title: Transposon-mediated generation of BCR-ABL1-expressing transgenic cell lines for unbiased sensitivity testing of tyrosine kinase inhibitors.
abstract: Point mutations in the ABL1 kinase domain are an important mechanism of resistance to tyrosine kinase inhibitors (TKI) in BCR-ABL1-positive and, as recently shown, BCR-ABL1-like leukemias. The cell line Ba/F3 lentivirally transduced with mutant BCR-ABL1 constructs is widely used for in vitro sensitivity testing and response prediction to tyrosine kinase inhibitors. The transposon-based Sleeping Beauty system presented offers several advantages over lentiviral transduction including the absence of biosafety issues, faster generation of transgenic cell lines, and greater efficacy in introducing large gene constructs. Nevertheless, both methods can mediate multiple insertions in the genome. Here we show that multiple BCR-ABL1 insertions result in elevated IC50 levels for individual TKIs, thus overestimating the actual resistance of mutant subclones. We have therefore established flow-sorting-based fractionation of BCR-ABL1-transformed Ba/F3 cells facilitating efficient enrichment of cells carrying single-site insertions, as demonstrated by FISH-analysis. Fractions of unselected Ba/F3 cells not only showed a greater number of BCR-ABL1 hybridization signals, but also revealed higher IC50 values for the TKIs tested. The data presented highlight the need to carefully select transfected cells by flow-sorting, and to control the insertion numbers by FISH and real-time PCR to permit unbiased in vitro testing of drug resistance.
authors: Children's Cancer Research Institute, Vienna, Austria.
authors: Konstantin
authors: K
authors: Byrgazov
authors: Children's Cancer Research Institute, Vienna, Austria.
authors: Chantal Blanche
authors: CB
authors: Lucini
authors: Children's Cancer Research Institute, Vienna, Austria.
authors: Bettina
authors: B
authors: Berkowitsch
authors: Children's Cancer Research Institute, Vienna, Austria.
authors: Margit
authors: M
authors: Koenig
authors: Children's Cancer Research Institute, Vienna, Austria.
authors: Oskar A
authors: OA
authors: Haas
authors: Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria.
authors: Gregor
authors: G
authors: Hoermann
authors: Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Vienna, Austria.
authors: Peter
authors: P
authors: Valent
authors: Children's Cancer Research Institute, Vienna, Austria.|Department of Pediatrics, Medical University of Vienna, Austria.
authors: Thomas
authors: T
authors: Lion
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004251:DNA Transposable Elements; D016044:Fusion Proteins, bcr-abl / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*
chemical_list: D004251:DNA Transposable Elements; D047428:Protein Kinase Inhibitors; D016044:Fusion Proteins, bcr-abl
keywords: BCR-ABL1; Ba/F3; transposon-based gene transfer; tyrosine kinase inhibitors