The role of BET proteins in the differential regulation of myogenesis: Implications for muscle-wasting disorders.

Loss of muscle mass is a common feature of a wide variety of diseases. For example, in the progressive muscle wasting condition Duchenne Muscular Dystrophy (DMD), the loss of the dystrophin gene renders muscle fibres sensitive to contractile damage. Damaged fibres are subsequently lost due to necrosis, and muscle stem cells compensate by generating new muscle. This leads to cycles of degeneration and regeneration and ultimately to functional exhaustion of the muscle stem cell pool. Fatality results as a consequence of the failure of the heart to support blood circulation, or the diaphragm to support breathing. As such, muscle regeneration can be considered an important component of DMD pathology, and strategies which promote regeneration may be able to slow the muscle-wasting observed in these patients. Similarly, promoting muscle regeneration may also be able to reverse the muscle-wasting pathologies associated with aging (sarcopenia), diseases such as cancer and AIDS (cachexia), and diabetes/obesity.

We have identified BET proteins as novel regulators of muscle growth and differentiation. BET proteins bind to the genome in order to coordinate gene expression. Of key importance is the observation that different members of the BET family exert opposite effects on muscle cell differentiation in cultured cells. Specifically, inhibition of the BET protein Brd4 blocks differentiation, whereas inhibition of Brd3 enhances differentiation. The proposed Programme of work aims to characterise these findings in detail. Specifically, we will investigate the mechanisms behind these phenomena, characterise the genomic binding sites of BET proteins, validate these findings in primary human and mouse cells, and investigate the role of BET proteins in in vivo transgenic and muscle injury model systems. This Programme of work has the potential to reveal fundamental new insights into muscle function with important implications for the treatment of muscle-wasting disorders.

To investigate the therapeutic potential of selective BET inhibition, we will test the possibility that disruption of Brd3 could be used as a means of promoting muscle growth using a mouse model of DMD. Given the intense interest in BET proteins in the cancer field, there are a number of highly potent small molecule BET inhibitors available to researchers. However, an important limitation of these compounds is their inability to distinguish between different BET family members (e.g. Brd3 and Brd4, which we have shown have opposite effects on myogenesis). We therefore propose the use of antisense oligonucleotides to selectively target Brd3 for downregulation via one of two mechanisms. Firstly, we will use a splice corruption approach which forces the target Brd3 messenger RNA to be incorrectly processed. We have extensive experience using state-of-the art antisense technology (i.e. peptide-morpholino conjugates) which can be effectively delivered to dystrophic mouse muscle in vivo to modulate gene expression in this manner. Secondly, we will use a conventional target degradation approach (i.e. gapmer oligonucleotides) to directly reduce the levels of Brd3 messenger RNA in mouse muscle. The use of oligonucleotides allows for highly specific targeting of a single BET family member (i.e. Brd3) without off-target inhibition of the other family members (i.e. Brd4 and Brd2) - which is currently not possible with existing small molecule inhibitors. The overall aim of the Programme is therefore to harness new discoveries in basic muscle biology in order to facilitate the development of novel therapeutics and improve the lives of patients with muscle-wasting diseases.

The function of BET proteins has so far not been studied in the context of muscle differentiation, although they have been intensely studies in recent years in the context of cancer. BET proteins are ideal targets for small molecule inhibitors as their internal bormodomain motifs consist of hydrophobic pockets which readily accommodate drug molecules in order to directly disrupt their chromatin binding activity. However, the bromodomain motifs are highly conserved between the BET proteins and therefore small molecules BET inhibitors cannot discriminate between family members. As a result, it is likely that key features of BET protein function have been overlooked. Using these same small molecules inhibitors, we have identified BET proteins as important regulators of myoblast proliferation and differentiation. Using RNAi, we have discovered differential roles for BET proteins Brd3 and Brd4 in the regulation of myogenic differentiation in culture. This finding points to a previously unappreciated facet of fundamental muscle biology, and provides a rationale for therapeutic targeting of Brd3 in order to promote muscle growth in the case of muscle-wasting disorders, or for inducing differentiation in rhabdomyosarcoma cells in order to slow cancer progression. The proposed Programme of work will further characterise BET gene function using inducible knockdown and overexpression systems, primary human and mouse cultures, ChIP-seq using anti-BET antibodies, and BET protein-complex composition/post-translational modification by TiO2 phosphoproteomics. Additionally, we will use genetic conditional knockout models to ablate BET expression in satellite cells and challenge muscle with notexin injection to determine the role of different BET proteins in the regenerative response. Lastly, we will explore oligonucleotide approaches to disrupt Brd3 expression in order to promote muscle growth as a therapeutic strategy for treating muscle-wasting disorders/rhabdomyosarcoma.

We expect the proposed research will have a major impact on our fundamental understanding the molecular control of myogenesis. This will be of wide interest to those studying muscle biology, transcriptional regulation, and BET gene function in the context of muscle- and non-muscle-associated diseases (e.g. cancer). Our hope is that the knowledge gained from the proposed work will contribute to the development of new therapies for muscle-wasting disorders and rhabdomyosarcoma, and thereby improve health outcomes for affected patients. The Programme is also expected to generate material of industrial/commercial value, and have an impact on the next generation of scientists working in neuromuscular research, policy makers, and patient-based organisations.

The proposed Programme will also have a significant industrial / commercial impact. Numerous companies are currently developing novel therapies for inherited muscle wasting conditions such as DMD and related diseases (e.g. BioMarin, Sarepta Therapeutics, Pfizer, Shire, Isis Pharmaceuticals and Summit Plc). We will work closely with industry as the Programme develops to capitalise on the applications of targeting BET proteins for muscle-wasting disorders. In doing so we will build on the relationships and links which have been established over the course of the last 5 years, working with small biotechnology companies e.g. Sarepta Therapeutics and larger industry partners e.g. Pfizer to advance our fundamental science and ensure that our knowledge, know-how and relevant intellectual property are exploited to achieve the desired impact. All intellectual property developed during the course of the Programme is managed by Isis Innovation, the technology transfer company of the University of Oxford, who will assess, protect and commercialise the IP generated from the project before engaging with potential industrial end-users.

A significant impact of the present Project will be in the training and development of scientific researchers. The staff working on the grant will be trained not only in the molecular, cellular, bioinformatics and neuromuscular techniques associated with the research Project but also in the rigour of reporting required for the development of new methods, new technologies and ultimately new medicines for treating human disease. Scientific staff will receive training in relation to conference presentations, interacting with industry and receive numerous opportunities to develop skills in public engagement. This will lead to highly effective communications skills and also to employment prospects not only in academia but also in the biotechnology and large pharma sectors .

Finally, perhaps the most important set of beneficiaries who will be impacted by the outcomes of our work are DMD and muscle-wasting disease patients themselves, their families and the many associated advocacy groups that work tirelessly and campaign strongly for effective therapies to treat these diseases. All will be interested to understand the potential of targeting BET proteins as a novel approach for stimulating muscle growth and differentiation applicable to treating these severe diseases and how and over what timescale these are likely to confer benefit.