Publications by authors named "Hana Raslova"

55 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

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

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

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

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

Filamin A: key actor in platelet biology.

Blood 2019 10;134(16):1279-1288

INSERM UMR_S 1176, Université Paris-Sud, Université Paris-Saclay, Le Kremlin Bicêtre, France; and.

Filamins (FLNs) are large dimeric actin-binding proteins that regulate actin cytoskeleton remodeling. In addition, FLNs serve as scaffolds for signaling proteins, such as tyrosine kinases, GTPases, or phosphatases, as well as for adhesive receptors, such as integrins. Thus, they connect adhesive receptors to signaling pathways and to cytoskeleton. There are 3 isoforms of FLN (filamin a [FLNa], FLNb, FLNc) that originate from 3 homologous genes. FLNa has been the recent focus of attention because its mutations are responsible for a wide spectrum of defects called filaminopathies A, affecting brain (peri-ventricular nodular heterotopia), heart (valve defect), skeleton, gastrointestinal tract, and, more recently, the megakaryocytic lineage. This review will focus on the physiological and pathological roles of FLNa in platelets. Indeed, FLNa mutations alter platelet production from their bone marrow precursors, the megakaryocytes, yielding giant platelets in reduced numbers (macrothrombocytopenia). In platelets per se, FLNa mutations may lead to impaired αIIbβ3 integrin activation or in contrast, increased αIIbβ3 activation, potentially enhancing the risk of thrombosis. Experimental work delineating the interaction of FLNa with its platelet partners, including αIIbβ3, the von Willebrand factor receptor GPIb-IX-V, the tyrosine kinase Syk, and the signaling pathway of the collagen receptor GPVI, will also be reviewed.
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http://dx.doi.org/10.1182/blood.2019000014DOI Listing
October 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

Downregulation of TREM-like transcript-1 and collagen receptor α2 subunit, two novel RUNX1-targets, contributes to platelet dysfunction in familial platelet disorder with predisposition to acute myelogenous leukemia.

Haematologica 2019 06 13;104(6):1244-1255. Epub 2018 Dec 13.

Instituto de Investigaciones Médicas Alfredo Lanari, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina

Germline mutations lead to thrombocytopenia and platelet dysfunction in familial platelet disorder with predisposition to acute myelogenous leukemia (AML). Multiple aspects of platelet function are impaired in these patients, associated with altered expression of genes regulated by We aimed to identify -targets involved in platelet function by combining transcriptome analysis of patient and sh-transduced megakaryocytes (MK). Down-regulated genes included TREM-like transcript (TLT)-1 (TREML1) and the integrin subunit alpha (α)-2 (ITGA2) of collagen receptor α2-beta (β)-1, which are involved in platelet aggregation and adhesion, respectively. binding to regions enriched for H3K27Ac marks was demonstrated for both genes using chromatin immunoprecipitation. Cloning of these regions upstream of the respective promoters in lentivirus allowing mCherry reporter expression showed that RUNX1 positively regulates and , and this regulation was abrogated after deletion of RUNX1 sites. TLT-1 content was reduced in patient MK and platelets. A blocking anti-TLT-1 antibody was able to block aggregation of normal but not patient platelets, whereas recombinant soluble TLT-1 potentiated fibrinogen binding to patient platelets, pointing to a role for TLT-1 deficiency in the platelet function defect. Low levels of α2 integrin subunit were demonstrated in patient platelets and MK, coupled with reduced platelet and MK adhesion to collagen, both under static and flow conditions. In conclusion, we show that gene expression profiling of RUNX1 knock-down or mutated MK provides a suitable approach to identify novel RUNX1 targets, among which downregulation of and clearly contribute to the platelet phenotype of familial platelet disorder with predisposition to AML.
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http://dx.doi.org/10.3324/haematol.2018.188904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545826PMC
June 2019

Clinic, pathogenic mechanisms and drug testing of two inherited thrombocytopenias, ANKRD26-related Thrombocytopenia and MYH9-related diseases.

