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.

Fig. 1

An elevated level of constitutive calcium entry (CE) is relevant for chronic lymphocytic leukemia (CLL) clinical outcome. a- Representative kinetic plots of CE in representative healthy control B cells (n = 8, Ctrl), CE- B-CLL (n = 13) and CE+ B-CLL (n = 17) samples. A Cox regression model of progression free survival was used to dichotomize CLL patients in CE- and CE+ (dash line in the islet). Kaplan-Meier plots showing: b- time to progression free survival; c- time to treatment free survival; and d- lymphocyte doubling time between CE+ and CE- CLL groups. P values are indicated when significant

Fig. 2

Constitutive calcium entry (CE) is independent from anti-IgM capacity to induce Ca²⁺ signaling in chronic lymphocytic leukemia (CLL). a- Normalized ratio of peak to baseline Ca²⁺ flux in response to anti-IgM stimulation of healthy control (n = 13, Ctrl), CE- CLL (n = 13) and CE+ CLL (n = 16) samples. b- Bivariate analysis by Kaplan-Meier curves of Ca²⁺ signaling in the context of CLL subsets according to the CE (+/−) and anti-IgM capacity to induce Ca²⁺ signaling (IgM +/−). A Cox regression model of progression free survival (PFS) was used to dichotomize CLL patients in CE- and CE+, on one hand, and IgM- and IgM+, on the other hand. c/d- the effects of Ibrutinib (BTK inhibitor, 2.5 μM) and LY294002 (PI3K inhibitor, 5 μM) on CE in CE+/IgM+ B-CLL samples (n = 3). e/f- the effects of Ibrutinib (BTK inhibitor) and LY294002 (PI3K inhibitor) on anti-IgM capacity to induce Ca²⁺ signaling in CE+/IgM+ B-CLL samples (n = 3). P values are indicated when significant

Fig. 3

Constitutive calcium entry (CE) is related to STIM1, Orai1 and TRPC1. a/b- The effects of the Orai1 channel inhibitor Syntha(S)66 on CE (a) and on anti-IgM Ca²⁺ response (b) in CE+/IgM+ CLL samples (n = 3). c- siRNA control analysis by FACS. The mean fluorescence intensities (MFI) of representative CE+ transfected B-CLL cells are shown in the upper left corner of each plot for the siRNA control, and in the lower left corner for the specific siRNAs. d- Average time course of CE in siRNA transfected CE+ B-CLL cells (n = 3). P values are indicated when significant

Fig. 4

B-CLL cells from CE+ CLL patients (n = 11) display increased Orai1 and STIM1 protein levels compared with those from CE- CLL patients (n = 8). Left panels, representative Western blot images for Orai1(a), TRPC1 (b), and STIM1 (c). Quantification after normalization to GAPDH protein expression is depicted (d). P values are indicated when significant

Fig. 5

The pool of STIM1 in plasma membrane controls constitutive calcium entry (CE). a- Cytoplasmic (after permeabilization, total STIM1[t]) and plasma membrane staining of STIM1 (STIM1_PM) on B-CLL cells from CE+ (n = 7) and CE- (n = 12) CLL patients. The isotype control is shown (black line). The mean fluorescence intensities (MFI) of representative CE- B-CLL cells are shown in the upper left corner of each plot in blue, and in the lower left corner in blue for CE+ B-CLL cells. b- Correlation between CE and tSTIM1_T (upper panel) or STIM1_PM (lower panel). c- Receiver operating curves (ROC) were generated to determine the area under the curve (AUC) and the optimal cut-off value to discriminate STIM1 high from STIM1 low patients. d- The effects of the anti-STIM1 mAb clone GOK on CE in CLL samples (n = 6). e- No effect of the anti-STIM1 mAb on anti-IgM Ca²⁺ response in CLL samples (n = 10). The r² coefficient and P values are indicated when significant

Fig. 6

In the whole CLL cohort (n = 74), an elevated level of STIM1 at plasma membrane (STIM1PM) is relevant for CLL clinical outcome and influence in vitro cell survival. a Kaplan-Meier plots showing progression free survival and treatment free survival for STIM1PM dichotomize into high and low levels. b Increase in the density of STIM1PM improves the efficacy of rituximab (RTX) in the STIM1_PM high CLL subgroup (n = 9) when used in combination with the anti-STIM1 mAb (both 10 μg/mL, 48 h), effect which was not observed in the STIM1_PM low CLL subgroup (n = 8). P values are indicated when significant