Volume 25, Issue 6, Pages 1395-1407 (June 2017) SU9516 Increases α7β1 Integrin and Ameliorates Disease Progression in the mdx Mouse Model of Duchenne Muscular Dystrophy  Apurva Sarathy, Ryan D. Wuebbles, Tatiana M. Fontelonga, Ashley R. Tarchione, Lesley A. Mathews Griner, Dante J. Heredia, Andreia M. Nunes, Suzann Duan, Paul D. Brewer, Tyler Van Ry, Grant W. Hennig, Thomas W. Gould, Andrés E. Dulcey, Amy Wang, Xin Xu, Catherine Z. Chen, Xin Hu, Wei Zheng, Noel Southall, Marc Ferrer, Juan Marugan, Dean J. Burkin  Molecular Therapy  Volume 25, Issue 6, Pages 1395-1407 (June 2017) DOI: 10.1016/j.ymthe.2017.03.022 Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 1 SU9516 Increases α7 Integrin in Myogenic Cell Lines through Inhibition of the SPAK/OSR1 Pathway (A and B) SU9516 shows an increase in β-galactosidase activity in (A) α7+/lacZ myoblasts and (B) α7+/lacZ myotubes over a wide range of concentrations. (C and D) Western blot analysis confirmed that treatment with 12 μM SU9516 increased the levels of α7B integrin after 48 hr in (C) C2C12 myotubes (n = 3, Cohen’s d = 4.75) and (D) telomerized human DMD patient myotubes over a wide range of concentrations (n = 3/concentration [conc.], η2 = 0.82). (E) STOCK1S-50699 showed an increase in β-galactosidase activity and obtained maximum fold fluorescence levels, nearly as high as SU9516 in α7+/lacZ-treated myotubes (n = 10/conc.). (F) Western blot analysis in patient human DMD myotubes treated with STOCK1S-50699 showed a 1.8-fold increase in α7B integrin levels (n = 3, Cohen’s d = 2.39). ***p < 0.001, **p < 0.01, *p < 0.05. Values are the mean ± SEM for replicates. Molecular Therapy 2017 25, 1395-1407DOI: (10.1016/j.ymthe.2017.03.022) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 2 SU9516 Expediates Differentiation in C2C12 Myoblasts C2C12 myoblasts were differentiated with 12 μM SU9516 or DMSO. After 72 hr, C2C12 myoblasts (cells were treated in three different chamber slides) were fixed and immunostained for MHC. (A) Myofibers stained at 72 hr (green, MHC; blue, DAPI). Magnification, 40×. Scale bar, 100 μm. (B) At 72 hr, myotube diameters were increased with SU9516 treatment versus DMSO (n = 45–47 myofibers counted per group). (C) SU9516-treated myogenic cells showed a greater fusion index, defined as the ratio of the number of nuclei in MHC-positive myotubes to the total number of nuclei in one field for five random microscopic fields (n = 35 myofibers evaluated per group). (D) SU9516 treatment increased the fraction of total myotubes with higher numbers of nuclei (n = 35 myofibers evaluated per group). The results represent means and SEM for myofibers treated and evaluated in three different chamber slides. (E) SU9516-treated C2C12 cells showed an early increase in α7B integrin with differentiation versus DMSO (n = 3 per time point, Cohen’s d < 0.8 at 24 hr, d = 5.15 at 48 hr, d = 3.78 at 72 hr). *p < 0.05, **p < 0.01, ***p < 0.001. Values are the mean ± SEM for replicates. Molecular Therapy 2017 25, 1395-1407DOI: (10.1016/j.ymthe.2017.03.022) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 3 SU9516 Increases α7β1 Integrin Levels in the Skeletal Muscle of mdx Mice (A–C) Western blot analysis performed in the diaphragm and the gastrocnemius muscle of 10-week-old mdx mice showed (A) an increase in the levels of α7B and β1D integrin in the diaphragm of the SU9516-treated mdx mice. This increase is quantified in (B), where an ∼2-fold increase in α7B (n = 4, Cohen’s d = 3.04) and (C) an ∼3.4-fold increase in β1D were observed in the diaphragm of SU9516-treated mdx mice (n = 4, Cohen’s d = 2.74). (D) Western blot analysis in the gastrocnemius showed an increase in the levels of α7B and β1D integrin in SU9516-treated mdx mice. (E and F) These increases were quantified in (E), where an ∼2-fold increase in α7B (n = 6, Cohen’s d = 2.80) and (F) an ∼1.7-fold increase in β1D (n = 4, Cohen’s d = 2.03) were observed in SU9516-treated mdx mice. (G and H) Immunofluorescence performed on 10-μm cryosections for α7B and β1D integrin in (G) the diaphragm and (H) the gastrocnemius muscle of mdx mice showed sarcolemmal localization. *p < 0.05, ***p < 0.001. Values are the mean ± SEM for replicates. Molecular Therapy 2017 25, 1395-1407DOI: (10.1016/j.ymthe.2017.03.022) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 4 SU9516 Improves In Vivo Outcome Measures and Diaphragm Muscle Function in mdx Mice All comparisons were made across WT, vehicle-treated mdx and SU9516-treated mdx mice. (A) SU9516-treated mice showed a smaller gain in body mass compared with vehicle-treated controls in weeks 9 and 10. WT animals showed the least increase in body weight over time (η2treatment = 0.07). (B) Weekly forelimb grip strength. SU9516 treated mdx mice showed greater muscle strength compared with vehicle-treated mdx mice at weeks 8 and 9 (η2treatment = 0.13). (C) At 10 weeks of age, mouse diaphragm muscle function was assessed. At a 100-Hz tetanic stimulus, SU9516-treated mdx mice showed greater isometric tetanic tension compared with vehicle-treated controls (η2 = 0.59). (D) A force-frequency protocol to measure tetanic tension generated by the diaphragm showed that SU9516-treated diaphragms produced higher tension compared with vehicle-treated diaphragms when stimulated between 100- to 150-Hz frequencies (η2treatment = 0.19). (E) Mdx diaphragms in both SU9516- and vehicle-treated groups fatigued to ∼80% of the initial force (η2 = 0.57). (F) SU9516 diaphragms recovered by ∼8% compared with vehicle (η2treatment = 0.66) 10 min after recovery from fatigue. Tukey post hoc test annotations: *p < 0.05, **p < 0.01, ***p < 0.01 for SU9516-mdx versus Vehicle-mdx treated mdx mice. #p < 0.05, ##p < 0.01, ### p < 0.001. #### p < 0.0001 vehicle-mdx versus WT. +p < 0.05, ++p < 0.01 for WT versus SU9516-mdx mice. Values are the mean ± SEM for replicates. Molecular Therapy 2017 25, 1395-1407DOI: (10.1016/j.ymthe.2017.03.022) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 5 SU9516 Promotes Myofiber Regeneration through Inhibition of the p65-NF-κB Pathway in mdx Skeletal Muscle (A) SU9516-treated mdx diaphragms showed a 5.5% increase in the percentage of centrally nucleated fibers over vehicle-treated diaphragms (n = 5/mdx treatment group, Cohen’s d = 1.72). (B) The percentage of eMHC-positive fibers in the diaphragm of SU9516-treated mdx was higher than vehicle-treated mdx by 3.3% (n = 5/mdx treatment group, Cohen’s d = 1.9). (C) eMHC staining in immunofluorescence images of diaphragms from vehicle- and SU9516-treated mdx mice. SU9516-treated diaphragms show an increase in eMHC-positive fibers. (D) SU9516 treatment decreases the level of p-p65-NF-κB by ∼2.83-fold in lysates of mdx diaphragm compared with vehicle-treated controls (n = 3/ mdx treatment group, Cohen’s d = 4.05). (E) Western blot analysis for total phosphorylated p65-NF-κB showed that SU9516 treatment decreases the level of p-p65-NF-κB by >3-fold in human DMD patient myotubes compared with the vehicle-treated counterparts (n = 3 treatments per vehicle or SU9516 group, Cohen’s d = 11.12). *p < 0.05, **p < 0.01. Values are the mean ± SEM for replicates. Molecular Therapy 2017 25, 1395-1407DOI: (10.1016/j.ymthe.2017.03.022) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 6 SU9516 Ameliorates Pathology in mdx Skeletal Muscle (A) Myofiber size distribution in diaphragms of WT and vehicle- and SU9516-treated mice was assessed utilizing 10-μm cryosections stained with wheat germ agglutinin, followed by minimum Ferret’s diameter measurements. The distribution of fiber size in SU9516-treated diaphragms shifted toward WT fiber size distribution; i.e., larger myofibers. (B) The percent of fibrotic area, as quantified by Sirius Red staining, showed a decrease in collagen-positive areas within the SU9516-treated mdx diaphragm cross-sections compared with vehicle-treated mdx mice (n = 3/mdx treatment group, Cohen’s d = 3.14, *p < 0.05, **p < 0.01). (C) Proposed model for the mechanism of action by which SU9516 ameliorates dystrophic pathology and enhances α7β1 integrin in dystrophic muscle fibers. Values are the mean ± SEM for replicates. Molecular Therapy 2017 25, 1395-1407DOI: (10.1016/j.ymthe.2017.03.022) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions