Megakaryocyte maturation involves activation of the adaptive unfolded protein response.

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.

Deletion of Grin1 in mouse megakaryocytes reveals NMDA receptor role in platelet function and proplatelet formation.

The process of proplatelet formation (PPF) requires coordinated interaction between megakaryocytes (MKs) and the extracellular matrix (ECM), followed by a dynamic reorganization of the actin and microtubule cytoskeleton. Localized fluxes of intracellular calcium ions (Ca2+) facilitate MK-ECM interaction and PPF. Glutamate-gated N-methyl-D-aspartate receptor (NMDAR) is highly permeable to Ca2+. NMDAR antagonists inhibit MK maturation ex vivo; however, there are no in vivo data. Using the Cre-loxP system, we generated a platelet lineage-specific knockout mouse model of reduced NMDAR function in MKs and platelets (Pf4-Grin1-/- mice). Effects of NMDAR deletion were examined using well-established assays of platelet function and production in vivo and ex vivo. We found that Pf4-Grin1-/- mice had defects in megakaryopoiesis, thrombopoiesis, and platelet function, which manifested as reduced platelet counts, lower rates of platelet production in the immune model of thrombocytopenia, and prolonged tail bleeding time. Platelet activation was impaired to a range of agonists associated with reduced Ca2+ responses, including metabotropic like, and defective platelet spreading. MKs showed reduced colony and proplatelet formation. Impaired reorganization of intracellular F-actin and α-tubulin was identified as the main cause of reduced platelet function and production. Pf4-Grin1-/- MKs also had lower levels of transcripts encoding crucial ECM elements and enzymes, suggesting NMDAR signaling is involved in ECM remodeling. In summary, we provide the first genetic evidence that NMDAR plays an active role in platelet function and production. NMDAR regulates PPF through a mechanism that involves MK-ECM interaction and cytoskeletal reorganization. Our results suggest that NMDAR helps guide PPF in vivo.

N-methyl-D-aspartate receptor regulates the circadian clock in megakaryocytic cells and impacts cell proliferation through BMAL1.

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.

Why was the study done? Time of day impacts platelet function and production. Our bodies are informed about external time by the brain, but all other cells including platelet precursors megakaryocytes also have their own clock.Circadian disruption contributes to disorders such as thrombosis (e.g. stroke and heart attacks) and blood cancers (e.g. leukemia). However, the mechanism of circadian control in megakaryocytes remains poorly elucidated.N-methyl-D-aspartate receptor (NMDAR) regulates circadian clock in the brain and is expressed in megakaryocytes, thus we hypothesized that NMDAR also regulates circadian clock in megakaryocytic cells.What did the researchers do and find? We used Meg-01 cell line, its genetically modified version with deleted NMDAR, and data from murine megakaryocytes to determine the NMDAR impact on the clock in these cells.We found that megakaryocytic cells had all the machinery required to maintain their own circadian clock. NMDAR deletion disrupted circadian clock in megakaryocytic cells.Manipulation of circadian clock in Meg-01 cells (through BMAL1 overexpression) impacted proliferation and survival of cells.What do the results mean? Megakaryocytic cells have their own circadian clock regulated by NMDAR, and its disruption impacts cell proliferation.What is the objective influence on the wider field? It is possible that deregulated function of megakaryocytes that occurs in disease can be corrected through the modulation of NMDAR or other components of the cellular circadian clock, thus further studies to develop and test such strategies in disease models are warranted.

Ionotropic glutamate receptors in platelets: opposing effects and a unifying hypothesis.

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.

Platelet-Reactive Antibodies in Patients after Ischaemic Stroke-An Epiphenomenon or a Natural Protective Mechanism.

