Poster: A Novel Cell-Based Screening Approach for the Identification of FOXA 1 Pathway Inhibitors for the Treatment of Tamoxifen-Resistant Breast Cancer
Poster presentation at ELRIG Drug Discovery 2012, Manchester, UK.
Winner of a SLAS Young Scientist Award.
Molecular Pharmacology Department, Cancer Research Technology Development Laboratory, at Wolfson Institute for Biomedical Research, University College London, England
The oestrogen receptor (ER) is the major driver of ER-positive breast cancers. While these cancers are generally treated with endocrine therapies including tamoxifen, resistance often occurs demonstrating a need for an alternative strategy. Dr Jason Carroll's CR-UK funded research identified FOXA1 as a novel pioneer factor critical for ER-mediated gene expression. Depletion of FOXA1 by RNAi not only prevents the majority ER-target gene expression, but reduces the growth of both tamoxifen-sensitive and resistant breast cancer cell lines. The FOXA1/ER pathway has thus stepped into the spotlight as an exciting new target for the treatment of tamoxifen-resistant breast cancer. At CRT we have been working in collaboration with Dr Carroll's lab to develop a cell-based drug discovery program for the identification of FOXA1 pathway inhibitors.
Our cell-based assay cascade allows us to screen for inhibitors of the FOXA1 pathway in a high-throughput manner whilst removing non-specific compounds or ER modulators. The primary assay consists of FOXA1/ER binding elements coupled to a luciferase reporter developed using the proprietary know-how from the Carroll laboratory. This cell-based assay was validated at CRT using FOXA1 RNAi and ER antagonists. After miniaturisation to 384well format, scale up of stably transfected MCF7 cells culture and automation of screening process, this biological system was used to screen CRT’s full chemical collection, 150 000 compounds, at 3uM. The implementation of acoustic technology within CRT’s HTS platform supported the integration of fully automated nanolitre range compound transfer in order to improve the quality and the flexibility of our HTS processes.
This approach resulted in a successful HTS campaign leading to the identification of 442 compounds showing inhibitory action of reporter ranging from 68 to 142%, compared to Fulvestrant reference compound at 3uM.
The confirmation of hit specificity is underway. The hits from the primary screen would include specific FOXA1 pathway inhibitors but also non-specific ER pathway and general transcription inhibitors. To exclude these hits, two counter-screens have been utilised: a counterscreen CMV-driven luciferase reported assay aiming to identify general inhibitors of transcription ; a Biochemical-based luciferase assay to identify compounds interfering with reporter read out technology.
Putative hits will then be progressed to biochemical ER binding assay and cell viability profiling against the FOXA1 dependent and nondependent cell lines MCF7 and MBA-MB-231 respectively. Finally, validated hits will be further characterised using mRNA expression and chromatin immunoprecipitation analysis in Jason Carroll’s laboratory. This drug discovery program demonstrates the opportunity to realise the potential of CRUK funded research for the treatment of breast cancer.