Abstract 1348: A novel bicistronic reporter-based high-throughput screening platform for discovery of transcription-targeted lead compounds

Abstract

Abstract Given that aberrant gene expression is widely spread in cancer, there is an imperative need of developing reliable high-throughput drug screening (HTS) platforms in search of lead compounds that can rectify abnormal expression of cancer-driving genes for treating this deadly disease. Although it has been demonstrated that reporter gene assays are useful in the identification of transcription-targeted lead compounds, these assays are often unreliable and can yield high rates of false compounds due to the facts that reporter genes are driven by cloned promoters and localized in foreign chromatin environments. While cloned promoters lack essential cis-regulatory elements (e.g., enhancers) far removed from transcription start sites, foreign chromatin environment can alter gene expression in a way atypical to endogenous genes. To address these limitations, we employed emerging genome-editing tools to develop a novel reporter-based HTS assay whereby the reporter gene is inserted immediately downstream of the coding region of an endogenous gene, and thereby co-expressed bicistronically with the endogenous gene as a single transcript under the control of the native transcriptional regulatory machinery. As a proof-of-principle, we first employed the CRISPR-Cas9 system to knock a fused selection gene tk-ble (encoding Zeocin resistance for selection for targeting events and thymidine kinase for negative selection for the removal of the selection gene, respectively) flanked by a wild-type and a mutated FRT fragment into the eukaryotic translation initiation factor 4E (EIF4E) gene locus, and then replaced the selection gene with a firefly luciferase (FLuc), or a Renilla luciferase (RLuc), by cassette exchange mediated by the recombinase Flp. We demonstrated that the FLuc gene was expressed as single transcripts with EIF4E by Northern blotting, and responded to chemical treatments in a way analog to the endogenous EIF4E gene. Using the cells carrying bicistronic FLuc reporter, we screened a chemical library containing 4,800 compounds for small molecules inhibitory for EIF4E expression, and identified 11 hits, out of which 6 compounds were validated to decrease EIF4E expression by qRT-PCR. While the 5 false-positive compounds were likely yielded due to their direct inhibition of FLuc activity, 4 of these false positives could be readily identified by an orthogonal assay using recombinant cells carrying RLuc in the same EIF4E locus. Therefore, our results have demonstrated that screening assays based on bicistronic in-situ reporters are more reliable, and powerful in searching for transcription-targeted lead compounds with high confidence. Citation Format: Liwei Lang, Chunhong Yan. A novel bicistronic reporter-based high-throughput screening platform for discovery of transcription-targeted lead compounds. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1348.