Distinct and Targetable Role of Calcium and Calcium Sensing Receptor in Leukaemia

Abstract

Calcium, the most abundant mineral in the body, is a key component of bones and is released by bone remodelling. Calcium ions play a role in the localisation, engraftment and the adhesion of normal haematopoietic stem cells (HSC) to extracellular matrix (ECM) proteins in the bone marrow microenvironment (BMM) via the calcium sensing receptor (CaSR), thereby maintaining normal haematopoiesis. However, the role of this heterotrimeric G-protein-coupled receptor and its associated pathways in the local BMM for the development of leukaemia is poorly understood. We hypothesized that calcium ions, subject to a fine balance between osteoblasts and osteoclasts and released from bone, and/or CaSR contribute to development, progression and response to therapy in leukaemia and might be targetable via CaSR. By imaging of a genetically-encoded calcium indicator using intravital microscopy (IVM), we have shown that the local calcium concentration forms a gradient in the BMM with the highest concentrations close to the endosteal area. Furthermore, calcium levels differ between different leukaemias, with the calcium concentration of mice and human patients being highest in acute myeloid leukaemia (AML). Additionally, CaSR was expressed at a higher level on AML compared to chronic myeloid leukaemia (CML) cells. Using genetic deletion or overexpression or pharmacological inhibition of CaSR, we revealed that this receptor influences the localization of CML and AML cells in the BMM. Furthermore, we showed that the secondary messenger intracellular calcium (iCa 2+) can be regulated by CaSR in different leukaemia cell lines which have differential sensitivity to extracellular calcium (eCa 2+). Exposure of the human cell lines K562 (BCR-ABL1 +) and THP1 (MLL-AF9 +) to increasing calcium concentrations revealed that CaSR expression, migration and adhesion to the extracellular matrix (ECM) protein fibronectin were not altered in CML, but significantly increased in AML. Genetic deletion or overexpression of Casr in leukaemia-initiating cells in murine models revealed that CaSR acts as tumour suppressor in BCR-ABL1-driven CML and B-cell acute lymphoblastic leukaemia (B-ALL) and as oncogene in AML. Conversely, overexpression of CaSR in CaSR-deficient AML-initiating cells ‘rescued’ the disease. Limiting dilution transplantation of CaSR-deficient AML-initiating cells to assess leukaemic stem cell number and/or function revealed a 6.5-fold reduction of leukemic stem cells. Treatment of mice with the CaSR agonist cinacalcet and imatinib prolonged survival of mice with CML, while treatment with a CaSR antagonist NPS-2143 significantly reduced tumour burden and prolonged survival of mice with AML in syngeneic and xenogeneic transplantation experiments. In summary, our results suggest that calcium ions, likely originating from the BMM and/or CaSR strongly and differentially influence leukaemia localisation and progression. As an adjunct to existing treatment strategies, targeting of CaSR with specific pharmacologic agonists may be beneficial in CML and B-ALL, while CaSR antagonists may prolong survival of patients with AML.