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Endoplasmic reticulum stress triggers the unfolded protein response (UPR) to promote cell survival or apoptosis. Transient endoplasmic reticulum stress activation has been reported to trigger megakaryocyte production, and UPR activation has been reported as a feature of megakaryocytic cancers. However, the role of UPR signaling in megakaryocyte biology is not fully understood. We studied the involvement of UPR in human megakaryocytic differentiation using PMA (phorbol 12‐myristate 13‐acetate)‐induced maturation of megakaryoblastic cell lines and thrombopoietin‐induced differentiation of human peripheral blood‐derived progenitors. Our results demonstrate that an adaptive UPR is a feature of megakaryocytic differentiation and that this response is not associated with ER stress‐induced apoptosis. Differentiation did not alter the response to the canonical endoplasmic reticulum stressors DTT or thapsigargin. However, thapsigargin, but not DTT, inhibited differentiation, consistent with the involvement of Ca2+ signaling in megakaryocyte differentiation. [ABSTRACT FROM AUTHOR]

Peripheral circadian clocks control cell proliferation and survival, but little is known about their role and regulation in megakaryocytic cells. N-methyl-D-aspartate receptor (NMDAR) regulates the central clock in the brain. The purpose of this study was to determine whether NMDAR regulates the megakaryocytic cell clock and whether the megakaryocytic clock regulates cell proliferation and cell death. We found that both the Meg-01 megakaryocytic cell line and native murine megakaryocytes expressed circadian clock genes. Megakaryocyte-directed deletion of Grin1 in mice caused significant disruption of the circadian rhythm pathway at the transcriptional level and increased expression of BMAL1 at the protein level. Similarly, both pharmacological (MK-801) and genetic (GRIN-/-) inhibition of NMDAR in Meg-01 cells in vitro resulted in widespread changes in clock gene expression including increased expression of BMAL1, the core clock transcription factor. BMAL1 overexpression reduced Meg-01 cell proliferation and altered the time-dependent expression of the cell cycle regulators MYC and WEE1, whereas BMAL1 knockdown led to increased cell death in Meg-01-GRIN1-/- cells. Our results demonstrate that NMDAR regulates the circadian clock in megakaryocytic cells and that the circadian clock component BMAL1 contributes to the control of Meg-01 cell proliferation and survival. [ABSTRACT FROM AUTHOR]

Myeloid leukemia associated with Down syndrome (ML-DS) has a unique molecular landscape that differs from other subtypes of acute myeloid leukemia. ML-DS is often preceded by a myeloproliferative neoplastic condition called transient abnormal myelopoiesis (TAM) that disrupts megakaryocytic and erythroid differentiation. Over the last two decades, many genetic and epigenetic changes in TAM and ML-DS have been elucidated. These include overexpression of molecules and micro-RNAs located on chromosome 21, GATA1 mutations, and a range of other somatic mutations and chromosomal alterations. In this review, we summarize molecular changes reported in TAM and ML-DS and provide a comprehensive discussion of these findings. Recent advances in the development of CRISPR/Cas9-modified induced pluripotent stem cell-based disease models are also highlighted. However, despite significant progress in this area, we still do not fully understand the pathogenesis of ML-DS, and there are no targeted therapies. Initial diagnosis of ML-DS has a favorable prognosis, but refractory and relapsed disease can be difficult to treat; therapeutic options are limited in Down syndrome children by their stronger sensitivity to the toxic effects of chemotherapy. Because of the rarity of TAM and ML-DS, large-scale multicenter studies would be helpful to advance molecular characterization of these diseases at different stages of development and progression. [ABSTRACT FROM AUTHOR]

Ionotropic glutamate receptors include α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR), kainate receptors (KAR), and N-methyl-D-aspartate receptors (NMDAR). All function as cation channels; AMPAR and KAR are more permeable to sodium and NMDAR to calcium ions. Compared to the brain, receptor assemblies in platelets are unusual, suggesting distinctive functionalities. There is convincing evidence that AMPAR and KAR amplify platelet function and thrombus formation in vitro and in vivo. Transgenic mice lacking GluA1 and GluK2 (AMPAR and KAR subunits, respectively) have longer bleeding times and prolonged time to thrombosis in an arterial model. In humans, rs465566 KAR gene polymorphism associates with altered in vitro platelet responses suggesting enhanced aspirin effect. The NMDAR contribution to platelet function is less well defined. NMDA at low concentrations (≤10 μM) inhibits platelet aggregation and high concentrations (≥100 μM) have no effect. However, open NMDAR channel blockers interfere with platelet activation and aggregation induced by other agonists in vitro; anti-GluN1 antibodies interfere with thrombus formation under high shear rates ex vivo; and rats vaccinated with GluN1 develop iron deficiency anemia suggestive of mild chronic bleeding. In this review, we summarize data on glutamate receptors in platelets and propose a unifying model that reconciles some of the opposing effects observed. [ABSTRACT FROM AUTHOR]