Eur J Med Genet 2018 Nov 13;61(11):715-722. Epub 2018 Mar 13.

University of Pavia, Pavia, Italy; IRCCS Policlinico San Matteo Foundation, Pavia, Italy. Electronic address:

Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count resulting in impaired hemostasis. Patients can have spontaneous hemorrhages and/or excessive bleedings provoked by hemostatic challenges as trauma or surgery. To date, ITs encompass 32 different rare monogenic disorders caused by mutations of 30 genes. This review will focus on the major discoveries that have been made in the last years on the diagnosis, treatment and molecular mechanisms of ANKRD26-Related Thrombocytopenia and MYH9-Related Diseases. Furthermore, we will discuss the use a Thrombopoietin mimetic as a novel approach to treat the thrombocytopenia in these patients. We will propose the use of a new 3D bone marrow model to study the mechanisms of action of these drugs and to test their efficacy and safety in patients. The overall purpose of this review is to point out that important progresses have been made in understanding the pathogenesis of ANKRD26-Related Thrombocytopenia and MYH9-Related Diseases and new therapeutic approaches have been proposed and tested. Future advancement in this research will rely in the development of more physiological models to study the regulation of human platelet biogenesis, disease mechanisms and specific pharmacologic targets.
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http://dx.doi.org/10.1016/j.ejmg.2018.01.014DOI Listing
November 2018

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

New pathogenic mechanisms induced by germline erythropoietin receptor mutations in primary erythrocytosis.

Haematologica 2018 04 21;103(4):575-586. Epub 2017 Dec 21.

INSERM, UMR 1170, Gustave Roussy, Laboratoire d'Excellence GR-Ex, Villejuif, France

Primary familial and congenital polycythemia is characterized by erythropoietin hypersensitivity of erythroid progenitors due to germline nonsense or frameshift mutations in the erythropoietin receptor gene. All mutations so far described lead to the truncation of the C-terminal receptor sequence that contains negative regulatory domains. Their removal is presented as sufficient to cause the erythropoietin hypersensitivity phenotype. Here we provide evidence for a new mechanism whereby the presence of novel sequences generated by frameshift mutations is required for the phenotype rather than just extensive truncation resulting from nonsense mutations. We show that the erythropoietin hypersensitivity induced by a new erythropoietin receptor mutant, p.Gln434Profs*11, could not be explained by the loss of negative signaling and of the internalization domains, but rather by the appearance of a new C-terminal tail. The latter, by increasing erythropoietin receptor dimerization, stability and cell-surface localization, causes pre-activation of erythropoietin receptor and JAK2, constitutive signaling and hypersensitivity to erythropoietin. Similar results were obtained with another mutant, p.Pro438Metfs*6, which shares the same last five amino acid residues (MDTVP) with erythropoietin receptor p.Gln434Profs*11, confirming the involvement of the new peptide sequence in the erythropoietin hypersensitivity phenotype. These results suggest a new mechanism that might be common to erythropoietin receptor frameshift mutations. In summary, we show that primary familial and congenital polycythemia is more complex than expected since distinct mechanisms are involved in the erythropoietin hypersensitivity phenotype, according to the type of erythropoietin receptor mutation.
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http://dx.doi.org/10.3324/haematol.2017.176370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5865417PMC
April 2018

Critical role of the HDAC6-cortactin axis in human megakaryocyte maturation leading to a proplatelet-formation defect.

Nat Commun 2017 11 27;8(1):1786. Epub 2017 Nov 27.

Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée par la Ligue Nationale contre le Cancer, 94805, Villejuif, France.

