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Leukemia is a cancer of the blood and bone marrow characterized by an uncontrolled proliferation and accumulation of abnormal white blood cells. Leukemia can be classified based on the course of the disease (acute or chronic) and the blood cell type involved (myeloid or lymphocytic), leading to four main subtypes: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML). Leukemia represents 2.5% of all new cancer cases per year, and survival rates in some leukemias remain low at 40%.
The bone marrow microenvironment (BMM) is a system within the bone marrow comprising cellular and acellular components, all of which play a major role in hematopoiesis, providing the physical space where hematopoietic stem cells (HSCs) reside. The BMM interacts with HSCs, offering a “niche” for those cells and in case of leukemia, the BMM has a supportive role in disease maintenance and progression by supporting Leukemia stem cells (LSCs). One of the components of the BMM are calcium ions. Calcium is the most abundant mineral in the body, a key component of bones and is released by parathyroid hormone (PTH) induced bone remodeling. Calcium ions play a role in the localization, engraftment and adhesion of normal HSC to extracellular matrix (ECM) proteins in the BMM via the calcium sensing receptor (CaSR), thereby maintaining normal hematopoiesis. In addition of a major regulator of calcium homeostasis, CaSR contribute to the development of different cancers, functioning as either tumor suppressor or oncogene, depending on the involved tissue. However, the role of CaSR and its associated pathways in the local BMM for the development of leukemia is poorly understood. We hypothesized that calcium ions released from bone, subject to a fine balance between osteoblasts and osteoclasts, and/or CaSR, contribute to development, progression and response to therapy.
We have shown that the local calcium concentration forms a gradient in the bone marrow niche and in mice with CML is similarly low as in control mice, but significantly higher in mice suffering from BCR ABL1 driven B ALL or MLL AF9 driven AML. Similarly, the calcium concentration in the human BMM was found to be higher in AML than in other leukemias. Regarding the function of calcium in leukemia cells, we found that AML and CML cells respond differently to calcium exposure, with AML cells exhibiting regulation of cellular processes such as adhesion to the ECM protein fibronectin and migration toward CXCL 12, whereas CML cells remained mostly unaltered. Using genetic deletion or overexpression of CaSR in murine models of leukemia, we observed that CaSR acts as tumor suppressor in BCR-ABL1 driven CML and B ALL and as oncogene in AML.
Focusing on AML, our data shows that deficiency of CaSR on LICs leads, on one hand to increased apoptosis, and on the other hand to reduced cell cycle, reactive oxygen species (ROS) production and DNA damage in vivo, which may explain the observed prolongation of survival of mice. Complementary, in vitro experiments demonstrated that cells overexpressing CaSR have a distinct, cancer promoting phenotype compared to wildtype cells. Overexpression of CaSR led to an increase in proliferation, cell cycle, ROS production, DNA damage and reduced apoptosis. We have identified CaSR mediated pathways in AML and shown that CaSR enhances leukemia progression by activating MAPK/ERK and Wnt β catenin signaling. In addition, the CaSR interacting protein filamin A (FLNA) was shown to contribute to aggressive disease in vitro and in vivo. Furthermore, the mechanism underlying the role of CaSR in AML pathogenesis and possible regulation of LSCs was studied. Our findings demonstrated that CaSR ablation reduces myeloid progenitor function and proved that CaSR is required for maintenance of LSC pool by regulating its frequency and function. Further supporting the role of CaSR in LSC maintenance, genes associated with AML stemness and self renewal capacity were upregulated when CaSR was overexpressed and downregulated when CaSR was depleted. Given the role of CaSR in AML, the CaSR antagonist NPS 2143 was tested in vivo. The combination treatment of NPS 2143 with the standard of care, ara C, significantly reduced the tumor burden and prolonged the survival of mice with AML in syngeneic and xenotransplantation experiments. Based on the finding that CaSR functions as a tumor suppressor in CML, treatment of mice with the CaSR agonist cinacalcet in combination with imatinib prolonged survival of mice with CML compared to treatment with the mice given vehicle.
Our results suggest that calcium ions stemming from the calcium-rich BMM via CaSR strongly and differentially influence leukemia progression. As an adjunct to existing treatment therapies, targeting of CaSR with specific pharmacologic antagonists may prolong survival of patients with AML.
