Re-Sensitization of Azacytidine-Resistant MDS/AML Cells Via Modulation of KEAP1-NRF2 Redox Pathway

Introduction: The hypomethylation therapy by the cytidine analogue pro-drug 5-azacytidine (AZA) significantly extends the life expectancy of patients with myeloid malignancies, as shown in randomized trials in patients with myelodysplastic syndromes (MDS). Although AZA induces more durable hematologic responses, the rate and duration of remissions are insufficient, and most patients eventually progress on therapy, highlighting the need to better understand the mechanisms of drug resistance.

Our and others’ previous data suggest that AZA resistance is the result of the interplay between multiple molecular mechanisms [Minařík L. et al. 2022]. Recent evidence highlights the important role of oxidative/reductive (redox) homeostasis in the development of resistance [Pollyea, et al. 2018, Jones CL. et al. 2019, Gu, X. et al. 2021]. Redox homeostasis is controlled by redox sensor Kelch like associated protein 1 (KEAP1), whose oxidation of cysteine under elevated oxidative stress stabilizes the transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), leading to cytoprotection against oxidative stress. In this work we aimed to investigate the role of the KEAP1-NRF2 pathway in the AZA efficacy.

Methods: We derived AZA-resistant clones (AZA-R) from the human OCI-M2 MDS/acute myeloid leukemia (AML) cell line. The IC50 of AZA-R estimated by WST assay ranged from 4-16 µM. We applied CM₂-H₂DCFDA and ThiolTracker fluorescent probes to determine the oxidative state and glutathione levels (GSH) of AZA-sensitive (AZA-S) and AZA-R cells. To evaluate functional impact of altered redox homeostasis in AZA-R, we applied a quantitative mass spectrometry-based proteomic approach to identify protein targets of oxidative modifications in AZA-S and AZA-R after AZA treatment. The consequence of altered redox homeostasis was determined by RNA sequencing of AZA-S and AZA-R cells after 24h treatment with 1 µM AZA. The findings were functionally validated.

Results: We showed that the redox state of AZA-S was increased by AZA, while the basal oxidative levels of AZA-R cells were 2-fold higher than those of AZA-S cells and were not further altered by AZA. Similarly, AZA induced GSH levels in AZA-S but not in AZA-R cells. Treatment of AZA-S with N-acetylcysteine (NAC) ameliorated oxidative stress and alleviated AZA toxicity, suggesting that AZA-induced oxidative stress is a key factor in its cytotoxic effect. Conversely, a reduction in GSH levels in these cells led to an increase in oxidative state, which was accompanied by enhanced sensitivity to AZA.

Proteomic analysis showed that AZA treatment of AZA-S cells resulted in altered cysteine oxidation of 20% of cysteine proteins (578 out of 2853). Resistance to AZA was associated with oxidation state of 14% of cysteine proteins, among which the key redox sensor KEAP1 was significantly differently oxidized between AZA-R and AZA-S. Transcriptomic profiling revealed that 75 out of 478 NRF2 target genes were differentially expressed between AZA-S and AZA-R. NRF2 protein levels were significantly lower in the nucleus of AZA-R MDS/AML myeloblasts by Western blotting. These data indicate a loss of the dynamic response of AZA-R cells to oxidative stress by the KEAP1-NRF2 system.

Therefore, we aimed to activate NRF2 in AZA-R by treatment with KEAP1 inhibitors (iKEAP1). We found that NRF2 activation in these cells modifies the redox balance and restores the sensitivity of AZA-R cells to AZA, presumably by restoring the antioxidant properties of the cells. Finally, we transplanted AZA-S and AZA-R cells into immunodeficient mice in order to validate our data in vivo. Generated Cell line-Derived Xenograft (CDX) AZA-R models were treated with iKEAP and indeed iKEAP1 markedly prolonged event-free survival and overall survival of MDS/AML mice upon AZA treatment.

Conclusions: We demonstrate that the mechanism of AZA resistance involves the cellular adaptation to oxidative stress and that modulation of the KEAP1-NRF2 cellular antioxidant response pathway can efficiently re-sensitize AZA-resistant cells in vitro. We also demonstrated our findings of enhanced AZA efficacy by KEAP1 inhibition in vivo in an experimental CDX mouse model. This preliminary evidence supports the initiation of a randomized clinical trial to block KEAP1-NRF2 pathway in patients with high-risk-MDS progressing on AZA therapy.

No relevant conflicts of interest to declare.