Glutamate-mediated calcium entry in megakaryocytic cells: evidence for a hijacking effect to support leukaemia growth

Abstract

We have previously reported a novel mechanism for calcium (Ca2+) entry into leukaemic megakaryoblasts that increases cell proliferation. This mechanism engages N-methyl-D-aspartate receptors (NMDARs) that are glutamate-gated calcium ion channels located in the plasma cell membrane. Intriguingly, we saw that NMDAR inhibition reduces proliferation of leukaemic cells, suggesting a novel way to interfere with megakaryocytic cancers. Here, we continued to interrogate NMDAR functionality and its contribution to the growth of normal and leukaemic megakaryocytes. Meg-01, Set-2 and K-562 cells were used as models of leukaemic megakaryoblasts. NMDAR effects in cultured cells were tested using well-established NMDAR agonists and antagonists. The role of NMDARs in normal megakaryocytes was examined using a conditional knock-out mouse model (Pf4-Grin1-/- mice) and in ex vivo cultures of primary mouse megakaryocytes. The NMDAR-mediated Ca2+ fluxes were detected in megakaryocytic cells loaded with Fluo-4-AM. When cultured in the presence of NMDAR inhibitors, leukaemic cells underwent differentiation and acquired cytoplasmic vacuoles. This pro-differentiation effect was associated with mild induction of apoptosis but more evidence for autophagy. In contrast, NMDAR antagonists reduced megakaryocytic differentiation of normal lineage-negative mouse progenitors and diminished proplatelet formation ex vivo. Further, Pf4-Grin1-/- mice had lower platelet counts. In conclusion, glutamate-mediated Ca2+ entry appears hijacked in leukaemic cells away from supporting differentiation towards supporting proliferation. We are now using a CRISPR/Cas9 system to confirm effects of pharmacological modulators in cultured cells and characterising mechanisms through which Pf4-Grin1-/- mice develop thrombocytopaenia. Elucidation of these effects may help design new strategies to modulate human megakaryopoiesis. Funding: Child Cancer Foundation (project 12/17); Leukaemia and Blood Cancer New Zealand and donation from Anne, David and Victoria Norman.