STIM1 at the plasma membrane as a new target in progressive chronic lymphocytic leukemia

Abstract

BackgroundDysregulation in calcium (Ca2+) signaling is a hallmark of chronic lymphocytic leukemia (CLL). While the role of the B cell receptor (BCR) Ca2+ pathway has been associated with disease progression, the importance of the newly described constitutive Ca2+ entry (CE) pathway is less clear. In addition, we hypothesized that these differences reflect modifications of the CE pathway and Ca2+ actors such as Orai1, transient receptor potential canonical (TRPC) 1, and stromal interaction molecule 1 (STIM1), the latter being the focus of this study.MethodsAn extensive analysis of the Ca2+ entry (CE) pathway in CLL B cells was performed including constitutive Ca2+ entry, basal Ca2+ levels, and store operated Ca2+ entry (SOCE) activated following B cell receptor engagement or using Thapsigargin. The molecular characterization of the calcium channels Orai1 and TRPC1 and to their partner STIM1 was performed by flow cytometry and/or Western blotting. Specific siRNAs for Orai1, TRPC1 and STIM1 plus the Orai1 channel blocker Synta66 were used. CLL B cell viability was tested in the presence of an anti-STIM1 monoclonal antibody (mAb, clone GOK) coupled or not with an anti-CD20 mAb, rituximab. The Cox regression model was used to determine the optimal threshold and to stratify patients.ResultsSeeking to explore the CE pathway, we found in untreated CLL patients that an abnormal CE pathway was (i) highly associated with the disease outcome; (ii) positively correlated with basal Ca2+ concentrations; (iii) independent from the BCR-PLCγ2-InsP3R (SOCE) Ca2+ signaling pathway; (iv) supported by Orai1 and TRPC1 channels; (v) regulated by the pool of STIM1 located in the plasma membrane (STIM1PM); and (vi) blocked when using a mAb targeting STIM1PM. Next, we further established an association between an elevated expression of STIM1PM and clinical outcome. In addition, combining an anti-STIM1 mAb with rituximab significantly reduced in vitro CLL B cell viability within the high STIM1PM CLL subgroup.ConclusionsThese data establish the critical role of a newly discovered BCR independent Ca2+ entry in CLL evolution, provide new insights into CLL pathophysiology, and support innovative therapeutic perspectives such as targeting STIM1 located at the plasma membrane.