Thrombocytopenia is a major side effect of a new class of anticancer agents that target histone deacetylase (HDAC). Their mechanism is poorly understood. Here, we show that HDAC6 inhibition and genetic knockdown lead to a strong decrease in human proplatelet formation (PPF). Unexpectedly, HDAC6 inhibition-induced tubulin hyperacetylation has no effect on PPF. The PPF decrease induced by HDAC6 inhibition is related to cortactin (CTTN) hyperacetylation associated with actin disorganization inducing important changes in the distribution of megakaryocyte (MK) organelles. CTTN silencing in human MKs phenocopies HDAC6 inactivation and knockdown leads to a strong PPF defect. This is rescued by forced expression of a deacetylated CTTN mimetic. Unexpectedly, unlike human-derived MKs, HDAC6 and CTTN are shown to be dispensable for mouse PPF in vitro and platelet production in vivo. Our results highlight an unexpected function of HDAC6-CTTN axis as a positive regulator of human but not mouse MK maturation.
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http://dx.doi.org/10.1038/s41467-017-01690-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5702605PMC
November 2017

Megakaryocyte and polyploidization.

Exp Hematol 2018 01 27;57:1-13. Epub 2017 Oct 27.

INSERM UMR1170, Equipe labellisée LNNC, Gustave Roussy, Villejuif, France; Université Paris Saclay, UMR1170, Gustave Roussy, France; Université Paris-Diderot, Paris, France; Institut National de la Transfusion Sanguine, Paris, France. Electronic address:

In mammals, platelets are produced in the blood by cytoplasmic fragmentation of megakaryocytes (MKs). Platelet production is thus dependent on both the MK number and size. During differentiation, MKs switch from a division by mitosis to polyploidization by endomitosis to increase their size. The endomitotic process includes several successive rounds of DNA replication with an entry in mitosis with a failure in late cytokinesis and a defect in karyokinesis. This leads to a giant cell with a modal ploidy at 16N and one multilobulated nucleus. The entire genome is duplicated several times and all alleles remain functional producing a hypermetabolic cell. A defect in abscission explains the cytokinesis failure and is related to an altered accumulation of actomyosin at the cleavage furrow as a consequence of both a low local RhoA activity and silencing of the MYH10 gene. This mechanism is regulated by transcription factors that govern differentiation explaining the intricacies of both processes. However, the endomitotic cell cycle regulation is still incompletely understood, particularly mitosis entry, escape to the tetraploid checkpoint, and defect in karyokinesis. Polyploidization is regulated during ontogeny, the first embryonic MKs being 2N. The molecular mechanism of this embryo-fetal/adult transition is beginning to be understood. In physiological conditions, MK ploidy is increased by an enhanced platelet demand through the thrombopoietin/myeloproliferative leukemia axis. In numerous hematologic malignancies, MK ploidy decreases, but it is always associated with a defect in MK differentiation. It has been proposed that polyploidization induction could be a treatment for some malignant MK disorders.
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http://dx.doi.org/10.1016/j.exphem.2017.10.001DOI Listing
January 2018

Mutations of the integrin αIIb/β3 intracytoplasmic salt bridge cause macrothrombocytopenia and enlarged platelet α-granules.

Am J Hematol 2018 02 17;93(2):195-204. Epub 2017 Nov 17.

Institut Hospitalo-Universitaire de Rythmologie et de Modélisation Cardiaque, Plateforme Technologique d'Innovation Biomédicale, Hôpital Xavier Arnozan, Pessac, France.

