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.

BIOCEV 1st Faculty of Medicine Charles University Vestec 25250 Czech Republic

BIOCEV 1st Faculty of Medicine Charles University Vestec 25250 Czech Republic; Clinic Haematology General Faculty Hospital Prague 12808 Czech Republic

BIOCEV 1st Faculty of Medicine Charles University Vestec 25250 Czech Republic; Faculty of Science Charles University Prague 128 00 Czech Republic

Clinic Haematology General Faculty Hospital Prague 12808 Czech Republic

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Saygin C., Carraway H.E. Current and emerging strategies for management of myelodysplastic syndromes. Blood Rev. 2021;48 doi: 10.1016/j.blre.2020.100791. PubMed DOI

Santini V. How I treat MDS after hypomethylating agent failure. Blood. 2019;133:521–529. doi: 10.1182/blood-2018-03-785915. PubMed DOI

DiNardo C.D., Jonas B.A., Pullarkat V., Thirman M.J., Garcia J.S., Wei A.H., Konopleva M., Döhner H., Letai A., Fenaux P., et al. Azacitidine and venetoclax in previously untreated acute myeloid leukemia. N. Engl. J. Med. 2020;383:617–629. doi: 10.1056/NEJMoa2012971. PubMed DOI

Pullarkat V., Pratz K.W., Döhner H., Recher C., Thirman M.J., DiNardo C.D., Fenaux P., Schuh A.C., Wei A.H., Pigneux A., et al. Venetoclax and azacitidine in untreated patients with therapy-related acute myeloid leukemia, antecedent myelodysplastic syndromes or chronic myelomonocytic leukemia. Blood Cancer J. 2025;15:49. doi: 10.1038/s41408-025-01263-3. PubMed DOI PMC

Konopleva M., Pollyea D.A., Potluri J., Chyla B., Hogdal L., Busman T., McKeegan E., Salem A.H., Zhu M., Ricker J.L., et al. Efficacy and biological correlates of response in a phase II study of venetoclax monotherapy in patients with acute myelogenous leukemia. Cancer Discov. 2016;6:1106–1117. doi: 10.1158/2159-8290.Cd-16-0313. PubMed DOI PMC

Nwosu G.O., Ross D.M., Powell J.A., Pitson S.M. Venetoclax therapy and emerging resistance mechanisms in acute myeloid leukaemia. Cell Death Dis. 2024;15:413. doi: 10.1038/s41419-024-06810-7. PubMed DOI PMC

Agrawal K., Das V., Vyas P., Hajdúch M. Nucleosidic DNA demethylating epigenetic drugs - a comprehensive review from discovery to clinic. Pharmacol. Ther. 2018;188:45–79. doi: 10.1016/j.pharmthera.2018.02.006. PubMed DOI

Agarwal R.P., Olivero O.A. Genotoxicity and mitochondrial damage in human lymphocytic cells chronically exposed to 3'-azido-2',3'-dideoxythymidine. Mutat. Res. 1997;390:223–231. doi: 10.1016/s1383-5718(97)00014-4. PubMed DOI

Aimiuwu J., Wang H., Chen P., Xie Z., Wang J., Liu S., Klisovic R., Mims A., Blum W., Marcucci G., Chan K.K. RNA-dependent inhibition of ribonucleotide reductase is a major pathway for 5-azacytidine activity in acute myeloid leukemia. Blood. 2012;119:5229–5238. doi: 10.1182/blood-2011-11-382226. PubMed DOI PMC

Palii S.S., Van Emburgh B.O., Sankpal U.T., Brown K.D., Robertson K.D. DNA methylation inhibitor 5-Aza-2'-deoxycytidine induces reversible genome-wide DNA damage that is distinctly influenced by DNA methyltransferases 1 and 3B. Mol. Cell Biol. 2008;28:752–771. doi: 10.1128/mcb.01799-07. PubMed DOI PMC

Schaefer M., Hagemann S., Hanna K., Lyko F. Azacytidine inhibits RNA methylation at DNMT2 target sites in human cancer cell lines. Cancer Res. 2009;69:8127–8132. doi: 10.1158/0008-5472.Can-09-0458. PubMed DOI

