Genome-wide CRISPR screening reveals genetic modifiers of mutant EGFR dependence in human NSCLC.

Hao Zeng,Alicia Lindeman,Mika Manser,Shumei Qiu,Bo Lu,Damien Begue,Raffaella Zamponi,Frederic Sigoillot,Johnny Castillo-Cabrera,John S Reece-Hoyes,Qiong Wang,Zinger Yang,Carsten Russ,Youzhen Wang,Debora Bonenfant,Feng Cong,Xiaomo Jiang,Vaik Strande

doi:10.7554/elife.50223

Hao Zeng, Alicia Lindeman + Show 16 more

Open Access

https://doi.org/10.7554/elife.50223

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Journal: eLife	Publication Date: Nov 19, 2019
Citations: 32	License type: CC BY 4.0

Affiliation: Novartis (United States)

Abstract

EGFR-mutant NSCLCs frequently respond to EGFR tyrosine kinase inhibitors (TKIs). However, the responses are not durable, and the magnitude of tumor regression is variable, suggesting the existence of genetic modifiers of EGFR dependency. Here, we applied a genome-wide CRISPR-Cas9 screening to identify genetic determinants of EGFR TKI sensitivity and uncovered putative candidates. We show that knockout of RIC8A, essential for G-alpha protein activation, enhanced EGFR TKI-induced cell death. Mechanistically, we demonstrate that RIC8A is a positive regulator of YAP signaling, activation of which rescued the EGFR TKI sensitizing phenotype resulting from RIC8A knockout. We also show that knockout of ARIH2, or other components in the Cullin-5 E3 complex, conferred resistance to EGFR inhibition, in part by promoting nascent protein synthesis through METAP2. Together, these data uncover a spectrum of previously unidentified regulators of EGFR TKI sensitivity in EGFR-mutant human NSCLC, providing insights into the heterogeneity of EGFR TKI treatment responses.

Highlights

Lung cancer is the leading cause of cancer-related mortality worldwide, with non-small-cell lung cancer (NSCLC) being the most common subtype (Bray et al, 2018; Herbst et al, 2018)
Thereafter, the drug tolerant ‘persister’ (DTP) cells commenced cell proliferation in the presence of erlotinib, yielding colonies of cells referred to as ‘drug tolerant expanded persister’ (DTEP) cells or drug resistant cells (Figure 1C). These data suggested that epidermal growth factor receptor (EGFR) inhibition in the cultured cells mimics clinical observations of the incomplete response and/or innate resistance to EGFR tyrosine kinase inhibitors (TKIs) treatment, allowing the assay window to screen for mediators of EGFR TKI sensitivity
We intentionally applied high dose of erlotinib (1 mM) to allow the survival of a small subpopulation of DTP cells and development of drug resistance in the long-term. This strategy ensured that genes whose deletion synergize with or confer resistance to erlotinib could be negatively or positively selected from the screen, respectively, following erlotinib treatment compared to DMSO treatment

Summary

Introduction

Lung cancer is the leading cause of cancer-related mortality worldwide, with non-small-cell lung cancer (NSCLC) being the most common subtype (Bray et al, 2018; Herbst et al, 2018). Activating mutations in the kinase domain of epidermal growth factor receptor (EGFR) are present in about 10% to 40% of NSCLC patients, most frequently in-frame deletions in exon 19 (ex del) and a missense arginine-to-leucine mutation at codon 858 (L858R) (Sharma et al, 2007; Pao and Chmielecki, 2010). Acquired resistance inevitably develops, leading to disease progression in almost all patients (Pao and Chmielecki, 2010). Secondary EGFR on-target mutations, most frequently T790M mutation, account for about half of relapsed tumours with acquired resistance (Kobayashi et al, 2005; Pao et al, 2005; Sequist et al, 2011).

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