Abstract
Acute myeloid leukaemia (AML) is a cancer affecting haematopoietic stem and progenitor cells and is characterised by disrupted differentiation, metabolic rewiring, and epigenetic dysregulation. AML incidence is highest in people aged over 60, for whom conventional chemotherapy is often unsuitable, creating a need for alternative treatment options. In these cases, key therapeutic objectives aimed at slowing disease progression and overcoming chemoresistance include promoting leukaemic cell differentiation and reducing proliferation and viability. Azacitidine is a hypomethylating agent that captures the methylating enzyme DNA methyltransferase-1 (DNMT1) and targets the epigenetic regulation of leukaemic cell fate. While azacitidine has demonstrated efficacy in advancing these therapeutic objectives, clinical responses remain variable. Using two compounds targeting redox and metabolic pathways, which are closely linked to epigenetic regulation, I investigated the effects of combination therapy with azacitidine on treatment outcomes for AML.
Firstly, ascorbate (vitamin C) is a potent antioxidant and a co-factor for ten-eleven translocation methylcytosine dioxygenase 2 (TET2) enzymes, which catalyse the first step of DNA demethylation. Secondly, metformin is a frontline diabetes drug that inhibits complex I of the electron transport chain, thereby inhibiting oxidative phosphorylation. I assessed the responses of two AML cell lines, SKM-1 and OCI-AML3, to repeated doses of azacitidine (0.5 μM), ascorbate (200 μM), metformin (5 mM), azacitidine and ascorbate combined, and azacitidine and metformin combined, over a series of seven-day extended culture experiments. The effects of treatment on cellular outcomes were characterised by measuring cell proliferation, viability, morphology, surface marker expression, mitochondrial activity, mitochondrial redox status, and DNA methylation.
I found that azacitidine had cytostatic, differentiating, and hypomethylating effects on SKM-1 and OCI-AML3 cells. Treatment with repeated doses of 0.5 μM azacitidine in extended culture experiments reduced cell proliferation but preserved a high proportion of viability. Metformin had a cytotoxic effect on cells, and the combination of metformin with azacitidine resulted in significant inhibition of leukaemic cell viability not seen with azacitidine monotherapy. In contrast, ascorbate increased levels of 5-hydroxymethyl-2’-deoxycytidine in cell DNA, which was consistent with the enhancement of TET2 activity. When ascorbate and azacitidine were combined, neither compound interfered with the DNA methylation outcomes of the other, indicating complementary hypomethylating action across multiple cellular pathways. In summary, both ascorbate and metformin are promising candidates for the enhancement of azacitidine treatment through the targeting of leukaemia-specific vulnerabilities.