Disrupted filamin A/αIIbß3 interaction induces macrothrombocytopenia by increasing RhoA activity.

Filamin A (FLNa) links the cell membrane with the cytoskeleton and is central in several cellular processes. Heterozygous mutations in the X-linked FLNA 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/αIIbß3 interaction in the downregulation of RhoA activity. This was confirmed by experiments based on the overexpression of FLNa mutants deleted in the αIIbß3-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/αIIbß3 interaction.

Multilayer intraclonal heterogeneity in chronic myelomonocytic leukemia.

The functional diversity of cells that compose myeloid malignancies, i.e., the respective roles of genetic and epigenetic heterogeneity in this diversity, remains poorly understood. This question is addressed in chronic myelomonocytic leukemia, a myeloid neoplasm in which clinical diversity contrasts with limited genetic heterogeneity. To generate induced pluripotent stem cell clones, we reprogrammed CD34+ cells collected from a patient with a chronic myelomonocytic leukemia in which whole exome sequencing of peripheral blood monocyte DNA had identified 12 gene mutations, including a mutation in KDM6A and two heterozygous mutations in TET2 in the founding clone and a secondary KRAS(G12D) mutation. CD34+ cells from an age-matched healthy donor were also reprogrammed. We captured a part of the genetic heterogeneity observed in the patient, i.e. we analyzed five clones with two genetic backgrounds, without and with the KRAS(G12D) mutation. Hematopoietic differentiation of these clones recapitulated the main features of the patient's disease, including overproduction of granulomonocytes and dysmegakaryopoiesis. These analyses also disclosed significant discrepancies in the behavior of hematopoietic cells derived from induced pluripotent stem cell clones with similar genetic background, correlating with limited epigenetic changes. These analyses suggest that, beyond the coding mutations, several levels of intraclonal heterogeneity may participate in the yet unexplained clinical heterogeneity of the disease.

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

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.

Carboxyl-terminal-dependent recruitment of nonmuscle myosin II to megakaryocyte contractile ring during polyploidization.

Endomitosis is a unique megakaryocyte (MK) differentiation process that is the consequence of a late cytokinesis failure associated with a contractile ring defect. Evidence from in vitro studies has revealed the distinct roles of 2 nonmuscle myosin IIs (NMIIs) on MK endomitosis: only NMII-B (MYH10), but not NMII-A (MYH9), is localized in the MK contractile ring and implicated in mitosis/endomitosis transition. Here, we studied 2 transgenic mouse models in which nonmuscle myosin heavy chain (NMHC) II-A was genetically replaced either by II-B or by a chimeric NMHCII that combined the head domain of II-A with the rod and tail domains of II-B. This study provides in vivo evidence on the specific role of NMII-B on MK polyploidization. It demonstrates that the carboxyl-terminal domain of the heavy chains determines myosin II localization to the MK contractile ring and is responsible for the specific role of NMII-B in MK polyploidization.

The formin DIAPH1 (mDia1) regulates megakaryocyte proplatelet formation by remodeling the actin and microtubule cytoskeletons.

Megakaryocytes are highly specialized precursor cells that produce platelets via cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) requires profound changes in microtubule and actin organization. In this work, we demonstrated that DIAPH1 (mDia1), a mammalian homolog of Drosophila diaphanous that works as an effector of the small GTPase Rho, negatively regulates PPF by controlling the dynamics of the actin and microtubule cytoskeletons. Moreover, we showed that inhibition of both DIAPH1 and the Rho-associated protein kinase (Rock)/myosin pathway increased PPF via coordination of both cytoskeletons. We provide evidence that 2 major effectors of the Rho GTPase pathway (DIAPH1 and Rock/myosin II) are involved not only in Rho-mediated stress fibers assembly, but also in the regulation of microtubule stability and dynamics during PPF.

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

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.

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

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.

Role of Rho-GTPases in megakaryopoiesis.

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 RhoA or Cdc42 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.Abbreviations: 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 ß1VEGF: Vascular endothelial growth factorWAS: Wiskott Aldrich syndromeWASP: Wiskott Aldrich syndrome proteinWAVE: WASP-family verprolin-homologous proteinWIP: WASP-interacting proteinYAP: Yes-associated protein.

Induced Pluripotent Stem Cells Enable Disease Modeling and Drug Screening in Calreticulin del52 and ins5 Myeloproliferative Neoplasms.

Mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and primary myelofibrosis patients. To address the contribution of the human CALR mutants to the pathogenesis of myeloproliferative neoplasms (MPNs) in an endogenous context, we modeled the CALRdel52 and CALRins5 mutants by induced pluripotent stem cell (iPSC) technology using CD34+ progenitors from 4 patients. We describe here the generation of several clones of iPSC carrying heterozygous CALRdel52 or CALRins5 mutations. We showed that CALRdel52 induces a stronger increase in progenitors than CALRins5 and that both CALRdel52 and CALRins5 mutants favor an expansion of the megakaryocytic lineage. Moreover, we found that both CALRdel52 and CALRins5 mutants rendered colony forming unit-megakaryocyte (CFU-MK) independent from thrombopoietin (TPO), and promoted a mild constitutive activation level of signal transducer and activator of transcription 3 in megakaryocytes. Unexpectedly, a mild increase in the sensitivity of colony forming unit-granulocyte (CFU-G) to granulocyte-colony stimulating factor was also observed in iPSC CALRdel52 and CALRins5 compared with control iPSC. Moreover, CALRdel52-induced megakaryocytic spontaneous growth is more dependent on Janus kinase 2/phosphoinositide 3-kinase/extracellular signal-regulated kinase than TPO-mediated growth and opens a therapeutic window for treatments in CALR-mutated MPN. The iPSC models described here represent an interesting platform for testing newly developed inhibitors. Altogether, this study shows that CALR-mutated iPSC recapitulate MPN phenotypes in vitro and may be used for drug screening.

Miniaturized 3D bone marrow tissue model to assess response to Thrombopoietin-receptor agonists in patients.

Thrombocytopenic disorders have been treated with the Thrombopoietin-receptor agonist Eltrombopag. Patients with the same apparent form of thrombocytopenia may respond differently to the treatment. We describe a miniaturized bone marrow tissue model that provides a screening bioreactor for personalized, pre-treatment response prediction to Eltrombopag for individual patients. Using silk fibroin, a 3D bone marrow niche was developed that reproduces platelet biogenesis. Hematopoietic progenitors were isolated from a small amount of peripheral blood of patients with mutations in ANKRD26 and MYH9 genes, who had previously received Eltrombopag. The ex vivo response was strongly correlated with the in vivo platelet response. Induced Pluripotent Stem Cells (iPSCs) from one patient with mutated MYH9 differentiated into functional megakaryocytes that responded to Eltrombopag. Combining patient-derived cells and iPSCs with the 3D bone marrow model technology allows having a reproducible system for studying drug mechanisms and for individualized, pre-treatment selection of effective therapy in Inherited Thrombocytopenias.

Platelets are tiny cell fragments essential for blood to clot. They are created and released into the bloodstream by megakaryocytes, giant cells that live in the bone marrow. In certain genetic diseases, such as Inherited Thrombocytopenia, the bone marrow fails to produce enough platelets: this leaves patients extremely susceptible to bruising, bleeding, and poor clotting after an injury or surgery. Certain patients with Inherited Thrombocytopenia respond well to treatments designed to boost platelet production, but others do not. Why these differences exist could be investigated by designing new test systems that recreate the form and function of bone marrow in the laboratory. However, it is challenging to build the complex and poorly understood bone marrow environment outside of the body. Here, Di Buduo et al. have developed an artificial three-dimensional miniature organ bioreactor system that recreates the key features of bone marrow. In this system, megakaryocytes were grown from patient blood samples, and hooked up to a tissue scaffold made of silk. The cells were able to grow as if they were in their normal environment, and they could shed platelets into an artificial bloodstream. After treating megakaryocytes with drugs to stimulate platelet production, Di Buduo et al. found that the number of platelets recovered from the bioreactor could accurately predict which patients would respond to these drugs in the clinic. This new test system enables researchers to predict how a patient will respond to treatment, and to tailor therapy options to each individual. This technology could also be used to test new drugs for Inherited Thrombocytopenias and other blood-related diseases; if scaled-up, it could also, one day, generate large quantities of lab-grown blood cells for transfusion.

Megakaryocyte endomitosis is a failure of late cytokinesis related to defects in the contractile ring and Rho/Rock signaling.

Megakaryocyte (MK) is the naturally polyploid cell that gives rise to platelets. Polyploidization occurs by endomitosis, which was a process considered to be an incomplete mitosis aborted in anaphase. Here, we used time-lapse confocal video microscopy to visualize the endomitotic process of primary human megakaryocytes. Our results show that the switch from mitosis to endomitosis corresponds to a late failure of cytokinesis accompanied by a backward movement of the 2 daughter cells. No abnormality was observed in the central spindle of endomitotic MKs. A furrow formation was present, but the contractile ring was abnormal because accumulation of nonmuscle myosin IIA was lacking. In addition, a defect in cell elongation was observed in dipolar endomitotic MKs during telophase. RhoA and F-actin were partially concentrated at the site of furrowing. Inhibition of the Rho/Rock pathway caused the disappearance of F-actin at midzone and increased MK ploidy level. This inhibition was associated with a more pronounced defect in furrow formation as well as in spindle elongation. Our results suggest that the late failure of cytokinesis responsible for the endomitotic process is related to a partial defect in the Rho/Rock pathway activation.

Megakaryocyte and polyploidization.

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.

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

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.

Presence of a defect in karyokinesis during megakaryocyte endomitosis.

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.

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

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.