Rare gain-of-function mutations within the ITGA2B or ITGB3 genes have been recognized to cause macrothrombocytopenia (MTP). Here we report three new families with autosomal dominant (AD) MTP, two harboring the same mutation of ITGA2B, αIIbR995W, and a third family with an ITGB3 mutation, β3D723H. In silico analysis shows how the two mutated amino acids directly modify the salt bridge linking the intra-cytoplasmic part of αIIb to β3 of the integrin αIIbβ3. For all affected patients, the bleeding syndrome and MTP was mild to moderate. Platelet aggregation tended to be reduced but not absent. Electron microscopy associated with a morphometric analysis revealed large round platelets; a feature being the presence of abnormal large α-granules with some giant forms showing signs of fusion. Analysis of the maturation and development of megakaryocytes reveal no defect in their early maturation but abnormal proplatelet formation was observed with increased size of the tips. Interestingly, this study revealed that in addition to the classical phenotype of patients with αIIbβ3 intracytoplasmic mutations there is an abnormal maturation of α-granules. It is now necessary to determine if this feature is a characteristic of all mutations disturbing the αIIb R995/β3 D723 salt bridge.
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http://dx.doi.org/10.1002/ajh.24958DOI Listing
February 2018

A new heterozygous mutation in GP1BA gene responsible for macrothrombocytopenia.

Br J Haematol 2018 11 30;183(3):503-506. Epub 2017 Oct 30.

UMR1062 INSERM, Medicine Faculty, Aix Marseille University, Marseille, France.

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http://dx.doi.org/10.1111/bjh.14986DOI Listing
November 2018

ETO2-GLIS2 Hijacks Transcriptional Complexes to Drive Cellular Identity and Self-Renewal in Pediatric Acute Megakaryoblastic Leukemia.

Cancer Cell 2017 03;31(3):452-465

INSERM U1170, Equipe Labellisée Ligue Contre le Cancer, Gustave Roussy Institute, 39 rue Camille Desmoulins, 94800 Villejuif, France; Gustave Roussy, 94800 Villejuif, France; Université Paris Diderot, 75013 Paris, France; Université Paris-Sud, 91405 Orsay, France. Electronic address:

Chimeric transcription factors are a hallmark of human leukemia, but the molecular mechanisms by which they block differentiation and promote aberrant self-renewal remain unclear. Here, we demonstrate that the ETO2-GLIS2 fusion oncoprotein, which is found in aggressive acute megakaryoblastic leukemia, confers megakaryocytic identity via the GLIS2 moiety while both ETO2 and GLIS2 domains are required to drive increased self-renewal properties. ETO2-GLIS2 directly binds DNA to control transcription of associated genes by upregulation of expression and interaction with the ETS-related ERG protein at enhancer elements. Importantly, specific interference with ETO2-GLIS2 oligomerization reverses the transcriptional activation at enhancers and promotes megakaryocytic differentiation, providing a relevant interface to target in this poor-prognosis pediatric leukemia.
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http://dx.doi.org/10.1016/j.ccell.2017.02.006DOI Listing
March 2017

An incomplete trafficking defect to the cell-surface leads to paradoxical thrombocytosis for human and murine MPL P106L.

Blood 2016 12 10;128(26):3146-3158. Epub 2016 Nov 10.

INSERM, UMR 1170, Équipe labellisée Ligue Nationale contre le Cancer, Gustave Roussy, Villejuif, France.

The mechanisms behind the hereditary thrombocytosis induced by the thrombopoietin (THPO) receptor MPL P106L mutant remain unknown. A complete trafficking defect to the cell surface has been reported, suggesting either weak constitutive activity or nonconventional THPO-dependent mechanisms. Here, we report that the thrombocytosis phenotype induced by MPL P106L belongs to the paradoxical group, where low MPL levels on platelets and mature megakaryocytes (MKs) lead to high serum THPO levels, whereas weak but not absent MPL cell-surface localization in earlier MK progenitors allows response to THPO by signaling and amplification of the platelet lineage. MK progenitors from patients showed no spontaneous growth and responded to THPO, and MKs expressed MPL on their cell surface at low levels, whereas their platelets did not respond to THPO. Transduction of MPL P106L in CD34 cells showed that this receptor was more efficiently localized at the cell surface on immature than on mature MKs, explaining a proliferative response to THPO of immature cells and a defect in THPO clearance in mature cells. In a retroviral mouse model performed in Mpl mice, MPL P106L could induce a thrombocytosis phenotype with high circulating THPO levels. Furthermore, we could select THPO-dependent cell lines with more cell-surface MPL P106L localization that was detected by flow cytometry and [I]-THPO binding. Altogether, these results demonstrate that MPL P106L is a receptor with an incomplete defect in trafficking, which induces a low but not absent localization of the receptor on cell surface and a response to THPO in immature MK cells.
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http://dx.doi.org/10.1182/blood-2016-06-722058DOI Listing
December 2016

