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

Abstract

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.

Figure 1. The HCSA recapitulates results from traditional clonogenic survival assay and allows the identification of radiation sensitizers

(A). Comparison in techniques between traditional and HCSA methods. The workflow on the left panel depicts the typical workflow of the traditional assay, whereas the right panel depicts workflow for the HCSA. The length of arrows corresponds to the relative length of time it takes to execute each of the steps. (B). Left panel: crystal violet stain and low magnification images of the triplicate wells from a 96 well plate with cells seeded at various densities per well. Right panel: INCell6000 images of the corresponding wells. (C) A comparison of the colonies formed by two lung cancer cell lines with different morphologies, one the H460 which forms tighter aggregates whereas the A549 expresses mesenchymal characteristics and grows colonies in patterns that are more spread out. The image software was able to enumerate colonies of both types, as depicted by the image analysis. (D) Comparison in survival curves between the traditional and HCSA methods. HCSA recapitulates a similar survival curve as produced by the traditional method. (E) The survival fraction at 2 Gy (SF2) comparing various cell types: glioblastoma (U251), prostate cancer (DU145, PC3), and pancreatic cancer (Miapaca2).

Figure 2. Validation of the HCSA method to identify radiation sensitizers using vorinostat and PI-103

(A) One micromolar vorinostat was used for the tCSA method, but at such doses no clonogens were produced in the HCSA, and therefore lower doses (200 and 400 nM) were used (B–C). HCSA had very similar enhancement effects compared to tCSA, with a dose-dependent radiation enhancement effect. DER = Dose Enhancement Ratio as a radiation dose of control over drug at survival fraction 0.5 (SF0.5). (D) 500 nM PI103 was added to seeded cells for 6 hours prior to irradiation at indicated doses. Media was changed at 72 hours of total drug exposure. (E) PI103 at 25 nM or 50 nM (F) was added to seeded cells for 6 hours prior to irradiation at corresponding doses. Cells were not perturbed until crystal violet staining on day 5

Figure 3. HCSA enables the identification of classes of radiation sensitizers through drug screening

(A) Representative screening results of plate one of the Custom Clinical Collection (CC1) and plate two of the collection (CC2) on the H460 cells, with the identity of the drugs on the right side of each row of cells. The columns represent one drug concentration or sham control, and each row depicts one particular drug. #N/A are blanks with 0.1% DMSO or RPMI. A green block represents single agent activity without apparent radiation enhancement effect, a pink block represents drugs that are weakly enhancing (<1 log10 shifted) and red block represents drugs that are strongly enhancing (≥1 log10 shifted). (B) List of drugs that are identified in the screen to have significant enhancement effects. Drugs within the same class are listed together. The inset figure illustrates with asterisks a few of the proteins along the signaling cascade of activated KRAS that radiosensitize when blocked. (C) Examples of three classes of radiosensitizing drugs identified in the screen, namely the HDAC inhibitors, PARP inhibitors and MEK inhibitors.

Figure 4. The MEK inhibitor trametinib is a selective radiation sensitizer in KRAS mutant lung cancer cells

(A) Traditional CSA was used to determine the relative potency of trametinib (MEKi) in various lung cancer cell lines, with or without KRAS mutations. This effect seems to be selective in cells lines with KRAS mutations, also the potency differ between the KRAS mutant lines. No effect was apparent in the wild type cells. (B) Radiation induced pERK activation that was seen in the H460 but not in the WT H661 cell line. This activation was fully blocked by treating cells with trametinib. (C) Cell cycle analysis in two KRAS mutant and two KRAS WT cells. Significant S phase prolongation and diminution of G1/S cells was apparent in the KRAS mutant cells treated with trametinib and radiation, but not seen with either agent alone or in the WT cell lines. (D) Xenograft model of H460 cells to determine the amount of growth delay with combined treatments versus single agent alone. Drug treatment lasted 14 days, the first 5 were combined with radiation at 2 Gy per day. Drugs was given by oral gavage at 2 mg/kg 4 hours prior to radiation administration.