title: Targeted next-generation sequencing of DNA regions proximal to a conserved GXGXXG signaling motif enables systematic discovery of tyrosine kinase fusions in cancer.
abstract: Tyrosine kinase (TK) fusions are attractive drug targets in cancers. However, rapid identification of these lesions has been hampered by experimental limitations. Our in silico analysis of known cancer-derived TK fusions revealed that most breakpoints occur within a defined region upstream of a conserved GXGXXG kinase motif. We therefore designed a novel DNA-based targeted sequencing approach to screen systematically for fusions within the 90 human TKs; it should detect 92% of known TK fusions. We deliberately paired 'in-solution' DNA capture with 454 sequencing to minimize starting material requirements, take advantage of long sequence reads, and facilitate mapping of fusions. To validate this platform, we analyzed genomic DNA from thyroid cancer cells (TPC-1) and leukemia cells (KG-1) with fusions known only at the mRNA level. We readily identified for the first time the genomic fusion sequences of CCDC6-RET in TPC-1 cells and FGFR1OP2-FGFR1 in KG-1 cells. These data demonstrate the feasibility of this approach to identify TK fusions across multiple human cancers in a high-throughput, unbiased manner. This method is distinct from other similar efforts, because it focuses specifically on targets with therapeutic potential, uses only 1.5 µg of DNA, and circumvents the need for complex computational sequence analysis.
authors.affiliation: Weill Graduate School of Medical Sciences, Cornell University, New York, NY 10021, USA.
authors.forename: Juliann
authors.initials: J
authors.lastname: Chmielecki
authors.forename: Martin
authors.initials: M
authors.lastname: Peifer
authors.forename: Peilin
authors.initials: P
authors.lastname: Jia
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Socci
authors.forename: Katherine
authors.initials: K
authors.lastname: Hutchinson
authors.forename: Agnes
authors.initials: A
authors.lastname: Viale
authors.forename: Zhongming
authors.initials: Z
authors.lastname: Zhao
authors.forename: Roman K
authors.initials: RK
authors.lastname: Thomas
authors.forename: William
authors.initials: W
authors.lastname: Pao
mesh_terms: D020816:Amino Acid Motifs; D045744:Cell Line, Tumor; D056905:Chromosome Breakpoints; D002874:Chromosome Mapping; D017124:Conserved Sequence; D003598:Cytoskeletal Proteins / Q000235:genetics; D006801:Humans; D050506:Mutant Chimeric Proteins / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics*; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D020539:Sequence Analysis, Protein; D015398:Signal Transduction / Q000235:genetics
chemical_list: C088832:CCDC6 protein, human; D003598:Cytoskeletal Proteins; D050506:Mutant Chimeric Proteins; D009363:Neoplasm Proteins; C496348:FGFR1 protein, human; D011505:Protein-Tyrosine Kinases; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D051496:Receptor, Fibroblast Growth Factor, Type 1