ORCID Profile
0000-0002-7022-3352
Current Organisation
The University of Auckland
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Publisher: Georg Thieme Verlag KG
Date: 04-2020
Abstract: 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.
Publisher: Cold Spring Harbor Laboratory
Date: 08-11-2017
DOI: 10.1101/214585
Abstract: Pancreatic neuroendocrine tumors (pNETs) are uncommon cancers arising from pancreatic islet cells. Analysis of gene mutation, copy number and RNA expression of 57 sporadic pNETs showed that pNET genomes are dominated by aneuploidy. Remarkably, ~25% of pNETs had genomes characterized by recurrent loss of heterozygosity (LoH) of the same 10 chromosomes, accompanied by bi-allelic MEN1 inactivation, and these cases had generally poor clinical outcome. Another ~25% of all pNETs had chromosome 11 LoH and bi-allelic MEN1 inactivation, lacking the recurrent LoH pattern – these had universally good clinical outcome. Some level of aneuploidy was common, and overall ~80% of pNETs had LoH of ≥1 chromosome. This aneuploidy led to changes in RNA expression at the level of whole chromosomes and allowed pathogenic germline variants (e.g. ATM ) to be expressed unopposed, inactivating downstream tumor suppressor pathways. Some pNETs appear to utilize VHL gene methylation or mutation to activate pseudo-hypoxia. Contrary to expectation neither tumor morphology within well-differentiated pNETs nor single gene mutation had significant associations with clinical outcome, nor did expression of RNAs reflecting the activity of immune, differentiation, proliferative or tumor suppressor pathways. MEN1 was the only statistically significant recurrently mutated driver gene in pNETs. Only one pNET had clearly oncogenic and actionable SNVs (in PTEN and FLCN ) confirmed by corroborating RNA expression changes. The two distinct patterns of aneuploidy described here, associated with markedly poor and good clinical outcome respectively, define a novel oncogenic mechanism and the first route to genomic precision oncology for this tumor type.
Publisher: American Society of Hematology
Date: 28-04-2022
Abstract: 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.
Publisher: Springer Science and Business Media LLC
Date: 20-07-2018
DOI: 10.1038/S41525-018-0058-3
Abstract: Pancreatic neuroendocrine tumors (pNETs) are uncommon cancers arising from pancreatic islet cells. Here we report the analysis of gene mutation, copy number, and RNA expression of 57 sporadic well-differentiated pNETs. pNET genomes are dominated by aneuploidy, leading to concordant changes in RNA expression at the level of whole chromosomes and chromosome segments. We observed two distinct patterns of somatic pNET aneuploidy that are associated with tumor pathology and patient prognosis. Approximately 26% of the patients in this series had pNETs with genomes characterized by recurrent loss of heterozygosity (LoH) of 10 specific chromosomes, accompanied by bi-allelic MEN1 inactivation and generally poor clinical outcome. Another ~40% of patients had pNETs that lacked this recurrent LoH pattern but had chromosome 11 LoH, bi-allelic MEN1 inactivation, and universally good clinical outcome. The somatic aneuploidy allowed pathogenic germline variants (e.g., ATM ) to be expressed unopposed, with RNA expression patterns showing inactivation of downstream tumor suppressor pathways. No prognostic associations were found with tumor morphology, single gene mutation, or expression of RNAs reflecting the activity of immune, differentiation, proliferative or tumor suppressor pathways. In pNETs, single gene mutations appear to be less important than aneuploidy, with MEN1 the only statistically significant recurrently mutated driver gene. In addition, only one pNET in the series had clearly actionable single nucleotide variants (SNVs) (in PTEN and FLCN ) confirmed by corroborating RNA expression changes. The two clinically relevant patterns of LoH described here define a novel oncogenic mechanism and a plausible route to genomic precision oncology for this tumor type.
No related grants have been discovered for Nicholas Knowlton.