MYOD1L122R induces cancer stem cell pathways to impart chemotherapy and radiation-resistance in aggressive rhabdomyosarcoma [ChIP-seq]

Rhabdomyosarcoma (RMS) is a common pediatric soft tissue sarcoma of skeletal muscle. Worse outcomes are associated with specific subtypes of RMS, including the 10% of fusion-negative tumors that harbor point mutations in the DNA binding domain of MYOD1 (myogenic differentiation 1) transcription factor. Due to the rarity of these patient samples and dearth of cell lines/animal models, the molecular function of MYOD1L122R remains unknown. Here, we create the first animal model of this disease showing that MYOD1L122R is not oncogenic but collaborates with RAS activation to create highly aggressive RMS in transgenic zebrafish, akin to what is reported in human disease. Limiting dilution cell transplantation revealed that MYOD1L122R also elevated the overall frequency of tumor propagating cells in the zebrafish model. We next generated isogenic knock-in MYOD1L122R human cell line models and showed that these cells were refractory to standard of care vincristine, actinomycin, and cyclophosphamide while also elevating the overall frequency of tumor propagating cells. Indeed, single-cell RNA transcriptional profiling and single-cell assay for transposase-accessible chromatin with sequencing (ATAC-Seq) of patient and patient-derived xenografts confirmed the elevation of cancer stem cell programs within MYOD1L122R tumors. Using unbiased whole-genome ChIP and RNA sequencing, we next identified the shared and differential genomic binding regions by MYOD1L122R as compared to wildtype MYOD1. Most notably, MYOD1L122R bound uniquely to and transcriptionally upregulated mesenchymal pathway enriched genes that are known to correlate with RMS cancer stemness. Finally, mechanistic studies uncovered that MYOD1L122R binds to the regulatory element of ROR2 (receptor tyrosine kinase like orphan receptor 2), upregulating its expression, which then turns on a novel WNT11-ROR2-VANGL2 axis to induce non-canonical WNT signaling and elevate cancer stemness.