William Vainchenker

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.

Activating mutations in human acute megakaryoblastic leukemia

Oncogenic activation of tyrosine kinase signaling pathway is recurrent in human leukemia. To gain insight into the oncogenic process leading to acute megakaryoblastic leukemia (AMKL), we performed sequence analyses of a subset of oncogenes known to be activated in human myeloid and myeloproliferative disorders. In a series of human AMKL samples from both Down syndrome and non-Down syndrome patients, mutations were identified within KIT, FLT3, JAK2, JAK3, and MPL genes, with a higher frequency in DS than in non-DS patients. The novel mutations were analyzed using BaF3 cells, showing that JAK3 mutations were activating mutations. Finally, we report a novel constitutively active MPL mutant, MPLT487A, observed in a non-Down syndrome childhood AMKL that induces a myeloproliferative disease in mouse bone marrow transplantation assay.

From hematopoietic stem cells to platelets

Summary.u2002 Megakaryocytopoiesis is the process that leads to the production of platelets. This process involves the commitment of multipotent hematopoietic stem cells toward megakaryocyte (MK) progenitors, the proliferation and differentiation of MK progenitors, the polyploidization of MK precursors and the maturation of MK. Mature MK produce platelets by cytoplasmic fragmentation occurring through a dynamic and regulated process, called proplatelet formation, and consisting of long pseudopodial elongations that break in the blood flow. Recent insights have demonstrated that the MK and erythroid lineages are tightly associated at both the cellular and molecular levels, especially in the transcription factors that regulate their differentiation programs. Megakaryocytopoiesis is regulated by two types of transcription factors, those regulating the differentiation process, such as GATA‐1, and those regulating proplatelet formation, such as NF‐E2. The humoral factor thrombopoietin (TPO) is the primary regulator of MK differentiation and platelet production through the stimulation of its receptor MPL. Numerous acquired or congenital pathologies of the MK lineage are now explained by molecular abnormalities in the activity of the transcription factors involved in megakaryocytopoiesis, in the Tpo or c‐mpl genes, as well as in signaling molecules associated with MPL. The recent development of MPL agonists may provide efficient agents for the treatment of some thrombocytopenias.

EKLF restricts megakaryocytic differentiation at the benefit of erythrocytic differentiation.

Previous observations suggested that functional antagonism between FLI-1 and EKLF might be involved in the commitment toward erythrocytic or megakaryocytic differentiation. We show here, using inducible shRNA expression, that EKLF knockdown in mouse erythroleukemia (MEL) cells decreases erythrocytic and increases megakaryocytic as well as Fli-1 gene expression. Chromatin immunoprecipitation analyses revealed that the increase in megakaryocytic gene expression is associated with a marked increase in RNA pol II and FLI-1 occupancy at their promoters, albeit FLI-1 protein levels are only minimally affected. Similarly, we show that human CD34(+) progenitors infected with shRNA lentivirus allowing EKLF knockdown generate an increased number of differentiated megakaryocytic cells associated with increased levels of megakaryocytic and Fli-1 gene transcripts. Single-cell progeny analysis of a cell population enriched in bipotent progenitors revealed that EKLF knockdown increases the number of megakaryocytic at the expense of erythrocytic colonies. Taken together, these data indicate that EKLF restricts megakaryocytic differentiation to the benefit of erythrocytic differentiation and suggest that this might be at least partially mediated by the inhibition of FLI-1 recruitment to megakaryocytic and Fli-1 gene promoters.

Regulation of megakaryocyte maturation and platelet formation

Summary.u2002 Each day in every human, approximately 1u2003×u20031011 platelets are produced by the cytoplasmic fragmentation of megakaryocytes (MK), their marrow precursor cells. Platelets are the predominating factor in the process of hemostasis and thrombosis. Recent studies have shown that platelets also play a hitherto unsuspected role in several other processes such as inflammation, innate immunity, neoangiogenesis and tumor metastasis. The late phases of MK differentiation identified by polyploidization, maturation and organized fragmentation of the cytoplasm leading to the release of platelets in the blood stream represent a unique model of differentiation. The molecular and cellular mechanisms regulating platelet biogenesis are better understood and may explain several platelet disorders. This review focuses on MK polyploidization, and platelet formation, and discusses their alteration in some platelet disorders.

A Senescence-Like Cell-Cycle Arrest Occurs During Megakaryocytic Maturation: Implications for Physiological and Pathological Megakaryocytic Proliferation

During normal megakaryocyte development, in response to thrombopoetin, mature cells enter a senescence-like state in which they shed platelets; this state, characterized by cell cycle arrest, is defective in malignant megakaryocytes.

