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

TY - JOUR

T1 - Targeting Endogenous K-RAS for Degradation through the Affinity-Directed Protein Missile System

AU - Roth, Sascha

AU - Macartney, Thomas

AU - Konopacka, Agnieszka

AU - Chan, Kwok-Ho

AU - Zhou, Houjiang

AU - Queisser, Markus A.

AU - Sapkota, Gopal

N1 - Funding: MRC (Grant MC_UU_00018/6 and MC_UU_12016/3)

PY - 2020/9/17

Y1 - 2020/9/17

N2 - 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 (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.

AB - 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 (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.

KW - cancer targets

KW - monobody

KW - nanobody

KW - oncogene

KW - PROTAC

KW - protein degradation

KW - RAS

KW - RAS/MAPK signaling

KW - targeted proteolysis

KW - ubiquitin proteasome system

UR - https://www.scopus.com/pages/publications/85089297813

U2 - 10.1016/j.chembiol.2020.06.012

DO - 10.1016/j.chembiol.2020.06.012

M3 - Article

C2 - 32668202

AN - SCOPUS:85089297813

SN - 2451-9456

VL - 27

SP - 1151-1163.e6

JO - Cell Chemical Biology

JF - Cell Chemical Biology

IS - 9

ER -