Valencia A., Masala E., Rossi A., Martino A., Sanna A., Buchi F., Canzian F., Cilloni D., Gaidano V., Voso M.T., et al. Expression of nucleoside-metabolizing enzymes in myelodysplastic syndromes and modulation of response to azacitidine. Leukemia. 2014;28:621–628. doi: 10.1038/leu.2013.330. PubMed DOI PMC

Gruber E., Franich R.L., Shortt J., Johnstone R.W., Kats L.M. Distinct and overlapping mechanisms of resistance to azacytidine and guadecitabine in acute myeloid leukemia. Leukemia. 2020;34:3388–3392. doi: 10.1038/s41375-020-0973-z. PubMed DOI

Qin T., Jelinek J., Si J., Shu J., Issa J.P. Mechanisms of resistance to 5-aza-2'-deoxycytidine in human cancer cell lines. Blood. 2009;113:659–667. doi: 10.1182/blood-2008-02-140038. PubMed DOI PMC

Cheng J.X., Chen L., Li Y., Cloe A., Yue M., Wei J., Watanabe K.A., Shammo J.M., Anastasi J., Shen Q.J., et al. RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia. Nat. Commun. 2018;9:1163. doi: 10.1038/s41467-018-03513-4. PubMed DOI PMC

Montes P., Guerra-Librero A., García P., Cornejo-Calvo M.E., López M.D.S., Haro T., Martínez-Ruiz L., Escames G., Acuña-Castroviejo D. Effect of 5-Azacitidine treatment on redox status and inflammatory condition in MDS patients. Antioxidants. 2022;11 doi: 10.3390/antiox11010139. PubMed DOI PMC

Nadasi E., Clark J.S., Szanyi I., Varjas T., Ember I., Baliga R., Arany I. Epigenetic modifiers exacerbate oxidative stress in renal proximal tubule cells. Anticancer Res. 2009;29:2295–2299. PubMed

Pollyea D.A., Stevens B.M., Jones C.L., Winters A., Pei S., Minhajuddin M., D'Alessandro A., Culp-Hill R., Riemondy K.A., Gillen A.E., et al. Venetoclax with azacitidine disrupts energy metabolism and targets leukemia stem cells in patients with acute myeloid leukemia. Nat. Med. 2018;24:1859–1866. doi: 10.1038/s41591-018-0233-1. PubMed DOI PMC

Fandy T.E., Jiemjit A., Thakar M., Rhoden P., Suarez L., Gore S.D. Decitabine induces delayed reactive oxygen species (ROS) accumulation in leukemia cells and induces the expression of ROS generating enzymes. Clin. Cancer Res. : Off. J. Am. Assoc. Cancer Res. 2014;20:1249–1258. doi: 10.1158/1078-0432.Ccr-13-1453. PubMed DOI PMC

Bossis G., Sarry J.E., Kifagi C., Ristic M., Saland E., Vergez F., Salem T., Boutzen H., Baik H., Brockly F., et al. The ROS/SUMO axis contributes to the response of acute myeloid leukemia cells to chemotherapeutic drugs. Cell Rep. 2014;7:1815–1823. doi: 10.1016/j.celrep.2014.05.016. PubMed DOI

Cui Q., Wang J.Q., Assaraf Y.G., Ren L., Gupta P., Wei L., Ashby C.R., Jr., Yang D.H., Chen Z.S. Modulating ROS to overcome multidrug resistance in cancer. Drug Resist. Updates : Rev Commentaries Antimicrobial Anticancer Chemother. 2018;41:1–25. doi: 10.1016/j.drup.2018.11.001. PubMed DOI

Jones C.L., Stevens B.M., D'Alessandro A., Culp-Hill R., Reisz J.A., Pei S., Gustafson A., Khan N., DeGregori J., Pollyea D.A., Jordan C.T. Cysteine depletion targets leukemia stem cells through inhibition of electron transport complex II. Blood. 2019;134:389–394. doi: 10.1182/blood.2019898114. PubMed DOI PMC

Wang K., Jiang J., Lei Y., Zhou S., Wei Y., Huang C. Targeting metabolic-redox circuits for cancer therapy. Trends Biochem. Sci. 2019;44:401–414. doi: 10.1016/j.tibs.2019.01.001. PubMed DOI

