Publications by authors named "Najet Debili"

59 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

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

Murine platelet production is suppressed by S1P release in the hematopoietic niche, not facilitated by blood S1P sensing.

Blood Adv 2019 06;3(11):1702-1713

INSERM U970, Paris Cardiovascular Research Centre, Paris, France.

The bioactive lipid mediator sphingosine 1-phosphate (S1P) was recently assigned critical roles in platelet biology: whereas S1P receptor-mediated S1P gradient sensing was reported to be essential for directing proplatelet extensions from megakaryocytes (MKs) toward bone marrow sinusoids, MK sphingosine kinase 2 (Sphk2)-derived S1P was reported to further promote platelet shedding through receptor-independent intracellular actions, and platelet aggregation through S1P Yet clinical use of S1P pathway modulators including fingolimod has not been associated with risk of bleeding or thrombosis. We therefore revisited the role of S1P in platelet biology in mice. Surprisingly, no reduction in platelet counts was observed when the vascular S1P gradient was ablated by impairing S1P provision to plasma or S1P degradation in interstitial fluids, nor when gradient sensing was impaired by deletion selectively in MKs. Moreover, S1P expression and signaling were both undetectable in mature MKs in situ, and MK deletion did not affect platelet aggregation or spreading. When deletion was induced in hematopoietic progenitor cells, platelet counts were instead significantly elevated. Isolated global Sphk2 deficiency was associated with thrombocytopenia, but this was not replicated by MK-restricted deletion and was reversed by compound deletion of either or , suggesting that this phenotype arises from increased S1P export and S1P activation secondary to redistribution of sphingosine to Sphk1. Consistent with clinical observations, we thus observe no essential role for S1P in facilitating platelet production or activation. Instead, S1P restricts megakaryopoiesis through S1P, and can further suppress thrombopoiesis through S1P when aberrantly secreted in the hematopoietic niche.
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http://dx.doi.org/10.1182/bloodadvances.2019031948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6560353PMC
June 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

Dynamins 2 and 3 control the migration of human megakaryocytes by regulating CXCR4 surface expression and ITGB1 activity.

Blood Adv 2018 12;2(23):3540-3552

Department of Medicine and.

Megakaryocyte (MK) migration from the bone marrow periosteal niche toward the vascular niche is a prerequisite for proplatelet extension and release into the circulation. The mechanism for this highly coordinated process is poorly understood. Here we show that dynasore (DNSR), a small-molecule inhibitor of dynamins (DNMs), or short hairpin RNA knockdown of DNM2 and DNM3 impairs directional migration in a human MK cell line or MKs derived from cultured CD34 cells. Because cell migration requires actin cytoskeletal rearrangements, we measured actin polymerization and the activity of cytoskeleton regulator RhoA and found them to be decreased after inhibition of DNM2 and DNM3. Because SDF-1α is important for hematopoiesis, we studied the expression of its receptor CXCR4 in DNSR-treated cells. CXCR4 expression on the cell surface was increased, at least partially because of slower endocytosis and internalization after SDF-1α treatment. Combined inhibition of DNM2 and DNM3 or forced expression of dominant-negative Dnm2-K44A or GTPase-defective DNM3 diminished β1 integrin (ITGB1) activity. DNSR-treated MKs showed an abnormally clustered staining pattern of Rab11, a marker of recycling endosomes. This suggests decreased recruitment of the recycling pathway in DNSR-treated cells. Altogether, we show that the GTPase activity of DNMs, which governs endocytosis and regulates cell receptor trafficking, exerts control on MK migration toward SDF-1α gradients, such as those originating from the vascular niche. DNMs play a critical role in MKs by triggering membrane-cytoskeleton rearrangements downstream of CXCR4 and integrins.
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http://dx.doi.org/10.1182/bloodadvances.2018021923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290100PMC
December 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

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

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

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

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

Germline duplication of ATG2B and GSKIP predisposes to familial myeloid malignancies.

Nat Genet 2015 Oct 17;47(10):1131-40. Epub 2015 Aug 17.

INSERM, Unité Mixte de Recherche (UMR) 1170, Villejuif, France.

