Next-Generation cDNA Screening for Oncogene and Resistance Phenotypes

Nobuaki Shindoh,Yuka Yoda,Edward A Fox,Scott J Rodig,Akinori Yoda,Timothy J Sullivan,Liat Bird,Andrew A Lane,David M Weinstock,Nadja Kopp,Oliver Weigert,Sumitra Deb

doi:10.1371/journal.pone.0049201

Nobuaki Shindoh, Yuka Yoda + Show 10 more

Open Access

https://doi.org/10.1371/journal.pone.0049201

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Abstract

There is a pressing need for methods to define the functional relevance of genetic alterations identified by next-generation sequencing of cancer specimens. We developed new approaches to efficiently construct full-length cDNA libraries from small amounts of total RNA, screen for transforming and resistance phenotypes, and deconvolute by next-generation sequencing. Using this platform, we screened a panel of cDNA libraries from primary specimens and cell lines in cytokine-dependent murine Ba/F3 cells. We demonstrate that cDNA library-based screening can efficiently identify DNA and RNA alterations that confer either cytokine-independent proliferation or resistance to targeted inhibitors, including RNA alterations and intergenic fusions. Using barcoded next-generation sequencing, we simultaneously deconvoluted cytokine-independent clones recovered after transduction of 21 cDNA libraries. This approach identified multiple gain-of-function alleles, including KRAS G12D, NRAS Q61K and an activating splice variant of ERBB2. This approach has broad applicability for identifying transcripts that confer proliferation, resistance and other phenotypes in vitro and potentially in vivo.

Highlights

Thousands of somatic variants have recently been catalogued across multiple cancer types
We previously discovered a mutant allele of the CRLF2 cytokine receptor by screening cDNA libraries generated from primary acute lymphoblastic leukemias (ALL), which multiple groups concurrently identified as an important oncogene in poor-risk ALL [8,9]
We developed quantitative PCR approaches using primers that amplify the 59 region of housekeeping genes to confirm that similar frequencies of long (e.g. REV3L, 10719 bp) and short (e.g. GAPDH: 1401 bp) fulllength cDNAs are present (Figure S1)

Summary

Introduction

Thousands of somatic variants have recently been catalogued across multiple cancer types. To deconvolute the integrated cDNA from pools of clones, we developed methods for next-generation sequencing and bioinformatic analysis that greatly improve the efficiency of cDNA library-based screening. We developed quantitative PCR (qPCR) approaches using primers that amplify the 59 region of housekeeping genes to confirm that similar frequencies of long (e.g. REV3L, 10719 bp) and short (e.g. GAPDH: 1401 bp) fulllength cDNAs are present (Figure S1).

Results

Conclusion