Genome-wide CRISPR-cas9 knockout screening identifies GRB7 as a driver for MEK inhibitor resistance in KRAS mutant colon cancer

Chune Yu,Yufei Xu,Hubing Shi,Jing Yu,Xuelei Ma,Huiling Wang,Jie Xu,Dan Luo,Xiaobo Zheng,Min Zhang,Jing Jing,Ke Jiang,Guangchao Xu,Jiaxin Wang

doi:10.1038/s41388-021-02077-w

Abstract

Targeting the KRAS pathway is a promising but challenging approach for colorectal cancer therapy. Despite showing potent efficacy in BRAF-mutated melanoma, MEK inhibitors appeared to be tolerated by colorectal cancer cells due to their intrinsic compensatory signaling. Here, we performed genome-wide CRISPR/Cas9 screening in the presence of MEK inhibitor to identify genes that are synthetically lethal with MEK inhibition in CRC models harboring KRAS mutations. Several genes were identified as potential functional drivers, which were significantly enriched in the GRB7-mediated RTK pathway. Loss-of-function and gain-of-function assays validated that GRB7 potently rendered CRC cells primary resistance to MEK inhibitors through the RTK pathway. Mass spectrum analysis of GRB7 immunoprecipitates revealed that PLK1 was the predominant interacting kinase of GRB7. Inhibition of PLK1 suppressed downstream signaling of RTK, including FAK, STAT3, AKT, and 4EBP1. The combination of PLK1 and MEK inhibitors synergistically inhibited CRC cell proliferation and induced apoptosis in vitro and in vivo. In conclusion, we identified GRB7-PLK1 as a pivotal axis mediating RTKs, resulting in MEK inhibitor tolerance. PLK1 is therefore a promising target for synergizing MEK inhibitors in the clinical treatment of CRC patients harboring KRAS mutations.

Highlights

Colorectal cancer (CRC) is one of the most common malignancies, with the third highest incidence and death rate in both women and men in the United States [1]
RTK pathway mediates MEK inhibitor (MEKi) resistance in KRAS mutant colon cancer To identify the critical driver genes involved in MEKi resistance, we designed a combination strategy with genome-wide CRISPR/Cas9 knockout library screening and transcriptomic analysis of KRASmutated cell lines (Fig. 1A)
growth factor receptor-bound protein 7 (GRB7) mediates MEKi resistance in KRAS-mutated CRC cells To validate the function of GRB7 in terms of driving primary resistance to MAPKi using an orthogonal approach, we evaluated CRC cell proliferation in the presence of MEKi with shRNAmediated perturbation of GRB7

Summary

Introduction

Colorectal cancer (CRC) is one of the most common malignancies, with the third highest incidence and death rate in both women and men in the United States [1]. 40% of CRCs harbor oncogenic KRAS mutations, mainly G12D, G12V, and G13D, which can sustainedly activate the MAPK signaling pathway [2, 3]. Given the fact that KRAS mutations are often associated with poor prognosis in CRC patients [4], therapeutic regimens targeting KRAS or its signaling are regarded as one of the most desired strategies in clinics [5]. AMG510 and MRTX849, which target KRAS G12C have entered clinical trials, these inhibitors produced less profound activity in CRC than non-small cell lung cancer patients [6,7,8,9,10]. The KRAS G12C mutation accounts for only one to three percent of all CRC patients, which largely limits the potential populations who could benefit [2, 11]. A strategy that directly targets a broad spectrum of KRAS mutations remains challenging

Methods

Results

Conclusion