No major predisposition gene for familial myeloproliferative neoplasms (MPN) has been identified. Here we demonstrate that the autosomal dominant transmission of a 700-kb duplication in four genetically related families predisposes to myeloid malignancies, including MPN, frequently progressing to leukemia. Using induced pluripotent stem cells and primary cells, we demonstrate that overexpression of ATG2B and GSKIP enhances hematopoietic progenitor differentiation, including of megakaryocytes, by increasing progenitor sensitivity to thrombopoietin (TPO). ATG2B and GSKIP cooperate with acquired JAK2, MPL and CALR mutations during MPN development. Thus, the germline duplication may change the fitness of cells harboring signaling pathway mutations and increases the probability of disease development.
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http://dx.doi.org/10.1038/ng.3380DOI Listing
October 2015

Level of RUNX1 activity is critical for leukemic predisposition but not for thrombocytopenia.

Blood 2015 Feb 9;125(6):930-40. Epub 2014 Dec 9.

Institut National de la Santé et de la Recherche Médicale, Villejuif, France; Gustave Roussy, Villejuif, France; Université Paris Sud, Villejuif, France;

To explore how RUNX1 mutations predispose to leukemia, we generated induced pluripotent stem cells (iPSCs) from 2 pedigrees with germline RUNX1 mutations. The first, carrying a missense R174Q mutation, which acts as a dominant-negative mutant, is associated with thrombocytopenia and leukemia, and the second, carrying a monoallelic gene deletion inducing a haploinsufficiency, presents only as thrombocytopenia. Hematopoietic differentiation of these iPSC clones demonstrated profound defects in erythropoiesis and megakaryopoiesis and deregulated expression of RUNX1 targets. iPSC clones from patients with the R174Q mutation specifically generated an increased amount of granulomonocytes, a phenotype reproduced by an 80% RUNX1 knockdown in the H9 human embryonic stem cell line, and a genomic instability. This phenotype, found only with a lower dosage of RUNX1, may account for development of leukemia in patients. Altogether, RUNX1 dosage could explain the differential phenotype according to RUNX1 mutations, with a haploinsufficiency leading to thrombocytopenia alone in a majority of cases whereas a more complete gene deletion predisposes to leukemia.
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http://dx.doi.org/10.1182/blood-2014-06-585513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4347283PMC
February 2015

p19 INK4d controls hematopoietic stem cells in a cell-autonomous manner during genotoxic stress and through the microenvironment during aging.

Stem Cell Reports 2014 Dec 20;3(6):1085-102. Epub 2014 Nov 20.

Institut National de la Santé et de la Recherche Médicale, U1009, Equipe labellisée Ligue Nationale contre le Cancer, 114 rue Edouard Vaillant, 94805 Villejuif, France; University Paris Sud, 114, rue Edouard Vaillant, 94805 Villejuif, France; Gustave Roussy, IFR54, 114, rue Edouard Vaillant, 94805 Villejuif, France. Electronic address:

Hematopoietic stem cells (HSCs) are characterized by the capacity for self-renewal and the ability to reconstitute the entire hematopoietic compartment. Thrombopoietin maintains adult HSCs in a quiescent state through the induction of cell cycle inhibitors p57(Kip2) and p19(INK4d). Using the p19(INK4d-/-) mouse model, we investigated the role of p19(INK4d) in basal and stress-induced hematopoiesis. We demonstrate that p19(INK4d) is involved in the regulation of HSC quiescence by inhibition of the G0/G1 cell cycle transition. Under genotoxic stress conditions, the absence of p19(INK4d) in HSCs leads to accelerated cell cycle exit, accumulation of DNA double-strand breaks, and apoptosis when cells progress to the S/G2-M stages of the cell cycle. Moreover, p19(INK4d) controls the HSC microenvironment through negative regulation of megakaryopoiesis. Deletion of p19(INK4d) results in megakaryocyte hyperproliferation and increased transforming growth factor β1 secretion. This leads to fibrosis in the bone marrow and spleen, followed by loss of HSCs during aging.
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http://dx.doi.org/10.1016/j.stemcr.2014.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4264042PMC
December 2014

A new form of macrothrombocytopenia induced by a germ-line mutation in the PRKACG gene.

Blood 2014 Oct 24;124(16):2554-63. Epub 2014 Jul 24.

Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 1009, Université Paris-Sud 11, Equipe Labellisée Ligue Contre le Cancer, Villejuif, France; Gustave Roussy, Villejuif, France;

Macrothrombocytopenias are the most important subgroup of inherited thrombocytopenias. This subgroup is particularly heterogeneous because the affected genes are involved in various functions such as cell signaling, cytoskeleton organization, and gene expression. Herein we describe the clinical and hematological features of a consanguineous family with a severe autosomal recessive macrothrombocytopenia associated with a thrombocytopathy inducing a bleeding tendency in the homozygous mutated patients. Platelet activation and cytoskeleton reorganization were impaired in these homozygous patients. Exome sequencing identified a c.222C>G mutation (missense p.74Ile>Met) in PRKACG, a gene encoding the γ-catalytic subunit of the cyclic adenosine monophosphate-dependent protein kinase, the mutated allele cosegregating with the macrothrombocytopenia. We demonstrate that the p.74Ile>Met PRKACG mutation is associated with a marked defect in proplatelet formation and a low level in filamin A in megakaryocytes (MKs). The defect in proplatelet formation was rescued in vitro by lentiviral vector-mediated overexpression of wild-type PRKACG in patient MKs. We thus conclude that PRKACG is a new central actor in platelet biogenesis and a new gene involved in inherited thrombocytopenia with giant platelets associated with a thrombocytopathy.
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http://dx.doi.org/10.1182/blood-2014-01-551820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4199957PMC
October 2014

TET2 deficiency inhibits mesoderm and hematopoietic differentiation in human embryonic stem cells.

Stem Cells 2014 Aug;32(8):2084-97

Institut National de la Santé et de la Recherche Médicale, UMR 1009, Laboratory of Excellence GR-Ex, 114 rue Edouard Vaillant, Villejuif, Paris, France; Institut Gustave Roussy, Villejuif, Paris, France; Université Paris Sud 11, Orsay, France.

Ten-eleven-translocation 2 (TET2) belongs to the TET protein family that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine and plays a central role in normal and malignant adult hematopoiesis. Yet the role of TET2 in human hematopoietic development remains largely unknown. Here, we show that TET2 expression is low in human embryonic stem cell (ESC) lines and increases during hematopoietic differentiation. shRNA-mediated TET2 knockdown had no effect on the pluripotency of various ESCs. However, it skewed their differentiation into neuroectoderm at the expense of endoderm and mesoderm both in vitro and in vivo. These effects were rescued by reintroducing the targeted TET2 protein. Moreover, TET2-driven differentiation was dependent on NANOG transcriptional factor. Indeed, TET2 bound to NANOG promoter and in TET2-deficient cells the methylation of the NANOG promoter correlated with a decreased in NANOG expression. The altered differentiation resulting from TET2 knockdown in ESCs led to a decrease in both the number and the cloning capacities of hematopoietic progenitors. These defects were due to an increased apoptosis and an altered gene expression profile, including abnormal expression of neuronal genes. Intriguingly, when TET2 was knockdown in hematopoietic cells, it increased hematopoietic development. In conclusion, our work suggests that TET2 is involved in different stages of human embryonic development, including induction of the mesoderm and hematopoietic differentiation.
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http://dx.doi.org/10.1002/stem.1718DOI Listing
August 2014

Thrombocytopenia-associated mutations in the ANKRD26 regulatory region induce MAPK hyperactivation.

J Clin Invest 2014 Feb 16;124(2):580-91. Epub 2014 Jan 16.

Point mutations in the 5' UTR of ankyrin repeat domain 26 (ANKRD26) are associated with familial thrombocytopenia 2 (THC2) and a predisposition to leukemia. Here, we identified underlying mechanisms of ANKRD26-associated thrombocytopenia. Using megakaryocytes (MK) isolated from THC2 patients and healthy subjects, we demonstrated that THC2-associated mutations in the 5' UTR of ANKRD26 resulted in loss of runt-related transcription factor 1 (RUNX1) and friend leukemia integration 1 transcription factor (FLI1) binding. RUNX1 and FLI1 binding at the 5' UTR from healthy subjects led to ANKRD26 silencing during the late stages of megakaryopoiesis and blood platelet development. We showed that persistent ANKRD26 expression in isolated MKs increased signaling via the thrombopoietin/myeloproliferative leukemia virus oncogene (MPL) pathway and impaired proplatelet formation by MKs. Importantly, we demonstrated that ERK inhibition completely rescued the in vitro proplatelet formation defect. Our data identify a mechanism for development of the familial thrombocytopenia THC2 that is related to abnormal MAPK signaling.
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http://dx.doi.org/10.1172/JCI71861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3904625PMC
February 2014

Crucial role for endoplasmic reticulum stress during megakaryocyte maturation.

