Targeting mitochondrial and glutathione metabolism sensitizes leukemia cells to DNA-hypomethylating agents and venetoclax combination therapy

Acute myeloid leukemia (AML) is the most common acute leukemia diagnosed in adults. DNA-hypomethylating agents (HMAs), such as decitabine and azacitidine, are widely used to treat AML and myelodysplastic syndromes (MDS). Although HMAs demonstrate only modest efficacy as monotherapies, clinical responses improve substantially when HMAs are combined with other therapeutic agents, notably venetoclax (VEN) in older patients, FLT3 inhibitors (FLT3i) for FLT3-mutated AML, and IDH inhibitors (IDHi) for IDH1/2-mutated disease. Nevertheless, drug resistance frequently emerges, emphasizing the need to elucidate resistance mechanisms and develop new therapeutic strategies. The typical schedule of HMA treatment, several consecutive days of sequential dosing followed by a rest interval, suggests that epigenetic memory may contribute to resistance. To investigate potential mechanisms of resistance, we examined the effects of transient low-dose decitabine in AML cell lines, observing that three consecutive treatment days markedly suppressed cell proliferation and viability. However, after three weeks of drug-free recovery, surviving cells regained robust growth, indicating that a subset of cells adapts and potentially acquires relapse-associated drug-resistance. We found that decitabine induces persistent mitochondrial dysfunction, characterized by altered mitochondrial networks, ultrastructure, and activity, that remains evident even after drug withdrawal and likely contributes to initial growth inhibition. Notably, surviving cells exhibit strong enrichment of the glutamate-glutathione metabolic pathway, suggesting a compensatory mechanism that mitigates mitochondrial stress. Integrated RNA sequencing and metabolomic profiling identified a key enzyme in the glutamate-glutathione metabolic pathway for this metabolic adaptation. In human AML xenograft models, inhibition of this enzyme significantly restored sensitivity to HMA (decitabine or azacytidine)-VEN combination therapy in resistant or poorly responsive AML cells. Together, our findings identify glutathione-based metabolic adaption as a potential mechanism of HMA resistance and highlight targeting mitochondrial and glutathione metabolism as a promising therapeutic strategy to enhance the efficacy of HMA-containing combination regimens.