Ischaemic brain damage induces autoimmune responses, including the production of autoantibodies with potential neuroprotective effects. Platelets share unexplained similarities with neurons, and the formation of anti-platelet antibodies has been documented in neurological disorders. The aim of this study was to investigate the presence of anti-platelet antibodies in the peripheral blood of patients after ischaemic stroke and determine any clinical correlations. Using a flow cytometry-based platelet immunofluorescence method, we detected platelet-reactive antibodies in 15 of 48 (31%) stroke patients and two of 50 (4%) controls (p < 0.001). Western blotting revealed heterogeneous reactivities with platelet proteins, some of which overlapped with brain proteins. Stroke patients who carried anti-platelet antibodies presented with larger infarcts and more severe neurological dysfunction, which manifested as higher scores on the National Institutes of Health Stroke Scale (NIHSS; p = 0.009), but they had a greater recovery in the NIHSS by the time of hospital discharge (day 7 ± 2) compared with antibody-negative patients (p = 0.043). Antibodies from stroke sera reacted more strongly with activated platelets (p = 0.031) and inhibited platelet aggregation by up to 30.1 ± 2.8% (p < 0.001), suggesting the potential to interfere with thrombus formation. In conclusion, platelet-reactive antibodies can be found in patients soon after ischaemic stroke and correlate with better short-term outcomes, suggesting a potential novel mechanism limiting thrombosis.

Inhibition of NMDA receptor function with an anti-GluN1-S2 antibody impairs human platelet function and thrombosis.

GluN1 is a mandatory component of N-methyl-D-aspartate receptors (NMDARs) best known for their roles in the brain, but with increasing evidence for relevance in peripheral tissues, including platelets. Certain anti-GluN1 antibodies reduce brain infarcts in rodent models of ischaemic stroke. There is also evidence that human anti-GluN1 autoantibodies reduce neuronal damage in stroke patients, but the underlying mechanism is unclear. This study investigated whether anti-GluN1-mediated neuroprotection involves inhibition of platelet function. Four commercial anti-GluN1 antibodies were screened for their abilities to inhibit human platelet aggregation. Haematological parameters were examined in rats vaccinated with GluN1. Platelet effects of a mouse monoclonal antibody targeting the glycine-binding region of GluN1 (GluN1-S2) were tested in assays of platelet activation, aggregation and thrombus formation. The epitope of anti-GluN1-S2 was mapped and the mechanism of antibody action modelled using crystal structures of GluN1. Our work found that rats vaccinated with GluN1 had a mildly prolonged bleeding time and carried antibodies targeting mostly GluN1-S2. The monoclonal anti-GluN1-S2 antibody (from BD Biosciences) inhibited activation and aggregation of human platelets in the presence of adrenaline, adenosine diphosphate, collagen, thrombin and a protease-activated receptor 1-activating peptide. When human blood was flowed over collagen-coated surfaces, anti-GluN1-S2 impaired thrombus growth and stability. The epitope of anti-GluN1-S2 was mapped to α-helix H located within the glycine-binding clamshell of GluN1, where the antibody binding was computationally predicted to impair opening of the NMDAR channel. Our results indicate that anti-GluN1-S2 inhibits function of human platelets, including dense granule release and thrombus growth. Findings add to the evidence that platelet NMDARs regulate thrombus formation and suggest a novel mechanism by which anti-GluN1 autoantibodies limit stroke-induced neuronal damage.

Insights into the role of JAK2-I724T variant in myeloproliferative neoplasms from a unique cohort of New Zealand patients.

OBJECTIVES: This study aimed to compile bioinformatic and experimental information for JAK2 missense variants previously reported in myeloproliferative neoplasms (MPN) and determine if germline JAK2-I724T, recently found to be common in New Zealand Polynesians, associates with MPN. METHODS: For all JAK2 variants found in the literature, gnomAD_exome allele frequencies were extracted and REVEL scores were calculated using the dbNSFP database. We investigated the prevalence of JAK2-I724T in a cohort of 111 New Zealand MPN patients using a TaqMan assay, examined its allelic co-occurrence with JAK2-V617F using Oxford Nanopore sequencing, and modelled the impact of I724T on JAK2 using I-Mutant and ChimeraX software. RESULTS: Several non-V617F JAK2 variants previously reported in MPN had REVEL scores greater than 0.5, suggesting pathogenicity. JAK2-I724T (REVEL score 0.753) was more common in New Zealand Polynesian MPN patients (n = 2/27; 7.4%) than in other New Zealand patients (n = 0/84; 0%) but less common than expected for healthy Polynesians (n = 56/377; 14.9%). Patients carrying I724T (n = 2), one with polycythaemia vera and one with essential thrombocythaemia, had high-risk MPN. Both patients with JAK2-I724T were also positive for JAK2-V617F, found on the same allele as I724T, as well as separately. In silico modelling did not identify noticeable structural changes that would give JAK2-I724T a gain-of-function. CONCLUSION: Several non-canonical JAK2 variants with high REVEL scores have been reported in MPN, highlighting the need to further understand their relationship with disease. The JAK2-I724T variant does not drive MPN, but additional investigations are required to exclude any potential modulatory effect on the MPN phenotype.

