MEK inhibition induces myogenic differentiation in RAS-driven rhabdomyosarcoma.

Fusion-negative rhabdomyosarcoma (FN-RMS), which lacks PAX3/7 gene rearrangement, arises from skeletal muscle precursor cells that fail to differentiate despite expression of the myogenic master transcription factor, MYOD1. These tumors frequently harbor mutations in RAS isoforms (NRAS, HRAS, or KRAS), but the role of RAS in blocking myogenic differentiation is incompletely understood. In this study, we used a combination of high-throughput drug screening, transcriptomics, and epigenomics approaches to investigate the role of RAS in FN-RMS differentiation and survival. Oncogenic RAS was required for FN-RMS survival and activated the MAPK pathway to block myoblast differentiation. Consistent with these findings, the MEK inhibitor, trametinib, selectively reduced FN-RMS cell viability; upregulated the prodifferentiation myogenic transcription factor, MYOG; and induced myogenic differentiation. Mechanistically, we found that ERK2, a downstream target of MEK, bound to myogenic differentiation genes, including the promoter of MYOG, where it phosphorylated RNA polymerase II, resulting in RNA polymerase II stalling and transcriptional repression. MEK inhibition resulted in release of ERK2 from the MYOG promoter, facilitating MYOG transcription. Accordingly, trametinib treatment also resulted in MYOG-dependent chromatin remodeling, leading to the establishment of super-enhancers at genes required for late myogenic differentiation (including MYH3) and the loss of RAS-dependent super-enhancers at proliferation genes, such as MYC. In vivo, MEK inhibition induced myogenic differentiation FN-RMS cells to suppress their growth as xenografts. We then performed a combinatorial drug screen and identified combinations that might improve the therapeutic efficacy of trametinib. Excitingly, the most synergistic combination in vitro, trametinib and the multikinase inhibitor, BMS-754807, also induced tumor regression in mouse xenograft models of FN- RMS. Synergy was similarly observed between trametinib and the IGF1R monoclonal antibody, ganitumab, establishing the combination of MEK and IGF1R inhibition as synergistic in FN-RMS. Therefore, in addition to uncovering a mechanism by which RAS signaling suppresses MYOG expression to block MYOG-dependent chromatin remodeling and cellular differentiation in FN-RMS, these findings suggest that patients with FN-RMS may benefit from combination therapy with MEK and IGF1R inhibitors. Citation Format: Marielle E. Yohe, Berkley E. Gryder, Hsien-Chao Chou, Young K. Song, Xiaohu Zhang, Donna Butcher, Kristine A. Isanogle, Christina M. Robinson, Xiaoling Luo, Jin-Qiu Chen, Elijah J. Edmondson, Simone Difilippantionio, Craig J. Thomas, Javed Khan. MEK inhibition induces myogenic differentiation in RAS-driven rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B17.