutants in mice.

Oncogene 2019 03 19;38(10):1651-1660. Epub 2018 Oct 19.

INSERM, UMR 1170, Gustave Roussy, Villejuif, France.

Frameshift mutations in the calreticulin (CALR) gene are present in 30% of essential thrombocythemia and myelofibrosis patients. The two most frequent mutations are CALR del52 (type 1, approximately 60%) and CALR ins5 (type 2, around 30%), but many other rarer mutations exist accounting each for less than 2% of all CALR mutations. Most of them are structurally classified as type 1-like and type 2-like CALR mutations according to the absence or presence of a residual wild-type calcium-binding motif and the modification of the alpha-helix structure. Yet, several key questions remain unanswered, especially the reason of such low frequencies of these other mutations. In an attempt to investigate specific pathogenic differences between type 1-like and type 2-like CALR mutations and del52 and ins5, we modeled two type 1-like (del34 and del46) and one type 2-like (del19) mutations in cell lines and in mice. All CALR mutants constitutively activate JAK2 and STAT5/3/1 in a similar way in the presence of the thrombopoietin receptor (MPL) and induced cytokine-independent cell growth but to a lesser extent with rare mutants over time. This correlates with reduced expression levels of rare CALR mutants compared to del52 and ins5. Lethally irradiated mice that were engrafted with bone marrow transduced with the different CALR mutations developed thrombocytosis, but to a much lesser extent with ins5 and the type 2-like CALR mutation. In contrast to type 2-like mice, type 1-like mice developed marked myelofibrosis and splenomegaly 10 months after engraftment. Similar to del52, type 1-like CALR mutations induced an expansion at an early stage of hematopoiesis compared to ins5 and type 2-like mutation. Thus, type 1-like and type 2-like CALR mutants structurally and functionally resemble del52 and ins5 mutants, respectively.
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http://dx.doi.org/10.1038/s41388-018-0538-zDOI Listing
March 2019

Disrupted filamin A/αβ interaction induces macrothrombocytopenia by increasing RhoA activity.

Blood 2019 04 2;133(16):1778-1788. Epub 2019 Jan 2.

Unité Mixte de Recherche (UMR) 1170, INSERM, Equipe Labelllisée Ligue Nationale Contre le Cancer, Gustave Roussy Cancer Campus, Université Paris-Sud, Université Paris-Saclay, Villejuif, France.

Filamin A (FLNa) links the cell membrane with the cytoskeleton and is central in several cellular processes. Heterozygous mutations in the X-linked gene are associated with a large spectrum of conditions, including macrothrombocytopenia, called filaminopathies. Using an isogenic pluripotent stem cell model derived from patients, we show that the absence of the FLNa protein in megakaryocytes (MKs) leads to their incomplete maturation, particularly the inability to produce proplatelets. Reduction in proplatelet formation potential is associated with a defect in actomyosin contractility, which results from inappropriate RhoA activation. This dysregulated RhoA activation was observed when MKs were plated on fibrinogen but not on other matrices (fibronectin, vitronectin, collagen 1, and von Willebrand factor), strongly suggesting a role for FLNa/αβ interaction in the downregulation of RhoA activity. This was confirmed by experiments based on the overexpression of FLNa mutants deleted in the αβ-binding domain and the RhoA-interacting domain, respectively. Finally, pharmacological inhibition of the RhoA-associated kinase ROCK1/2 restored a normal phenotype and proplatelet formation. Overall, this work suggests a new etiology for macrothrombocytopenia, in which increased RhoA activity is associated with disrupted FLNa/αβ interaction.
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http://dx.doi.org/10.1182/blood-2018-07-861427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484462PMC
April 2019

Mutations in the gene cause severe congenital neutropenia as well as Shwachman-Diamond-like syndrome.

Blood 2018 09 18;132(12):1318-1331. Epub 2018 Jun 18.

French Registry of Chronic Neutropenia, Trousseau Hospital, Paris, France.

Congenital neutropenias (CNs) are rare heterogeneous genetic disorders, with about 25% of patients without known genetic defects. Using whole-exome sequencing, we identified a heterozygous mutation in the gene, encoding the signal recognition particle (SRP) 54 GTPase protein, in 3 sporadic cases and 1 autosomal dominant family. We subsequently sequenced the gene in 66 probands from the French CN registry. In total, we identified 23 mutated cases (16 sporadic, 7 familial) with 7 distinct germ line mutations including a recurrent in-frame deletion (Thr117del) in 14 cases. In nearly all patients, neutropenia was chronic and profound with promyelocytic maturation arrest, occurring within the first months of life, and required long-term granulocyte colony-stimulating factor therapy with a poor response. Neutropenia was sometimes associated with a severe neurodevelopmental delay (n = 5) and/or an exocrine pancreatic insufficiency requiring enzyme supplementation (n = 3). The SRP54 protein is a key component of the ribonucleoprotein complex that mediates the co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum (ER). We showed that SRP54 was specifically upregulated during the in vitro granulocytic differentiation, and that mutations or knockdown led to a drastically reduced proliferation of granulocytic cells associated with an enhanced P53-dependent apoptosis. Bone marrow examination of -mutated patients revealed a major dysgranulopoiesis and features of cellular ER stress and autophagy that were confirmed using -mutated primary cells and knockdown cells. In conclusion, we characterized a pathological pathway, which represents the second most common cause of CN with maturation arrest in the French CN registry.
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http://dx.doi.org/10.1182/blood-2017-12-820308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6536700PMC
September 2018

