Stem cell based high-throughput screen to identify inhibitors of DUX4

First step towards developing a targeted therapy for any diseases is to generate model system for studding the mechanism and for testing various therapeutic approaches. Fascioscapulohumeral muscular dystrophy (FSHD) is diseases which rather affect muscle stem cells then mature myofibers. Most likely the pathological effect is in prenatal and postnatal stage, during muscle formation and muscle regeneration. To address the both stages we engineered various stem cell line suitable to study gain of function during early embryogenesis (human and mouse ES cells) and myogenesis in adults (C2C12). In the cell lines we introduced a single gene ,DUX4, which is believe to trigger the molecular cascade of FSHD pathology. We toke the advantage of conditional cell toxicity of DUX4 and we developed a small molecule screening platform for identifying inhibitors of DUX4. Assay based on rapid cell death within 24 hours induced by high levels of DUX4 was used for high throughput screen of 44,000 chemicals. The assay was proved to be easy control by titration of doxycyline, adaptable to miniaturized formats, robust, rapid, with low false positive rates and high signal to noise ratios. We identified more than 1280 compounds with significant rescue ability. To narrow down to direct DUX4 inhibitors, we have conducted serial follow up assays, including secondary screens to eliminate compounds which interfere with the rtTA/TRE inducible gene expression system, to distinguish common anti oxidants and/or anti stressors, to confirm reversion of toxicity in different non myogenic cell types. Several classes of compounds reverted toxicity indirectly, including antioxidants. By developing HTS platform and established cell based systems for follow up studies we established the base for the most crucial topic and urgent needs of FSHD patients: specific and direct pharmacological therapy.