Hypomethylation therapy by 5-azacytidine (AZA) is indicated upon progression of onco-hematological diseases: myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). AZA represents a backbone treatment option in MDS/AML that significantly prolongs patient survival, but early development of resistance seriously complicates the therapy. AZA inhibits DNA methylation, induces DNA damage as well as the production of reactive oxygen species (ROS). Our and others data have shown that ROS, through oxidative modifications of thiols in proteins, fine-tune intrinsic stress and survival cell programs of cells. Therefore, we hypothesized that AZA induces protein oxidative modifications that control adaptive processes when MDS/AML cells respond to AZA.

In this study, we applied a quantitative mass spectrometry-based proteomic approach to identify protein targets of oxidative modifications in a cellular MDS/AML model of AZA resistance (Minarik, Pimkova et al., 2022) and in primary patient samples, and complemented the results with transcriptomic analysis. Identification of the redox state of 8,686 cysteine sites in 3,380 proteins revealed that AZA preferentially regulated the redox state of proteins that are activated in response to the DNA damage and contain a zinc finger domain essential for DNA/RNA binding, and that oxidation induced cell death is one of the mechanisms of action of AZA. MDS/AML OCI-M2 cell and AZA-resistant primary myeloblasts are characterized by high glutathione (GSH) levels and low S-glutathionylation of proteins. GSH depletion by buthionine sulfoximine increased protein S-glutathionylation and restored response to AZA in AZA resistant cells thus validating our assumptions.

This study unveils a role of redox homeostasis in the toxicity and mechanism of adaptation to the hypomethylation therapy. This is the first work that comprehensively described the effect of AZA treatment on oxidative modifications of proteins in MDS/AML cell line responding and not responding to treatment and compared these data with redox changes on proteins in patients relapsing on AZA. Remarkably we found that AZA-induced redox changes of the proteome in MDS/AML cell line forms a substantial part of AZA toxicity and also contribute significantly to the development of AZA resistance.