JAK inhibitors for the treatment of myeloproliferative neoplasms and other disorders.

F1000Res 2018 17;7:82. Epub 2018 Jan 17.

Signal Transduction & Molecular Hematology Unit, Ludwig Institute for Cancer Research, Brussels, Belgium.

JAK inhibitors have been developed following the discovery of the V617F in 2005 as the driver mutation of the majority of non- myeloproliferative neoplasms (MPNs). Subsequently, the search for JAK2 inhibitors continued with the discovery that the other driver mutations ( and ) also exhibited persistent JAK2 activation. Several type I ATP-competitive JAK inhibitors with different specificities were assessed in clinical trials and exhibited minimal hematologic toxicity. Interestingly, these JAK inhibitors display potent anti-inflammatory activity. Thus, JAK inhibitors targeting preferentially JAK1 and JAK3 have been developed to treat inflammation, autoimmune diseases, and graft-versus-host disease. Ten years after the beginning of clinical trials, only two drugs have been approved by the US Food and Drug Administration: one JAK2/JAK1 inhibitor (ruxolitinib) in intermediate-2 and high-risk myelofibrosis and hydroxyurea-resistant or -intolerant polycythemia vera and one JAK1/JAK3 inhibitor (tofacitinib) in methotrexate-resistant rheumatoid arthritis. The non-approved compounds exhibited many off-target effects leading to neurological and gastrointestinal toxicities, as seen in clinical trials for MPNs. Ruxolitinib is a well-tolerated drug with mostly anti-inflammatory properties. Despite a weak effect on the cause of the disease itself in MPNs, it improves the clinical state of patients and increases survival in myelofibrosis. This limited effect is related to the fact that ruxolitinib, like the other type I JAK2 inhibitors, inhibits equally mutated and wild-type JAK2 (JAK2WT) and also the JAK2 oncogenic activation. Thus, other approaches need to be developed and could be based on either (1) the development of new inhibitors specifically targeting V617F or (2) the combination of the actual JAK2 inhibitors with other therapies, in particular with molecules targeting pathways downstream of JAK2 activation or the stability of JAK2 molecule. In contrast, the strong anti-inflammatory effects of the JAK inhibitors appear as a very promising therapeutic approach for many inflammatory and auto-immune diseases.
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http://dx.doi.org/10.12688/f1000research.13167.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773931PMC
January 2018

The severe phenotype of Diamond-Blackfan anemia is modulated by heat shock protein 70.

Blood Adv 2017 10 10;1(22):1959-1976. Epub 2017 Oct 10.

Université Paris 7 Denis Diderot-Sorbonne Paris Cité, Paris, France.

Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome that exhibits an erythroid-specific phenotype. In at least 70% of cases, DBA is related to a haploinsufficient germ line mutation in a ribosomal protein (RP) gene. Additional cases have been associated with mutations in GATA1. We have previously established that the RPL11 phenotype is more severe than RPS19 phenotype because of delayed erythroid differentiation and increased apoptosis of RPL11 erythroid progenitors. The HSP70 protein is known to protect GATA1, the major erythroid transcription factor, from caspase-3 mediated cleavage during normal erythroid differentiation. Here, we show that HSP70 protein expression is dramatically decreased in RPL11 erythroid cells while being preserved in RPS19 cells. The decreased expression of HSP70 in RPL11 cells is related to an enhanced proteasomal degradation of polyubiquitinylated HSP70. Restoration of HSP70 expression level in RPL11 cells reduces p53 activation and rescues the erythroid defect in DBA. These results suggest that HSP70 plays a key role in determining the severity of the erythroid phenotype in RP-mutation-dependent DBA.
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http://dx.doi.org/10.1182/bloodadvances.2017008078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728147PMC
October 2017

CK2: a key regulator of thrombopoiesis.

Blood 2017 12;130(25):2695-2697

INSERM; GUSTAVE ROUSSY; UNIVERSITÉ PARIS-SACLAY.

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http://dx.doi.org/10.1182/blood-2017-10-808568DOI Listing
December 2017