UC San Diego

Cullin-RING ligases (CRLs) are one of the main families of E3 ligases in the Ubiquitin-Proteasome degradation system, which is responsible for the degradation of ~80% of muscle proteins. CRL substrate adaptors have been established as players in muscle atrophy and nemaline myopathies. This led to us to investigate whether CRLs are important for muscle development. C2C12 muscle cells were treated with MLN4924, an inhibitor of CRL activation. Treatment at the start of myoblast differentiation showed a defect in myotube formation due to an absence of cell commitment to the differentiation program, indicating that CRLs are important for early myogenesis. We obtained similar results for satellite cells isolated from mouse skeletal muscles, confirming the absolute necessity of CRL activity for normal myogenesis. The inhibition of CRL activity also led

to a defect in acetylcholine receptor aggregation. Similarly, a receptor defect has also been observed in CRL-linked nemaline myopathies, again indicating a possible role for these E3-ligases in the disease etiology. Currently, MLN4924 is used as a cancer drug that is undergoing clinical trials. However, cancer patients typically experience cachexia, or muscle wasting, which would likely be further exacerbated by the use of MLN4924. We observed a rescue of the MLN4924 induced differentiation-blockage several days after removal of the inhibitor. We also found that treatment with 5’Aza’dC, a drug that stimulates Myogenin expression, alleviates the phenotype. Our data indicate significant roles for CRLs during muscle differentiation as well as negative side effects for the use of MLN4924 in patients.