A high-throughput assay for small molecule destabilizers of the KRAS oncoprotein

Abstract

Mutations in the Ras family of small GTPases, particularly KRAS, occur at high frequencies in cancer and represent a major unmet therapeutic need due to the lack of effective targeted therapies. Past efforts directed at inhibiting the activity of the Ras oncoprotein have proved difficult. We propose an alternative approach to target Ras by eliminating Ras protein from cells with pharmacological means. In this study, we developed a cell-based, high-content screening platform to identify small molecules that could promote the degradation of the KRAS oncoprotein. We generated an EGFP-KRASG12V fluorescence reporter system and implemented it for automated screening in 1536-well plates using high-throughput cellular imaging. We screened a library of clinically relevant compounds at wide dose range and identified Ponatinib and AMG-47a as two candidate compounds that selectively reduced the levels of EGFP-KRASG12V protein but did not affect EGFP protein in cells. This proof-of-principle study demonstrates that it is feasible to use a high-throughput screen to identify compounds that promote the degradation of the Ras oncoprotein as a new approach to target Ras.

Figure 1. Design and validation of the EGFP-KRAS^G12V reporter cell line.

A. The EGFP-KRAS^G12V reporter was expressed under a tetracycline-inducible promoter. Small molecules that cause the destabilization and/or degradation of EGFP-KRAS^G12V can be detected by measuring the changes in fluorescence signal in the cell. A control reporter expressing only EGFP from the same vector (not illustrated) was also constructed for use in counter screen. B. Fluorescent microscopy image of HeLa cells expressing EGFP-KRAS^G12V 48 hours after induction with 500 ng/mL doxycycline indicate that the fusion protein is enriched at the plasma membrane. C. Western blot of whole cell lysates showing the functional activation of ERK 1/2 and Akt, as measured by their phosphorylation, in HeLa cells following EGFP-KRAS^G12V induction. The induction level of EGFP-KRAS^G12V approached maximum at 500 ng/mL doxycycline. D. Flow cytometry quantification of dose-response induction of EGFP-KRAS^G12V in HeLa cells 48 hours after doxycycline. The fluorescence signal of EGFP-KRAS^G12V approached maximum at 500 ng/mL doxycycline.

Figure 2. Optimization of screen parameters in 1536-well plates.

A. Comparison of signal and background from wells containing induced and uninduced EGFP-KRAS^G12V cells, respectively, as measured by the Acumen ^eX3 microplate cytometer (left) and the IN Cell Aanalyzer 2000 high-content imaging platform (right). Within each platform the data was normalized using the associated software for the calculation of Z′ score. Each data point represents a single well. B. Optimized workflow for the primary screening and data analysis using the IN Cell Analyzer 2000 platform.

Figure 3. Assessment of the sensitivity and reproducibility of the primary screen.

A. Correlation of corresponding wells from duplicate plates in the library. Wells with fewer than 150 cells were excluded from this and all subsequent analysis. Regression line and associated R² value are shown. B. Dose-response curves of Torin-1 treated wells from all plates showing the high reproducibility of its activity in the screen (error bars represent SD from all Torin-1 wells within each plate). C. Z′ factors and Torin-1 IC₅₀ values of all library plates (A and B are duplicates plates). D. Graph of EGFP-KRAS^G12V fluorescence signal and cell count of all wells in the library. Data series are colored to show the distribution of different control wells. Wells with fewer than 150 cells were excluded from the analysis due to high EGFP signal variation at very low cell density and for the purpose of filtering out compound doses that are overtly toxic.

Figure 4. Validation of hit compounds by flow cytometry.

A. Mean EGFP-KRAS^G12V signal and cell count from wells treated with Torin-1, AMG-47a and Ponatinib from the primary screen. B–D Flow cytometry quantification of HeLa cells expressing either EGFP-KRAS^G12V (B), EGFP (C) or EGFP-KRAS^WT after 48 hours exposure to compounds. (*p<0.05 and **p<0.001, two-tailed Student's t-test. Error bars represent SEM of at least three independent experiments).

Figure 5. Validation of hit compounds by western blot.

A. Levels of EGFP-KRAS^G12V protein in HeLa cells treated with a KRAS siRNA (siKRAS), AMG-47a and Ponatinib were probed with KRAS antibody in whole cell lysates. Numbers below the blot indicated relative protein levels. The siKRAS treated samples were normalized to siNEG control, and the compound treated samples were normalized to DMSO control. B. Levels of EGFP protein in HeLa cells treated with AMG-47a, Ponatinib and Torin-1 were probed with EGFP antibody in whole cell lysates. Numbers below the blot indicate relative protein levels normalized to DMSO control.