Arterioscler Thromb Vasc Biol 2013 Dec 10;33(12):2750-8. Epub 2013 Oct 10.

From the Inserm U770, Université Paris-Sud, Le Kremlin-Bicêtre, France (J.J.L., C.C., L.A., E.P., S.D., J.E., R.B.); INSERM UMR1009, Institut Gustave Roussy, Université Paris-Sud, Villejuif, France (A.P., K.L., N.D.); and Centre de Référence des Pathologies Plaquettaires, Plateforme Technologique d'Innovation Biomédicale, Hôpital Xavier Arnozan, Pessac, France (P.N.).

Objective: Apoptotic-like phase is an essential step for the platelet formation from megakaryocytes. How controlled is this signaling pathway remained poorly understood. The aim of this study was to determine whether endoplasmic reticulum (ER) stress-induced apoptosis occurs during thrombopoiesis.

Approach And Results: Investigation of ER stress and maturation markers in different models of human thrombopoiesis (CHRF, DAMI, MEG-01 cell lines, and hematopoietic stem cells: CD34(+)) as well as in immature pathological platelets clearly indicated that ER stress occurs transiently during thrombopoiesis. Direct ER stress induction by tunicamycin, an inhibitor of N-glycosylation, or by sarco/endoplasmic reticulum Ca(2+) ATPase type 3b overexpression, which interferes with reticular calcium, leads to some degree of maturation in megakaryocytic cell lines. On the contrary, exposure to salubrinal, a phosphatase inhibitor that prevents eukaryotic translation initiation factor 2α-P dephosphorylation and inhibits ER stress-induced apoptosis, decreased both expression of maturation markers in MEG-01 and CD34(+) cells as well as numbers of mature megakaryocytes and proplatelet formation in cultured CD34(+) cells.

Conclusions: Taken as a whole, our research suggests that transient ER stress activation triggers the apoptotic-like phase of the thrombopoiesis process.
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http://dx.doi.org/10.1161/ATVBAHA.113.302184DOI Listing
December 2013

Heterozygous and homozygous JAK2(V617F) states modeled by induced pluripotent stem cells from myeloproliferative neoplasm patients.

PLoS One 2013 16;8(9):e74257. Epub 2013 Sep 16.

Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 1009, Laboratory of Excellence, Globule rouge-Excellence (GR-Ex), Villejuif, France ; University Paris-Sud 11, Le Kremlin-Bicêtre, France ; Institut Gustave Roussy, Villejuif, France.

JAK2(V617F) is the predominant mutation in myeloproliferative neoplasms (MPN). Modeling MPN in a human context might be helpful for the screening of molecules targeting JAK2 and its intracellular signaling. We describe here the derivation of induced pluripotent stem (iPS) cell lines from 2 polycythemia vera patients carrying a heterozygous and a homozygous mutated JAK2(V617F), respectively. In the patient with homozygous JAK2(V617F), additional ASXL1 mutation and chromosome 20 allowed partial delineation of the clonal architecture and assignation of the cellular origin of the derived iPS cell lines. The marked difference in the response to erythropoietin (EPO) between homozygous and heterozygous cell lines correlated with the constitutive activation level of signaling pathways. Strikingly, heterozygous iPS cells showed thrombopoietin (TPO)-independent formation of megakaryocytic colonies, but not EPO-independent erythroid colony formation. JAK2, PI3K and HSP90 inhibitors were able to block spontaneous and EPO-induced growth of erythroid colonies from GPA(+)CD41(+) cells derived from iPS cells. Altogether, this study brings the proof of concept that iPS can be used for studying MPN pathogenesis, clonal architecture, and drug efficacy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0074257PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774801PMC
May 2014

Loss of the F-BAR protein CIP4 reduces platelet production by impairing membrane-cytoskeleton remodeling.

Blood 2013 Sep 23;122(10):1695-706. Epub 2013 Jul 23.

Department of Pediatrics, Robert H. Lurie Comprehensive Cancer Center and Children's Memorial Hospital, Northwestern Feinberg School of Medicine, Chicago, IL 60611, USA.

