HSP90 inhibition partially rescues alternative splicing dysregulation in cell models of myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are multisystemic diseases caused by the expression of toxic expansion RNAs that sequester muscleblind-like (MBNL) proteins, resulting in extensive alternative splicing dysregulation. Given that there are no current disease-modifying treatments for DM, we sought to identify compounds that rescue the underlying splicing dysregulation. A medium throughput splicing screen utilizing DM1 patient-derived fibroblasts was developed and used to screen 1584 compounds from the NIH NCI Diversity Set VI, leading to the identification of macbecin II, an HSP90 inhibitor. Macbecin II-mediated HSP90 inhibition corrects several MBNL-regulated splicing events in DM1 myotubes, and a structurally distinct HSP90 inhibitor, CCT018159, produces similar effects. Using RT-PCR splicing analysis, siRNA knockdown, RT-qPCR, immunoblotting, and RNA fluorescence in situ hybridization we examined the effects of HSP90 inhibition in DM models. HSP90 inhibition increases MBNL1 and MBNL2 transcript levels, increases MBNL2 protein, and reduces toxic CUG RNA and nuclear foci in DM1 cell models. An analysis of individual HSP90 isoforms reveals that knocking down HSP90AA1, HSP90AB1, and TRAP1 partially improves splicing defects, whereas HSP90B1 knockdown exacerbates mis-splicing. We also show that treatment with HSP90 inhibitors corrects mis-splicing in DM2 myotubes. Together, these data identify HSP90 as a modifier of RNA toxicity and alternative splicing in DM and support further evaluation of HSP90-directed therapeutic strategies.