Abstract 4371: Store-operated Ca2+ entry and NFATC1-activation control oncogenic signaling in B-cell transformation and leukemogenesis
Authors: Kume Kohei , Chen Liting , Lee Jaewoong , Müschen Markus 
Published: August 2018

Abstract
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). We previously showed that oncogenic signaling from the BCR-ABL1 kinase in Ph+ ALL results in autonomous oscillatory Ca2+ signaling. However, the significance of Ca2+ oscillations remained unclear. Here we examined the function of Orai1 and Stim1, the central mediators of SOCE, and the role of Ca2+ oscillations in a BCR-ABL1-driven B-cell leukemia model.
Results: To investigate the consequence of inducible deletion of SOCE-effector genes, Orai1, Stim1, we generated BCR-ABL1- and CreERT2-transduced Orai1f/f and Stim1/2f/f B-cell leukemia. Both Orai1 and Stim1 deletion impaired SOCE and disrupted the patterns 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-deleted B-cell leukemia 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 deletion induced apoptosis in the BCR-ABL1-transformed cells. Studying NFATc1 activity by Western blot, as reflected by nuclear localization, in B-cell leukemia before and after Orai1 and Stim1 deletion revealed that Cre-mediated deletion of Orai1 and Stim1 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 CreERT2-transduced Nfatc1f/f B-cell leukemia and observed its effect on cell growth. As expected, inducible ablation of Nfatc1 resulted in rapid depletion of B-cell leukemia 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 xenografts of B cell ALL tested, including samples from patients who relapsed after initially successful chemotherapy. These results suggest that the Ca2+-NFATC1 signal may suppress apoptosis and promote drug resistance and relapse in human B-cell leukemia.
Conclusions: Taken together, our findings showed that SOCE has a critical role in NFATC1 activation and survival of BCR-ABL1-driven pre-B ALL cells. Orai1- and Stim1-mediated autonomous Ca2+ oscillatory signal is required for BCR-ABL1-dependent proliferation. This signal may bypass NFATC1 activation, which suppresses apoptosis and enhances proliferation.
Citation Format: Kohei Kume, Liting Chen, Jaewoong Lee, Markus Müschen. Store-operated Ca2+ entry and NFATC1-activation control oncogenic signaling in B-cell transformation and leukemogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4371.