Megakaryocytes generate platelets through extensive reorganization of the cytoskeleton and plasma membrane. Cdc42 interacting protein 4 (CIP4) is an F-BAR protein that localizes to membrane phospholipids through its BAR domain and interacts with Wiskott-Aldrich Syndrome Protein (WASP) via its SRC homology 3 domain. F-BAR proteins promote actin polymerization and membrane tubulation. To study its function, we generated CIP4-null mice that displayed thrombocytopenia similar to that of WAS(-) mice. The number of megakaryocytes and their progenitors was not affected. However, the number of proplatelet protrusions was reduced in CIP4-null, but not WAS(-), megakaryocytes. Electron micrographs of CIP4-null megakaryocytes showed an altered demarcation membrane system. Silencing of CIP4, not WASP, expression resulted in fewer proplatelet-like extensions. Fluorescence anisotropy studies showed that loss of CIP4 resulted in a more rigid membrane. Micropipette aspiration demonstrated decreased cortical actin tension in megakaryocytic cells with reduced CIP4 or WASP protein. These studies support a new biophysical mechanism for platelet biogenesis whereby CIP4 enhances the complex, dynamic reorganization of the plasma membrane (WASP independent) and actin cortex network (as known for WASP and cortical actin) to reduce the work required for generating proplatelets. CIP4 is a new component in the highly coordinated system of megakaryocytic membrane and cytoskeletal remodeling affecting platelet production.
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http://dx.doi.org/10.1182/blood-2013-03-484550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3765055PMC
September 2013

Defective tubulin organization and proplatelet formation in murine megakaryocytes lacking Rac1 and Cdc42.

Blood 2013 Oct 16;122(18):3178-87. Epub 2013 Jul 16.

University of Würzburg, Department of Experimental Biomedicine, University Hospital and Rudolf Virchow Center, Deutsche Forschungsgemeinschaft Research Center for Experimental Biomedicine, Würzburg, Germany;

Blood platelets are anuclear cell fragments that are essential for blood clotting. Platelets are produced by bone marrow megakaryocytes (MKs), which extend protrusions, or so-called proplatelets, into bone marrow sinusoids. Proplatelet formation requires a profound reorganization of the MK actin and tubulin cytoskeleton. Rho GTPases, such as RhoA, Rac1, and Cdc42, are important regulators of cytoskeletal rearrangements in platelets; however, the specific roles of these proteins during platelet production have not been established. Using conditional knockout mice, we show here that Rac1 and Cdc42 possess redundant functions in platelet production and function. In contrast to a single-deficiency of either protein, a double-deficiency of Rac1 and Cdc42 in MKs resulted in macrothrombocytopenia, abnormal platelet morphology, and impaired platelet function. Double-deficient bone marrow MKs matured normally in vivo but displayed highly abnormal morphology and uncontrolled fragmentation. Consistently, a lack of Rac1/Cdc42 virtually abrogated proplatelet formation in vitro. Strikingly, this phenotype was associated with severely defective tubulin organization, whereas actin assembly and structure were barely affected. Together, these results suggest that the combined action of Rac1 and Cdc42 is crucial for platelet production, particularly by regulating microtubule dynamics.
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http://dx.doi.org/10.1182/blood-2013-03-487942DOI Listing
October 2013

Presence of a defect in karyokinesis during megakaryocyte endomitosis.

Cell Cycle 2012 Dec 16;11(23):4385-9. Epub 2012 Nov 16.

INSERM, UMR 1009, Institut Gustave Roussy, Villejuif, France.

Megakaryocyte is the naturally polyploid cell that gives rise to platelets. Polyploidization occurs by endomitosis, a process corresponding to a late failure of cytokinesis with a backward movement of the daughter cells. Generally, a pure defect in cytokinesis produces a multinucleated cell, but megakaryocytes are characterized by a single polylobulated nucleus with a 2 (N) ploidy. Here, we show the existence of a defect in karyokinesis during the endomitotic process. From late telophase until the reversal of cytokinesis, some dipolar mitosis/endomitosis and most multipolar endomitosis present a thin DNA link between the segregated chromosomes surrounded by an incomplete nuclear membrane formation, which implies that sister chromatid separation is not complete. This observation may explain why polyploid megakaryocytes display a single polylobulated nucleus along with an increase in ploidy.
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http://dx.doi.org/10.4161/cc.22712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3552921PMC
December 2012

Dysmegakaryopoiesis of FPD/AML pedigrees with constitutional RUNX1 mutations is linked to myosin II deregulated expression.

Blood 2012 Sep 16;120(13):2708-18. Epub 2012 Aug 16.

Institut National de la Santé et de la Recherche Médicale, Villejuif, France.

