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

Area of Science:

• Molecular Biology
• Oncology
• Drug Discovery

Background:

• K-RAS is a frequently mutated oncogene, posing challenges for conventional inhibitor development.
• Targeted proteolysis offers a new modality to address previously undruggable targets.
• AdPROM system utilizes peptidic binders for targeted protein degradation.

Purpose of the Study:

• To develop and evaluate AdPROMs for targeted proteolysis of endogenous RAS proteins.
• To assess the efficacy of GFP- and H/K-RAS-targeting AdPROMs in cancer cell lines.

Main Methods:

• CRISPR/Cas9 gene editing to knock in a GFP tag on the native K-RAS gene in A549 cells (A549^GFPKRAS).
• Construction of AdPROMs with high-affinity GFP or H/K-RAS binders.
• Expression of AdPROMs in engineered and native cell lines to induce proteasomal degradation.

Main Results:

• GFP-targeting AdPROM induced robust proteasomal degradation of endogenous GFP-K-RAS in A549^GFPKRAS cells.
• Anti-HRAS-targeting AdPROM degraded both GFP-tagged and untagged K-RAS, as well as untagged H-RAS, in various cell lines.
• Demonstrated successful targeted degradation of endogenous RAS proteins.

Conclusions:

• Endogenous RAS proteins can be effectively targeted for proteolysis using the AdPROM system.
• This approach provides a potential alternative therapeutic strategy for targeting undruggable RAS mutations.
• AdPROM-mediated targeted proteolysis shows promise for cancer therapy.