Dasatinib as a treatment for Duchenne muscular dystrophy

Human Molecular Genetics, 2016, Vol. 25, No. 2 266–274 doi: 10.1093/hmg/ddv469 Advance Access Publication Date: 24 November 2015 Original Article ORIGINAL ARTICLE Dasatinib as a treatment for Duchenne muscular dystrophy Leanne Lipscomb, Robert W. Piggott, Tracy Emmerson and Steve J. Winder* *To whom correspondence should be addressed. Tel: +44 1142222332; Email: s.winder@sheffield.ac.uk Abstract Identification of a systemically acting and universal small molecule therapy for Duchenne muscular dystrophy would be an enormous advance for this condition. Based on evidence gained from studies on mouse genetic models, we have identified tyrosine phosphorylation and degradation of β-dystroglycan as a key event in the aetiology of Duchenne muscular dystrophy. Thus, preventing tyrosine phosphorylation and degradation of β-dystroglycan presents itself as a potential therapeutic strategy. Using the dystrophic sapje zebrafish, we have investigated the use of tyrosine kinase and other inhibitors to treat the dystrophic symptoms in this model of Duchenne muscular dystrophy. Dasatinib, a potent and specific Src tyrosine kinase inhibitor, was found to decrease the levels of β-dystroglycan phosphorylation on tyrosine and to increase the relative levels of nonphosphorylated β-dystroglycan in sapje zebrafish. Furthermore, dasatinib treatment resulted in the improved physical appearance of the sapje zebrafish musculature and increased swimming ability as measured by both duration and distance of swimming of dasatinib-treated fish compared with control animals. These data suggest great promise for pharmacological agents that prevent the phosphorylation of β-dystroglycan on tyrosine and subsequent steps in the degradation pathway as therapeutic targets for the treatment of Duchenne muscular dystrophy. Introduction The zebrafish Danio rerio has rapidly been adopted as an organism of choice for all aspects of the drug discovery pipeline (1–3). The zebrafish system offers unique advantages for drug screening in a vertebrate model organism, and in particular, muscular dystrophies are especially amenable due to their early, robust and readily recognizable phenotypes (4,5). The small size, embryonic status, low cost and ease of drug delivery directly via the water, makes zebrafish a very attractive model for whole-organism screening. Zebrafish show a typical vertebrate development pattern, and in the mutants, perturbation of muscle architecture and muscle function is readily observable even in the embryonic stages (4–6). In addition, of the genes known to be mutated in human forms of muscular dystrophy, many are represented in the zebrafish genome and those investigated so far exhibit dystrophic phenotypes in zebrafish (7,8). Although candidate compounds identified in fish would need to be validated in mammals before being taken on to human therapy, the low cost and speed of candidate drug screening, far outweigh any disadvantages. Recent unbiased screens for DMD therapeutics have also validated this approach and identified a number of compounds that appear effective in reducing dystrophic symptoms in zebrafish (9,10). In particular, the identification of PDE5 inhibitors appears to be useful in this regard as they have also been shown to be effective in mdx mice (11,12). Previous studies from the Lisanti group and ourselves suggested that tyrosine phosphorylation of dystroglycan is an important mechanism for controlling the association of dystroglycan with its cellular binding partners, dystrophin and utrophin, and also as a signal for degradation of dystroglycan (13–15). The Lisanti group further demonstrated that inhibition of the Received: September 22, 2015. Revised and Accepted: November 9, 2015 © The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 266 Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK Human Molecular Genetics, 2016, Vol. 25, No. 2 Figure 1. Levels of β-dystroglycan and β-dystroglycan phosphorylated on tyrosine in sapje and sibling larvae. Western blots of lysates of individual 3, 4 and 5 dpf Results Homozygous sapje zebrafish show a progressive loss of muscle organization visible from 3 days post-fertilization (dpf ) onwards (6,20). Concomitant with the loss of muscle organization, as observed by birefringence or fluorescence in whole embryos, is a progressive loss of immunoreactivity from the myosepta of other DGC components such as dystroglycan, compared with siblings (Supplementary Material, Fig. S1). The loss of other DGC components in the absence of dystrophin is common with other models of Duchenne muscular dystrophy (DMD) such as the mdx mouse (21), and in people with DMD (22). In order to more reliably quantify the extent of dystroglycan loss in sapje embryos, we performed quantitative western blotting of sapje and sibling larvae at 3, 4 and 5 dpf and examined the levels of β-dystroglycan, and β-dystroglycan phosphorylated on tyrosine, normalized to tubulin levels. As can be seen in Figure 1A, and in keeping with the immunofluorescence (IF) results in Supplementary Material, Figure S1, there is a progressive and significant loss of β-dystroglycan from 3 to 5 days in sapje larvae relative to siblings. In contrast to non-phosphorylated dystroglycan, tyrosine-phosphorylated β-dystroglycan does not decline until Day 5 (Fig. 1B and C). Therefore, there is a loss of non-phosphorylated β-dystroglycan that may contribute initially to the levels of tyrosine phosphorylated β-dystroglycan, but by 5 dpf, both nonphosphorylated and phosphorylated dystroglycan are significantly reduced. Also noticeable in Figure 1A, upper panels, is the appearance of higher molecular weight bands of tyrosine phosphorylated β-dystroglycan with a mass of ∼53 and 63 kDa, equivalent to 10 or 20 kDa heavier than the main 43 kDa β-dystroglycan band. Thus, the absence of dystrophin in sapje fish leads to a decrease in 43 kDa β-dystroglycan, with the concomitant appearance of slower migrating phosphorylated β-dystroglycan species. These data suggest a mechanism whereby in the absence of dystrophin, β-dystroglycan is more prone to phosphorylation on tyrosine; this results in a relative decrease in un-phosphorylated β-dystroglycan levels, and a concomitant sibling and sapje larvae western blotted with antibodies against phosphorylated β-dystroglycan [ p-β-DG, (A) top], non-phosphorylated β-dystroglycan [β-DG, (A) middle] and α-tubulin was used as a loading control [α-tub, (A) bottom]. Numbers represent relative position of molecular weight markers in kDa. (B and C) The integrated density of the blots probed against β-DG and p-β-DG shown in (A), quantified relative to α-tubulin levels in each sample. (...truncated)