Sies H., Mailloux R.J., Jakob U. Fundamentals of redox regulation in biology. Nat. Rev. Mol. Cell Biol. 2024;25:701–719. doi: 10.1038/s41580-024-00730-2. PubMed DOI PMC

Paulsen C.E., Carroll K.S. Cysteine-mediated redox signaling: chemistry, biology, and tools for discovery. Chem. Rev. 2013;113:4633–4679. doi: 10.1021/cr300163e. PubMed DOI PMC

Reina S., Pittalà M.G.G., Guarino F., Messina A., De Pinto V., Foti S., Saletti R. Cysteine oxidations in mitochondrial membrane proteins: the case of VDAC isoforms in mammals. Front. Cell Dev. Biol. 2020;8:397. doi: 10.3389/fcell.2020.00397. PubMed DOI PMC

Zhang J., Ye Z.W., Singh S., Townsend D.M., Tew K.D. An evolving understanding of the S-glutathionylation cycle in pathways of redox regulation. Free Radic. Biol. Med. 2018;120:204–216. doi: 10.1016/j.freeradbiomed.2018.03.038. PubMed DOI PMC

Mailloux R.J., Willmore W.G. S-glutathionylation reactions in mitochondrial function and disease. Front. Cell Dev. Biol. 2014;2:68. doi: 10.3389/fcell.2014.00068. PubMed DOI PMC

Shakir S., Vinh J., Chiappetta G. Quantitative analysis of the cysteine redoxome by iodoacetyl tandem mass tags. Anal. Bioanal. Chem. 2017;409:3821–3830. doi: 10.1007/s00216-017-0326-6. PubMed DOI PMC

Pimkova K., Jassinskaja M., Munita R., Ciesla M., Guzzi N., Cao Thi Ngoc P., Vajrychova M., Johansson E., Bellodi C., Hansson J. Quantitative analysis of redox proteome reveals oxidation-sensitive protein thiols acting in fundamental processes of developmental hematopoiesis. Redox Biol. 2022;53 doi: 10.1016/j.redox.2022.102343. PubMed DOI PMC

Minařík L., Pimková K., Kokavec J., Schaffartziková A., Vellieux F., Kulvait V., Daumová L., Dusilková N., Jonášová A., Vargová K.S., et al. Analysis of 5-Azacytidine resistance models reveals a set of targetable pathways. Cells. 2022;11 doi: 10.3390/cells11020223. PubMed DOI PMC

Ho T.C., LaMere M., Stevens B.M., Ashton J.M., Myers J.R., O'Dwyer K.M., Liesveld J.L., Mendler J.H., Guzman M., Morrissette J.D., et al. Evolution of acute myelogenous leukemia stem cell properties after treatment and progression. Blood. 2016;128:1671–1678. doi: 10.1182/blood-2016-02-695312. PubMed DOI PMC

Tyanova S., Temu T., Sinitcyn P., Carlson A., Hein M.Y., Geiger T., Mann M., Cox J. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods. 2016;13:731–740. doi: 10.1038/nmeth.3901. PubMed DOI

Sherman B.T., Huang da W., Tan Q., Guo Y., Bour S., Liu D., Stephens R., Baseler M.W., Lane H.C., Lempicki R.A. DAVID knowledgebase: a gene-centered database integrating heterogeneous gene annotation resources to facilitate high-throughput gene functional analysis. BMC Bioinf. 2007;8:426. doi: 10.1186/1471-2105-8-426. PubMed DOI PMC

Supek F., Bosnjak M., Skunca N., Smuc T. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS One. 2011;6 doi: 10.1371/journal.pone.0021800. PubMed DOI PMC

Xu S., Hu E., Cai Y., Xie Z., Luo X., Zhan L., Tang W., Wang Q., Liu B., Wang R., et al. Using clusterProfiler to characterize multiomics data. Nat. Protoc. 2024;19:3292–3320. doi: 10.1038/s41596-024-01020-z. PubMed DOI

Wu G., Dawson E., Duong A., Haw R., Stein L. ReactomeFIViz: a Cytoscape app for pathway and network-based data analysis. F1000Research. 2014;3:146. doi: 10.12688/f1000research.4431.2. PubMed DOI PMC