Acquired TET2 mutation in one patient with familial platelet disorder with predisposition to AML led to the development of pre-leukaemic clone resulting in T2-ALL and AML-M0.

J Cell Mol Med 2017 06 20;21(6):1237-1242. Epub 2016 Dec 20.

INSERM UMR 1170, Gustave Roussy, Université Paris-Saclay, Equipe Labellisée par la Ligue Nationale Contre le Cancer, Villejuif, France.

Familial platelet disorder with predisposition to acute myeloid leukaemia (FPD/AML) is characterized by germline RUNX1 mutations, thrombocytopaenia, platelet dysfunction and a risk of developing acute myeloid and in rare cases lymphoid T leukaemia. Here, we focus on a case of a man with a familial history of RUNX1 mutation who developed at the age of 42 years a T2-ALL and, 2 years after remission, an AML-M0. Both AML-M0 and T2-ALL blast populations demonstrated a loss of 1p36.32-23 and 17q11.2 regions as well as other small deletions, clonal rearrangements of both TCRγ and TCRδ and a presence of 18 variants at a frequency of more than 40%. Additional variants were identified only in T2-ALL or in AML-M0 evoking the existence of a common original clone, which gave rise to subclonal populations. Next generation sequencing (NGS) performed on peripheral blood-derived CD34 cells 5 years prior to T2-ALL development revealed only the missense TET2 mutation at a frequency of 1%, which increases to more than 40% in fully transformed leukaemic T2-ALL and AML-M0 clones. This result suggests that TET2 mutation in association with germline RUNX1 mutation leads to amplification of a haematopoietic clone susceptible to acquire other transforming alterations.
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http://dx.doi.org/10.1111/jcmm.13051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5431233PMC
June 2017

Eltrombopag, a potent stimulator of megakaryopoiesis.

Haematologica 2016 12;101(12):1443-1445

INSERM, UMR1170, Gustave Roussy, Villejuif, France

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http://dx.doi.org/10.3324/haematol.2016.153668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5479610PMC
December 2016

Activity of nonmuscle myosin II isoforms determines localization at the cleavage furrow of megakaryocytes.

Blood 2016 12 13;128(26):3137-3145. Epub 2016 Oct 13.

Institut National de la Santé et de la Recherche Médicale, Unité mixte de recherche (UMR) 1170, Villejuif, France.

Megakaryocyte polyploidy is characterized by cytokinesis failure resulting from defects in contractile forces at the cleavage furrow. Although immature megakaryocytes express 2 nonmuscle myosin II isoforms (MYH9 [NMIIA] and MYH10 [NMIIB]), only NMIIB localizes at the cleavage furrow, and its subsequent absence contributes to polyploidy. In this study, we tried to understand why the abundant NMIIA does not localize at the furrow by focusing on the RhoA/ROCK pathway that has a low activity in polyploid megakaryocytes. We observed that under low RhoA activity, NMII isoforms presented different activity that determined their localization. Inhibition of RhoA/ROCK signaling abolished the localization of NMIIB, whereas constitutively active RhoA induced NMIIA at the cleavage furrow. Thus, although high RhoA activity favored the localization of both the isoforms, only NMIIB could localize at the furrow at low RhoA activity. This was further confirmed in erythroblasts that have a higher basal RhoA activity than megakaryocytes and express both NMIIA and NMIIB at the cleavage furrow. Decreased RhoA activity in erythroblasts abolished localization of NMIIA but not of NMIIB from the furrow. This differential localization was related to differences in actin turnover. Megakaryocytes had a higher actin turnover compared with erythroblasts. Strikingly, inhibition of actin polymerization was found to be sufficient to recapitulate the effects of inhibition of RhoA/ROCK pathway on NMII isoform localization; thus, cytokinesis failure in megakaryocytes is the consequence of both the absence of NMIIB and a low RhoA activity that impairs NMIIA localization at the cleavage furrow through increased actin turnover.
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http://dx.doi.org/10.1182/blood-2016-04-711630DOI Listing
December 2016

Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors.

Haematologica 2017 02 23;102(2):282-294. Epub 2016 Sep 23.

Aix Marseille Univ, INSERM, INRA, NORT, Marseille, France

Variants in ETV6, which encodes a transcription repressor of the E26 transformation-specific family, have recently been reported to be responsible for inherited thrombocytopenia and hematologic malignancy. We sequenced the DNA from cases with unexplained dominant thrombocytopenia and identified six likely pathogenic variants in ETV6, of which five are novel. We observed low repressive activity of all tested ETV6 variants, and variants located in the E26 transformation-specific binding domain (encoding p.A377T, p.Y401N) led to reduced binding to corepressors. We also observed a large expansion of megakaryocyte colony-forming units derived from variant carriers and reduced proplatelet formation with abnormal cytoskeletal organization. The defect in proplatelet formation was also observed in control CD34 cell-derived megakaryocytes transduced with lentiviral particles encoding mutant ETV6. Reduced expression levels of key regulators of the actin cytoskeleton CDC42 and RHOA were measured. Moreover, changes in the actin structures are typically accompanied by a rounder platelet shape with a highly heterogeneous size, decreased platelet arachidonic response, and spreading and retarded clot retraction in ETV6 deficient platelets. Elevated numbers of circulating CD34 cells were found in p.P214L and p.Y401N carriers, and two patients from different families suffered from refractory anemia with excess blasts, while one patient from a third family was successfully treated for acute myeloid leukemia. Overall, our study provides novel insights into the role of ETV6 as a driver of cytoskeletal regulatory gene expression during platelet production, and the impact of variants resulting in platelets with altered size, shape and function and potentially also in changes in circulating progenitor levels.
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http://dx.doi.org/10.3324/haematol.2016.147694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5286936PMC
February 2017

Uncoupling of the Hippo and Rho pathways allows megakaryocytes to escape the tetraploid checkpoint.

Haematologica 2016 12 11;101(12):1469-1478. Epub 2016 Aug 11.

Institut National de la Santé et la Recherche Médicale (INSERM) UMR1170, Equipe Labellisée par la Ligue Nationale Contre le Cancer, Villejuif, France

Megakaryocytes are naturally polyploid cells that increase their ploidy by endomitosis. However, very little is known regarding the mechanism by which they escape the tetraploid checkpoint to become polyploid. Recently, it has been shown that the tetraploid checkpoint was regulated by the Hippo-p53 pathway in response to a downregulation of Rho activity. We therefore analyzed the role of Hippo-p53 pathway in the regulation of human megakaryocyte polyploidy. Our results revealed that Hippo-p53 signaling pathway proteins are present and are functional in megakaryocytes. Although this pathway responds to the genotoxic stress agent etoposide, it is not activated in tetraploid or polyploid megakaryocytes. Furthermore, Hippo pathway was observed to be uncoupled from Rho activity. Additionally, polyploid megakaryocytes showed increased expression of YAP target genes when compared to diploid and tetraploid megakaryocytes. Although p53 knockdown increased both modal ploidy and proplatelet formation in megakaryocytes, YAP knockdown caused no significant change in ploidy while moderately affecting proplatelet formation. Interestingly, YAP knockdown reduced the mitochondrial mass in polyploid megakaryocytes and decreased expression of PGC1α, an important mitochondrial biogenesis regulator. Thus, the Hippo pathway is functional in megakaryocytes, but is not induced by tetraploidy. Additionally, YAP regulates the mitochondrial mass in polyploid megakaryocytes.
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http://dx.doi.org/10.3324/haematol.2016.149914DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5479626PMC
December 2016

P53 activation inhibits all types of hematopoietic progenitors and all stages of megakaryopoiesis.