Stroke patients develop antibodies that react with components of N-methyl-D-aspartate receptor subunit 1 in proportion to lesion size.

BACKGROUND AND PURPOSE: Antibodies against neuronal antigens develop in patients after stroke and some may serve as biomarkers of neuronal injury. We aimed to determine whether antibodies against subunit 1 (GluN1) of the N-methyl-D-aspartate receptor also develop after stroke and if so, whether they correlate with stroke characteristics. METHODS: Forty-eight patients with ischemic stroke and 96 healthy controls were tested for the presence of serum antibodies targeting GluN1. Testing was conducted using 20-kDa recombinant GluN1-S2 peptide (by ELISA and Western blotting) and on rat brain tissue (by Western blotting and immunohistochemistry). Clinical examinations and computed tomographic brain scans were performed to assess clinical state and infarct size and location. RESULTS: Of the 48 patients with ischemic stroke, 21 (44%) had antibodies that reacted with the recombinant GluN1-S2. There was no evidence of antibody binding to intact GluN1 in brain tissue. Western blot appearances suggested reactivity with GluN1 degradation products. Patients with anti-GluN1-S2 antibodies were more likely to have higher National Institutes of Health Stroke Scale scores, larger infarcts, and more frequent cortical involvement. Of the 96 controls, only 3 (3%), all aged>50 years, had antibodies that reacted with GluN1-S2 at low levels. CONCLUSIONS: Antibodies that bind recombinant GluN1-S2 peptides (but not the intact GluN1 protein) develop transiently in patients after stroke in proportion to infarct size, suggesting that these antibodies are raised secondarily to neuronal damage. The anti-GluN1-S2 antibodies may provide useful information about the presence and severity of cerebral infarction. This will require confirmation in larger studies.

Advances in molecular characterization of pediatric acute megakaryoblastic leukemia not associated with Down syndrome; impact on therapy development.

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.

Advances in molecular characterization of myeloid proliferations associated with Down syndrome.

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 multi-center studies would be helpful to advance molecular characterization of these diseases at different stages of development and progression.

Deregulated calcium signaling in blood cancer: Underlying mechanisms and therapeutic potential.

Intracellular calcium signaling regulates diverse physiological and pathological processes. In solid tumors, changes to calcium channels and effectors via mutations or changes in expression affect all cancer hallmarks. Such changes often disrupt transport of calcium ions (Ca2+) in the endoplasmic reticulum (ER) or mitochondria, impacting apoptosis. Evidence rapidly accumulates that this is similar in blood cancer. Principles of intracellular Ca2+ signaling are outlined in the introduction. We describe different Ca2+-toolkit components and summarize the unique relationship between extracellular Ca2+ in the endosteal niche and hematopoietic stem cells. The foundational data on Ca2+ homeostasis in red blood cells is discussed, with the demonstration of changes in red blood cell disorders. This leads to the role of Ca2+ in neoplastic erythropoiesis. Then we expand onto the neoplastic impact of deregulated plasma membrane Ca2+ channels, ER Ca2+ channels, Ca2+ pumps and exchangers, as well as Ca2+ sensor and effector proteins across all types of hematologic neoplasms. This includes an overview of genetic variants in the Ca2+-toolkit encoding genes in lymphoid and myeloid cancers as recorded in publically available cancer databases. The data we compiled demonstrate that multiple Ca2+ homeostatic mechanisms and Ca2+ responsive pathways are altered in hematologic cancers. Some of these alterations may have genetic basis but this requires further investigation. Most changes in the Ca2+-toolkit do not appear to define/associate with specific disease entities but may influence disease grade, prognosis, treatment response, and certain complications. Further elucidation of the underlying mechanisms may lead to novel treatments, with the aim to tailor drugs to different patterns of deregulation. To our knowledge this is the first review of its type in the published literature. We hope that the evidence we compiled increases awareness of the calcium signaling deregulation in hematologic neoplasms and triggers more clinical studies to help advance this field.

