Chemical Genetic Screens Identify Kinase Inhibitor Combinations that Target Anti-Apoptotic Proteins for Cancer Therapy

Abstract

The study presented here provides a framework for the discovery of unique inhibitor combinations that target the apoptosis network for cancer therapy. A pair of doxycycline (Dox)-inducible cell lines that specifically report on the ability of an inhibitor to induce apoptosis by targeting either the Mcl-1 arm or the Bcl-2/Bcl-xL/Bcl-w arm were used. Cell-based assays were optimized for high throughput screening (HTS) with caspase 3/7 as a read out. HTS with a 355-member kinase inhibitor library and the panel of Dox-inducible cell lines revealed that cyclin dependent kinase (CDK) inhibitors induced apoptosis by targeting the Mcl-1 arm, whereas PI3K inhibitors induced apoptosis by targeting the Bcl-2/Bcl-xL/Bcl-w arm. Validation studies identified unique combinations that synergistically inhibited growth and induced apoptosis in a panel of cancer cell lines. Since these inhibitors have been or are currently in clinical trials as single agents, the combinations can be rapidly translated to the clinics.

Figure 1.

A strategy to identify kinase inhibitor combinations to target proteins in the apoptotic pathway. (A) Overview of the approach. (B) Dox-inducible cell lines to identify Bcl-2/Bcl-xL/Bcl-w pathway and Mcl-1 pathway inhibitors.

Figure 2.

Validation of the Dox-inducible cell lines for (A) the expression of Dox-inducible gene products and (B) the induction of apoptosis through chemical inactivation of Mcl-1 and Bcl-2/Bcl-xL/Bcl-w using camptothecin and ABT-263 respectively.

Figure 3.

Summary of the HTS. (A) Venn diagram representation of the clustering of hits from the screen. (B) Fold change caspase activation normalized to DMSO.

Figure 4.

Validation of hits. (A) Western blot analyses for PARP cleavage in the Dox-inducible HeLa cell lines treated with 0.5 μM for 6h. (B) Average combination index (CI) values at effective dose (ED)90, ED95 and ED99 in three cancer cell lines. (C). Western blot analyses of PARP cleavage and Mcl-1 after individual (P276–00 and PF05212384) and combination treatment.