Sun M.A., Wang Y., Cheng H., Zhang Q., Ge W., Guo D. RedoxDB--a curated database for experimentally verified protein oxidative modification. Bioinformatics. 2012;28:2551–2552. doi: 10.1093/bioinformatics/bts468. PubMed DOI

Perez-Riverol Y., Csordas A., Bai J., Bernal-Llinares M., Hewapathirana S., Kundu D.J., Inuganti A., Griss J., Mayer G., Eisenacher M., et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 2019;47:D442–D450. doi: 10.1093/nar/gky1106. PubMed DOI PMC

Messina S., De Simone G., Ascenzi P. Cysteine-based regulation of redox-sensitive Ras small GTPases. Redox Biol. 2019;26 doi: 10.1016/j.redox.2019.101282. PubMed DOI PMC

Alnajjar K.S., Sweasy J.B. A new perspective on oxidation of DNA repair proteins and cancer. DNA Repair (Amst) 2019;76:60–69. doi: 10.1016/j.dnarep.2019.02.006. PubMed DOI PMC

Zhou X., Cooper K.L., Huestis J., Xu H., Burchiel S.W., Hudson L.G., Liu K.J. S-nitrosation on zinc finger motif of PARP-1 as a mechanism of DNA repair inhibition by arsenite. Oncotarget. 2016;7:80482–80492. doi: 10.18632/oncotarget.12613. PubMed DOI PMC

Pettinati I., Brem J., Lee S.Y., McHugh P.J., Schofield C.J. The chemical biology of Human Metallo-β-Lactamase fold proteins. Trends Biochem. Sci. 2016;41:338–355. doi: 10.1016/j.tibs.2015.12.007. PubMed DOI PMC

Birkenmeier G., Stegemann C., Hoffmann R., Günther R., Huse K., Birkemeyer C. Posttranslational modification of human glyoxalase 1 indicates redox-dependent regulation. PLoS One. 2010;5 doi: 10.1371/journal.pone.0010399. PubMed DOI PMC

Hara T., Toyoshima M., Hisano Y., Balan S., Iwayama Y., Aono H., Futamura Y., Osada H., Owada Y., Yoshikawa T. Glyoxalase I disruption and external carbonyl stress impair mitochondrial function in human induced pluripotent stem cells and derived neurons. Transl. Psychiatry. 2021;11:275. doi: 10.1038/s41398-021-01392-w. PubMed DOI PMC

Mashimo M., Onishi M., Uno A., Tanimichi A., Nobeyama A., Mori M., Yamada S., Negi S., Bu X., Kato J., et al. The 89-kDa PARP1 cleavage fragment serves as a cytoplasmic PAR carrier to induce AIF-mediated apoptosis. J. Biol. Chem. 2021;296 doi: 10.1074/jbc.RA120.014479. PubMed DOI PMC

Udensi U.K., Tchounwou P.B. Dual effect of oxidative stress on leukemia cancer induction and treatment. J. Exp. Clin. Cancer Res. 2014;33:106. doi: 10.1186/s13046-014-0106-5. PubMed DOI PMC

Chen Y., Liang Y., Luo X., Hu Q. Oxidative resistance of leukemic stem cells and oxidative damage to hematopoietic stem cells under pro-oxidative therapy. Cell Death Dis. 2020;11:291. doi: 10.1038/s41419-020-2488-y. PubMed DOI PMC

Jing Q., Zhou C., Zhang J., Zhang P., Wu Y., Zhou J., Tong X., Li Y., Du J., Wang Y. Role of reactive oxygen species in myelodysplastic syndromes. Cell. Mol. Biol. Lett. 2024;29:53. doi: 10.1186/s11658-024-00570-0. PubMed DOI PMC

Zhou X., Cooper K.L., Sun X., Liu K.J., Hudson L.G. Selective sensitization of zinc finger protein oxidation by reactive oxygen species through arsenic binding. J. Biol. Chem. 2015;290:18361–18369. doi: 10.1074/jbc.M115.663906. PubMed DOI PMC

Yang C., Qu J., Cheng Y., Tian M., Wang Z., Wang X., Li X., Zhou S., Zhao B., Guo Y., et al. YY1 drives PARP1 expression essential for PARylation of NONO in mRNA maturation during neuroblastoma progression. J. Transl. Med. 2024;22:1153. doi: 10.1186/s12967-024-05956-4. PubMed DOI PMC

