Abstract 876: Genome-scale genetic knockout screen identifies modifiers of EGFR dependence in non-small cell lung cancer cells
Authors: DiMaina Jon , Duckworth Chris , Cheung Hiu Wing 
Published: July 2016

Abstract
Non-small cell lung cancers with epidermal growth factor receptor (EGFR) gene mutations can exhibit a strong dependence on its signaling for growth and survival. They are also sensitive to EGFR tyrosine kinase inhibitors (TKIs), which provide superior clinical benefits to conventional chemotherapy. However, despite initial response, most patients experience relapse with resistant tumors within a year. This study aims to identify modifiers of dependence on mutant EGFR signaling and the mechanisms by which they do so in order to improve therapeutic strategies and outcomes.
We first performed a genome-scale CRISPR-Cas9 genetic knockout screen. In this system, gene silencing occurs by site-specific double-strand-breaks induced by the Cas9 nuclease that is localized to its target by a single-guide RNA (sgRNA). The error-prone repair pathway introduces insertion/deletion mutations leading to frameshifts or premature stop codons. We constructed a pooled sgRNA library targeting more than 18,000 protein-coding human genes with multiple sgRNA vectors. The cell line HCC827 was used in our screen as it is EGFR-mutant and sensitive to EGFR TKIs. We transduced HCC827 lung cancer cells with the pooled sgRNA library and cultured them in the presence of Erlotinib, an EGFR TKI, or DMSO control for 17 days. At the end of the treatment period we identified sgRNAs that were enriched in Erlotinib-treated groups over control groups, indicating genes whose loss-of-function confer TKI resistance. We applied the RNAi gene enrichment ranking (RIGER) algorithm to identify gene hits with enrichment of multiple sgRNAs.
Top-ranked candidates include previously confirmed genes PTEN, NF1, NF2, TSC1, and TSC2, validating this system as a means to identify modifiers of EGFR dependence in HCC827 cells. A novel candidate gene is the E3 ubiquitin ligase HUWE1. We showed that suppression of HUWE1 by sgRNAs or inducible short hairpin RNA (shRNA) in HCC827 cells re-activated AKT and ERK1/2 signaling pathways and increased cell proliferation and survival in response to EGFR inhibition. These findings were confirmed in vivo by implanting mouse xenografts of HCC827 cells expressing shHUWE1 and monitoring tumor development in response to EGFR-TKI therapy. Tumors expressing HUWE1 led to durable tumor regression throughout the treatment period but tumors with suppressed HUWE1 continued to grow into large tumors. Immunohistochemistry analysis of excised tumors also revealed increased AKT and ERK1/2 signaling as well as increased proliferation and decreased apoptosis.
We have shown that dependence on EGFR signaling can be decreased in EGFR-mutant lung cancer cells through mechanisms that involve the activation of AKT and ERK1/2 signaling pathways. Ongoing studies involve identifying HUWE1 substrates/interactions that participate in tumor cell response to EGFR inhibitors, revealing a novel mechanism of resistance to EGFR-targeted therapy.
Citation Format: Jon DiMaina, Chris Duckworth, Hiu Wing Cheung. Genome-scale genetic knockout screen identifies modifiers of EGFR dependence in non-small cell lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 876.