Led by Dr. Chunhong Yan, Cancer Center researchers Drs. Liwei Lang and Han-Fei Ding and their colleagues have generated a novel strategy for identifying compounds that may be further developed as anti-cancer therapeutics. Since overexpression of the transcription factor eIF4E accounts for increased activity of genes needed for cancer cell growth and survival, efforts have been made to downregulate eIF4E as a cancer treatment strategy. The identification of compounds capable of this downregulation activity has been challenged by a lack of reliable high throughput screening assays. However, as published recently in Cell Press’ Chemistry & Biology, Dr. Yan’s team has created a bicistronic reporter assay to overcome many of the pitfalls of traditional assay systems. Importantly, it includes the capacity for conducting a subsequent orthogonal assay, which reduced the overall false positive rate to less than 10% in this proof-of-principle report.
Common pitfalls of traditional systems stem from the use of cloned, transgenic promoters that are randomly integrated into the genome, leading to contextual insufficiencies. In contrast, the assay developed by Dr. Yan’s group was designed to rely on the endogenous promoter to control the expression of an endogenous gene directly followed by an inserted IRES-reporter gene construct. This strategy thereby maintains the native environment in terms of epigenetic factors, chromatin, and distal cis-acting elements. Results of the proof-of-principle study confirmed that the insertion did not affect the expression of the native gene (EIF4E). The assay is based on CRISPR-Cas9-mediated genome editing tool and recombinase-mediated cassette-exchange technology.[Lang L, Ding HF, Chen X, Sun SY, Liu G, Yan C. 2015. Internal ribosome entry site-based bicistronic in situ reporter assays for discovery of transcription-targeted lead compounds. Chem Biol. 22(7):957-964.]