Small molecule kinase inhibitors alleviate different molecular features of myotonic dystrophy type 1

Expandable (CTG)n repeats in the 3′ UTR of the DMPK gene are a cause of myotonic dystrophy type 1 (DM1), which leads to a toxic RNA gain-of-function disease. Mutant RNAs with expanded CUG repeats are retained in the nucleus and aggregate in discrete inclusions. These foci sequester splicing factors of the MBNL family and trigger upregulation of the CUGBP family of proteins resulting in the mis-splicing of their target transcripts. To date, many efforts to develop novel therapeutic strategies have been focused on disrupting the toxic nuclear foci and correcting aberrant alternative splicing via targeting mutant CUG repeats RNA; however, no effective treatment for DM1 is currently available. Herein, we present results of culturing of human DM1 myoblasts and fibroblasts with two small-molecule ATP-binding site-specific kinase inhibitors, C16 and C51, which resulted in the alleviation of the dominant-negative effects of CUG repeat expansion. Reversal of the DM1 molecular phenotype includes a reduction of the size and number of foci containing expanded CUG repeat transcripts, decreased steady-state levels of CUGBP1 protein, and consequent improvement of the aberrant alternative splicing of several pre-mRNAs misregulated in DM1.


RNA FISH and Immunostaining.
Cells were fixed in 4% PFA/PBS at 4°C and washed three times in PBS followed by permeabilization in 2% prechilled acetone/PBS for 5 min. Pre-hybridization was performed in 30% formamide and 2xSSC buffer for 10 min followed by hybridization in a buffer containing 30% formamide, 2xSSC, 0.02% BSA, 66 µg/ml yeast tRNA, 10% dextran sulfate, 2 mM vanadyl ribonucleoside complex and 2 ng/µl DNA/LNA probes (CAG) 6 -CA labeled at the 5′end with Cy3 and modified at positions 2, 5, 8, 13, 16 and 19 with LNA. Post-hybridization washing was performed in 30% formamide and 2xSSC at 45°C for 30 min followed by 1xSSC at 37°C for the next 30 min.
Slides were either mounted in SlowFade Gold antifade reagent with DAPI (Invitrogen) for CUG exp RNA foci analysis or used for subsequent IF. For RNA FISH in combination with IF, the slides were incubated in blocking buffer (5% non-fat milk, 10% normal serum and 0.3% Triton X-100 in PBS) for 1 h at room temperature and treated with rabbit anti-MBNL1 primary antibody (A2764) overnight at 4°C. Afterwards, the slides were washed in PBS three times for 5 min each and then incubated with a secondary anti-rabbit antibody conjugated to Alexa Fluor 488 (Molecular Probes). Following three PBS washes, the slides were mounted in the antifade reagent containing DAPI.
The collected supernatant was centrifuged at 19,000 rpm for 20 min. The protein was purified using a HisTrap FF Crude column (GE Healthcare) on an FPLC system and PD-10 columns (GE Healthcare) following the manufacturer's protocols, and the total protein concentration was measured by the Bradford assay. Protein (1.5 µM) was stored at -20°C in 50 % glycerol and buffer containing 50 mM Tris (pH 8.0), 50 mM KCl, 300 mM NaCl, 1 mM MgCl 2 and 0.02% Tween-20.
The filter binding assay (FBA) was performed in a 30-µl volume of FBB filter binding (FB) buffer containing 50 mM Tris (pH 8.0), 50 mM KCl, 50 mM NaCl, and 1 mM MgCl 2 . As depicted in Figure

Fibroblasts.
Representative confocal microscopy images of RNA FISH/IF from cultured human DM1 fibroblasts (500 CUG RNA repeats) that were treated with C16 (0.5 μM). Untreated cells are shown as controls. Culturing with the compound resulted in displacing of MBNL1 from faint nuclear CUG exp RNA foci. Nuclei were stained with DAPI (blue), CUG exp RNA foci (red), MBNL1 (green). Scale bar, 10 µm.

Fibroblasts.
Representative confocal microscopy images of RNA FISH/IF from cultured human DM1 fibroblasts (500 CUG RNA repeats) that were treated with C51 (30 μM). Untreated cells are shown as controls. Culturing with the compound resulted in displacing of MBNL1 from faint nuclear CUG exp RNA foci. Nuclei were stained with DAPI (blue), CUG exp RNA foci (red), MBNL1 (green). Scale bar, 10 µm.   Representative agarose gel images of RT-PCR products of the endogenous SERCA1, LDB3 and DMD splicing isoforms in control human cells. Treatment with C16 and C51 did not affect splicing pattern of the analyzed pre-mRNAs in control fibroblasts and myoblasts. Bar graphs show mean values (±SEM) of the percentage of DM1specific splice isoforms relative to the total amount detected in both splice variants. Data was gathered in at least three independent experiments. Exon inclusion (+E); exon exclusion (-E).

Figure S-4D. Splicing Pattern of pre-mRNAs Not Regulated by MBNL1 and CUGBP1 after Treatment with C16
and C51.
Representative agarose gel images of RT-PCR products of several pre-mRNAs regulated by NOVA1, FOX2, PTBP1 and PTBP2 in DM1 human myoblasts after the treatment with C16 and C51 at indicated concentrations. No changes in the splicing pattern were detected upon the treatments with the exception of PPP3CB. Numbers below the gels indicate the ratio between bands representing exon exclusion to the total intensity of both splice products.