Najet Debili

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

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.

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.

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.

In Vitro Effects of Mpl Ligand on Human Hemopoietit Progenitor Cells

Regulation of megakaryocytopoiesis and platelet production is a complex phenomenon. Numerous pleiotropic cytokines act in vivo and in vitro on megakaryocytopoiesis (1). Historically, studies on the regulation of megakaryocytopoiesis were largely dominated by the concept of humoral regulation, a concept based on the model of erythropoiesis and formulated before the development of clonal assays for hemopoietic progenitors. In animals, induction of acute thrombocytopenia with antiplatelet antibodies rapidly results in an increase of platelet production as measured by isotopic techniques. Simultaneously, an increase in platelet size, along with megakaryocyte number, size, ploidy, and cytoplasmic maturation is observed. Despite 35 years of work, the factor responsible for these effects (thrombopoietin, TPO) (2) could not be purified to homogeneity (3), and TPO was thought to be a late differentiation factor that acted synergistically with other growth factor, such as interleukin (IL)-3 (1,3) (see Chapter 5).

Mpl Expression and Functional Role in Human Megakaryocytopoiesis

The c-mpl gene was discovered in 1990 as the cellular homologue of v-mpl, the oncogene of the murine myeloproliferative leukemia virus, MPLV (see Chapter 6) (1). In mice, v-mpl induces a lethal myeloproliferative disease involving multiple hemopoietic progenitors (2–4). In vitro, v-mpl transformed multipotential and lineage-committed progenitors, leading to acquisition of growth-factor independence for both proliferation and terminal maturation (1), These data showed that v-mpl was a potent deregulator of hemopoiesis, but they did not provide any information about the function of the normal c-mpl proto-oncogene.