Targeting Endogenous K-RAS for Degradation through the Affinity-Directed Protein Missile System

K-RAS is known as the most frequently mutated oncogene. However, the development of conventional K-RAS inhibitors has been extremely challenging, with a mutation-specific inhibitor reaching clinical trials only recently. Targeted proteolysis has emerged as a new modality in drug discovery to tackle undruggable targets. Our laboratory has developed a system for targeted proteolysis using peptidic high-affinity binders, called AdPROM. Here, we used CRISPR/Cas9 technology to knock in a GFP tag on the native K-RAS gene in A549 adenocarcinoma (A549(GFPKRAS)) cells and constructed AdPROMs containing high-affinity GFP or H/KRAS binders. Expression of GFP-targeting AdPROM in A549(GFPKRAS) led to robust proteasomal degradation of endogenous GFP-K-RAS, while expression of anti-HRAS-targeting AdPROM in different cell lines resulted in the degradation of both GFP-tagged and untagged K-RAS, and untagged H-RAS. Our findings imply that endogenous RAS proteins can be targeted for proteolysis, supporting the idea of an alternative therapeutic approach to these undruggable targets.