title: A high content clonogenic survival drug screen identifies mek inhibitors as potent radiation sensitizers for KRAS mutant non-small-cell lung cancer.
abstract: BACKGROUND
  Traditional clonogenic survival and high throughput colorimetric assays are inadequate as drug screens to identify novel radiation sensitizers. We developed a method that we call the high content clonogenic survival assay (HCSA) that will allow screening of drug libraries to identify candidate radiation sensitizers.
  
  
  METHODS
  Drug screen using HCSA was done in 96 well plates. After drug treatment, irradiation, and incubation, colonies were stained with crystal violet and imaged on the INCell 6000 (GE Health). Colonies achieving 50 or more cells were enumerated using the INCell Developer image analysis software. A proof-of-principle screen was done on the KRAS mutant lung cancer cell line H460 and a Custom Clinical Collection (146 compounds).
  
  
  RESULTS
  Multiple drugs of the same class were found to be radiation sensitizers and levels of potency seemed to reflect the clinical relevance of these drugs. For instance, several PARP inhibitors were identified as good radiation sensitizers in the HCSA screen. However, there were also a few PARP inhibitors not found to be sensitizing that have either not made it into clinical development, or in the case of BSI-201, was proven to not even be a PARP inhibitor. We discovered that inhibitors of pathways downstream of activated mutant KRAS (PI3K, AKT, mTOR, and MEK1/2) sensitized H460 cells to radiation. Furthermore, the potent MEK1/2 inhibitor tramenitib selectively enhanced radiation effects in KRAS mutant but not wild-type lung cancer cells.
  
  
  CONCLUSIONS
  Drug screening for novel radiation sensitizers is feasible using the HCSA approach. This is an enabling technology that will help accelerate the discovery of novel radiosensitizers for clinical testing.
authors.affiliation: Department of Radiation Oncology, Houston, TX. Electronic address: shlin@mdanderson.org.
authors.forename: Steven H
authors.initials: SH
authors.lastname: Lin
authors.affiliation: Department of Radiation Oncology, Houston, TX.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Thoracic/Head & Neck Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX.
authors.forename: Uma
authors.initials: U
authors.lastname: Giri
authors.affiliation: Center for Translational Cancer Research, Texas A&M Health Science Center, Institute of Biosciences and Technology, Houston, TX.
authors.forename: Clifford
authors.initials: C
authors.lastname: Stephan
authors.affiliation: Center for Translational Cancer Research, Texas A&M Health Science Center, Institute of Biosciences and Technology, Houston, TX.
authors.forename: Mary
authors.initials: M
authors.lastname: Sobieski
authors.affiliation: Center for Translational Cancer Research, Texas A&M Health Science Center, Institute of Biosciences and Technology, Houston, TX.
authors.forename: Ling
authors.initials: L
authors.lastname: Zhong
authors.affiliation: Department of Experimental Radiation Oncology, Houston, TX.
authors.forename: Kathy A
authors.initials: KA
authors.lastname: Mason
authors.affiliation: Department of Experimental Radiation Oncology, Houston, TX.
authors.forename: Jessica
authors.initials: J
authors.lastname: Molkentine
authors.affiliation: Department of Biostatistics, The University of Texas M.D. Anderson Cancer Center, Houston, TX.
authors.forename: Howard D
authors.initials: HD
authors.lastname: Thames
authors.affiliation: STCube Pharmaceuticals, Inc., Houston, TX.
authors.forename: Stephen S
authors.initials: SS
authors.lastname: Yoo
authors.affiliation: Department of Thoracic/Head & Neck Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX.
authors.forename: John V
authors.initials: JV
authors.lastname: Heymach
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001549:Benzamides / Q000494:pharmacology / Q000627:therapeutic use; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000235:genetics / Q000532:radiotherapy*; D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000532:radiotherapy*; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors; D048370:MAP Kinase Kinase 2 / Q000037:antagonists & inhibitors; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008297:Male; D051379:Mice; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D011728:Pyridones / Q000494:pharmacology / Q000627:therapeutic use; D011744:Pyrimidinones / Q000494:pharmacology / Q000627:therapeutic use; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology* / Q000627:therapeutic use; D014410:Tumor Stem Cell Assay; D018631:ras Proteins / Q000235:genetics
chemical_list: D000970:Antineoplastic Agents; D001549:Benzamides; C117307:KRAS protein, human; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D011728:Pyridones; D011744:Pyrimidinones; D011838:Radiation-Sensitizing Agents; C090712:iniparib; C560077:trametinib; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins