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.
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.
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. IntroductionFamilial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML, OMIM 601399) is an autosomal dominant disorder characterized by dysmegakaryopoiesis, qualitative and quantitative platelet defects, and a propensity to develop myelodysplastic syndromes (MDSs) and/or AML. Several types of heterozygous germ-line mutations or deletions in RUNX1, including missense, frameshift, and nonsense mutations or large intragenic deletion or single nucleotide deletion in the Runt domain have been identified in FPD/AML. The progression to AML is often linked to the somatic alteration of the second RUNX1 allele, 1 supporting the fact that RUNX1 acts as a tumor suppressor gene. RUNX1 (also known as AML1, PEBP2aB, or CBFA2) is 1 of the 3 DNA-binding ␣ subunits of the hematopoietic transcription complex called core binding factor (CBF). RUNX1 contains both a runt homology domain (RHD), which mediates DNA binding and heterodimerization with the core binding factor  (CBF) subunit to stabilize the interaction of the complex with DNA and to protect CBF from proteolytic degradation. The C-terminal domain of RUNX1 is responsible for transcriptional activation. RUNX1 can act as a repressor or an activator depending on the cellular context. It regulates positively different hematopoietic genes encoding cytokines and their receptors, such as IL-3, 2 GM-CSF, and M-CSF or negatively the CD4 gene contributing thus to impaired T-cell development. 3 Somatic alterations in RUNX1 are frequently found in AML, MDS, and chronic myelomonocytic leukemia (CMML).In different mouse models, RUNX1 was shown to be essential for establishing definitive hematopoiesis. 4 It is required for the generation of hematopoietic stem cells (HSCs) from the aorta, but not later on. Targeted deletion of RUNX1 in adult HSCs led to their ...
Summary. 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.
Acute megakaryoblastic leukemia (AMKL) is a form of acute myeloid leukemia (AML) associated with a poor prognosis. The genetics and pathophysiology of AMKL are not well understood. We generated a knockin mouse model of the one twenty-two-megakaryocytic acute leukemia (OTT-MAL) fusion oncogene that results from the t(1;22)(p13;q13) translocation specifically associated with a subtype of pediatric AMKL. We report here that OTT-MAL expression deregulated transcriptional activity of the canonical Notch signaling pathway transcription factor recombination signal binding protein for immunoglobulin κ J region (RBPJ) and caused abnormal fetal megakaryopoiesis. Furthermore, cooperation between OTT-MAL and an activating mutation of the thrombopoietin receptor myeloproliferative leukemia virus oncogene (MPL) efficiently induced a short-latency AMKL that recapitulated all the features of human AMKL, including megakaryoblast hyperproliferation and maturation block, thrombocytopenia, organomegaly, and extensive fibrosis. Our results establish that concomitant activation of RBPJ (Notch signaling) and MPL (cytokine signaling) transforms cells of the megakaryocytic lineage and suggest that specific targeting of these pathways could be of therapeutic value for human AMKL. IntroductionAcute megakaryoblastic leukemia (AMKL) is a heterogeneous subtype of acute myeloid leukemia (AML) and is more frequent in children than in adults (1-3). The molecular basis of AMKL is poorly understood in adults, whereas 2 major molecular subtypes are recognized in pediatric AMKL. The first group is represented by Down syndrome (DS) patients with both transient myeloproliferative disease (transient MPD) and AMKL who present with acquired GATA-binding protein 1 (GATA1) mutations, resulting in an N-terminal truncated GATA1 short (GATA1s) protein (4). The second group occurs in infants and is associated with the t(1;22)(p13;q13) chromosomal translocation, resulting in expression of the one twenty-two megakaryocytic acute leukemia (OTT-MAL) (also known as RBM15-MKL1) fusion protein (5-7).
The interaction of integrin αE(CD103)β7, often expressed on tumor-infiltrating T lymphocytes, with its cognate ligand, the epithelial cell marker E-cadherin on tumor cells, plays a major role in antitumor CTL responses. CD103 is induced on CD8 T cells upon TCR engagement and exposure to TGF-β1, abundant within the tumor microenvironment. However, the transcriptional mechanisms underlying the cooperative role of these two signaling pathways in inducing CD103 expression in CD8 T lymphocytes remain unknown. Using a human CTL system model based on a CD8+/CD103− T cell clone specific of a lung tumor–associated Ag, we demonstrated that the transcription factors Smad2/3 and NFAT-1 are two critical regulators of this process. We also identified promoter and enhancer elements of the human ITGAE gene, encoding CD103, involved in its induction by these transcriptional regulators. Overall, our results explain how TGF-β1 can participate in CD103 expression on locally TCR-engaged Ag-specific CD8 T cells, thus contributing to antitumor CTL responses and cancer cell destruction.
Summary. Each day in every human, approximately 1 · 10 11 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.
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