Abstract:
Background: Megakaryocytes are haematopoietic progenitors derived from the myeloid lineage that give rise to blood platelets. Megakaryocyte biology remains poorly understood and cancers derived from megakaryocytes frequently have poor outcomes. The importance of intracellular calcium signalling in megakaryocytes has been highlighted by the discovery that approximately 30% of patients with chronic megakaryocytic cancers contain mutations in the Calreticulin (CALR) gene that encodes a calcium binding protein. However, the mechanism through which CALR mutations affect intracellular calcium metabolism remains unknown. CALR is highly expressed in megakaryocytes. Its main role is to regulate the level of calcium stores in the endoplasmic reticulum (ER). Our group has identified a novel pathway for extracellular calcium entry into megakaryocytes that is driven by NMDA receptors (NMDAR). Papers from our group suggest that NMDAR-mediated calcium entry in megakaryocyte leukaemia cell line (Meg-01 cells) has been diverted to support cell proliferation, which raises the possibility that calcium pathways may provide a novel option to inhibit growth of megakaryocytic cancers. NMDAR antagonists limit proliferation and promote differentiation of leukaemic megakaryoblasts, and trigger cytoplasmic vacuoles. We hypothesised that leukaemic megakaryoblasts seized NMDAR-mediated calcium influx to support growth and proliferation in Meg- 01 cells. Objectives: The first objective of this thesis was to document loss of GluN1 expression (the obligate NMDAR subunit) in the genetically modified Meg-01 cells we recently developed (Meg-01-GRIN1-/- cells). The second objective was to investigate whether the vacuolation in Meg-01-GRIN1-/- cells could represent lysosomal-related organelles. Finally, I examined if there is a physical connection between NMDAR and CaMKII in Meg-01 cells. Methods: A new cell line model derived from Meg-01 cells was used in this thesis where GluN1 was deleted using CRISPR-Cas9 system; unmodified Meg-01 cells were used as controls. LC-MS/MS technique was employed to examine low quantities of GluN1 expression in Meg-01 and Meg-01- GRIN1-/- cells. Immunofluorescence was applied to examine the nature of vacuoles and granules that accumulated in Meg-01-GRIN1-/- cells; this employed anti-CD63 antibody. Western blotting was then used to semi-quantify the level of CD63 in Meg-01-GRIN1-/- cells compared with Meg-01 cells. Confocal microscopy was employed to examine a possible co-localisation between NMDAR and CaMKII. Western blotting technique was used to seek any difference in the levels of both total and phosphorylated CaMKII in Meg-01-GRIN1-/- cells. Results: Anti-GluN1 immunofluorescence confirmed the loss of GluN1 expression in Meg-01-GRIN1- /- cells. However, LC-MS/MS experiments did not detect GluN1 even in unmodified Meg-01 cells, highlighting the need to increase method sensitivity. CD63 expression was increased in Meg-01-GRIN1- /- cells, indicating accumulation of lysosomal-related organelles. Similar results were obtained for Meg- 01 cells cultured in the presence of MK-801. Confocal microscopy detected co-localisation between GluN1 and CaMKII, suggesting that GluN1 deletion impairs CaMKII signalling downstream of NMDAR. Conclusion: Loss of GluN1 in Meg-01 cells impairs biogenesis of lysosomal-related organelles that include dense granules. Cellular consequences of the NMDAR knockout may be direct due to reduced calcium entry into cells or indirect due to reduced CaMKII activity downstream of the NMDAR.