B-cell acute lymphoblastic leukaemia (B-ALL) is characterized by the overproduction of lymphoblasts in the bone marrow (BM), and it is the most common cancer in children while being comparatively uncommon in adults. On the other hand, in chronic myeloid leukaemia (CML), 70% of cases are found in patients older than 50 years, making it uncommon in children. All CML cases and up to 3% of paediatric B- ALL (and 25% of adult B-ALL) cases are due to fusion gene BCR-ABL1, which gives rise to the cytoplasmatic, constitutively active oncoprotein, tyrosine kinase BCR-ABL1 through a reciprocal translocation between chromosomes 9 and 22. The constitutively active BCR-ABL tyrosine kinase leads to deregulation of different signal transduction pathways such as cell growth, proliferation and cell survival. The role of the bone marrow microenvironment (BMM) can mediate disease initiation (only in mice), progression, therapy resistance, and relapse, as has been increasingly recognized over the last two decades. In general, the BMM is a very complex arrangement of various cell types such as osteoblasts, osteoclasts, endothelial cells, adipocytes, mesenchymal stromal cells, macrophages and several others. In addition, the BMM is composed of multiple chemical and mechanical factors and extra cellular matrix (ECM) proteins which contribute to the BMM’s features influencing leukaemia behaviour. Considering the incidence of B-ALL and CML in children and in adults respectively, we hypothesized that the young and/or an aged BMM might also play a previously unrecognized role in the aggressiveness of B-ALL and CML. We proposed that BM, transduced with BCR-ABL1-expressing retrovirus in the murine transduction/transplantation model of B-ALL, transplanted into young versus old recipient mice would lead to a more aggressive disease in young mice, and similarly CML would be more aggressive in old recipient mice. In close recapitulation with the human incidence, induction of CML led to a significantly shorted survival in old recipient mice. On the other hand, induction of B-ALL showed a shortened survival in young compared to old syngeneic mice, as well as in a xenotransplantation model. Among the highly heterogenous composition of the BMM, we implicate young BM macrophages as a supportive niche for B-ALL cells. The results were found to be mostly due to potential soluble factors differentially secreted from young and old macrophages. Therefore, we hypothesized that the chemokine CXCL13, which has been demonstrated to play a role in B cell migration and act as a diagnostic marker in the cerebrospinal fluid of patients with neuroborreliosis, might be responsible for the observed phenotype. CXCL13 was found to be more highly expressed in healthy and leukaemic young mice as well as in conditioned medium of young macrophages. Using a variety of in vitro experiments, CXCL13 showed to significantly increase the proliferation and the migration of leukaemia cells when exposed to young macrophages, and the phenotype was rescued while using a CXCL13 neutralizing antibody. The CXCL13 role was also confirmed in vivo, since macrophage ablation led to a prolongation of survival in young mice and a reduction of CXCL13 levels. The use of an additional mouse model, leukaemia cells with CXCR5 deficiency, led to a significant prolongation of survival of young mice, confirming the importance of the CXCL13-CXCR5 axis in B-ALL. In line with our murine results, we found that human macrophages and CXCL13 levels were higher in pediatric B-ALL patients than in adults. Consistent with our murine data, the expression level of CXCR5 may act as a prognostic marker in B-ALL, as well as a predictive marker for central nervous system relapse in human B-ALL. The overall findings show that a young BMM, and in particular macrophages, influences B-ALL progression. We specifically identified CXCL13, secreted by young macrophages, as a promoter of proliferation of B-ALL cells, influencing survival in B-ALL via CXCR5. The CXCR5-CXCL13 axis may be relevant in human B-ALL, and higher CXCR5 expression in human B-ALL may act as a predictive marker.
The endosteal bone marrow niche and vascular endothelial cells provide sanctuaries to leukemic cells. In murine chronic myeloid leukemia (CML) CD44 on leukemia cells and E-selectin on bone marrow endothelium are essential mediators for the engraftment of leukemic stem cells (LSC). We hypothesized that non-adhesion of CML-initiating cells to E-selectin on the bone marrow endothelium may lead to superior eradication of LSC in CML after treatment with imatinib than imatinib alone. Indeed, here we show that treatment with the E-selectin inhibitor GMI-1271 in combination with imatinib prolongs survival of mice with CML via decreased contact time of leukemia cells with bone marrow endothelium. Non-adhesion of BCR-ABL1+ cells leads to an increase of cell cycle progression and an increase of expression of the hematopoietic transcription factor and protooncogene Scl/Tal1 in leukemia-initiating cells (LIC). We implicate SCL/TAL1 as indirect phosphorylation target of BCR-ABL1 and as a negative transcriptional regulator of CD44 expression. We show that increased SCL/TAL1 expression is associated with improved outcome in human CML. These data demonstrate the BCR-ABL1-specific, cell-intrinsic pathways leading to altered interactions with the vascular niche via the modulation of adhesion molecules - a strategy therapeutically exploitable in future.