Glutaredoxin 2 is essential for AML survival through mitochondrial permeability transition pore regulation

Abstract

Patients with acute myeloid leukemia (AML) have a poor 5-year survival rate, highlighting the need for the identification of new approaches to target this disease. AML is highly dependent on glutathione (GSH) metabolism for survival. Although the metabolic role of GSH is well characterized in AML, the contribution of protein glutathionylation, a reversible modification that protects protein thiols from oxidative damage, remains largely unexplored. Therefore, we sought to elucidate the role of protein glutathionylation in AML pathogenesis. Here, we demonstrate that protein glutathionylation is essential for AML cell survival. Specifically, the loss of glutaredoxin 2 (GLRX2), an enzyme that removes GSH modifications, resulted in selective primary AML cell death while sparing normal human hematopoietic stem and progenitor cells. Unbiased proteomic analysis revealed increased mitochondrial protein glutathionylation upon GLRX2 depletion, accompanied by mitochondrial dysfunction, including impaired oxidative phosphorylation, reduced mitochondrial membrane potential, and increased opening of the mitochondrial permeability transition pore (mPTP). Further investigation identified adenosine triphosphate synthase subunit O (ATP5PO), a key regulator of mPTP opening and a component of the ATP synthase complex, as a critical GLRX2 target. Disruption of ATP5PO glutathionylation partially restored mPTP function and rescued AML cell viability after GLRX2 depletion. Moreover, both genetic and pharmacological inhibition of mPTP opening restored the leukemic potential of primary AML specimens in the absence of GLRX2. By disrupting glutathionylation-dependent mitochondrial homeostasis, this study reveals a novel vulnerability in AML that could inform future therapeutic strategies.

Graphical abstract

Figure 1.

GLRX2 KD reduces the growth of AML cells in vitro. (A) GLRX2 expression in LSCs and normal HSPCs. Single-cell RNA sequencing data from van Galen et al. Horizontal lines indicate the interquartile range; unpaired t test, ∗∗∗P < .001. (B) GLRX2 expression in bulk AML cells compared with normal hematopoietic cells. Data from Leucegene (GSE48173), TCGA, Beat-AML, and He et al, , , data sets. Horizontal lines indicate the interquartile range; unpaired t test, ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001. (C) GLRX2 protein expression in normal peripheral mobilized blood cells and primary AML samples (AML1-7). (D) Experimental design of GLRX2 KD experiments. MOLM13, PL21, and MV411 were assessed with viability assay or plated for proliferation assay, and colony-forming unit assay 96 hours after transduction. Created with BioRender. Ling C. (2025) BioRender.com/k96q695. (E) Representative immunoblot measuring protein expression of GLRX2 in MOLM13, PL21, and MV411 96 hours after lentivirus transduction delivering control short hairpin RNA (shRNA) or GLRX2 targeting shRNAs (shRNA1 and shRNA2; n = 3-4 biological replicates). (F) Representative experiment of viable cell counts of MOLM13, PL21, and MV411 after GLRX2 KD. Statistical significance was determined from 3 biological replicates calculating area under the curve. Mean ± standard deviation (SD); ordinary 1-way analysis of variance (ANOVA), ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001. (G) Colony-forming ability of MOLM13, PL21, and MV411 upon GLRX2 KD. Mean ± SD (n = 3 biological replicates); ordinary 1-way ANOVA, ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001. (H) Representative staining of annexin-V and DAPI in control or GLRX2-KD MOLM13 cells. (I) Bar graphs show early apoptotic (annexin-V⁺/DAPI⁻) and late apoptotic (annexin-V⁺/DAPI⁺) MOLM13, PL21, and MV411 cells. Mean ± SD (n = 3 biological replicates); total apoptotic (annexin-V⁺) population compared with ordinary 1-way ANOVA, ∗∗P < .01; ∗∗∗P < .001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LSCs, leukemia stem cells; ns, not significant; shControl, short hairpin control.

Figure 2.

