title: Next-generation cDNA screening for oncogene and resistance phenotypes.
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.
authors.affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America.
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Shindoh
authors.forename: Akinori
authors.initials: A
authors.lastname: Yoda
authors.forename: Yuka
authors.initials: Y
authors.lastname: Yoda
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Sullivan
authors.forename: Oliver
authors.initials: O
authors.lastname: Weigert
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Lane
authors.forename: Nadja
authors.initials: N
authors.lastname: Kopp
authors.forename: Liat
authors.initials: L
authors.lastname: Bird
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Rodig
authors.forename: Edward A
authors.initials: EA
authors.lastname: Fox
authors.forename: David M
authors.initials: DM
authors.lastname: Weinstock
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D000069347:Erlotinib Hydrochloride; D015723:Gene Library; D018734:Genes, erbB-2; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing / Q000379:methods*; D014644:Genetic Variation; D051379:Mice; D009857:Oncogenes; D010641:Phenotype; D020033:Protein Isoforms; D011799:Quinazolines / Q000494:pharmacology
chemical_list: D020033:Protein Isoforms; D011799:Quinazolines; D000069347:Erlotinib Hydrochloride