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

Abstract

Background: Dysregulation 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.

Methods: An 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.

Results: Seeking 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 (STIM1_PM); and (vi) blocked when using a mAb targeting STIM1_PM. Next, we further established an association between an elevated expression of STIM1_PM and clinical outcome. In addition, combining an anti-STIM1 mAb with rituximab significantly reduced in vitro CLL B cell viability within the high STIM1_PM CLL subgroup.

Conclusions: These 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.

Trial registration: ClinicalTrials.gov NCT03294980.

Keywords: CLL; Constitutive Ca2+ entry; Disease outcome; STIM1.