FPD/AML is a familial platelet disorder characterized by platelet defects, predisposition to acute myelogenous leukemia (AML) and germ-line heterozygous RUNX1 alterations. Here we studied the in vitro megakaryopoiesis of 3 FPD/AML pedigrees. A 60% to 80% decrease in the output of megakaryocytes (MKs) from CD34(+) was observed. MK ploidy level was low and mature MKs displayed a major defect in proplatelet formation. To explain these defects, we focused on myosin II expression as RUNX1 has been shown to regulate MYL9 and MYH10 in an inverse way. In FPD/AML MKs, expression of MYL9 and MYH9 was decreased, whereas MYH10 expression was increased and the MYH10 protein was still present in the cytoplasm of mature MKs. Myosin II activity inhibition by blebbistatin rescued the ploidy defect of FPD/AML MKs. Finally, we demonstrate that MYH9 is a direct target of RUNX1 by chromatin immunoprecipitation and luciferase assays and we identified new RUNX1 binding sites in the MYL9 promoter region. Together, these results demonstrate that the defects in megakaryopoiesis observed in FPD/AML are, in part, related to a deregulation of myosin IIA and IIB expression leading to both a defect in ploidization and proplatelet formation.
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http://dx.doi.org/10.1182/blood-2012-04-422337DOI Listing
September 2012

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization.

Nat Commun 2012 Mar 6;3:717. Epub 2012 Mar 6.

INSERM UMR 1009, Institut Gustave Roussy, 114, rue Edouard Vaillant, Villejuif 94805, France.

Megakaryocytes are unique mammalian cells that undergo polyploidization (endomitosis) during differentiation, leading to an increase in cell size and protein production that precedes platelet production. Recent evidence demonstrates that endomitosis is a consequence of a late failure in cytokinesis associated with a contractile ring defect. Here we show that the non-muscle myosin IIB heavy chain (MYH10) is expressed in immature megakaryocytes and specifically localizes in the contractile ring. MYH10 downmodulation by short hairpin RNA increases polyploidization by inhibiting the return of 4N cells to 2N, but other regulators, such as of the G1/S transition, might regulate further polyploidization of the 4N cells. Conversely, re-expression of MYH10 in the megakaryocytes prevents polyploidization and the transition of 2N to 4N cells. During polyploidization, MYH10 expression is repressed by the major megakaryocyte transcription factor RUNX1. Thus, RUNX1-mediated silencing of MYH10 is required for the switch from mitosis to endomitosis, linking polyploidization with megakaryocyte differentiation.
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http://dx.doi.org/10.1038/ncomms1704DOI Listing
March 2012

Compound inheritance of a low-frequency regulatory SNP and a rare null mutation in exon-junction complex subunit RBM8A causes TAR syndrome.

Nat Genet 2012 Feb 26;44(4):435-9, S1-2. Epub 2012 Feb 26.

Department of Haematology, University of Cambridge, Cambridge, UK.

The exon-junction complex (EJC) performs essential RNA processing tasks. Here, we describe the first human disorder, thrombocytopenia with absent radii (TAR), caused by deficiency in one of the four EJC subunits. Compound inheritance of a rare null allele and one of two low-frequency SNPs in the regulatory regions of RBM8A, encoding the Y14 subunit of EJC, causes TAR. We found that this inheritance mechanism explained 53 of 55 cases (P < 5 × 10(-228)) of the rare congenital malformation syndrome. Of the 53 cases with this inheritance pattern, 51 carried a submicroscopic deletion of 1q21.1 that has previously been associated with TAR, and two carried a truncation or frameshift null mutation in RBM8A. We show that the two regulatory SNPs result in diminished RBM8A transcription in vitro and that Y14 expression is reduced in platelets from individuals with TAR. Our data implicate Y14 insufficiency and, presumably, an EJC defect as the cause of TAR syndrome.
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http://dx.doi.org/10.1038/ng.1083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3428915PMC
February 2012

Thrombocytopenia resulting from mutations in filamin A can be expressed as an isolated syndrome.

Blood 2011 Nov 29;118(22):5928-37. Epub 2011 Sep 29.

Centre de Référence des Pathologies Plaquettaires, Plateforme Technologique d'Innovation Biomédicale, Hôpital Xavier Arnozan, Pessac, France.

