High content clonogenic survival assay for drug library screening identifies potent radiation sensitizers for non-small cell lung cancer
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AbstractRadiotherapy is a critical component in the definitive treatment of locally-advanced non-small cell lung cancer (LA-NSCLC), and while concurrent platinum-based chemoradiation is the standard approach, improvements have been minimal. Molecularly-targeted therapy to further enhance radiation effects beyond platinum-sensitization is needed to further improve radiotherapy efficacy. A systematic approach needs to be developed that can rapidly identify novel drugs that can be combined to enhance radiation effects. However, current approaches using traditional clonogenic survival assay (tCSA) and colorimetric assays are inadequate for high throughput drug screens to identify novel radiation sensitizers. To enable screens adapted for radiation drug screening, we have developed a method which we coin the High Throughput Clonogenic Survival Assay (HCSA) that allows rapid screening of drug libraries to identify potent radiation sensitizers. As a proof-of-principle, we used the Kras mutated lung cancer cell line H460, and validated the hits using traditional assays. Drugs at various concentrations or vehicle were added to cells seeded overnight in 96 well plates at low densities, and after 6 hours, plates were irradiated at various doses. After incubation, colonies were stained and imaged on the INCell 6000 (GE Health), and colony count determined using the INCell Developer image analysis software. Drug screens were done using a Custom Clinical Collection of 150 compounds. We compared the HCSA method to tCSA and found that HCSA recapitulates a similar cell survival curve and survival fraction at 2 Gy (SF2) values for several cancer cell types. Drug screens identified several compounds that were cytotoxic to the KRAS mutant H460 cells alone at 1 M (inhibitors to Src/Abl, IGF1R, ROCK1, AKT, PI3K, c-MET) and at low nM concentrations (HSP90 and HDAC inhibitors). 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 late generation PARP inhibitors were found to be good sensitizers in the HCSA, but the ones that have not made it into clinical development, or in the case of BSI-201, was proven to not even be a PARP inhibitor, do not come out in the screen. We also discovered that several inhibitors of pathways downstream of activated mutant KRAS (PI3K, AKT, mTOR, and MEK1/2) sensitized H460 cells to radiation, which is biologically relevant for this Kras mutant cell line. We believe these results demonstrate the promise of HCSA as a high throughput drug screening platform to identify novel radiation sensitizers. This will greatly expedite the discovery of novel targets and drugs for clinical translation to enhance the effects of radiotherapy in lung cancer.Citation Format: Steven H. Lin, Hui Liu, Yawei Qiao, Uma Giri, Jing Zhang, Clifford Stephan, Mary Sobieski, John V. Heymach, Stephen S. Yoo. High content clonogenic survival assay for drug library screening identifies potent radiation sensitizers for non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4898. doi:10.1158/1538-7445.AM2014-4898
