PAX-fusion negative rhabdomyosarcoma (RMS) arises from skeletal muscle precursors that have failed to differentiate normally despite the expression of the myogenic master transcription factor, MYOD1. The cure rate for relapsed or refractory fusion negative RMS is poor despite aggressive multi-modality treatment. Novel treatment approaches such as the use of targeted therapies including those that induce skeletal muscle differentiation might improve overall survival for patients with fusion negative RMS. Genetic studies have shown that the most common single nucleotide variant in fusion negative RMS is an oncogenic change in one of the RAS isoforms, namely NRAS, HRAS or KRAS. In this study, we hypothesized that targeting aberrant RAS activity releases the differentiation block in fusion negative RMS and sought to unravel the underlying epigenetic mechanisms through which RAS signaling drives oncogenic transcription in RMS.

To achieve this goal, we combined high-throughput drug screening with biochemical, RNAseq and ChIPseq assays across a panel of RMS cell lines driven by oncogenic RAS mutations. Critically, we demonstrated that expression of oncogenic RAS was necessary for survival of these RAS-mutated RMS cells. In addition, overexpression of mutant RAS isoforms in C2C12 myoblasts inhibited myogenic differentiation induced by low-serum conditions. This differentiation block was mediated primarily by engagement of the RAF-MEK-ERK MAP kinase pathway. In corroboration with these observations, an unbiased screen of the ability of small molecules to impact cell viability demonstrated that inhibitors of the MAP kinase pathway were the most potently selective class of molecules for RAS-mutated RMS. In particular, trametinib, an allosteric, non-ATP competitive inhibitor of MEK1/2, was the most consistently potent MEK inhibitor in RAS-mutated RMS cell lines. Trametinib treatment induced G1 arrest and skeletal muscle differentiation in RAS-mutated RMS cell lines. Trametinib also slowed tumor growth and prolonged survival in xenograft models of RAS-mutated RMS.

To determine the mechanism by which MEK inhibition induced skeletal muscle differentiation in RAS-mutated RMS, we analyzed changes in gene expression, transcription factor deposition and histone modification in RMS cells treated with trametinib. Trametinib treatment increased expression of myogenic transcription factors, such as MYOG and MEF2C, and decreased expression of transcription factors important for proliferation, such as MYC and ID3, in RAS-mutated RMS cells. ChIPseq experiments demonstrated that this transcriptional reprogramming was driven in part by changes in the active enhancer landscape, since H3K27ac deposition at MYH3, TTNT2 and other muscle-specific loci increased with trametinib treatment. Both MYC and MYOD1 bound the active enhancers induced by trametinib treatment in RAS-mutated RMS, despite an overall decrease in MYC expression. Finally, we found significant ERK2 deposition on the MYOG promoter in the untreated cells. ERK2 is known to recruit the Polycomb repressive machinery at developmental loci in embryonic stem cells and therefore aberrant ERK2 activity may facilitate repression of MYOG expression in RAS-mutated RMS.

In summary, our data support a model of RAS-driven RMS in which aberrant ERK activity drives tumor cell proliferation, in part through increased expression and stability of MYC, and prevents myogenic differentiation, in this case through alterations in the enhancer landscape and interactions with the Polycomb repressive machinery. Future work is aimed at identifying rational combinations of trametinib and direct epigenetic modulators that synergistically drive RAS-mutated RMS differentiation with the goal of providing measurable clinical benefit in relapsed or refractory RAS-mutated RMS.

Citation Format: Marielle E. Yohe, Berkley E. Gryder, Jack F. Shern, Young K. Song, Hongling Liao, Hsein-Chao Chou, Sivasish Sindiri, Arnulfo Mendoza, Xiaohu Zhang, Rajarashi Guha, Diana C. Haines, James P. Madigan, Jun S. Wei, Marc Ferrer, Craig J. Thomas, Javed Khan. Reprogramming RAS-driven rhabdomyosarcoma via MEK inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A25.