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.

Automated summary

The lack of detailed understanding of the mechanism of action of many cancer drugs is hindering their optimal use. This study used a high-resolution implementation of the proteome-wide cellular thermal shift assay to investigate the protein interaction changes induced by the antimetabolite 5-fluorouracil (5-FU) and related nucleosides. The findings revealed the expected effects on the known main target and enzymes in specific pathways, but also identified novel protein interactions and pathways associated with 5-FU, including RNA-modification and-processing pathways. The study further highlighted key components of the 5-FU mechanism of action, indicating that the inhibition of a specific target is not necessary for cell proliferation.