Molecular and Structural Characterization of Lenalidomide-Mediated Sequestration of eIF3i

Lenalidomide is a ligand of the E3 ligase substrate adapter cereblon (CRBN) that achieves its clinical effects in part by the promotion of substrate recruitment and degradation. In contrast to prior targets, eIF3i is recruited but not degraded upon complex formation with lenalidomide and CRBN, although the structural details and mechanistic outcomes of this interaction are unresolved. Here, we characterize the structural basis and mechanistic outcomes of lenalidomide-induced sequestration of eIF3i from the eIF3 complex. Identification of the binding interface on eIF3i by a covalent lenalidomide probe and mass spectrometry rationalizes the sequestration event. We further connect eIF3i and CRBN to lenalidomide-driven effects on angiogenic markers, Akt1 phosphorylation, and associated antiangiogenesis phenotypes. Finally, we find that eIF3i sequestration is observed in MM.1S and MOLM13 cells after the degradation of other substrates, such as IKZF1. The defined binding interface elucidated by chemical proteomics and the observation of eIF3i sequestration as a lenalidomide function open future directions in designing new chemical adapters for protein sequestration as a strategy to selectively control protein functions.