Abstract A027: Rescuing MYMK/MYMX-Dependent Cell Fusion to Promote Myogenic Differentiation in Rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS) arises from a skeletal muscle lineage but fails to complete terminal myogenic differentiation, including cell–cell fusion. Productive cell fusion during skeletal muscle formation requires the coordinated activity of the fusogenic membrane proteins myomaker (MYMK) and myomixer (MYMX). We investigated whether RMS cells are arrested in differentation at a step close to myogenic cell fusion and that restoring balanced MYMK/MYMX expression would rescue cell–cell fusion, promote myogenic differentiation, and reduce tumorigenic behavior. We first assessed fusogen expression in fusion-negative (FN) RD and fusion-positive (FP) Rh30 RMS cells, using normal myoblasts as controls. Western blotting and qPCR showed reciprocal fusogen imbalance where Rh30 exhibited a relatively MYMK-low/MYMX-high signature, whereas RD cells were MYMK-high/MYMX-low. This pattern showed little correction after differentiation, unlike myoblast controls, which induced both fusogens during myotube formation. To confirm the observation of muscle fusogen imbalance in RMS cells, we interrogated 460 publicly available RNA-sequencing data sets. Bulk RNA sequencing showed that FN & FP RMS tumors variably but consistently expressed MYMK, MYMX, MYOD1, and MYOG. We next used single-cell data to assess whether fusogen expression was coordinated within individual cells. Compared with normal developing muscle, differentiated RMS cells were less often positive for both MYMK and MYMX at the same time. MYMK/MYMX double-positive cells represented 20.7% of differentiated RMS cells, compared with 46.6% of human developmental muscle cells and 65.4% of mouse developmental muscle cells. Thus, our data indicate that RMS cells express the core myogenic and fusogenic machinery but inefficiently achieve the paired MYMK/MYMX state needed for cell–cell fusion. We then tested whether restoring fusogen pairing could overcome the block for cell fusion in RMS cells. We generated fusogen rescue models expressing MYMK, MYMX, or MYMK/MYMX. In Rh30 cells, MYMK or MYMK/MYMX overexpression was associated with enlarged multinucleated cells that frequently stained strongly for MyHC. In RD cells, MYMK/MYMX overexpression—but not MYMX alone—produced MyHC-positive multinucleated structures with a more elongated, myotube-like morphology. Furthermore, overexpresion of MYMK or MYMK/MYMX in RD and Rh30 cells was associated with reduced confluency and transiently increased cytotoxicity. Together, these data support a model in which RMS cells fail to fuse because of an imbalance in the expression of MYMK and MYMX during differentiation. Restoring a MYMK-inclusive fusogen state promotes MyHC-positive multinucleation in both FN- and FP-RMS models, providing functional evidence that the terminal fusion step is an actionable bottleneck in RMS differentiation. Ongoing cytoplasmic mixing assays, cell-cycle profiling, and xenograft studies will determine whether these structures represent productive fusion, durable differentiation, and reduced tumorigenic potential.