Endocrine therapy is the main therapeutic approach for estrogen receptor-positive (ER+) breast cancer, the most frequent breast cancer subtype in women. However, endocrine resistance inevitably arises, with ER reactivation being a key resistance mechanism. Overcoming primary or acquired resistance to endocrine treatment remains a major challenge. To identify novel therapeutic approaches towards regulation of ER we leveraged a fragment-based ligand discovery platform. The platform consists of a library of ~460 small molecule fully functionalised fragments (FFFs) that was designed to facilitate downstream target deconvolution through chemical proteomics. The FFF screening platform was coupled to a high-content phenotypic assay to identify fragments that reduce ER protein stability. Confirmed fragment ‘hits’ found to downregulate ER levels were selected for target deconvolution by in-cell photoaffinity chemoproteomics in an attempt to identify fragment binders. Chemoproteomic analysis characterised 8 proteins that were found to be significantly associated with the fragment and hence putatively modulate ER stability. The identified targets were assessed by orthogonal CRISPRn screening, which highlighted one gene that phenocopied the FFF hit in down-regulating ER protein levels and also reduced the long term proliferation of ER+ breast cancer cells upon knockout. Our observations are consistent with literature demonstrating that inhibition of the protein complex that our target belongs to downregulates ER levels and proliferation in breast cancer cells, whilst enhancing the effectiveness of endocrine therapies in resistance models. Collectively, this work emphasises the power of the FFF platform in identifying high confidence and therapeutically relevant targets that validate across multiple screening modalities.
Co-authors: Patrick O’Shea, Ghaith Hamza, Steven Novick, Jenna Bradley, Sinead Knight, Adam Hendricks. Ian Storer, Larissa Carnevalli, Laura Rosenberg, Davide Gianni
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