A high content clonogenic survival drug screen identifies mek inhibitors as potent radiation sensitizers for KRAS mutant non-small-cell lung cancer

J Thorac Oncol. 2014 Jul;9(7):965-973. doi: 10.1097/JTO.0000000000000199.


Introduction: 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antineoplastic Agents / pharmacology*
  • Antineoplastic Agents / therapeutic use
  • Benzamides / pharmacology
  • Benzamides / therapeutic use
  • Carcinoma, Non-Small-Cell Lung / drug therapy
  • Carcinoma, Non-Small-Cell Lung / genetics
  • Carcinoma, Non-Small-Cell Lung / radiotherapy*
  • Cell Line, Tumor
  • Drug Screening Assays, Antitumor / methods*
  • Humans
  • Lung Neoplasms / drug therapy
  • Lung Neoplasms / genetics
  • Lung Neoplasms / radiotherapy*
  • MAP Kinase Kinase 1 / antagonists & inhibitors
  • MAP Kinase Kinase 2 / antagonists & inhibitors
  • MAP Kinase Signaling System / drug effects*
  • Male
  • Mice
  • Poly(ADP-ribose) Polymerase Inhibitors
  • Protein Kinase Inhibitors / pharmacology
  • Protein Kinase Inhibitors / therapeutic use
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins p21(ras)
  • Pyridones / pharmacology
  • Pyridones / therapeutic use
  • Pyrimidinones / pharmacology
  • Pyrimidinones / therapeutic use
  • Radiation-Sensitizing Agents / pharmacology*
  • Radiation-Sensitizing Agents / therapeutic use
  • Tumor Stem Cell Assay
  • ras Proteins / genetics


  • Antineoplastic Agents
  • Benzamides
  • KRAS protein, human
  • Poly(ADP-ribose) Polymerase Inhibitors
  • Protein Kinase Inhibitors
  • Proto-Oncogene Proteins
  • Pyridones
  • Pyrimidinones
  • Radiation-Sensitizing Agents
  • iniparib
  • trametinib
  • MAP Kinase Kinase 1
  • MAP Kinase Kinase 2
  • Proto-Oncogene Proteins p21(ras)
  • ras Proteins