P1004: ACTIVATING MUTATIONS IN JAK2 AND CALR DIFFERENTIALLY AFFECT INTRACELLULAR CALCIUM LEVELS AND CALCIUM FLUX

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Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: In myeloproliferative neoplasms (MPNs), mutations in Janus kinase 2 (JAK2), calreticulin (CALR), and the thrombopoietin receptor (MPL) activate common Janus kinase/signal transducer and activator of transcription signaling (JAK/STAT) pathways. These mutations confer hypersensitivity in cytokine-induced proliferation and drive clonal expansion of hematopoietic progenitor cells. However, the exact mechanisms of action of these mutants have not been fully comprehended. Calcium (Ca2+) is an important intracellular secondary messenger, which regulates diverse essential cellular functions, like activation of integrins, cell migration, exocytosis and many more. One major Ca2+ influx pathway in many cell types is store-operated calcium entry (SOCE) through calcium release-activated calcium (CRAC) channels. Although, Phospholipase Cγ1 (PLCγ1) which controls Ca2+ signaling pathways, has been implicated in the formation of early erythropoiesis, the role of mutated JAK2 and CALR on SOCE has been remained elusive. Aims: The purpose of this study was to determine the effect of JAK2-V617F and CALR mutations on SOCE and key signaling pathways in the absence or presence of erythropoietin (EPO) or thrombopoietin (TPO), respectively. Methods: 32D cells (JAK2WT/EpoR, JAK2-V617F/EpoR; CALRWT/MPL, CALR-ins5/MPL and del52/MPL) were labeled with Fura-2 AM for 30 minutes and then seeded in 0.01% poly-L-lysine pre-coated 96-well plates. Intracellular Ca2+ measurements were performed using Synergy H1 plate reader. Total Ca2+ measurement time frame was from 0sec to 1080sec, where basal Ca2+ concentration was measured between 0sec-108sec. Subsequently, cells were stimulated with EPO/TPO in Ca2+-free Ringer solution and Ca2+ measurement was performed from 109sec-594sec (store depletion), followed by addition of 2mM Ca2+ Ringer solution to induce SOCE where the measurement was acquired from 595sec-1080sec. Fura-2 emission ratios (F340/380) were quantified by analyzing the integrated Ca2+ signal. Results: Upon EPO stimulation, SOCE measured as area under the curve (AUC) was significantly increased in 32D JAK2V617F cells (p=0.035) as compared to 32D-WT cells. Conversely, under unstimulated conditions 32D-WT cells (p=0.001) showed significantly higher basal levels of cytosolic Ca2+ compared to 32D JAK2-V617F cells. Furthermore, we measured the Ca2+ levels following store depletion and SOCE in TPO stimulated CALR cells. The Ca2+ concentration during store depletion (Max peak: p=0.04; AUC: p=0.05) and SOCE (Max peak: p=0.06; AUC: p=0.07) was higher in WT CALR cells in comparison with CALR-ins5 and del52 cells. To dissect the underlying molecular mechanisms, we analyzed the key downstream components of EPO/JAK2 and TPO/MPL signaling pathways. Upon stimulation, essential elements of the Ca2+ signaling pathways such as PLCγ1 and IP3, were found to be activated in these cell lines. A substantial change in the dynamics of phosphorylated states of PLCγ1, IP3, STAT3, STAT5, AKT, MEK, and ERK was found in 32D JAK2-V617F and CALR mutated cells. Summary/Conclusion: The mutations in JAK2 and CALR differentially affect store-operated calcium entry. We here provide novel evidence that the JAK2-V617F mutation induces an essential change in the regulatory mechanism of EpoR/JAK2-dependent intracellular Ca2+ homeostasis upon EPO stimulation. This affects both basal and SOCE induced Ca2+ levels, cytokine dependent PLCγ1 signalling pathways, and cellular proliferation. These findings explain the clinically observed differential response of JAK2V617F vs. WT progenitor cells to cytokines in MPN. Keywords: Calcium, Intracellular signaling, Myeloproliferative disorder

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