title: Dual ALK and EGFR inhibition targets a mechanism of acquired resistance to the tyrosine kinase inhibitor crizotinib in ALK rearranged lung cancer.
abstract: BACKGROUND
  The multitargeted tyrosine kinase inhibitor (TKI) crizotinib is active against ALK translocated non-small-cell lung cancer (NSCLC); however acquired resistance invariably develops over time. ALK mutations have previously been implicated in only a third of resistant tumors. We sought to evaluate alternative mechanisms of resistance and preclinical strategies to overcome these in a cell line driven by EML4-ALK.
  
  
  METHODS
  We selected the NSCLC cell line NCI-H3122 (H3122: EML4-ALK E13;A20) and derived resistant variants that were able to grow in the presence of 1 μM crizotinib. These were analyzed for ALK mutations, sensitivity to crizotinib in combination with other TKIs, and for activation of alternative tyrosine kinases.
  
  
  RESULTS
  All H3122 crizotinib resistant (CR) clones lacked amplification or mutations in the kinase domain of ALK. To evaluate if possible alternative kinases functioned as "bypass" tracks for downstream signaling activation in these resistance cells, we performed of phosho-receptor tyrosine kinase array that demonstrated that CR clones had higher phospho-EGFR signals than H3122 cells before and after exposure to crizotinib. A functional approach of dual ALK TKI (with crizotinib) with combinatory TKI inhibition was used as a secondary screen for possible targets. Crizotinib+erlotinib (reversible EGFR TKI) and crizotinib+afatinib (irreversible EGFR/ERBB2 TKI) were able to inhibit the growth of H3122 CR clones, confirming EGFR activation as a mechanism of resistance. The removal of crizotinib from the culture media re-sensitized CR cells to crizotinib.
  
  
  CONCLUSIONS
  We identified activation of EGFR as a mechanism of resistance to crizotinib in preclinical models of ALK translocated NSCLC. If EGFR activation is confirmed as a predominant mechanism of ALK TKI-induced resistance in patient-derived tumors, the use of ALK plus EGFR TKIs could be explored for this important cohort of NSCLCs.
authors.affiliation: Department of Medicine, Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Norihiro
authors.initials: N
authors.lastname: Yamaguchi
authors.affiliation: Department of Medicine, Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Antonio R
authors.initials: AR
authors.lastname: Lucena-Araujo
authors.affiliation: Department of Medicine, Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Sohei
authors.initials: S
authors.lastname: Nakayama
authors.affiliation: Department of Medicine, Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Lorena L
authors.initials: LL
authors.lastname: de Figueiredo-Pontes
authors.affiliation: Department of Medicine, Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Gonzalez
authors.affiliation: Department of Medicine, Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yasuda
authors.affiliation: Department of Medicine, Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Susumu
authors.initials: S
authors.lastname: Kobayashi
authors.affiliation: Department of Medicine, Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. Electronic address: dbcosta@bidmc.harvard.edu.
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Costa
mesh_terms: D000077716:Afatinib; D000077548:Anaplastic Lymphoma Kinase; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000235:genetics; D048708:Cell Growth Processes / Q000187:drug effects; D045744:Cell Line, Tumor; D000077547:Crizotinib; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D005784:Gene Amplification; D015321:Gene Rearrangement; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000235:genetics; D046228:Microarray Analysis; D009154:Mutation / Q000235:genetics; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use*; D011720:Pyrazoles / Q000627:therapeutic use*; D011725:Pyridines / Q000627:therapeutic use*; D011799:Quinazolines / Q000627:therapeutic use; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: C522504:EML4-ALK fusion protein, human; D015514:Oncogene Proteins, Fusion; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011725:Pyridines; D011799:Quinazolines; D000077716:Afatinib; D000077547:Crizotinib; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; C512478:EGFR protein, human; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases
keywords: Anaplastic lymphoma kinase; Crizotinib; Epidermal growth factor receptor; Kinase inhibitor; Non-small-cell lung cancer; Tyrosine kinase