Opposing Calcium-Dependent Effects of GsMTx4 in Acute Lymphoblastic Leukemia: In Vitro Proliferation vs. In Vivo Survival Advantage

Abstract

Mechanogated (MG) ion channels play a crucial role in mechano-transduction and immune cell regulation, yet their impact on blood cancers, particularly acute lymphoblastic leukemia (ALL), remains poorly understood. This study investigates the pharmacological effects of GsMTx4, an MG channel inhibitor, in human ALL cells both in vitro and in vivo. Unexpectedly, we found that GsMTx4 remarkably increased basal calcium (Ca²⁺) levels in ALL cells through constitutive Ca²⁺ entry and enhanced store-operated Ca²⁺ influx upon thapsigargin stimulation. This increase in basal Ca²⁺ signaling promoted ALL cell viability and proliferation in vitro. Notably, chelating intracellular Ca²⁺ with BAPTA-AM reduces GsMTx4-mediated leukemia cell viability and proliferation. However, in vivo, GsMTx4 decreases cytosolic Ca²⁺ levels in Nalm-6 GFP⁺ cells isolated from mouse blood, effectively countering leukemia progression and significantly extending survival in NSG mice transplanted with leukemia cells (median survival: GsMTx4 vs. control, 37.5 days vs. 29 days, p = 0.0414). Our results highlight the different properties of GsMTx4 activity in in vitro and in vivo models. They also emphasize that Ca²⁺ signaling is a key vulnerability in leukemia, where its precise modulation dictates disease progression. Thus, targeting Ca²⁺ channels could offer a novel therapeutic strategy for leukemia by exploiting Ca²⁺ homeostasis.

Keywords: ALL; Ca2+ signaling; GsMTx4; cell survival.

Figure 1

Mechanogated ion channel inhibitor (GsMTx4) enhances basal Ca²⁺ levels in ALL cell lines. The basal Ca²⁺ level was used as a read-out to measure the impact of GsMTX4 on constitutive Ca²⁺ channel activity in ALL cells. (A–E) ALL cell lines pre-treated with control (Ctr, DMSO) and GsMTx4 using the ratiometric Ca²⁺ indicator Fura-2 QBT. The basal Ca²⁺ level was evaluated before normalization as the average of initial F₃₄₀ nm/F₃₈₀ nm values. Data are mean ± SEM (n = 3). Statistical significance was analyzed by two-tailed, unpaired Student’s t-tests.

Figure 2

GsMTx4 increases Mn²⁺ influx in ALL cell lines. The quenching of Fura-2 fluorescence by Mn²⁺ in ALL cell lines was measured at 360 nm (the Ca²⁺-independent excitation wavelength of Fura-2). (A–E) ALL cells before exposure to Mn²⁺ (10 μM) were preincubated during Fura-2 GBT loading with GsMTx4. Results express normalized fluorescence and quenching peak quantification. Data are mean ± SEM (n = 5). Statistical significance was analyzed by two-tailed, unpaired Student’s t-tests. * p < 0.05, *** p < 0.001, and **** p < 0.0001.

Figure 3

GsMTx4 potentiates store-operated Ca²⁺ entry (SOCE) in ALL cell lines. (A–C) Representative traces and quantification of SOCE peak upon store depletion in 0 mM Ca²⁺ with 2 µM TG (thapsigargin) followed by addition of 1.8 mM Ca²⁺ to the solution. Cells before exposure to TG in Ca²⁺ buffer were preincubated during Fura-2 QBT loading with GsMTx4. Data are mean ± SEM (n = 4). Statistical significance was analyzed by two-tailed, unpaired Student’s t-tests.

Figure 4

GsMTx4 stimulates proliferation and viability of ALL cells through Ca²⁺ signaling. (A) ALL cell lines were treated with GsMTx4 for 48 h. Then, the proliferation rate was determined by CCk-8 staining. The percentage was established after normalizing on control (Ctr) cells. (B,C) ALL cell lines were treated with GsMTx4 for 48 h in the absence or presence of BAPTA-AM (1 μM). (B) Cell proliferation rate was determined by CCK-8 assay. (C) Cell viability was determined by flow cytometry after annexin V/PI staining. Viable cells represent the percentage of annexin V-FITC/PI- negative population. Data are mean ± SEM (n = 3). Statistical significance was analyzed by two-tailed, unpaired Student’s t-tests; ns, not significant.

Figure 5

GsMTx4 decreases cytosolic Ca²⁺ levels of ALL cells and prolongs overall survival of NSG mice in vivo. (A) Experimental design of NSG mice treatment experiments. BLI, bioluminescence imaging. (B) Flow cytometry Ca²⁺ assessment in Nalm-6 GFP+ cells in the blood at day 16. (C) An example of bioluminescence imaging of Nalm-6 GFP/luc at days 14 and 28 of tumor challenge. (D) Kaplan–Meier survival analysis of mice transplanted with Nalm-6 GFP-luc cells and treated with Ctr (DMSO, n = 4) and GsMTx4 (n = 4). Statistical significance was calculated with the log-rank (Mantel–Cox) test, (* p = 0.041).