GLRX2 KD selectively impairs primary AML specimens in vitro and in vivo. (A) CFU assay of AML8, AML15, and AML16 with individual GLRX KD. Mean± SD (n = 3 biological replicates); repeated measures (RM) 1-way ANOVA, ∗∗P < .01. (B) Experimental design of the clonogenic assay. Ten primary AML and 1 NBM specimens were transfected with scramble or GLRX2 targeting siRNA and plated in MethoCult medium 24 hours after transfection. Created with BioRender. Ling C. (2025) BioRender.com/k96q695. (C) Expression of GLRX2 determined by western blot in 3 bulk primary AML specimens (AML13-15) 48 hours after transfection. (D-E) Colony-forming potential of 10 bulk AML samples (AML8-17) and 1 NBM sample. Mean ± SD; unpaired t test, ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (F) Human engraftment evaluation in the femur of humanized mice. Three primary AML and 3 NBM specimens were injected into NSG-SGM3 mice through tail vein injection 24 hours after transfection with scramble or GLRX2 targeting siRNA. Eight to 10 weeks after injection, bone marrow cells were collected from femurs to assess disease burden and differentiation. One representative mouse from AML and NBM specimens is shown. Created with BioRender. Ling C. (2025) BioRender.com/k96q695. (G) Engraftment of 3 primary AML specimens (AML18-20) in NSG-SGM3 mice after GLRX2 KD. Each point represents a single mouse. Mean ± SD; unpaired t test, ∗P < .05; ∗∗∗∗P < .0001. (H) Engraftment of 3 primary NBM specimens in NSG-SGM3 mice after GLRX2 KD. Each point represents a single mouse. Mean ± SD; unpaired t test. CFU, colony-forming unit; FSC-A, forward scatter area; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, not significant; PE, phycoerythrin; PECY7, phycoerythrin-cyanine 7; Scr, scrambled siRNA.

Figure 3.

Identification of glutathionylated proteins. (A) Schematic of methods for BioGEE switch assay. Created with BioRender. Ling C. (2025) BioRender.com/a9h05yv. (B) Western blot probed with streptavidin IRDye to identify glutathionylated proteins enriched from BioGEE switch assay in MOLM13 cell line (n = 1). (C) Summary of 49 hits grouped by function identified from control and GLRX2 KD MOLM13 cells detected by liquid chromatography–tandem mass spectrometry–based BioGEE switch assay. Significant hits were defined as proteins with <5% false discovery rate (FDR) and a greater than fourfold change upon GLRX2 KD compared with nontargeting control shRNA–expressing cells. (D) Selected gene ontology terms with <5% FDR and −log₁₀-adjusted P value >3 in the GLRX2-regulated proteins. DTT, dithiothreitol; HS, sulfhydryl group; LC-MS/MS, liquid chromatography-tandem mass spectrometry; NEM, N-ethylmaleimide; NEM-S, N-ethylmaleimide bound to sulfur atom; SH, free thiol; SSG-Bio, biotinylated GSH ethyl ester; SSG, glutathionylation; TCA, tricarboxylic acid cycle.

Figure 4.

GLRX2 regulates ATP5PO to mediates mPTP opening. (A) Representative oxygen consumption curve from viable MOLM13 5 days after GLRX2 KD. For all Seahorse experiments, viable cells were enriched using a dead cell removal bead kit before analysis. (B) Basal respiration in viable MOLM13, PL21, and MV411 5 days after GLRX2 KD. Mean ± SD (n = 3-4 biological replicates); ordinary 1-way ANOVA, ∗P < .05; ∗∗P < .01. (C) Oxygen consumption rate measured in primary AML specimens 8, 10, 11, and 12, 2 days after GLRX2 KD using the Seahorse assay. Mean ± SD; unpaired t test, ∗∗P < .01. (D) Effects of GLRX2 KD 5 days after transduction on ATP production in viable MOLM13, PL21, and MV411 measured using the Seahorse Mito Stress Test. Mean ± SD (n = 3-4 biological replicates); ordinary 1-way ANOVA, ∗P < .05; ∗∗P < .01. (E) Total cellular ATP level determined by mass spectrometry in MOLM13, PL21, and MV411, 5 days after GLRX2 KD. Mean ± SD (n = 4 technical replicates); ordinary 1-way ANOVA, ∗∗P < .01; ∗∗∗∗P < .0001. (F) Representative data of MOLM13 calcein blue signaling treated with CoCl₂, and ionomycin in DAPI⁻ population (n = 3 biological replicates). (G) mPTP opening in DAPI⁻ MOLM13, PL21, and MV411 4 days after expressing shControl or shRNAs against GLRX2. Mean ± SD (n = 3 biological replicates); ordinary 1-way ANOVA, ∗∗P < .01; ∗∗∗P < .001, ∗∗∗∗P < .0001. (H) mPTP opening measured in 4 DAPI⁻ primary AML specimens (AML14-17) 2 days after GLRX2 KD. Mean ± SD; unpaired t test, ∗∗P < .01. (I) Mitochondrial membrane potential in DAPI⁻ MOLM13, PL21, and MV411 4 days after GLRX2 KD. Mean ± SD (n = 3 biological replicates); ordinary 1-way ANOVA, ∗∗∗P < .001; ∗∗∗∗P < .0001. (J) Mitochondrial membrane potential measured in DAPI⁻ primary AML specimens 14, 15, and 17, 2 days after GLRX2 KD. Mean ± SD; unpaired t test, ∗∗P < .01. AU, arbitrary units; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; MFI, mean fluorescence intensity; OCR, oxygen consumption rate; Scr, scrambled siRNA; TMRE, tetramethylrhodamine, ethyl ester.

Figure 5.

