Abstract
BackgroundFacioscapulohumeral muscular dystrophy (FSHD) is caused by epigenetic alterations at the D4Z4 macrosatellite repeat locus on chromosome 4, resulting in inappropriate expression of the DUX4 protein. The DUX4 protein is therefore the primary molecular target for therapeutic intervention.MethodsWe have developed a high-throughput screen based on the toxicity of DUX4 when overexpressed in C2C12 myoblasts, and identified inhibitors of DUX4-induced toxicity from within a diverse set of 44,000 small, drug-like molecules. A total of 1,280 hits were then subjected to secondary screening for activity against DUX4 expressed by 3T3 fibroblasts, for absence of activity against the tet-on system used to conditionally express DUX4, and for potential effects on cellular proliferation rate.ResultsThis allowed us to define a panel of 52 compounds to use as probes to identify essential pathways of DUX4 activity. We tested these compounds for their ability to protect wild-type cells from other types of cell death-inducing insults. Remarkably, we found that 60% of the DUX4 toxicity inhibitors that we identified also protected cells from tert-butyl hydrogen peroxide, an oxidative stress-inducing compound. Compounds did not protect against death induced by caspase activation, DNA damage, protein misfolding, or ER stress. Encouragingly, many of these compounds are also protective against DUX4 expression in human cells.ConclusionThese data suggest that oxidative stress is a dominant pathway through which DUX4-provoked toxicity is mediated in this system, and we speculate that enhancing the oxidative stress response pathway might be clinically beneficial in FSHD.
Highlights
Facioscapulohumeral muscular dystrophy (FSHD) is caused by epigenetic alterations at the D4Z4 macrosatellite repeat locus on chromosome 4, resulting in inappropriate expression of the DUX4 protein
Because the phenotype requires both activity of the DUX4 protein and transmission of a signal through one or more cell death pathways, we reasoned that such a screen would identify both compounds that interfere directly with the activity of the DUX4 protein, as well as compounds that interfere with essential DUX4 downstream pathways leading to cell death
We used an assay based on colorimetric detection of ATP levels in the cells to quantify viability after DUX4 expression, and optimized cell number, dox concentration, and time of exposure, to develop a robust high-throughput screening assay with a Z-factor value of 0.74 (n = 8) (Figure 1B)
Summary
Facioscapulohumeral muscular dystrophy (FSHD) is caused by epigenetic alterations at the D4Z4 macrosatellite repeat locus on chromosome 4, resulting in inappropriate expression of the DUX4 protein. Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common degenerative myopathies It is caused by epigenetic alterations within D4Z4, the macrosatellite repeat at the end of the long arm of chromosome 4 [1,2,3,4], which arise due to contractions in repeat number [5,6] or second site mutation of regulators of D4Z4 [7]. Because the phenotype requires both activity of the DUX4 protein and transmission of a signal through one or more cell death pathways, we reasoned that such a screen would identify both compounds that interfere directly with the activity of the DUX4 protein, as well as compounds that interfere with essential DUX4 downstream pathways leading to cell death Since these pathways are not well understood, such compounds could serve as useful probes into the molecular mechanism by which DUX4 causes cell death
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