Oncotarget 2016 May;7(22):31980-92

INSERM, UMR 1170, Laboratory of Excellence GR-Ex, Villejuif, France.

TP53 also known as p53 is a tumor suppressor gene mutated in a variety of cancers. P53 is involved in cell cycle, apoptosis and DNA repair mechanisms and is thus tightly controlled by many regulators. Recently, strategies to treat cancer have focused on the development of MDM2 antagonists to induce p53 stabilization and restore cell death in p53 non-mutated cancers. However, some of these molecules display adverse effects in patients including induction of thrombocytopenia. In the present study, we have explored the effect of SAR405838 not only on human megakaryopoiesis but also more generally on hematopoiesis. We compared its effect to MI-219 and Nutlin, which are less potent MDM2 antagonists than SAR405838. We found that all these compounds induce a deleterious effect on all types of hematopoietic progenitors, as well as on erythroid and megakaryocytic differentiation. Moreover, they inhibit both early and late stages of megakaryopoiesis including ploidization and proplatelet formation. In conclusion, MDM2 antagonists induced a major hematopoietic defect in vitro as well as an inhibition of all stages of megakaryopoiesis that may account for in vivo thrombocytopenia observed in treated patients.
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http://dx.doi.org/10.18632/oncotarget.7881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5077990PMC
May 2016

Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants.

Blood 2016 Mar 14;127(10):1325-35. Epub 2015 Dec 14.

Ludwig Institute for Cancer Research Brussels, Brussels, Belgium; Université Catholique de Louvain and de Duve Institute, Brussels, Belgium;

Mutations in the calreticulin gene (CALR) represented by deletions and insertions in exon 9 inducing a -1/+2 frameshift are associated with a significant fraction of myeloproliferative neoplasms (MPNs). The mechanisms by which CALR mutants induce MPN are unknown. Here, we show by transcriptional, proliferation, biochemical, and primary cell assays that the pathogenic CALR mutants specifically activate the thrombopoietin receptor (TpoR/MPL). No activation is detected with a battery of type I and II cytokine receptors, except granulocyte colony-stimulating factor receptor, which supported only transient and weak activation. CALR mutants induce ligand-independent activation of JAK2/STAT/phosphatydylinositol-3'-kinase (PI3-K) and mitogen-activated protein (MAP) kinase pathways via TpoR, and autonomous growth in Ba/F3 cells. In these transformed cells, no synergy is observed between JAK2 and PI3-K inhibitors in inhibiting cytokine-independent proliferation, thus showing a major difference from JAK2V617F cells where such synergy is strong. TpoR activation was dependent on its extracellular domain and its N-glycosylation, especially at N117. The glycan binding site and the novel C-terminal tail of the mutant CALR proteins were required for TpoR activation. A soluble form of TpoR was able to prevent activation of full-length TpoR provided that it was N-glycosylated. By confocal microscopy and subcellular fractionation, CALR mutants exhibit different intracellular localization from that of wild-type CALR. Finally, knocking down either MPL/TpoR or JAK2 in megakaryocytic progenitors from patients carrying CALR mutations inhibited cytokine-independent megakaryocytic colony formation. Taken together, our study provides a novel signaling paradigm, whereby a mutated chaperone constitutively activates cytokine receptor signaling.
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http://dx.doi.org/10.1182/blood-2015-11-681932DOI Listing
March 2016

Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis.

Blood 2016 Mar 25;127(10):1317-24. Epub 2015 Nov 25.

