Ca²⁺ Oscillations and NFATC1 -Activation Enable Oncogenic Signaling in Pre-B Cell Transformation and Leukemogenesis

Background: Nuclear factor of activated T cells (NFATC1) regulates activation and differentiation of B and T cells. Activation of NFATC1 requires sustained Ca²⁺ influx by store-operated Ca²⁺ entry (SOCE). Our group previously showed that oncogenic signaling from the BCR-ABL1 kinase in Ph⁺ ALL results in autonomous oscillatory Ca²⁺ signaling (Klein et al., J Exp Med 2004; Feldhahn et al., J Exp Med 2005). However, the significance of Ca²⁺ oscillations remained unclear. Here we examined the function of Orai and Stim1/2, the central mediators of SOCE, and the role of Ca²⁺ oscillations in the survival of BCR-ABL1 -driven pre-B ALL cells.

Results: To investigate the consequence of inducible deletion of SOCE-effector genes, Orai1, Stim1, and Stim2, we have generated BCR-ABL1 - and CreER^T2-transduced murine pre-B Orai1^f/f and Stim1/2^f/f cells. Both Orai1 - and Stim1/2 -deletion impaired Ca²⁺ influx from the extracellular space when Ca²⁺ uptake from cytosol to endoplasmic reticulum was pharmacologically inhibited. Consistently, both Orai1 - and Stim1/2 -deletion significantly attenuated the amplitude and frequency of autonomous Ca²⁺ oscillations, compared with that of empty vector controls. To determine whether the impaired Ca²⁺ signaling associated with cell survival and proliferation, we next analyzed their growth and colony-forming capacity. As expected, both Orai1 - and Stim1/2 -deleted pre-B ALL cells exhibited growth disadvantage and reduced colony-forming capacity, compared with empty vector controls. Flow cytometry studies of Annexin V revealed that both Orai1 - and Stim1/2 -deletion induced apoptosis in the BCR-ABL1 -transformed cells. These results suggest that autonomous Ca²⁺ oscillations are needed to circumvent apoptosis, and enhance cell growth and self-renewal in BCR-ABL-driven ALL cells. Interestingly, even in normal pre-B cells and in the absence of autonomous Ca²⁺ oscillations, both Orai1 - and Stim1/2 -deletion caused growth disadvantage suggesting the existence of similar cellular function to Ca²⁺ oscillations in normal pre-B cells. Subsequently, we studied NFATc1 activity by Western blot, as reflected by nuclear localization, in pre-B ALL cells before and after Orai1 - and Stim1/2 -deletion. NFATc1 is known as one of a central NFAT family member that is activated in lymphocytes upon BCR and TCR stimulation. While Orai1^fl/fl and Stim1/2^fl/fl B-lineage ALL cells transduced with empty vector controls displayed marked nuclear accumulation of NFATc1, Cre-mediated deletion of Orai1 and Stim1/2 strongly reduced nuclear accumulation of NFATc1, with slight increases in the cytoplasmic fraction. To examine the consequences of NFATc1 inhibition, we generated BCR-ABL1 - and CreER^T2-transduced murine pre-B Nfatc1^f/f and observed its effect on cell growth. As expected, inducible ablation of Nfatc1 resulted in rapid depletion of pre-B ALL cells from the cell culture compared to empty vector controls. Importantly, a NFAT-calcineurin association inhibitor, INCA-6, displayed growth inhibitory effects in all six patient-derived xenograft of B cell ALL tested, including samples from patients who relapsed after initially successful chemotherapy. These results suggest that the Ca²⁺-NFAT signal may suppress apoptosis, and promote drug-resistance and relapse in human pre-ALL cells.

Conclusions: Taken together, our findings showed that SOCE has a critical role in NFAT activation and survival of BCR-ABL1 -driven pre-B ALL cells. Orai1- and Stim1/2-mediated autonomous Ca²⁺ oscillatory signal is required for BCR-ABL1-dependent proliferation. This signal may bypass the NFAT activation which suppress apoptosis and enhance proliferation in BCR-ABL-driven ALL cells.