Abnormal Regulation of Intracellular Calcium in Human Megakaryocytes Contributes to the Pathophysiology of Calr-Mutant Myeloproliferative Neoplasms
Authors: Di Buduo Christian Andrea , Abbonante Vittorio , Rumi Elisa , Pietra Daniela , Moccia Francesco , Barosi Giovanni , Rosti Vittorio , Cazzola Mario , Balduini Alessandra 
Published: October 2019

Abstract
The BCR-ABL-negative Myeloproliferative Neoplasms (MPNs), are a group of clonal hematopoietic malignancies, that consist of three disorders: Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). Approximately one quarter of patients with ET or PMF carry a somatic mutation of CALR, the gene encoding for the endoplasmic reticulum (ER) chaperone calreticulin. A 52-bp deletion (Type I) and a 5-bp insertion (Type II mutation) are the most frequent lesions. Both generate a new carboxy-terminus of the mutant proteins, in which the ER retention signal KDEL is lost, causing abnormal interaction of the mutants with the thrombopoietin (TPO) receptor (MPL), thus constitutively activating its downstream signaling in megakaryocytes. CALR Type I mutation causes important changes in the charge of the carboxyl-terminal tail, which is responsible for calcium (Ca2+) binding activity. As a consequence, megakaryocytes from CALR Type I patients present enhanced activation of Store-Operated Ca2+ Entry (SOCE), the mechanisms that trigger extracellular Ca2+ inflow upon agonist-induced intracellular Ca2+ mobilization from the ER. A fine control of SOCE and overall Ca2+ homeostasis is fundamental to ensure proper megakaryocyte function. Despite this knowledge, the role of a crosstalk among MPL, CALR and SOCE in regulating megakaryopoiesis has never been hypothesised.
Our research analysed the activation of MPL downstream pathways and Ca2+ signalling in human cord blood and peripheral blood-cultured megakaryocytes, upon stimulation with recombinant human TPO (rhTPO). Additionally, we cultured megakaryocytes from the peripheral blood of patients carrying the CALR Type I mutation to investigate whether CALR and SOCE alterations determine abnormal activation of the MPL downstream pathway in MPNs.
We demonstrated that binding of rhTPO to MPL induces a series of downstream signaling events that lead to the activation of STAT5, AKT and ERK1/2 in human megakaryocytes. This activation relies on significant accumulation of inositol triphosphate (IP3), and consequent intracellular Ca2+ release from the ER followed by extracellular Ca2+ entry, indicative of activated SOCE. Pharmacologic inhibition of the IP3 receptor or SOCE, with 2-APB or BTP-2 respectively, resulted in the complete absence of rhTPO-induced MPL signaling activation. SOCE activation is thus dependent on the dissociation of CALR and STIM1, a protein of the SOCE machinery, which normally forms a complex in un-stimulated megakaryocytes. Importantly, we verified that in human megakaryocytes in rhTPO-starved conditions, CALR forms a molecular complex with its co-chaperone, ERp57, and STIM1, while they dissociate upon rhTPO stimulation. Conversely, we observed that CALR, ERp57 and STIM1 are constitutively dissociated in megakaryocytes from CALR Type I patients, even in complete absence of rhTPO. This dissociation resulted in markedly increased TPO-induced cytosolic Ca2+ flows with a consequent abnormal megakaryocyte proliferation that could be counteracted by pharmacological inhibition of SOCE with BTP-2.
Our findings provide the first evidence that CARL Type I, in addition to MPL activation, concurs to the alteration of SOCE, which sustains the phosphorylation of STAT5, AKT and ERK1/2, and induces megakaryocyte proliferation. Further investigation is needed to understand whether this altered megakaryocyte function is determined by the decrease in CALR levels or by the presence of CALR mutants in the ER. Nevertheless, the abnormal regulation of Ca2+ flows may represent a potentially actionable target.

Disclosures
No relevant conflicts of interest to declare.