Identification of novel cancer therapeutic targets using a designed and pooled shRNA library screen

David Oliver,Lillian O Perinskaya,Alexander Gasparian,Michael D Wyatt,Samuel Lee,Adrian Zenteno,Homayoun Valafar,Piaomu Liu,Mengqian Chen,Michael Shtutman,Ellen Gardiner,Phillip Buckhaults,Hao Ji,Edsel Peña,Eugenia Broude

doi:10.1038/srep43023

David Oliver, Lillian O Perinskaya + Show 13 more

Open Access

https://doi.org/10.1038/srep43023

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Abstract

Targeted cancer therapeutics aim to exploit tumor-specific, genetic vulnerabilities specifically affecting neoplastic cells without similarly affecting normal cells. Here we performed sequencing-based screening of an shRNA library on a panel of cancer cells of different origins as well as normal cells. The shRNA library was designed to target a subset of genes previously identified using a whole genome screening approach. This focused shRNA library was infected into cells followed by analysis of enrichment and depletion of the shRNAs over the course of cell proliferation. We developed a bootstrap likelihood ratio test for the interpretation of the effects of multiple shRNAs over multiple cell line passages. Our analysis identified 44 genes whose depletion preferentially inhibited the growth of cancer cells. Among these genes ribosomal protein RPL35A, putative RNA helicase DDX24, and coatomer complex I (COPI) subunit ARCN1 most significantly inhibited growth of multiple cancer cell lines without affecting normal cell growth and survival. Further investigation revealed that the growth inhibition caused by DDX24 depletion is independent of p53 status underlining its value as a drug target. Overall, our study establishes a new approach for the analysis of proliferation-based shRNA selection strategies and identifies new targets for the development of cancer therapeutics.

Highlights

Targeted cancer therapeutics aim to exploit tumor-specific, genetic vulnerabilities affecting neoplastic cells without affecting normal cells
We constructed a library of genetic suppressor element (GSE) composed of cDNA fragments from the human transcriptome prepared from a mixture of normalized cDNAs from 18 cell lines derived from different types of cancers[8]
Target genes were identified by mapping fragments back to the human genome. 221 genes were targeted by at least 1 GSE enriched more than 1.5 fold in two or three tumor cell lines relative to normal cells (Supplemental Dataset S1)

Summary

Introduction

Targeted cancer therapeutics aim to exploit tumor-specific, genetic vulnerabilities affecting neoplastic cells without affecting normal cells. We performed BrdU suicide selection of a genome wide GSE library in a panel of normal and cancer cell lines to select tumor-specific target genes[8]. We re-sequenced and analyzed this dataset to reveal a large set of potential target genes To reduce this list to the most effective targets, we designed an shRNA library focused on the identified gene set and performed a proliferation-based selection of the library in four tumor cell lines and normal fibroblasts. The screening results were analyzed with a bootstrap likelihood ratio test (BLRT) statistical procedure we developed This screen and analysis procedure revealed a subset of 44 genes with additional evidence for cancer-specific growth inhibition. We show the association of the expression of the identified genes with development and mortality of human cancers and explore the mechanism of the effect of ARCN1 and DDX24 depletion in tumor cells

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