Functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders via chemical genetics [Hybrid capture]

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, we employ haploid genetics to show that hotspot mutations cluster in substrate receptors of hijacked ligases, where mutation type and frequency correlate with gene essentiality. A Hybrid capture assay reveals resistance-conferring mutations after degrader treatment. 29 putative target genes were selected to be sequenced. Target gene enrichment from gDNA of treated cells was performed, followed by amplification and sequencing to identify mutations. Total of 7 samples of KBM7 cells treated with BET PROTAC degraders ARV-771 or dBET6 (concentrations: 25x, 50x, 25x-50x pool). Amplicon next-generation sequencing of 29 genes after enrichment and amplification to detect genomic mutations.