AbstractBackgroundMonepantel is an anti‐helminthic drug that also has anti‐cancer properties. Despite several studies over the years, the molecular target of monepantel in mammalian cells is still unknown, and its mechanism‐of‐action is not fully understood, though effects on cell cycle, mTOR signalling and autophagy have been implicated.MethodsViability assays were performed on >20 solid cancer cell cells, and apoptosis assays were performed on a subset of these, including 3D cultures. Genetic deletion of BAX/BAK and ATG were used to establish roles of apoptosis and autophagy in killing activity. RNA‐sequencing was performed on four cell lines after monepantel treatment, and differentially regulated genes were confirmed by Western blotting.ResultsWe showed that monepantel has anti‐proliferative activity on a broad range of cancer cell lines. In some, this was associated with induction of apoptosis which was confirmed using a BAX/BAK‐deficient cell line. However, proliferation is still inhibited in these cells following monepantel treatment, indicating cell‐cycle disruption as the major anti‐cancer effect. Previous studies have also indicated autophagic cell death occurs following monepantel treatment. We showed autophagy induction in multiple cell lines; however, deletion of a key autophagy regulator ATG7 had minimal impact on monepantel’s anti‐proliferative activity, suggesting autophagy is associated with, but not required for its anti‐tumour effects. Transcriptomic analysis of four cell lines treated with monepantel revealed downregulation of many genes involved in the cell cycle, and upregulation of genes linked to ATF4‐mediated ER stress responses, especially those involved in amino‐acid metabolism and protein synthesis.ConclusionsAs these outcomes are all associated with mTOR signalling, cell cycle and autophagy, we now provide a likely triggering mechanism for the anti‐cancer activity of monepantel.