Previous whole-exome sequencing has demonstrated that melanoma tumors harbor mutations in the GRIN2A gene. GRIN2A encodes the regulatory GluN2A subunit of the glutamate-gated N-methyl-D-aspartate receptor (NMDAR), involvement of which in melanoma remains undefined. Here, we sequenced coding exons of GRIN2A in 19 lowpassage melanoma cell lines derived from patients with metastatic melanoma. Potential mutation impact was evaluated in silico, including within the GluN2A crystal structure, and clinical correlations were sought.We found that of 19 metastatic melanoma tumors, four (21%) carried five missense mutations in the evolutionarily conserved domains of GRIN2A; two were previously reported. Melanoma cells that carried these mutations were treatment-naïve. Sorting intolerant from tolerant analysis predicted that S349F, G762E, and P1132Lwould disrupt protein function.When modeled into the crystal structure of GluN2A, G762E was seen to potentially alter GluN1-GluN2A interactions and ligand binding, implying disruption to NMDAR functionality. Patients whose tumors carried non-synonymous GRIN2A mutations had faster disease progression and shorter overall survival (P <0.05). This was in contrast to the BRAF V600E mutation, found in 58% of tumors but showing no correlation with clinical outcome (P =0.963). Although numbers of patients in this study are small, and firm conclusions about the association between GRIN2A mutations and poor clinical outcome cannot be drawn, our results highlight the high prevalence of GRIN2A mutations in metastatic melanoma and suggest for the first time that mutated NMDARs impact melanoma progression. [ABSTRACT FROM AUTHOR]

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML) in which leukemic blasts have megakaryocytic features. AMKL makes up 4%-15% of newly diagnosed pediatric AML, typically affecting young children (less than 2 years old). AMKL associated with Down syndrome (DS) shows GATA1 mutations and has a favorable prognosis. In contrast, AMKL in children without DS is often associated with recurrent and mutually exclusive chimeric fusion genes and has an unfavorable prognosis. This review mainly summarizes the unique features of pediatric non-DS AMKL and highlights the development of novel therapies for high-risk patients. Due to the rarity of pediatric AMKL, large-scale multi-center studies are needed to progress molecular characterization of this disease. Better disease models are also required to test leukemogenic mechanisms and emerging therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li and Kalev‐Zylinska.)

Background: N -methyl-d-aspartate receptors (NMDARs) contribute calcium influx in megakaryocytic cells but their roles remain unclear; both pro- and anti-differentiating effects have been shown in different contexts., Objectives: The aim of this study was to clarify NMDAR contribution to megakaryocytic differentiation in both normal and leukemic cells., Methods: Meg-01, Set-2, and K-562 leukemic cell lines were differentiated using phorbol-12-myristate-13-acetate (PMA, 10 nmol L -1 ) or valproic acid (VPA, 500 μmol L -1 ). Normal megakaryocytes were grown from mouse marrow-derived hematopoietic progenitors (lineage-negative and CD41a-enriched) in the presence of thrombopoietin (30-40 nmol L -1 ). Marrow explants were used to monitor proplatelet formation in the native bone marrow milieu. In all culture systems, NMDARs were inhibited using memantine and MK-801 (100 μmol L -1 ); their effects compared against appropriate controls., Results: The most striking observation from our studies was that NMDAR antagonists markedly inhibited proplatelet formation in all primary cultures employed. Proplatelets were either absent (in the presence of memantine) or short, broad and intertwined (with MK-801). Earlier steps of megakaryocytic differentiation (acquisition of CD41a and nuclear ploidy) were maintained, albeit reduced. In contrast, in leukemic Meg-01 cells, NMDAR antagonists inhibited differentiation in the presence of PMA and VPA but induced differentiation when applied by themselves., Conclusions: NMDAR-mediated calcium influx is required for normal megakaryocytic differentiation, in particular proplatelet formation. However, in leukemic cells, the main NMDAR role is to inhibit differentiation, suggesting diversion of NMDAR activity to support leukemia growth. Further elucidation of the NMDAR and calcium pathways in megakaryocytic cells may suggest novel ways to modulate abnormal megakaryopoiesis.

