Abstract A35: A recurrent neomorphic mutation in MYOD1 defines a clinically aggressive subset of embryonal rhabdomyosarcoma
Authors: Shukla Neerav , Kohsaka Shinji , Ameur Nabahet , Yannes Angela , Viale Agnes , Qin Li-Xuan , Dogan Snjezana , Sciot Raf , Bridge Julia , Meyers Paul , Wexler Leonard , Fletcher Jonathan , Ladanyi Marc 
Published: May 2015

Abstract
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma diagnosed in children. To identify novel and recurrent oncogenic mutations in RMS, we performed whole exome sequencing on 20 RMS samples, including 9 alveolar RMS, and 11 embryonal RMS (ERMS). Two ERMS samples showed the same c.365T&gt;G point mutation in MYOD1, leading to an L122R substitution. Both were primary tumor samples and, in each case, normal tissue did not harbor the mutation, confirming its somatic nature. The L122R mutation occurs in the conserved basic region of MYOD1, at a highly specific residue that is a leucine in all myogenic bHLH transcription factors (MYOD1, MYF5, myogenin, MRF4) but is an arginine in MYC. Interestingly, previous studies of artificial mutants of the MYOD1 basic region found that MYOD1 L122R competes wild-type MYOD1 but without leading to target gene transactivation and also binds to MYC consensus sequences, regulating target genes similarly to MYC.
We genotyped an additional 97 ERMS samples for the MYOD1 L122R point mutation using a mass spectrometry-based platform. The mutation was detected in 8 additional cases, resulting in an overall prevalence of 10% (10/103) in our ERMS samples. Corresponding normal tissue was available in 6 of the 8 additional cases, and, again, no mutations were identified in germline DNA. The 10 ERMS tumors harboring the MYOD1 L122R mutation displayed highly cellularity and frequent spindle cell morphology with uniform strong positivity for MYOD1 by IHC, features associated with the adult spindle cell variant of ERMS. Notably, these ERMS with MYOD1 mutations were from patients diagnosed in later childhood or adulthood (mean age = 25; median age = 28), and were more likely to arise in the head/neck (8/10 vs 16/80; p=0.0003). Furthermore, the overall survival of this subgroup of patients was significantly poorer than for ERMS lacking this mutation (0% vs 48% at 10 yrs; p=0.02).
Transfection of MYOD1 L122R or wild type MYOD1 into C2C12 mouse myoblasts showed that MYOD1 L122R favors proliferation and transformation over myogenic differentiation. In a previous report, we had identified activating PIK3CA mutations in 3 of 60 ERMS samples. Remarkably, all 3 of these PIK3CA-mutated tumor samples are among the 10 MYOD1 L122R cases identified here. Therefore, we examined the combined effect of PIK3CA H1047R and MYOD1 L122R in soft-agar colony formation assays in C2C12 cells; this resulted in an increased number of colonies compared to either PIK3CA H1047R alone or PIK3CA H1047R + wild type MYOD1. Finally, expression profiling experiments in C2C12 cells and in BJ human fibroblasts comparing MYOD1 L122R, wild type MYOD1, and MYC showed that MYOD1 L122R shifts gene expression from a myogenic program to a MYC-like program.
In summary, we have identified a distinctive subset of ERMS with recurrent somatic mutations in MYOD1, a transcription factor that functions as a critical regulator of skeletal muscle differentiation, altering its function resulting in impaired myogenic differentiation and enhanced proliferation. The tumors tend to present in older patients, usually arise in the head/neck, overlap pathologically with the recently described spindle cell variant of ERMS, and have especially poor outcomes. This represents a novel, molecularly defined subset of RMS that should be considered for high risk protocols and targeted therapeutic development.
Citation Format: Neerav Shukla, Shinji Kohsaka, Nabahet Ameur, Angela Yannes, Agnes Viale, Li-Xuan Qin, Snjezana Dogan, Raf Sciot, Julia Bridge, Paul Meyers, Leonard Wexler, Jonathan Fletcher, Marc Ladanyi. A recurrent neomorphic mutation in MYOD1 defines a clinically aggressive subset of embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A35.