Abstract PR012: MYOD1L122R induces chemoresistance and elevates cancer stem cell programs through WNT11-ROR2-VANGL2 signaling in aggressive rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood or adolescents. The spindle sclerosing (ss-RMS) subtype has the worst outcome of all RMS patients and often harbor recurrent mutation of Leucine 122 to Arginine within the DNA binding domain of the transcription factor MYOD1. To date, the molecular function of MYOD1L122Rremains largely unknown. Methods: Here, we create the first animal model of MYOD1L122R using transgenic zebrafish. We also used CRISPR-homology-directed-repair and dox-inducible systems to express MYOD1L122R in human RMS cells. These models, along with patient tumors and patient-derived xenografts, were assessed by RNA- and ChIP-sequencing, single-cell RNA and ATAC (assay for transposase-accessible chromatin) sequencing, and immunohistochemistry to identify direct targets and downstream pathways regulated by MYOD1L122R. We also discovered that MYOD1L122R activates non-canonical WNT signaling to elevate cancer stemness and to impart chemo- and radiation-resistance through molecular mechanism studies. Results: Utilizing transgenic zebrafish models, we uncovered that MYOD1L122R is not oncogenic by itself but requires RAS pathway activation to generate rhabdomyosarcoma, akin to known gene associations found in ss-RMS patients. Limiting dilution cell transplantation revealed that MYOD1L122R increased the numbers of tumor propagating cells in zebrafish tumors. We next showed that engineered MYOD1L122R human tumors also have increased numbers of TPCs and were refractory to standard of care VAC (vincristine, actinomycin, and cyclophosphamide) and gamma-irradiation. Indeed, scRNA- and scATAC-seq of patient and patient-derived xenografts confirmed the elevation of cancer stem cell programs within MYOD1L122R tumors. We next identified the shared and differential genomic regions bound by MYOD1L122R using whole-genome ChIP and RNA sequencing. Most notably, MYOD1L122R bound to new DNA binding motifs shared with the MYC oncoprotein and transcriptionally upregulated cancer stem cell programs through distal enhancers. Unexpectedly, MYOD1L122R also bound to shared genomic regions with normal MYOD1 at transcription start sites/promoters to regulate muscle specific gene expression. Mechanistic studies revealed that MYOD1L122R binds to regulatory elements of ROR2 (receptor tyrosine kinase like orphan receptor 2), activating its expression and turning on the non-canonical WNT11-ROR2-VANGL2 pathway to elevate cancer stemness and to drive chemo- and radiation- resistance. Conclusions: MYOD1L122R is an RMS modifying gene that elevates cancer stemness and promotes chemo- and radiation- resistance through epigenetic reprogramming and activation of non-canonical WNT signaling pathway. The discovery of ROR2 in MYOD1L122R RMS patients grants future evaluation of this protein as a therapeutic target in combination with current standard of care. Citation Format: Yun Wei, Luis Antonio Corchete Sanchez, Diego Antelo, Sabateeshan Mathavarajah, Shuze Wang, Alexander Daiki Weissman, Jihee Lee, Miguel N. Rivera, Esther Rheinbay, David M. Langenau. MYOD1L122R induces chemoresistance and elevates cancer stem cell programs through WNT11-ROR2-VANGL2 signaling in aggressive rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr PR012.