Zebrafish runx1 promoter-EGFP transgenics mark discrete sites of definitive blood progenitors.

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.

The Epidemiology of Myeloproliferative Neoplasms in New Zealand between 2010 and 2017: Insights from the New Zealand Cancer Registry.

BACKGROUND: There is a paucity of data on ethnic disparities in patients with the classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs): polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF). METHODS: This study analysed the demographic data for PV, ET and PMF collected by the New Zealand Cancer Registry (NZCR) between 2010 and 2017. RESULTS: We found that the NZCR capture rates were lower than average international incidence rates for PV and ET, but higher for PMF (0.76, 0.99 and 0.82 per 100,000, respectively). PV patients were older and had worse outcomes than expected, which suggests these patients were reported to the registry at an advanced stage of their disease. Polynesian patients with all MPN subtypes, PV, ET and PMF, were younger than their European counterparts both at the time of diagnosis and death (p < 0.001). Male gender was an independent risk factor for mortality from PV and PMF (hazard ratios (HR) of 1.43 and 1.81, respectively; p < 0.05), and Maori ethnicity was an independent risk factor for mortality from PMF (HR: 2.94; p = 0.006). CONCLUSIONS: New Zealand Polynesian patients may have increased genetic predisposition to MPN, thus we advocate for modern genetic testing in this ethnic group to identify the cause. Further work is also required to identify modifiable risk factors for mortality in MPN, in particular those associated with male gender and Maori ethnicity; the results may benefit all patients with MPN.

Knockdown and overexpression of NR1 modulates NMDA receptor function.

The N-methyl-d-aspartate receptor (NMDAR) is critically involved in learning and memory, neuronal survival, as well as neuroexcitotoxicity and seizures. We hypothesize that even mild reductions in the numbers of hippocampal NMDARs could impair learning and memory, whereas increasing receptor activity would facilitate learning but reduce seizure threshold. We developed novel gene transfer strategies assisted by an adeno-associated viral vector 1/2 to bi-directionally modulate expression levels of the NR1 protein in rat hippocampus. Functional consequences of the altered NR1 expression were examined in the acute seizure model, and on normal processes of fear memory and neurogenesis. We found that knocking down NR1 protected against seizures at the expense of impaired learning, as predicted. Paradoxically, NR1 overexpression not only increased fear memory and neurogenesis, but also delayed onset of more severe seizures. In conclusion, the observed consequences of NR1 knockdown and overexpression underscore NMDAR requirement for neuronal plasticity, and are in agreement with its dichotomous functions.

N-Methyl-D-Aspartate Receptors in Hematopoietic Cells: What Have We Learned?

The N-methyl-D-aspartate receptor (NMDAR) provides a pathway for glutamate-mediated inter-cellular communication, best known for its role in the brain but with multiple examples of functionality in non-neuronal cells. Data previously published by others and us provided ex vivo evidence that NMDARs regulate platelet and red blood cell (RBC) production. Here, we summarize what is known about these hematopoietic roles of the NMDAR. Types of NMDAR subunits expressed in megakaryocytes (platelet precursors) and erythroid cells are more commonly found in the developing rather than adult brain, suggesting trophic functions. Nevertheless, similar to their neuronal counterparts, hematopoietic NMDARs function as ion channels, and are permeable to calcium ions (Ca2+). Inhibitors that block open NMDAR (memantine and MK-801) interfere with megakaryocytic maturation and proplatelet formation in primary culture. The effect on proplatelet formation appears to involve Ca2+ influx-dependent regulation of the cytoskeletal remodeling. In contrast to normal megakaryocytes, NMDAR effects in leukemic Meg-01 cells are diverted away from differentiation to increase proliferation. NMDAR hypofunction triggers differentiation of Meg-01 cells with the bias toward erythropoiesis. The underlying mechanism involves changes in the intracellular Ca2+ homeostasis, cell stress pathways, and hematopoietic transcription factors that upon NMDAR inhibition shift from the predominance of megakaryocytic toward erythroid regulators. This ability of NMDAR to balance both megakaryocytic and erythroid cell fates suggests receptor involvement at the level of a bipotential megakaryocyte-erythroid progenitor. In human erythroid precursors and circulating RBCs, NMDAR regulates intracellular Ca2+ homeostasis. NMDAR activity supports survival of early proerythroblasts, and in mature RBCs NMDARs impact cellular hydration state, hemoglobin oxygen affinity, and nitric oxide synthase activity. Overexcitation of NMDAR in mature RBCs leads to Ca2+ overload, K+ loss, RBC dehydration, and oxidative stress, which may contribute to the pathogenesis of sickle cell disease. In summary, there is growing evidence that glutamate-NMDAR signaling regulates megakaryocytic and erythroid cells at different stages of maturation, with some intriguing differences emerging in NMDAR expression and function between normal and diseased cells. NMDAR signaling may provide new therapeutic opportunities in hematological disease, but in vivo applicability needs to be confirmed.