GLRX2 mediates mPTP opening through reversible ATP5PO glutathionylation. (A) Tandem mass spectrum of the glutathionylated peptide GEVPC[305]TVTSASPLEEATLSELK from immunoprecipitated ATP5PO. The precursor ion with an m/z ratio of 856.07 is highlighted by the gray arrow, corresponding a GSH conjugated (+305 Da) triply charged peptide. (B) Quantitative measurement of ATP5PO glutathionylation, calculated by the ratio of modified to total ATP5PO MS1 peptide signal intensity 48 hours after doxycycline induction. Mean ± SD (n = 3 biological replicates); paired t test, ∗∗∗P < .005. (C) mPTP opening 4 days after GLRX2 KD in DAPI⁻ ATP5PO C141C wild-type (WT) and C141S (mutation) MOLM13 cells. Mean ± SD (n = 6 biological replicates); RM 1-way ANOVA, ∗P < .05; ∗∗P < .01. (D) Oxygen consumption curve of live ATP5PO C141C (WT) or C141S (mutation) MOLM13 with or without GLRX2 KD 5 days after transduction evaluated by Seahorse assay. (E) Mitochondrial membrane potential in DAPI⁻ ATP5PO C141C (WT) and C141S (mutation) MOLM13 cells 4 days after control or GLRX2 KD. Mean ± SD (n = 8 biological replicates); RM 1-way ANOVA. (F) Percentage of DAPI⁻ ATP5PO C141C (WT) and C141S (mutation) MOLM13 cells 4 days after transduction with control or GLRX2 shRNA. Mean ± SD (n = 3 biological replicates); ordinary 1-way ANOVA, ∗P < .05. (G) Opening of mPTP in GLRX2-KD MOLM13 cells is inhibited by 72-hour 250nM CsA treatment. Mean ± SD (n = 4 biological replicates); ordinary 1-way ANOVA, ∗∗∗∗P < .0001. (H) Colony-forming ability of MOLM13, PL21, and MV411 cells transduced with control or GLRX2-targeting shRNA in 250nM CsA-supplemented methylcellulose media. Mean ± SD (n = 4 biological replicates); ordinary 1-way ANOVA, ∗P < .05. FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; MFI, mean fluorescence intensity; MT, mutation; m/z, mass-to-charge; ns, not significant; NT, nontargeting shRNA; TMRE, tetramethylrhodamine, ethyl ester.

Figure 6.

GLRX2 regulates mPTP opening and OXPHOS in primary AML specimens. (A) Colony-forming ability of 5 bulk primary AML specimens (AML13-17) supplemented with 250nM CsA in methylcellulose media. Mean ± SD (n = 3 technical replicates); ordinary 1-way ANOVA, ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Number of colonies formed by AML17 with Scr and GLRX2 KD duplicated in Figure 2D. (B) mPTP opening in 3 DAPI⁻ primary AML specimens (AML13-15) transfected with scramble or GLRX2-targeting siRNAs, treated with or without CsA. Mean ± SD; ordinary 1-way ANOVA, ∗P < .05; ∗∗∗P < .001. (C) mPTP opening measured in 3 DAPI⁻ primary AML specimens (AML13-15) upon GLRX2 and CypD KD. Mean ± SD; ordinary 1-way ANOVA, ∗P < .01; ∗∗∗∗P < .0001. (D) Clonogenic assays of AML13-15 transfected with scramble, GLRX2, CypD, or GLRX2 + CypD siRNA. Mean ± SD (n = 3 technical replicates); ordinary 1-way ANOVA, ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (E) Engraftment of AML21 in NSG-SGM3 mice after GLRX2 and CypD KD. Each point represents a single mouse. Mean ± SD; ordinary 1-way ANOVA, ∗P < .05. (F) Kaplan-Meier survival curves for NSG-SGM3 mice engrafted with AML21 (patient-derived xenograft model) after GLRX2 and CypD KD. Curve comparison done between NT and each KD group with log-rank (Mantel-Cox) test. (G) mPTP opening measured in 4 viable primary AML specimen populations (AML13, 14, 17, and 19) treated with or without 2.5μM cytarabine upon siRNA-mediated GLRX2 KD. Mean ± SD; ordinary 1-way ANOVA, ∗P < .05; ∗∗P < .01. (H) Colony-forming potential of AML11-15, 17, and 19 transfected with scramble or siRNA targeting GLRX2, treated with or without 2.5μM cytarabine. Mean ± SD (n = 3 technical replicates); ordinary 1-way ANOVA, ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Number of colonies formed by AML11 and AML12 with Scr and GLRX2 KD conditions duplicated in Figure 2D. Comb, combination knockdown of CyPD and GLRX2; Cyt, cytarabine; MFI, mean fluorescence intensity; ns, not significant; NT, nontargeting shRNA; Scr, scrambled siRNA; Veh, vehicle.