P19INK4D links endomitotic arrest and megakaryocyte maturation and is regulated by AML-1

The molecular mechanisms that regulate megakaryocyte (MK) ploidization are poorly understood. Using MK differentiation from primary human CD34(+) cells, we observed that p19(INK4D) expression was increased both at the mRNA and protein levels during ploidization. p19(INK4D) knockdown led to a moderate increase (31.7% +/- 5%) in the mean ploidy of MKs suggesting a role of p19(INK4D) in the endomitotic arrest. This increase in ploidy was associated with a decrease in the more mature MK population (CD41(high)CD42(high)) at day 9 of culture, which was related to a delay in differentiation. Inversely, p19(INK4D) overexpression in CD34(+) cells resulted in a decrease in mean ploidy level associated with an increase in CD41 and CD42 expression in each ploidy class. Confirming these in vitro results, bone marrow MKs from p19(INK4D) KO mice exhibited an increase in mean ploidy level from 18.7N (+/- 0.58N) to 52.7N (+/- 12.3N). Chromatin immunoprecipitation assays performed in human MKs revealed that AML-1 binds in vivo the p19(INK4D) promoter. Moreover, AML-1 inhibition led to the p19(INK4D) down-regulation in human MKs. These results may explain the molecular link at the transcriptional level between the arrest of endomitosis and the acceleration of MK differentiation.

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.

Selective reduction of JAK2V617F-dependent cell growth by siRNA/shRNA and its reversal by cytokines.

The JAK(V617F) mutation is responsible for the majority of breakpoint cluster region (BCR)/Abelson (ABL)-negative myeloproliferative disorders. Ongoing clinical trials of Janus kinase 2 (JAK2) inhibitors in myeloproliferative disorder patients use small molecules targeting both wild-type and mutated JAK2. To selectively target malignant cells, we developed JAK2(V617F)-specific small interfering RNAs or short hairpin RNAs. Expression of these RNAs in cell lines or CD34(+) cells from patients reduced JAK2(V617F)-driven autonomous cell proliferation. Mechanisms of inhibition involved selective JAK2(V617F) protein down-regulation, and consequently, decrease in signal transducer and activator of transcription 5 phosphorylation, cell-cycle progression, and cell survival. However, the addition of high concentrations of cytokines to cell lines or erythropoietin to patient cells greatly reduced growth inhibition. Similarly, the efficacy of a JAK2 small molecule inhibitor on cell line and patient cell proliferation dose dependently decreased with the addition of cytokines. Our results demonstrate that it is possible to specifically target JAK2(V617F) by RNA interference (RNAi) strategies. In addition, cytokines partially reverse the inhibition induced by both RNAi and small molecule approaches. This strongly suggests that patient cytokine levels in current JAK2 inhibitor clinical trials modulate the outcome of these therapies.

LYL-1 deficiency induces a stress erythropoiesis

OBJECTIVEnLYL-1 is a transcription factor containing a basic helix-loop-helix motif closely related to SCL/TAL-1, a regulator of erythroid differentiation. Because LYL-1 is expressed in erythroid cell populations, we addressed its role in erythropoiesis using knockin mice.nnnMATERIALS AND METHODSnErythropoiesis of LYL-1(-/-) mice was studied by progenitor assays, flow cytometry, reconstitution assays, and functional tests. Expression of LYL-1, SCL, and GATA-1 was assessed at messenger RNA level by quantitative reverse transcription polymerase chain reaction.nnnRESULTSnLYL-1(-/-) mice displayed decreased erythropoiesis with a partial arrest in differentiation, and enhanced apoptosis associated with decreased Bcl-x(L) expression in the bone marrow (BM). In addition, LYL-1(-/-) BM cells were severely impaired in their abilities to reconstitute the erythroid lineage in competitive assays, suggesting a cell autonomous abnormality of erythropoiesis. In parallel, erythroid progenitor and precursor cells were significantly increased in the spleen of LYL-1(-/-) mice. Expression of LYL-1 was differentially regulated during maturation of erythroblasts and strikingly different between spleen- and BM-derived erythroblasts. Expression of LYL-1 decreased during erythroid differentiation in the spleen whereas it increased in the BM to reach the same level in mature erythroblasts as in the soleen. Loss of Lyl-1 expression was accompanied with an increase of SCL/TAL-1 and GATA-1 transcripts in spleen but not in BM-derived erythroblasts. Furthermore, phenylhydrazine-induced stress erythropoiesis was elevated in LYL-1(-/-) mice and mutant BM and spleen erythroid progenitors were hypersensitive to erythropoietin.nnnCONCLUSIONSnTaken together, these results suggest that LYL-1 plays a definite role in erythropoiesis, albeit with different effects in BM specifically regulating basal erythropoiesis, and spleen, controlling stress-induced erythropoiesis.