Treatment outcomes of patients with acute promyelocytic leukaemia between 2000 and 2017, a retrospective, single centre experience.

All-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are effective induction therapy for acute promyelocytic leukaemia (APL). However, early thrombo-haemorrhagic complications and mortality remain high. We aimed to investigate how the timing of ATRA initiation and the inclusion of ATO influence patient outcomes. Clinical records were retrospectively reviewed for all patients treated for APL in a single, tertiary centre during 2000-2017. Among 70 patients with APL, 36 (51.4%) presented with thrombo-haemorrhagic complications, and four (5.8%) died within 30 days. The median time to ATRA initiation was 11.2 (range 0-104) h from the time of admission. Patients requiring more transfusions started on ATRA sooner (P = 0.04). Patients with adverse early events did not start ATRA later (P = 0.99). Nevertheless, patients that required additional tests for diagnosis (PML immunofluorescence or molecular) started on ATRA later (28.5 versus 5.3 h; P < 0.0001), and had more thrombo-haemorrhagic complications (P = 0.04). Long-term survival was actually better in patients who started ATRA later (P = 0.03), which is likely explained by higher proportion of low risk patients in this group. Patients treated with ATO (n = 23) maintained higher fibrinogen levels and required less transfusions during induction (P < 0.05), with no disease-related deaths in this group over a median follow-up time of 37.8 months (interquartile range 44.9 months). In summary, fast ATRA initiation reduces early but not late adverse events in APL patients, and the inclusion of ATO helps further improve both early and late outcomes in APL.

N-Methyl-D-Aspartate Receptor Hypofunction in Meg-01 Cells Reveals a Role for Intracellular Calcium Homeostasis in Balancing Megakaryocytic-Erythroid Differentiation.

The release of calcium ions (Ca2+) from the endoplasmic reticulum (ER) and related store-operated calcium entry (SOCE) regulate maturation of normal megakaryocytes. The N-methyl-D-aspartate (NMDA) receptor (NMDAR) provides an additional mechanism for Ca2+ influx in megakaryocytic cells, but its role remains unclear. We created a model of NMDAR hypofunction in Meg-01 cells using CRISPR-Cas9 mediated knockout of the GRIN1 gene, which encodes an obligate, GluN1 subunit of the NMDAR. We found that compared with unmodified Meg-01 cells, Meg-01-GRIN1 -/- cells underwent atypical differentiation biased toward erythropoiesis, associated with increased basal ER stress and cell death. Resting cytoplasmic Ca2+ levels were higher in Meg-01-GRIN1 -/- cells, but ER Ca2+ release and SOCE were lower after activation. Lysosome-related organelles accumulated including immature dense granules that may have contributed an alternative source of intracellular Ca2+. Microarray analysis revealed that Meg-01-GRIN1 -/- cells had deregulated expression of transcripts involved in Ca2+ metabolism, together with a shift in the pattern of hematopoietic transcription factors toward erythropoiesis. In keeping with the observed pro-cell death phenotype induced by GRIN1 deletion, memantine (NMDAR inhibitor) increased cytotoxic effects of cytarabine in unmodified Meg-01 cells. In conclusion, NMDARs comprise an integral component of the Ca2+ regulatory network in Meg-01 cells that help balance ER stress and megakaryocytic-erythroid differentiation. We also provide the first evidence that megakaryocytic NMDARs regulate biogenesis of lysosome-related organelles, including dense granules. Our results argue that intracellular Ca2+ homeostasis may be more important for normal megakaryocytic and erythroid differentiation than currently recognized; thus, modulation may offer therapeutic opportunities.

