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

Sascha Röth,Thomas J Macartney,Agnieszka Konopacka,Kwok-Ho Chan,Houjiang Zhou,Markus A Queisser,Gopal P Sapkota

doi:10.1016/j.chembiol.2020.06.012

Abstract

SummaryK-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 (A549GFPKRAS) cells and constructed AdPROMs containing high-affinity GFP or H/K-RAS binders. Expression of GFP-targeting AdPROM in A549GFPKRAS 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.

Highlights

The three RAS oncogenes, H-RAS, K-RAS, and N-RAS, represent the most frequently mutated genes in cancer (Cox et al, 2014; Hobbs et al, 2016)
To explore targeted proteolysis of K-RAS using the affinitydirected protein missile (AdPROM) system, we used CRISPR/Cas9 technology to generate an A549 non-small cell lung carcinoma (NSCLC) cell line harboring a homozygous knockin of green fluorescent protein (GFP) cDNA at the N terminus of the native K-RAS gene (Figure S1)
By western blot analysis using both panRAS and K-RAS4B antibodies, the appearance of higher-molecular-weight GFP-K-RAS species with a concurrent disappearance of the native-molecular-weight K-RAS species was evident in the A549GFPKRAS cell line compared with wild-type (WT) A549 control cells (Figure 1B)

Summary

Introduction

The three RAS oncogenes, H-RAS, K-RAS, and N-RAS, represent the most frequently mutated genes in cancer (Cox et al, 2014; Hobbs et al, 2016) They encode four highly similar proteins, namely H-RAS, N-RAS, K-RAS4A, and K-RAS4B, which undergo C-terminal farnesylation (Reiss et al, 1990; Schaber et al, 1990). RAS proteins are small GTPases, which cycle between the GTPbound (active) and GDP-bound (inactive) states, controlled by guanosine nucleotide exchange factors and GTPase activating proteins (GAPs) (Vigil et al, 2010). Many pathogenic mutations in RAS genes impair GAP-mediated GTP hydrolysis, thereby favoring the persistence of the active RAS-GTP state, which triggers constitutive activation of downstream signaling resulting in unchecked proliferation of cancer cells (Hobbs et al, 2016; Marcus and Mattos, 2015)

Results

Discussion

Conclusion