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

• Published in: Blood 2019-11, Vol.134 (Supplement_1), p.1253-1253
• Main Authors: Kume, Kohei, Chen, Liting, Lee, Jaewoong, Müschen, Markus
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2019-130708

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 Stim1/2fl/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,