γ‐Secretase inhibitors in cancer clinical trials are pharmacologically and functionally distinct

Abstract

γ‐Secretase inhibitors ( GSI s) are being actively repurposed as cancer therapeutics based on the premise that inhibition of NOTCH 1 signaling in select cancers is therapeutic. Using novel assays to probe effects of GSI s against a broader panel of substrates, we demonstrate that clinical GSI s are pharmacologically distinct. GSI s show differential profiles of inhibition of the various NOTCH substrates, with some enhancing cleavage of other NOTCH substrates at concentrations where NOTCH 1 cleavage is inhibited. Several GSI s are also potent inhibitors of select signal peptide peptidase ( SPP / SPPL ) family members. Extending these findings to mammosphere inhibition assays in triple‐negative breast cancer lines, we establish that these GSI s have different functional effects. We also demonstrate that the processive γ‐secretase cleavage pattern established for amyloid precursor protein ( APP ) occurs in multiple substrates and that potentiation of γ‐secretase cleavage is attributable to a direct action of low concentrations of GSI s on γ‐secretase. Such data definitively demonstrate that the clinical GSI s are not biological equivalents, and provide an important framework to evaluate results from ongoing and completed human trials with these compounds. image γ‐Secretase inhibitors ( GSI s) have been repurposed to treat various human cancers. Using newly developed γ‐secretase cleavage assays and a breast cancer cell mammosphere inhibition assay, this study demonstrates that four clinical GSI s are neither pharmacologically nor functionally equivalent. In vitro and cell‐based assays for human NOTCH1–4 proteins enable facile study of γ‐secretase cleavage and the effects of GSIs, as well as establish sites of cleavage. Dose–response studies with clinical GSIs reveal markedly different pharmacologic properties. Studies in triple‐negative breast cancer cell lines show that the clinical GSIs are functionally distinct. Uncertainty remains regarding the properties of a GSI that are associated with potency in the mammosphere inhibition assay, and might be predictive of a therapeutic effect in human cancer.