GSTM5 inhibiting ferroptosis in myelodysplastic syndrome cells and its synergistic association with the NRF2/GPX4 pathway

Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis and dysregulated ferroptosis, with glutathione S-transferase mu 5 (GSTM5) being downregulated. This study aims to verify whether GSTM5 inhibits ferroptosis in MDS cells by activating the nuclear factor erythroid 2-related factor 2 (NRF2)/glutathione peroxidase 4 (GPX4) signaling pathway, by constructing GSTM5-overexpressing MDS cell lines combined with proteomics analysis.

A lentiviral expression plasmid, pCDH-GSTM5, was constructed using the full-length cDNA sequence of human GSTM5 cDNA, which was used to infect the human MDS cell line SKM-1. After screening, a stable overexpression cell line was obtained (GSTM5-LV group), and the cells transfected with the empty vector served as the negative control (NC-LV group). The protein and mRNA expression levels of GSTM5 and ferroptosis-related genes in the 2 groups were determined by Western blotting and RT-qPCR. The differentially expressed proteins (DEPs) between the 2 groups were identified with liquid chromatography-mass spectrometry (LC-MS/MS), followed by enrichment analyses, including GO, KEGG, and protein-protein interaction (PPI) network analyses. Principal component analysis (PCA) was applied to evaluate intergroup differences. An oxidative stress model was established by treating the 2 groups of cells with 10 μmol/L ferroptosis inducer Erastin. Then intracellular reactive oxygen species (ROS) levels, lipid peroxidation (BODIPY 581/591 C11 probe), and Fe²⁺ content were measured by flow cytometry and fluorescence microscopy, and cell cycle distribution was assessed by flow cytometry. Simultaneously, the protein and mRNA expression of solute carrier family 7 member 11 (SLC7A11), GPX4, and NRF2 were detected. Furthermore, after treatment of NRF2 agonist dimethyl fumarate (DMF), the protein and mRNA expression levels of SLC7A11, transferrin receptor protein 1 (TFRC), ferritin heavy chain (FTH1), and GPX4 were detected to evaluate the synergistic effect between GSTM5 and the NRF2 pathway. In addition, molecular docking was used to predict the interaction between the GSTM5 and NRF2 proteins. All experiments were performed independently in triplicate (n=3).

A total of 7377 proteins were identified by proteomics (FDR<0.01). Using P<0.05 and FC>1.5 or <0.67 as criteria, 350 DEPs were screened between the GSTM5-LV and NC-LV groups (212 upregulated and 138 downregulated), among which GSTM5, GPX4, and SLC7A11 were significantly upregulated, whereas TFRC was significantly downregulated. PCA showed a clear separation between the 2 groups. GO enrichment analysis indicated that the DEPs were mainly associated with translation and ribosome biogenesis, and were predominantly localized in the cytoplasm and ribosomes. KEGG pathway enrichment analysis showed that they were mainly enriched in the ribosome, ferroptosis, and glutathione metabolism pathways. Phenotypic experiments confirmed that, compared with the control group, GSTM5 overexpression significantly reduced intracellular ROS levels, lipid peroxidation, and Fe²⁺ accumulation in MDS cells (all P<0.05, with consistent results from fluorescence microscopy and flow cytometry), and induced cell cycle arrest at the G0/G1 and S phase, thereby inhibiting ferroptosis. In the 10 μmol/L Erastin-induced stress model, the increases in ROS, Fe²⁺, and lipid peroxidation in the GSTM5 overexpression group were significantly lower than those in NC-LV group (P<0.05), antagonizing Erastin-induced ferroptosis. Concurrently, GSTM5 upregulation was accompanied by increased protein and mRNA levels of NRF2 and its downstream target genes (GPX4, SLC7A11, and FTH1), and decreased TFRC expression (P<0.05). Molecular docking predicted a strong theoretical affinity between GSTM5 and NRF2 (lowest binding energy of -10.1 kcal/mol) with the formation of multiple hydrogen bonds. The NRF2 agonist DMF showed a synergistic effect with GSTM5 overexpression, with the combined treatment group exhibiting pronounced reductions in lipid peroxidation and Fe²⁺ (P<0.05).

GSTM5 overexpression significantly inhibits ferroptosis in MDS cells, and its mechanism may be related to promoting the activation of the NRF2/GPX4 pathway. GSTM5 exhibits a synergistic effect with the NRF2 agonist DMF, suggesting a possible functional association between the 2 molecules. This study preliminarily elucidates the protective role of GSTM5 against ferroptosis in MDS cells, providing a new theoretical basis and experimental foundation for ameliorating ineffective hematopoiesis in MDS.