Small Molecule Screening Identifies HSP90 as a Modifier of RNA Foci in Myotonic Dystrophy Type 1

Abstract

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by a CTG triplet repeat expansion within the 3' untranslated region of the DMPK gene. Expression of the expanded allele generates RNA containing long tracts of CUG repeats (CUGexp RNA) that form hairpin structures and accumulate in nuclear RNA foci; however, the factors that control DMPK expression and the formation of CUGexp RNA foci remain largely unknown. We performed an unbiased small molecule screen in an immortalized human DM1 skeletal muscle myoblast cell line and identified HSP90 as a modifier of endogenous RNA foci. Small molecule inhibition of HSP90 leads to enhancement of RNA foci and upregulation of DMPK mRNA levels. Knockdown and overexpression of HSP90 in undifferentiated DM1 myoblasts validated the impact of HSP90 with upregulation and downregulation of DMPK mRNA, respectively. Furthermore, we identified p-STAT3 as a downstream mediator of HSP90 impacting levels of DMPK mRNA and RNA foci. Interestingly, differentiated cells exhibited an opposite effect of HSP90 inhibition displaying downregulation of DMPK mRNA through a mechanism independent of p-STAT3 involvement. This study has revealed a novel mediator for DMPK mRNA and foci regulation in DM1 cells with the potential to identify targets for future therapeutic intervention.

Keywords: HSP90; Myotonic dystrophy; RNA foci; small molecule screening.

Figure 1:. Characterization of immortalized myoblast cell lines.

A-B.) DM480 cells exhibit RNA foci labelled using a (CAG)₅-TYE563 FISH probe; foci are quantified by mean foci count per nucleus and proportion of Foci+ nuclei. Scale bar = 10 μM. C-F.) Unaffected (MBC) and DM1 (DM480) cell lines show robust myogenic differentiation based on RNA and protein markers. G.) DM480 cells recapitulate DM1 misregulated alternative splicing patterns compared to MBC. n=3 independent cell cultures. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001.

Figure 1:. Characterization of immortalized myoblast cell lines.

A-B.) DM480 cells exhibit RNA foci labelled using a (CAG)₅-TYE563 FISH probe; foci are quantified by mean foci count per nucleus and proportion of Foci+ nuclei. Scale bar = 10 μM. C-F.) Unaffected (MBC) and DM1 (DM480) cell lines show robust myogenic differentiation based on RNA and protein markers. G.) DM480 cells recapitulate DM1 misregulated alternative splicing patterns compared to MBC. n=3 independent cell cultures. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001.

Figure 2:. Primary Screening identified four foci reducing and three foci enhancing compounds.

A. and B.) Diagram of primary and secondary screening layout. C.) Table of top foci reducing compounds and their targeted pathways. D.) Representative images from secondary screening of foci reducing compounds (5μM) and E.) quantification of proportion of foci+ cells (n = 2 plates; each an average of four fields of view (FOV) and mean foci count per nucleus (n ≥ 1922 nuclei) for the 5μM concentration. Quantification includes individual nuclei measurements across four FOV in duplicate for visualization purposes (AUC across all concentrations included in Supplemental Table 1) F.) Table of top foci enhancing compounds. G.) Representative images of foci enhancing compounds in secondary screening and H.) quantification of proportion of foci+ cells (n = 2 plates; each an average of four FOV), mean foci count per nucleus (n ≥ 1123 nuclei) and foci pixel intensity (n ≥ 1528 foci) for the 5μM concentration. Quantification includes individual nuclei/foci measurements across four FOV in duplicate for visualization purposes (AUC across all concentrations included in Supplemental Table 1). Scale bar = 10μm. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001.

Figure 2:. Primary Screening identified four foci reducing and three foci enhancing compounds.

A. and B.) Diagram of primary and secondary screening layout. C.) Table of top foci reducing compounds and their targeted pathways. D.) Representative images from secondary screening of foci reducing compounds (5μM) and E.) quantification of proportion of foci+ cells (n = 2 plates; each an average of four fields of view (FOV) and mean foci count per nucleus (n ≥ 1922 nuclei) for the 5μM concentration. Quantification includes individual nuclei measurements across four FOV in duplicate for visualization purposes (AUC across all concentrations included in Supplemental Table 1) F.) Table of top foci enhancing compounds. G.) Representative images of foci enhancing compounds in secondary screening and H.) quantification of proportion of foci+ cells (n = 2 plates; each an average of four FOV), mean foci count per nucleus (n ≥ 1123 nuclei) and foci pixel intensity (n ≥ 1528 foci) for the 5μM concentration. Quantification includes individual nuclei/foci measurements across four FOV in duplicate for visualization purposes (AUC across all concentrations included in Supplemental Table 1). Scale bar = 10μm. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001.

Figure 3:. Foci enhancing compounds show a differentiation dependent effect.

A-B) Foci reducing compounds show a consistent downregulation of DMPK mRNA levels in both MBC and DM480 cells before and after differentiation. C-D) RT-qPCR results of DMPK mRNA in undifferentiated and differentiated MBC and DM480 cells. E.) CUGexp RNA FISH labeling of differentiated DM480 cells following foci-enhancing compounds treatment and quantification of foci pixel intensity and mean foci count per nucleus. Scale bar = 10μm. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001.

Figure 4:. HSP90 mediates DMPK RNA upregulation and enhanced foci in undifferentiated DM480 cells.

A.) Western blots and quantification of HSP90 alpha and HSP90 beta siRNA knockdown in undifferentiated DM480 cells. B.) RT-qPCR for DMPK mRNA following downregulation of HSP90 alpha or HSP90 beta. C.) FISH labeling of CUGexp RNA foci following HSP90 alpha and HSP90 beta knockdown and quantification of pixel intensity. Scale bar = 10 μM. D.) Western blots and quantification of HSP90 plasmid overexpression in undifferentiated DM480. E.) RT-qPCR of DMPK mRNA following overexpression of HSP90 plasmids in undifferentiated DM480 cells. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001.

Figure 5:. STAT3 activity impacts DMPK mRNA and foci in undifferentiated DM1 cells.

A.) Westerns of undifferentiated and differentiated DM480 cells showing STAT3 and p-STAT3 protein levels and quantification. B.) Westerns showing STAT3 and p-STAT3 levels following treatments of foci-enhancing compounds and quantification. C.) RT-qPCR of DMPK mRNA following treatment of both undifferentiated and differentiated DM480 cells with Niclosamide. D.) CUGexp RNA FISH labeling following treatment with Niclosamide and quantification of foci pixel intensity and mean foci count per nucleus. Scale bar = 10μM. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001.