Filaminopathies A caused by mutations in the X-linked FLNA gene are responsible for a wide spectrum of rare diseases including 2 main phenotypes, the X-linked dominant form of periventricular nodular heterotopia (FLNA-PVNH) and the otopalatodigital syndrome spectrum of disorders. In platelets, filamin A (FLNa) tethers the principal receptors ensuring the platelet-vessel wall interaction, glycoprotein Ibα and integrin αIIbβ3, to the underlying cytoskeleton. Hemorrhage, coagulopathy, and thrombocytopenia are mentioned in several reports on patients with FLNA-PVNH. Abnormal platelet morphology in 2 patients with FLNA-PVNH prompted us to examine a third patient with similar platelet morphology previously diagnosed with immunologic thrombocytopenic purpura. Her enlarged platelets showed signs of FLNa degradation in Western blotting, and a heterozygous missense mutation in FLNA was detected. An irregular distribution of FLNa within the total platelet population was shown by confocal microscopy for all 3 patients. In vitro megakaryocyte cultures showed an abnormal differentiation, including an irregular distribution of FLNa with a frayed aspect, the presence of enlarged α-granules, and an abnormal fragmentation of the cytoplasm. Mutations in FLNA may represent an unrecognized cause of macrothrombocytopenia with an altered platelet production and a modified platelet-vessel wall interaction.
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http://dx.doi.org/10.1182/blood-2011-07-365601DOI Listing
November 2011

Down-regulation of the RUNX1-target gene NR4A3 contributes to hematopoiesis deregulation in familial platelet disorder/acute myelogenous leukemia.

Blood 2011 Dec 1;118(24):6310-20. Epub 2011 Jul 1.

Inserm, U1009, Villejuif, France.

RUNX1 encodes a DNA-binding α subunit of the core-binding factor, a heterodimeric transcription factor. RUNX1 is a master regulatory gene in hematopoiesis and its disruption is one of the most common aberrations in acute leukemia. Inactivating or dominant-negative mutations in the RUNX1 gene have been also identified in pedigrees of familial platelet disorders with a variable propensity to develop acute myeloid leukemia (FPD/AML). We performed analysis of hematopoiesis from 2 FPD/AML pedigrees with 2 distinct RUNX1 germline mutations, that is, the R139X in a pedigree without AML and the R174Q mutation in a pedigree with AML. Both mutations induced a marked increase in the clonogenic potential of immature CD34(+)CD38(-) progenitors, with some self-renewal capacities observed only for R174Q mutation. This increased proliferation correlated with reduction in the expression of NR4A3, a gene previously implicated in leukemia development. We demonstrated that NR4A3 was a direct target of RUNX1 and that restoration of NR4A3 expression partially reduced the clonogenic potential of patient progenitors. We propose that the down-regulation of NR4A3 in RUNX1-mutated hematopoietic progenitors leads to an increase in the pool of cells susceptible to be hit by secondary leukemic genetic events.
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http://dx.doi.org/10.1182/blood-2010-12-325555DOI Listing
December 2011

Aurora B is dispensable for megakaryocyte polyploidization, but contributes to the endomitotic process.

Blood 2010 Sep 14;116(13):2345-55. Epub 2010 Jun 14.

INSERM U1009, Institut Gustave Roussy, 39 rue Camille Desmoulins, Villejuif, France.

Polyploidization of megakaryocytes (MKs), the platelet precursors, occurs by endomitosis, a mitotic process that fails at late stages of cytokinesis. Expression and function of Aurora B kinase during endomitosis remain controversial. Here, we report that Aurora B is normally expressed during the human MK endomitotic process. Aurora B localized normally in the midzone or midbody during anaphase and telophase in low ploidy megakaryocytes and in up to 16N rare endomitotic MKs was observed. Aurora B was also functional during cytokinesis as attested by phosphorylation of both its activation site and MgcRacGAP, its main substrate. However, despite its activation, Aurora B did not prevent furrow regression. Inhibition of Aurora B by AZD1152-HQPA decreased cell cycle entry both in 2N to 4N and polyploid MKs and induced apoptosis mainly in 2N to 4N cells. In both MK classes, AZD1152-HQPA induced p53 activation and retinoblastoma hypophosphorylation. Resistance of polyploid MKs to apoptosis correlated to a high BclxL level. Aurora B inhibition did not impair MK polyploidization but profoundly modified the endomitotic process by inducing a mis-segregation of chromosomes and a mitotic failure in anaphase. This indicates that Aurora B is dispensable for MK polyploidization but is necessary to achieve a normal endomitotic process.
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http://dx.doi.org/10.1182/blood-2010-01-265785DOI Listing
September 2010