# Redox-dependent protein S-glutathionylation governs azacitidine sensitivity and resistance in AML

## TL;DR

This study shows that redox signaling, specifically protein S-glutathionylation, plays a key role in how AML cells respond to azacitidine, and targeting glutathione metabolism can restore drug sensitivity.

## Contribution

The study identifies redox-dependent S-glutathionylation as a novel mechanism of azacitidine resistance in AML and proposes glutathione metabolism as a therapeutic target.

## Key findings

- Azacitidine induces protein S-glutathionylation and cell death by disrupting redox homeostasis in sensitive AML cells.
- AZA-resistant cells maintain high glutathione levels and reduced S-glutathionylation, which can be reversed by glutathione depletion to restore AZA sensitivity.
- Primary AZA-relapsed AML samples show a redox profile similar to resistant cell lines, implicating the glyoxalase and mitochondrial systems in resistance.

## Abstract

Disruption of redox metabolism is a hallmark of drug-resistant cancer cells, representing a major obstacle to the effective treatment of acute myeloid leukemia (AML). While recent studies have highlighted the importance of redox balance in AML therapy, the specific contribution of protein redox signaling to resistance remains poorly understood. Defining these mechanisms could uncover therapeutic vulnerabilities of resistant AML cells and guide the development of novel combination strategies. Here, we performed comprehensive mass spectrometry–based redox and quantitative proteomic profiling of AML cell lines and patient samples sensitive or resistant to the hypomethylating agent azacitidine (AZA). We demonstrate that AZA disrupts redox homeostasis, which inactivates the glyoxalase system and DNA damage response, and thereby induces cell death. In contrast, AZA resistance is associated with a redox reset characterized by elevated glutathione levels and diminished protein S-glutathionylation. Importantly, AZA failed to induce oxidation of proteins in these pathways in resistant cells and patient-derived AML samples. Pharmacological inhibition of glutathione synthesis restored protein S-glutathionylation and resensitized resistant AML cells to AZA.

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•Azacitidine induces ROS generation and protein S-glutathionylation in sensitive AML cells, targeting redox-sensitive cysteine residues.•Selective oxidation of DNA repair proteins (PARP1) and metabolic enzymes (GLO1) leads to DNA damage and apoptosis as key components of azacitidine's mechanism of action.•Resistant cells evade AZA toxicity by maintaining a high GSH/GSSG ratio, upregulating glutaredoxin, and reducing global S-glutathionylation.•Glutathione depletion resensitizes resistant cells to AZA, while GSH supplementation protects sensitive cells, underscoring the central role of GSH metabolism.•The redox signature of primary AZA-relapsed cells aligned with azacitidine-resistant lines, implicating the mitochondrial and glyoxalase system in resistance.

Azacitidine induces ROS generation and protein S-glutathionylation in sensitive AML cells, targeting redox-sensitive cysteine residues.

Selective oxidation of DNA repair proteins (PARP1) and metabolic enzymes (GLO1) leads to DNA damage and apoptosis as key components of azacitidine's mechanism of action.

Resistant cells evade AZA toxicity by maintaining a high GSH/GSSG ratio, upregulating glutaredoxin, and reducing global S-glutathionylation.

Glutathione depletion resensitizes resistant cells to AZA, while GSH supplementation protects sensitive cells, underscoring the central role of GSH metabolism.

The redox signature of primary AZA-relapsed cells aligned with azacitidine-resistant lines, implicating the mitochondrial and glyoxalase system in resistance.