Autonomous Ca²⁺ Oscillations Reflect Oncogenic Signaling in B-ALL Cells

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 NRAS^G12D-driven B-ALL. Tamoxifen-inducible activation of Cre in Orai1^fl/fl and Stim1/2 ^fl/fl BCR-ABL1- and NRAS^G12D-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 NRAS^G12D-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 Nfatc1^flfl 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.