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Autonomous Ca2+ Oscillations Reflect Oncogenic BCR-Signaling in Multiple B-Cell Malignancies and Are Essential for Survival and Proliferation


 Background: Nuclear factor of activated T cells (NFAT) factors regulate activation and Ca2+ signaling in B-cells. Store-operated Ca2+ entry (SOCE) is regulated by Orai1 and Stim1 and upstream of NFAT. We previously reported on the observation of autonomous oscillatory Ca2+ signal activity in BCR-ABL1-driven B-ALL. Autonomous Ca2+ oscillations may provide oncogenic survival signals to B-ALL cells, however their significance and mechanism remained unclear.
 Results: Here we found that autonomous Ca2+ oscillations are common across multiple subtypes of B-cell lineage ALL and mature B-cell lymphoma and reflect downstream survival and proliferation signals from oncogenic BCR-signaling or oncogenes that mimic active BCR-signaling (e.g. ABL1-kinase fusions, CD79B mutation, EBV-encoded oncoprotein LMP2A, RAS-pathway lesions). By contrast, multiple myeloma and Hodgkin's lymphoma that lack BCR-expression and function also lack Ca2+ oscillations (Figure, top panel). As Orai1 and Stim1/2 are essential SOCE-effector genes, we performed genetic experiments to test the impact of Cre-mediated deletion of Orai1 and Stim1/2 in BCR-ABL1 and NRASG12D-dependent models of B-ALL. Inducible deletion of Orai1 or Stim1/2 not only abrogated SOCE but also autonomous Ca2+ oscillations. Signal amplitudes of residual autonomous Ca2+ oscillations were significantly reduced (P < 0.0001; Figure, bottom panel). Further, deletion of either Orai1 or Stim1/2 induced cell death and abrogated colony-forming capacity in both models of B-ALL. In agreement with these findings, NFATc1 no longer translocated to the nucleus upon Cre-mediated ablation of Orai1 or Stim1/2 induced, which reflected functional inactivation of NFATc1. These results demonstrated that Orai1- and Stim1/2-mediated SOCE signaling and autonomous Ca2+ oscillations are critical in BCR-ABL1 and NRASG12D-dependent B-ALL. A NFAT-calcineurin association inhibitor, INCA-6, was tested for its ability to suppress NFATc1 and autonomous Ca2+ signaling in patient-derived xenograft (PDX) models of B-ALL, mantle cell lymphoma and DLBCL. Treatment with INCA-6 suppressed survival and proliferation signals in all six PDX of B-ALL, mantle cell lymphoma and DLBCL but not multiple myeloma cells. Unlike myeloma cells, B-ALL, mantle cell and DLBCL cells expressed a functional (pre-)BCR. These findings suggest that the SOCE-NFAT pathway is linked to Ca2+ signaling downstream of a functional BCR- or oncogenic BCR-mimics. Furthermore, to determine whether high expression levels of ORAI1, STIM1, STIM2 and NFATC1 represents a biomarker of clinical outcome for patients with B-ALL, we segregated patients from two clinical trials (Children's Oncology Group P9906 (n=207) and Eastern Cooperative Oncology Group (E2993; n=215)) into two groups on the basis of higher or lower than median expression levels of ORAI1, STIM1, STIM2 and NFATC1 at the time of diagnosis. Higher than median expression levels of each of these four genes at the time of diagnosis predicted shorter overall and relapse-free survival (P < 0.02 or lower for each of these genes). These findings identify SOCE-NFAT signal as a novel biomarker with potential use in risk stratification of children and adults with B-ALL.
 Conclusions: We identified Orai1 and Stim1 as central mediators of SOCE. Most BCR-dependent B-cell malignancies are driven by oncogenic BCR-signals, which result in autonomous Ca2+ oscillations. While the significance of autonomous Ca2+-oscillations remains unclear, deletion of Orai1 and Stim1/2 resulted in a complete loss of Ca2+ oscillations, loss of NFATc1-activation and cell death. We conclude that previously unrecognized Ca2+-oscillations downstream of oncogenic BCR-signaling are required for survival and proliferation of B-ALL and B-cell lymphoma cells. Pharmacological inhibition of SOCE (Orai1 and Stim1/2) or NFATC1 (e.g. INCA-6) represents a selective strategy to disrupt autonomous Ca2+ oscillations and oncogenic BCR-signaling in a broad range of B-cell malignancies.
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 No relevant conflicts of interest to declare.
