High-throughput synthetic lethal drug screen to identify novel radiation sensitizers for KRAS mutant non-small cell lung cancer
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AbstractTraditional clonogenic survival and high throughput colorimetric assays are inadequate for drug screens to identify novel radiation sensitizers. To bridge this gap in knowledge, we have developed a method which we coin the High Throughput Clonogenic Survival Screen (HCS) that will allow high volume screening of drug libraries to identify potent radiation sensitizers, particularly for KRAS mutant lung cancer, a current unmet need. 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. Plates were kept in culture 4-6 days depending on the cell line. After incubation, colonies were stained with cresyl-violet, imaged on the INCell 6000 (GE Health), and colony count determined using the INCell Developer image analysis software. Colonies achieving 50 or more cells were tallied. Traditional clonogenic survival assays (tCSA) were performed by seeding cells into 6-well plates overnight in triplicate and adding drugs or vehicle for 6 hours prior to irradiation. Media was changed after 3 days, and plates were kept in culture for 10-14 days. Drug screens were done using the KRAS mutant lung cancer cell line H460 and the Custom Clinical Collection (145 compounds). We compared the HCS method to tCSA and found that HCS recapitulates a similar cell survival curve and survival fraction at 2 Gy (SF2) values for several cancer cell types. As a proof-of-principle to demonstrate that HCS could identify radiation sensitizers, we tested a known sensitizer vorinostat (SAHA) in both the tCSA and HCS assays. As expected, 1 M vorinostat caused a significant radiation sensitizing effect using tCSA, and was recapitulated, albeit at a lower concentration (100 nM), using the HCS assay. Drug screens (repeated 3 times) 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). The entire class of HDAC inhibitors (5 drugs) had some sensitizing effect to radiation (a leftward shift in the IC50 curve by ~10 fold). However, many drugs such as inhibitors to PARP, SRC, Chk1/2, DNA-PK, JNK, and MEK1/2, exhibited significant radiation sensitizing effect with minimal to no activity by themselves. We validated the sensitizing effect of the potent MEK1/2 inhibitor trametinib using tCSA, and found the effect was specific for KRAS mutant and not KRAS wild type cells. From these results, we believe high throughput drug screening for novel radiation sensitizers for KRAS mutant lung cancer is feasible using the HCS approach. This is an enabling technology that will allow synthetic lethal drug screens with radiation and accelerate the translation of drugs into clinical testing.Citation Format: Steven H. Lin, Jing Zhang, Uma Giri, Clifford Stephan, Mary Sobieski, Stephen S. Yoo, John V. Heymach. High-throughput synthetic lethal drug screen to identify novel radiation sensitizers for KRAS mutant non-small cell lung cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B08.
