CETSA interaction proteomics define specific RNA-modification pathways as key components of fluorouracil-based cancer drug cytotoxicity.

The optimal use of many cancer drugs is hampered by a lack of detailed understanding of their mechanism of action (MoA). Here, we apply a high-resolution implementation of the proteome-wide cellular thermal shift assay (CETSA) to follow protein interaction changes induced by the antimetabolite 5-fluorouracil (5-FU) and related nucleosides. We confirm anticipated effects on the known main target, thymidylate synthase (TYMS), and enzymes in pyrimidine metabolism and DNA damage pathways. However, most interaction changes we see are for proteins previously not associated with the MoA of 5-FU, including wide-ranging effects on RNA-modification and -processing pathways. Attenuated responses of specific proteins in a resistant cell model identify key components of the 5-FU MoA, where intriguingly the abrogation of TYMS inhibition is not required for cell proliferation.