Proteomics reveal extensive translatome reprogramming and biomarkers of rocaglate toxicity and resistance in cancer

Abstract

Translational activation is a major convergence point for oncogenic signals, and its direct targeted inhibition is an attractive cancer treatment strategy that bypasses signaling redundancies limiting the efficacy of many cancer drugs. Rocaglates are potent anti-cancer compounds historically identified as translation/eIF4A inhibitors. Prior studies focused exclusively on proteins whose expression is inhibited by rocaglates. However, this singular perspective alone cannot fully explain the potent toxicity of rocaglates across cancer types. The first rocaglate zotatifin recently entered phase I clinical evaluation for advanced solid tumor malignancies, and is receiving much attention as a potent inhibitor of SARS-CoV-2 replication and infectivity. The rapid push to develop rocaglates as cancer therapeutics and our ongoing efforts against the COVID-19 pandemic inject immense urgency for a more comprehensive understanding of rocaglate mechanisms. Here, we present evidence that rocaglates lead to complex global translational reprogramming, including the induction of an extensive population of unique proteins that mediate cellular rocaglate responses. This conceptually transforms their current one-dimensional definition as translation inhibitors. Using dedicated proteomic technologies including TMT-pSILAC to interrogate system-wide translatome reprogramming, and our recently developed MATRIX platform to capture blueprints of translation machinery adaptations, we discovered previously unrecognized biomarkers that mediate both rocaglate toxicity and resistance. As proof-of-concept, rocaglate-specific induction of GEF-H1 activates RHOA/JNK-dependent apoptosis across solid and blood cancer cells, whereas rocaglate-induced CD98hc up-regulation suppresses JNK signaling to exert pro-survival effects. Induction of these proteins depends on rocaglate-dependent augmentation of eEF1ε1 translational activity. These results represent the first characterization of rocaglate-inducible proteins with direct relevance to their potent in vivo toxicity. Overall, these findings transform our understanding of rocaglates, from pure translation inhibitors to comprehensive remodelers of the cellular protein synthesis landscape. No conflict of interest.