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

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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.