Early treatment of acute promyelocytic leukaemia is accurately guided by the PML protein localisation pattern: real-life experience from a tertiary New Zealand centre.

Current guidelines recommend that a rapid test be used to assist diagnosis of acute promyelocytic leukaemia (APL), but the choice of an assay is discretionary. PML immunofluorescence (PML IF) identifies the microparticulate pattern of the PML protein localisation, highly specific for APL. The aim of this study was to evaluate clinical utility of PML IF in a real-life setting based on a retrospective records review for all patients who had PML IF performed in our centre between 2000 and 2017. Final analysis included 151 patients, 70 of whom had APL. PML IF was reported on average 3 days faster than cytogenetics. Compared with genetic results, PML IF showed sensitivity of 96% and specificity of 100%. PML IF accurately predicted APL in four APL cases with cryptic karyotype/FISH and excluded APL in 98% cases tested based on the suspicious immunophenotype alone, 21/28 of whom had mutated NPM1. Results of PML IF influenced decision to start ATRA in 25 (36%) APL patients and led to its termination in six non-APL patients. In conclusion, PML IF is a fast and reliable test that facilitates accurate treatment decisions when APL is suspected. This performance of PML IF remains hard to match in a real-life setting.

Paradoxical facilitatory effect of low-dose alcohol consumption on memory mediated by NMDA receptors.

Epidemiological studies have suggested a negative correlation between alcohol intake and Alzheimer's disease. In vitro, ethanol negatively modulates NMDA receptor function. We hypothesized that chronic moderate alcohol intake leads to improved memory via adaptive responses in the expression of NMDA receptors and downstream signaling. We fed liquid diets containing no, moderate, or high amounts of ethanol to control and matched rats with hippocampal knock-down of the NR1 subunit. Rats with increased hippocampal NR1 expression were also generated to determine whether they had a phenotype similar to that of ethanol-fed animals. We found that moderate ethanol intake improved memory, increased NR1 expression, and changed some aspects of neurotrophin signaling. NR1 knock-down prevented ethanol's facilitatory effects, whereas hippocampal NR1 overexpression mimicked the effect of chronic low-dose ethanol intake on memory. In contrast, high-dose ethanol reduced neurogenesis, inhibited NR2B expression, and impaired visual memory. In conclusion, adaptive changes in hippocampal NMDA receptor expression may contribute to the positive effects of ethanol on cognition.

Distinctive features of polycythaemia vera in New Zealand Polynesians.

AIM: The aim of this study was to examine a potential ethnic disparity in the phenotype of polycythaemia vera (PV) between New Zealand European and Polynesian patients. METHOD: A retrospective review of medical records was conducted at Middlemore Hospital to identify adult patients with PV diagnosed between 1987 and 2007. Data extracted included diagnostic criteria, ethnicity, age, complications and survival. RESULTS: Eighty-eight adult patients with PV were identified during 1987-2007, 49 (55.7%) were Europeans and 36 (40.9%) Polynesians. The most striking finding was that Polynesian patients presented almost 14 years younger than Europeans (mean age of 54 years versus [vs] 68, respectively; P<.001). The white cell and platelet counts were higher in Polynesians compared with Europeans (mean white cell count of 22x109/L vs 13x109/L; mean platelet count of 648x109/L vs 512x109/L, respectively; P<.05 for both). The rate of JAK2 V617F mutation in Polynesians was 96%, equivalent to other large cohorts of European patients. The rates of long-term complications were comparable between Polynesians and Europeans, but the predicted impact on life expectancy was more severe for Polynesians. CONCLUSION: New Zealand Polynesian patients present with a distinctive PV phenotype. Their younger age at presentation suggests a different risk factor profile or a higher genetic susceptibility. We hope our observations initiate larger epidemiological and genetic studies to help elucidate the cause.