Crac Channel Deletion in Leukemic Cells Delays Progression of Leukemia and Prolongs Survival of Mice with Notch-1-Induced T-Cell Acute Lymphoblastic Leukemia

Abstract

Introduction: Ca2+ release-activated Ca2+ (CRAC) channels and their activators stromal interaction molecule (STIM) 1 and 2 are the main regulators of calcium entry in T Lymphocytes through a process known as store-operated Ca2+ entry (SOCE). SOCE results in the activation of calcineurin and other downstream signals with important effects on lymphocyte function. Notch-1 is a protein that is essential for T lymphocyte development. Activating mutations of Notch-1 occurs in about 60% of T-cell acute lymphoblastic leukemia (T-ALL). Introduction of constitutively active forms of Notch-1 in hematopoietic stem cells (HSC) induces T-ALL in mice, providing a useful animal model for the study of leukemia.Methods: To study the role of CRAC channels in T-ALL, we used a mouse model in which c-kit+ HSC from wild-type (WT) and STIM1/STIM2-deficient mice (DKO) were retrovirally transduced with the intracellular Notch-1 domain (ICN1). Transduced HSC were injected into lethally irradiated C57BL/6 mice. Following leukemia development, mice were analyzed for survival and cellular and molecular activity of leukemic cells using various techniques including histology, flow cytometry, RT-PCR and gene array expression analysis. In addition, we used the human T-ALL cell line CEM, in which we introduced a dominant negative form of the CRAC channel subunit ORAI1 (ORAI1-DN) that abolishes CRAC channel function and SOCE, for coculture with the human bone marrow stromal cell line HS5.Results: Mice injected with wild-type HSC transduced with ICN1 succumbed from T-ALL characterized by the presence of CD4+ CD8+ leukemic T cell blasts in the blood, bone marrow and infiltrating organs within 3 to 4 weeks after transfer of HSC. By contrast, mice that had received ICN1 transduced STIM1/2 deficient HSC lived approximately twice as long. The survival benefit was not due to differences in leukemic cell numbers or in proliferation and apoptosis of leukemic cells. Histologies of the bone marrow and spleen of WT leukemic mice showed necrotic lesions, pronounced neutrophil infiltration, the presence of histiocytes engulfing red blood cells (RBC) indicative of severe inflammation. No signs of necrosis and inflammation were present in DKO leukemic mice. Paralleling the inflammation and destruction of the bone marrow environment, WT leukemic mice showed greatly diminished presence of erythroid precursors (EP) in the bone marrow whereas EP frequencies in DKO leukemic mice were similar to those in non-leukemic mice. In line with findings in mice, we observed that human leukemic CEM T cells reduced the viability of HS5 stromal cells in a contact-dependent manner. This cytotoxic effect of CEM cells depended on CRAC channel function as CEM cells transduced with ORAI1-DN had little effect on HS5 viability.Conclusion: These results suggest that CRAC channels are important for the function of T-ALL cells and their effects on the organs they infiltrate, most notably the bone marrow. Inhibition of CRAC channel function prolongs survival of mice with T-ALL potentially by attenuating the cytotoxic effects of leukemic T cells on their environment and on hematopoiesis. Further studies are underway to understand the mechanisms by which CRAC channels regulate leukemic T cell function. DisclosuresFeske:Calcimedica: Consultancy, Equity Ownership, Honoraria, Patents & Royalties: CRAC Channel Inibitors.