Zhao H., Sifakis E.G., Sumida N., Millán-Ariño L., Scholz B.A., Svensson J.P., Chen X., Ronnegren A.L., Mallet de Lima C.D., Varnoosfaderani F.S., et al. PARP1- and CTCF-mediated interactions between active and repressed chromatin at the Lamina promote oscillating transcription. Mol. Cell. 2015;59:984–997. doi: 10.1016/j.molcel.2015.07.019. PubMed DOI

Vancurova M., Hanzlikova H., Knoblochova L., Kosla J., Majera D., Mistrik M., Burdova K., Hodny Z., Bartek J. PML nuclear bodies are recruited to persistent DNA damage lesions in an RNF168-53BP1 dependent manner and contribute to DNA repair. DNA Repair (Amst) 2019;78:114–127. doi: 10.1016/j.dnarep.2019.04.001. PubMed DOI

Antognelli C., Gambelunghe A., Talesa V.N., Muzi G. Reactive oxygen species induce apoptosis in bronchial epithelial BEAS-2B cells by inhibiting the antiglycation glyoxalase I defence: involvement of superoxide anion, hydrogen peroxide and NF-κB. Apoptosis. Int. J. Programm. Cell Death. 2014;19:102–116. doi: 10.1007/s10495-013-0902-y. PubMed DOI

Antognelli C., Palumbo I., Aristei C., Talesa V.N. Glyoxalase I inhibition induces apoptosis in irradiated MCF-7 cells via a novel mechanism involving Hsp27, p53 and NF-κB. Br. J. Cancer. 2014;111:395–406. doi: 10.1038/bjc.2014.280. PubMed DOI PMC

Liu Y., Li Q., Zhou L., Xie N., Nice E.C., Zhang H., Huang C., Lei Y. Cancer drug resistance: Redox resetting renders a way. Oncotarget. 2016;7:42740–42761. doi: 10.18632/oncotarget.8600. PubMed DOI PMC

Mailloux R.J., Treberg J.R. Protein S-glutathionlyation links energy metabolism to redox signaling in mitochondria. Redox Biol. 2016;8:110–118. doi: 10.1016/j.redox.2015.12.010. PubMed DOI PMC

Matsui R., Ferran B., Oh A., Croteau D., Shao D., Han J., Pimentel D.R., Bachschmid M.M. Redox regulation via Glutaredoxin-1 and protein S-Glutathionylation. Antioxidants Redox Signal. 2020;32:677–700. doi: 10.1089/ars.2019.7963. PubMed DOI PMC

Barajas-Espinosa A., Basye A., Jesse E., Yan H., Quan D., Chen C.A. Redox activation of DUSP4 by N-acetylcysteine protects endothelial cells from Cd2+-induced apoptosis. Free Radic. Biol. Med. 2014;74:188–199. doi: 10.1016/j.freeradbiomed.2014.06.016. PubMed DOI PMC

Lagadinou E.D., Sach A., Callahan K., Rossi R.M., Neering S.J., Minhajuddin M., Ashton J.M., Pei S., Grose V., O'Dwyer K.M., et al. BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells. Cell Stem Cell. 2013;12:329–341. doi: 10.1016/j.stem.2012.12.013. PubMed DOI PMC

Pei S., Minhajuddin M., Callahan K.P., Balys M., Ashton J.M., Neering S.J., Lagadinou E.D., Corbett C., Ye H., Liesveld J.L., et al. Targeting aberrant glutathione metabolism to eradicate human acute myelogenous leukemia cells. J. Biol. Chem. 2013;288:33542–33558. doi: 10.1074/jbc.M113.511170. PubMed DOI PMC

Chen X., Glytsou C., Zhou H., Narang S., Reyna D.E., Lopez A., Sakellaropoulos T., Gong Y., Kloetgen A., Yap Y.S., et al. Targeting mitochondrial structure sensitizes acute myeloid leukemia to venetoclax treatment. Cancer Discov. 2019;9:890–909. doi: 10.1158/2159-8290.Cd-19-0117. PubMed DOI PMC