Profiling the Landscape of Drug Resistance Mutations in Neosubstrates to Molecular Glue Degraders

Targeted protein
degradation (TPD) holds immense promise for drug
discovery, but mechanisms of acquired resistance to degraders remain
to be fully identified. Here, we used clustered regularly interspaced
short palindromic repeats (CRISPR)-suppressor scanning to identify
mechanistic classes of drug resistance mutations to molecular glue
degraders in GSPT1 and RBM39, neosubstrates targeted by E3 ligase
substrate receptors cereblon and DCAF15, respectively. While many
mutations directly alter the ternary complex heterodimerization surface,
distal resistance sites were also identified. Several distal mutations
in RBM39 led to modest decreases in degradation, yet can enable cell
survival, underscoring how small differences in degradation can lead
to resistance. Integrative analysis of resistance sites across GSPT1
and RBM39 revealed varying levels of sequence conservation and mutational
constraint that control the emergence of different resistance mechanisms,
highlighting that many regions co-opted by TPD are nonessential. Altogether,
our study identifies common resistance mechanisms for molecular glue
degraders and outlines a general approach to survey neosubstrate requirements
necessary for effective degradation.