GRIN2A mutations are frequent in melanoma tumours but their role in disease is not well understood. GRIN2A encodes a modulatory subunit of the N-methyl-d-aspartate receptor (NMDAR). We hypothesized that certain GRIN2A mutations increase NMDAR function and support melanoma growth through oncogenic effects. This hypothesis was tested using 19 low-passage melanoma cell lines, four of which carried novel missense mutations in GRIN2A that we previously reported. We examined NMDAR expression, function of a calcium ion (Ca 2+ ) channel and its contribution to cell growth using pharmacological modulators; findings were correlated with the presence or absence of GRIN2A mutations. We found that NMDAR expression was low in all melanoma cell lines, independent of GRIN2A mutations. In keeping with this, NMDAR-mediated Ca 2+ influx and its contribution to cell proliferation were weak in most cell lines. However, certain GRIN2A mutations and culture media with lower glutamate levels enhanced NMDAR effects on cell growth and invasion. The main finding was that G762E was associated with higher glutamate-mediated Ca 2+ influx and stronger NMDAR contribution to cell proliferation, compared with wild-type GRIN2A and other GRIN2A mutations. The pro-invasive phenotype of mutated cell lines was increased in culture medium containing less glutamate, implying environmental modulation of mutation effects. In conclusion, NMDAR ion channel function is low in cultured melanoma cells but supports cell proliferation and invasion. Selected GRIN2A mutations, such as G762E, are associated with oncogenic consequences that can be modulated by extracellular glutamate. Primary cultures may be better suited to determine the role of the NMDAR in melanoma in vivo., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The transcription factor Runx1 is essential for the development of definitive hematopoietic stem cells (HSCs) during vertebrate embryogenesis and is transcribed from 2 promoters, P1 and P2, generating 2 major Runx1 isoforms. We have created 2 stable runx1 promoter zebrafish-transgenic lines that provide insight into the roles of the P1 and P2 isoforms during the establishment of definitive hematopoiesis. The Tg(runx1P1:EGFP) line displays fluorescence in the posterior blood island, where definitive erythromyeloid progenitors develop. The Tg(runx1P2:EGFP) line marks definitive HSCs in the aorta-gonad-mesonephros, with enhanced green fluorescent protein-labeled cells later populating the pronephros and thymus. This suggests that a function of runx1 promoter switching is associated with the establishment of discrete definitive blood progenitor compartments. These runx1 promoter-transgenic lines are novel tools for the study of Runx1 regulation and function in normal and malignant hematopoiesis. The ability to visualize and isolate fluorescently labeled HSCs should contribute to further elucidating the complex regulation of HSC development.

Studies in zebrafish have potential to contribute to understanding of the vertebrate hematopoietic and vasculogenic systems. Our research has examined the roles of several molecules in pathways that lead to the development of blood and vessels in zebrafish, and has provided insights into the regulation of these processes. Gdf6a/radar, a member of the bone morphogenetic protein (BMP) family, is expressed in the zebrafish hypochord and primitive gut endoderm; structures that flank the developing dorsal aorta and posterior cardinal vein. This pattern of expression positions Gdf6a/radar as a candidate regulator of vasculogenesis. Support for such a role has come from experiments where Gdf6a/radar function was depleted with antisense morpholino oligonucleotides. This resulted in vascular leakiness, suggesting that Gdf6a/radar is involved in maintenance of vascular integrity. The transcription factor Runx1 is known to play a critical role in mammalian definitive hematopoiesis. When Runx1 expression domains and function were analyzed in zebrafish, the importance of this gene in definitive hematopoiesis was confirmed. However there was also evidence for a wider role, including involvement in vascular development and neuropoiesis. This work has laid the foundation for an ethylnitrosourea (ENU) mutagenesis screen based on runx1 whole-mount in situ hybridzation, that aims to identify genes operative in the runx1 pathway. An additional member of the Runx family, Runx3, is also involved in developmental hematopoiesis, with a function distinct from that of Runx1. We hypothesize that Runx1 and Runx3 form a continuum of transcriptional control within the hematopoietic system. An added attraction of zebrafish is that models of human disease can be generated, and we have shown that this system has potential for the study of Runx1-mediated leukemogenesis.