Febuxostat enhances the anti-tumor efficacy of 2-fluoroadenine and 5’-methylthioadenosine in MTAP-deleted cancer

Homozygous deletion of the methylthioadenosine phosphorylase gene (MTAP) is a frequent genetic alteration in cancer. The MTAP enzyme, which creates adenine from 5'-methylthioadenosine (MTA), is constitutively expressed in all tissues throughout the body. Previously we described a novel strategy to specifically target MTAP-deleted cancer cells by combining the anti-purine prodrug 2-fluoroadenine (2FA) with MTA. In vitro this combination efficiently kills MTAP- cancer cells, while leaving MTAP+ cells unharmed. However, in vivo the combination was less effective at tumor cell killing, resulting in slower growth, but not tumor regression. Here we show that this effectiveness difference is associated with a large difference in the amount of 2FA that is incorporated into 2FA-containing nucleotides. To explain this observation, we tested the hypothesis that 2FA was a target for the purine metabolic enzyme xanthine oxidase (XO), which is found in various extracellular fluids. In vitro, addition of XO protein to culture media effectively abolishes killing by 2FA and this effect can be fully reversed by addition febuxostat (FX), a xanthine oxidase inhibitor. In vivo, addition of FX to 2FA results in increased cell killing and toxicity and a 1000% increase in the amount of 2FA that is converted to its active form, 2-FA-monophosphate (2FAMP). Xenograft studies using MTAP- HT1080 and MiaPaCa-2 cell lines show that a 2FA/MTA/FX cocktail can cause tumor regression in vivo. These studies suggest that a combination of 2FA/MTA/FX may be useful in treating MTAP- cancer.