Inhibition of Rho-Associated Kinases ROCK1 and ROCK2 as a Therapeutic Strategy to Reactivate the Repressed<i>FXN</i>Gene in Friedreich Ataxia

Friedreich ataxia (FA) is an autosomal recessive disease characterized by progressive damage to the nervous system and severe cardiac abnormalities. The disease is caused by a GAA•TTC triplet repeat expansion in the first intron of the <i>FXN</i> gene, resulting in epigenetic repression of <i>FXN</i> transcription and reduction in FXN (frataxin) protein which results in mitochondrial dysfunction. Factors and pathways that promote <i>FXN</i> repression represent potential therapeutic targets whose inhibition would restore <i>FXN</i> transcription and frataxin protein levels. Here, we performed a candidate-based RNAi screen to identify kinases, a highly druggable class of proteins, that when knocked down upregulate <i>FXN</i> expression. Using this approach, we identified Rho kinase ROCK1 as a critical factor required for <i>FXN</i> repression. ShRNA-mediated knockdown of ROCK1, or the related kinase ROCK2, increases <i>FXN</i> mRNA and frataxin protein levels in FA patient-derived induced pluripotent stem cells (iPSCs) and differentiated neurons and cardiomyocytes to levels observed in normal cells. We demonstrate that small molecule ROCK inhibitors, including the FDA-approved drug belumosudil and fasudil, reactivate <i>FXN</i> expression in cultured FA iPSCs, neurons, cardiomyocytes, and FA patient primary fibroblasts and ameliorate the characteristic mitochondrial defects in these cell types. Remarkably, treatment of transgenic FA mice of both sexes with belumosudil or fasudil upregulates <i>FXN</i> expression, ameliorates the mitochondrial defects in the brain and heart tissues, and improves motor coordination and muscle strength. Collectively, our study identifies ROCK kinases as critical repressors of <i>FXN</i> expression and provides preclinical evidence that FDA-approved ROCK inhibitors may be repurposed for treatment of FA.