title: Using RNA-seq and targeted nucleases to identify mechanisms of drug resistance in acute myeloid leukemia.
abstract: The evolution from microarrays to transcriptome deep-sequencing (RNA-seq) and from RNA interference to gene knockouts using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and Transcription Activator-Like Effector Nucleases (TALENs) has provided a new experimental partnership for identifying and quantifying the effects of gene changes on drug resistance. Here we describe the results from deep-sequencing of RNA derived from two cytarabine (Ara-C) resistance acute myeloid leukemia (AML) cell lines, and present CRISPR and TALEN based methods for accomplishing complete gene knockout (KO) in AML cells. We found protein modifying loss-of-function mutations in Dck in both Ara-C resistant cell lines. CRISPR and TALEN-based KO of Dck dramatically increased the IC₅₀ of Ara-C and introduction of a DCK overexpression vector into Dck KO clones resulted in a significant increase in Ara-C sensitivity. This effort demonstrates the power of using transcriptome analysis and CRISPR/TALEN-based KOs to identify and verify genes associated with drug resistance.
authors.affiliation: Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Susan K
authors.initials: SK
authors.lastname: Rathe
authors.affiliation: 1] Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA [2] Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA [3] Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Branden S
authors.initials: BS
authors.lastname: Moriarity
authors.affiliation: Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Stoltenberg
authors.affiliation: Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Morito
authors.initials: M
authors.lastname: Kurata
authors.affiliation: Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Natalie K
authors.initials: NK
authors.lastname: Aumann
authors.affiliation: 1] Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA [2] Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA [3] Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Eric P
authors.initials: EP
authors.lastname: Rahrmann
authors.affiliation: Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Natashay J
authors.initials: NJ
authors.lastname: Bailey
authors.affiliation: Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Ellen G
authors.initials: EG
authors.lastname: Melrose
authors.affiliation: 1] Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA [2] Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Dominic A
authors.initials: DA
authors.lastname: Beckmann
authors.affiliation: Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Chase R
authors.initials: CR
authors.lastname: Liska
authors.affiliation: 1] Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA [2] Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA [3] Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA [4] Brain Tumor Program, University of Minnesota, Minneapolis, MN, USA [5] Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Largaespada
mesh_terms: D016475:3T3 Cells; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001483:Base Sequence; D045744:Cell Line, Tumor; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D003561:Cytarabine / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy* / Q000235:genetics*; D051379:Mice; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering; D017423:Sequence Analysis, RNA
chemical_list: D000964:Antimetabolites, Antineoplastic; D034741:RNA, Small Interfering; D003561:Cytarabine; D017346:Protein Serine-Threonine Kinases