INSERM, Unité Mixte de Recherche (UMR) 1170, Villejuif, France; Université Paris-Saclay, UMR 1170, Villejuif, France; Gustave Roussy, UMR 1170, Villejuif, France;

Frameshift mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and myelofibrosis patients. To address the contribution of the CALR mutants to the pathogenesis of myeloproliferative neoplasms, we engrafted lethally irradiated recipient mice with bone marrow cells transduced with retroviruses expressing these mutants. In contrast to wild-type CALR, CALRdel52 (type I) and, to a lesser extent, CALRins5 (type II) induced thrombocytosis due to a megakaryocyte (MK) hyperplasia. Disease was transplantable into secondary recipients. After 6 months, CALRdel52-, in contrast to rare CALRins5-, transduced mice developed a myelofibrosis associated with a splenomegaly and a marked osteosclerosis. Monitoring of virus-transduced populations indicated that CALRdel52 leads to expansion at earlier stages of hematopoiesis than CALRins5. However, both mutants still specifically amplified the MK lineage and platelet production. Moreover, a mutant deleted of the entire exon 9 (CALRdelex9) did not induce a disease, suggesting that the oncogenic property of CALR mutants was related to the new C-terminus peptide. To understand how the CALR mutants target the MK lineage, we used a cell-line model and demonstrated that the CALR mutants, but not CALRdelex9, specifically activate the thrombopoietin (TPO) receptor (MPL) to induce constitutive activation of Janus kinase 2 and signal transducer and activator of transcription 5/3/1. We confirmed in c-mpl- and tpo-deficient mice that expression of Mpl, but not of Tpo, was essential for the CALR mutants to induce thrombocytosis in vivo, although Tpo contributes to disease penetrance. Thus, CALR mutants are sufficient to induce thrombocytosis through MPL activation.
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http://dx.doi.org/10.1182/blood-2015-11-679571DOI Listing
March 2016

Presence of atypical thrombopoietin receptor (MPL) mutations in triple-negative essential thrombocythemia patients.

Blood 2016 Jan 8;127(3):333-42. Epub 2015 Oct 8.

INSERM, Unité Mixte de Recherche (UMR) 1170, Gustave Roussy, Villejuif, France; Université Paris-Saclay, UMR1170, Gustave Roussy, Villejuif, France; UMR1170, Gustave Roussy, Villejuif, France; Consultation d'Hématologie-Immunologie, Hôpital Saint Louis, Assistance Publique-Hôpitaux de Paris, Paris, France.

Mutations in signaling molecules of the cytokine receptor axis play a central role in myeloproliferative neoplasm (MPN) pathogenesis. Polycythemia vera is mainly related to JAK2 mutations, whereas a wider mutational spectrum is detected in essential thrombocythemia (ET) with mutations in JAK2, the thrombopoietin (TPO) receptor (MPL), and the calreticulin (CALR) genes. Here, we studied the mutational profile of 17 ET patients negative for JAK2V617F, MPLW515K/L, and CALR mutations, using whole-exome sequencing and next-generation sequencing (NGS) targeted on JAK2 and MPL. We found several signaling mutations including JAK2V617F at very low allele frequency, 1 homozygous SH2B3 mutation, 1 MPLS505N, 1 MPLW515R, and 2 MPLS204P mutations. In the remaining patients, 4 presented a clonal and 7 a polyclonal hematopoiesis, suggesting that certain triple-negative ETs are not MPNs. NGS on 26 additional triple-negative ETs detected only 1 MPLY591N mutation. Functional studies on MPLS204P and MPLY591N revealed that they are weak gain-of-function mutants increasing MPL signaling and conferring either TPO hypersensitivity or independence to expressing cells, but with a low efficiency. Further studies should be performed to precisely determine the frequency of MPLS204 and MPLY591 mutants in a bigger cohort of MPN.
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http://dx.doi.org/10.1182/blood-2015-07-661983DOI Listing
January 2016