Transposon-mediated generation of BCR-ABL1 -expressing transgenic cell lines for unbiased sensitivity testing of tyrosine kinase inhibitors

// Konstantin Byrgazov 1, * , Chantal Blanche Lucini 1, * , Bettina Berkowitsch 1 , Margit Koenig 1 , Oskar A. Haas 1 , Gregor Hoermann 2 , Peter Valent 3 , Thomas Lion 1, 4 1 Children’s Cancer Research Institute, Vienna, Austria 2 Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria 3 Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Vienna, Austria 4 Department of Pediatrics, Medical University of Vienna, Austria * These authors have contributed equally to this work Correspondence to: Thomas Lion, email: thomas.lion@ccri.at Keywords: BCR-ABL1, tyrosine kinase inhibitors, Ba/F3, transposon-based gene transfer Received: March 31, 2016     Accepted: October 17, 2016     Published: October 27, 2016 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 IC 50 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 IC 50 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.