Autonomous Ca2+ Oscillations Reflect Oncogenic Signaling in B-ALL Cells
Authors: Kume Kohei , Chen Liting , Lee Jaewoong , Müschen Markus 
Published: November 2019

Background: Engagement of the B-cell receptor (BCR) results in Ca2+ flux and is linked to B-cell survival based on store-operated Ca2+ entry (SOCE), which is triggered by ORAI1 and stromal interaction molecule-1 (STIM1). While normal B-cells release Ca2+ only in response to external stimuli (e.g. BCR-engagement), we recently observed that transformed B-cells exhibit autonomous oscillatory Ca2+ signals that are linked to oncogene activity. Studying a novel dual biosensor system to concurrently measure Ca2+ fluctuations and oncogenic kinase activity in the same B-ALL cells, we discovered that oncogenic kinase- and Ca2+-signals alternate between On- and Off-phases, during which kinase and Ca2+ signals are mutually exclusive.
Results: Consistent with a scenario in which autonomous Ca2+-signaling is linked to oncogenic signaling, we observed that high mRNA levels of Orai1 and Stim1 in patients with B-ALL were linked to poor clinical outcomes. To study the role of SOCE and its effectors Orai1 and Stim1 in oncogene signaling, we developed mouse models for inducible deletion of Orai1 and Stim1/2 in BCR-ABL1- and NRASG12D-driven B-ALL. Tamoxifen-inducible activation of Cre in Orai1fl/fl and Stim1/2 fl/fl BCR-ABL1- and NRASG12D-driven B-ALL cells induced excision of both SOCE-mediators and induced near-complete loss of Ca2+-signaling competence. While treatment with the Ca2+ pump inhibitor, thapsigargin, elicited a strong SOCE signal, Ca2+-signaling was entirely muted upon Orai1-deletion, while deletion of Stim1 diminished and substantially delayed the residual Ca2+-signal. Studying autonomous Ca2+-signaling activity in B-ALL cells, Orai1-deletion resulted in a complete loss of oscillatory Ca2+-signaling, while deletion of Stim1/2 distorted the morphology of the Ca2+-signals with lowered amplitude and greatly extended signal duration. While both Orai1 and Stim1 contribute to SOCE, these observations highlight distinct roles of the two molecules in autonomous Ca2+ oscillation. For both BCR-ABL1- and NRASG12D-driven B-ALL, ablation of both Orai1- and Stim1/2 had profound effects on cell viability and the ability to form leukemic colonies. These results are consistent with the hypothesis that SOCE and autonomous Ca2+-oscillations are essential for oncogenic signaling in B-ALL and thus enable colony formation, proliferation and survival.
To elucidate a functional link between autonomous Ca2+-oscillations and oncogenic signaling in B-ALL, we studied activation of Nfatc1, a central regulator of oncogene signaling and frequency-dependent decoder of Ca2+-oscillations. Upon Cre-mediated deletion of Orai1 and Stim1/2, however, Nfatc1 no longer translocated to the nucleus and was subject to increased proteasomal degradation. Establishing a functional link between Nfatc1-activation and survival and proliferation signaling in B-ALL cells, inducible activation of Nfatc1-deletion in BCR-ABL1-transformed Nfatc1flfl B-ALL cells phenocopied the effects observed with genetic deletion of Orai1 and Stim1/2. Interestingly, the NFATC1-inhibitor INCA6 exhibited strong cytotoxic responses in human B-ALL cells with strong Ca2+-oscillations, while Hodgkin's lymphoma and myeloma cells, lacking Ca2+-oscillations, were resistant to INCA6.
To directly determine how oncogenic signaling impinges on Ca2+-oscillations, we developed a dual biosensor to concurrently measure Ca2+ fluctuations (RCaMP; red) and oncogenic BCR-ABL1 kinase activity (CRKL-phosphorylation; green) in the same B-ALL cells. Simultaneous measurement of Ca2+ (red) and kinase (green) activity revealed that B-ALL cells continuously alternated between Red-only and Green-only, suggesting that kinase and Ca2+ signals both oscillate in a mutually exclusive manner.
Conclusions: Our findings show central roles of SOCE-effector genes, Orai1 and Stim1/2, in oncogenic signaling of B-ALL cells and activation of Nfatc1 as a driver of proliferation and survival signaling. Engineering of a dual biosensor for concurrent measurement of Ca2+ and oncogenic kinase activity revealed that B-ALL cells continuously alternate between Ca2+ and oncogenic kinase activity, with Ca2+-peaks marking the off-state of the oncogenic kinase.

Disclosures
No relevant conflicts of interest to declare.