Functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders [Saturating mutagenesis]

Targeted protein degradation is a novel pharmacology established by drugs that recruit target proteins to E3 ubiquitin ligases. Based on the structure of the degrader and the target, different E3 interfaces are critically involved, thus forming defined "functional hotspots". Understanding disruptive mutations in functional hotspots informs on the architecture of the assembly, and highlights residues susceptible to acquire resistance phenotypes. Here, deep mutational scanning revealed hotspots that are conserved or specific for chemically distinct degraders and targets and validated hotspots mutated in patients that relapse from degrader treatment. CRBN/VHL deficient RKO cells were transduced with the respective mutational library. VHL screens were performed in two infection replicates, CRBN screens in triplicates. Cells were treated for 7 days with DMSO or small-molecule degraders. Amplicon next-generation sequencing of the amplified CRBN and VHL cDNA harboring a mutation.