Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

A recurrent neomorphic mutation in MYOD1 defines a clinically aggressive subset of embryonal rhabdomyosarcoma associated with PI3K-AKT pathway mutations

Abstract

Rhabdomyosarcoma, a cancer of skeletal muscle lineage, is the most common soft-tissue sarcoma in children1. Major subtypes of rhabdomyosarcoma include alveolar (ARMS) and embryonal (ERMS) tumors2,3. Whereas ARMS tumors typically contain translocations generating PAX3-FOXO1 or PAX7-FOXO1 fusions that block terminal myogenic differentiation4,5,6, no functionally comparable genetic event has been found in ERMS tumors. Here we report the discovery, through whole-exome sequencing, of a recurrent somatic mutation encoding p.Leu122Arg in the myogenic transcription factor MYOD1 in a distinct subset of ERMS tumors with poor outcomes that also often contain mutations altering PI3K-AKT pathway components. Previous mutagenesis studies had shown that MYOD1 with a p.Leu122Arg substitution can block wild-type MYOD1 function and bind to MYC consensus sequences7, suggesting a possible switch from differentiation to proliferation. Our functional data now confirm this prediction. Thus, MYOD1 p.Leu122Arg defines a subset of rhabdomyosarcomas eligible for high-risk protocols and the development of targeted therapeutics.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Prevalence of MYOD1 p.Leu122Arg in rhabdomyosarcoma.
Figure 2: Distinctive clinicopathological features and outcome of ERMS with MYOD1 p.Leu122Arg.
Figure 3: Effects of MYOD1 Leu122Arg on cell growth and myogenic differentiation in vitro.
Figure 4: Effects of concurrent expression of PIK3CA His1047Arg on MYOD1 Leu122Arg–induced phenotypes in vitro and in vivo.
Figure 5: Aberrant transcriptional effects of MYOD1 Leu122Arg.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

Sequence Read Archive

References

  1. Malempati, S. & Hawkins, D.S. Rhabdomyosarcoma: review of the Children's Oncology Group (COG) Soft-Tissue Sarcoma Committee experience and rationale for current COG studies. Pediatr. Blood Cancer 59, 5–10 (2012).

    Article  Google Scholar 

  2. Xia, S.J., Pressey, J.G. & Barr, F.G. Molecular pathogenesis of rhabdomyosarcoma. Cancer Biol. Ther. 1, 97–104 (2002).

    Article  CAS  Google Scholar 

  3. Breneman, J.C. et al. Prognostic factors and clinical outcomes in children and adolescents with metastatic rhabdomyosarcoma—a report from the Intergroup Rhabdomyosarcoma Study IV. J. Clin. Oncol. 21, 78–84 (2003).

    Article  Google Scholar 

  4. Davis, R.J. et al. Fusion of PAX7 to FKHR by the variant t(1;13)(p36;q14) translocation in alveolar rhabdomyosarcoma. Cancer Res. 54, 2869–2872 (1994).

    CAS  PubMed  Google Scholar 

  5. Galili, N. et al. Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nat. Genet. 5, 230–235 (1993).

    Article  CAS  Google Scholar 

  6. Barr, F.G. et al. Rearrangement of the PAX3 paired box gene in the paediatric solid tumour alveolar rhabdomyosarcoma. Nat. Genet. 3, 113–117 (1993).

    Article  CAS  Google Scholar 

  7. Van Antwerp, M.E. et al. A point mutation in the MyoD basic domain imparts c-Myc-like properties. Proc. Natl. Acad. Sci. USA 89, 9010–9014 (1992).

    Article  CAS  Google Scholar 

  8. Shukla, N. et al. Oncogene mutation profiling of pediatric solid tumors reveals significant subsets of embryonal rhabdomyosarcoma and neuroblastoma with mutated genes in growth signaling pathways. Clin. Cancer Res. 18, 748–757 (2012).

    Article  CAS  Google Scholar 

  9. Taylor, J.G. et al. Identification of FGFR4-activating mutations in human rhabdomyosarcomas that promote metastasis in xenotransplanted models. J. Clin. Invest. 119, 3395–3407 (2009).

    CAS  PubMed  Google Scholar 

  10. Ledent, V., Paquet, O. & Vervoort, M. Phylogenetic analysis of the human basic helix-loop-helix proteins. Genome Biol. 3, RESEARCH0030 (2002).

    Article  Google Scholar 

  11. Nascimento, A.F. & Fletcher, C.D. Spindle cell rhabdomyosarcoma in adults. Am. J. Surg. Pathol. 29, 1106–1113 (2005).

    PubMed  Google Scholar 

  12. Carroll, S.J. & Nodit, L. Spindle cell rhabdomyosarcoma: a brief diagnostic review and differential diagnosis. Arch. Pathol. Lab. Med. 137, 1155–1158 (2013).

    Article  Google Scholar 

  13. Crescenzi, M., Crouch, D.H. & Tato, F. Transformation by myc prevents fusion but not biochemical differentiation of C2C12 myoblasts: mechanisms of phenotypic correction in mixed culture with normal cells. J. Cell Biol. 125, 1137–1145 (1994).

    Article  CAS  Google Scholar 

  14. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).

    Article  CAS  Google Scholar 

  15. Asp, P. et al. Genome-wide remodeling of the epigenetic landscape during myogenic differentiation. Proc. Natl. Acad. Sci. USA 108, E149–E158 (2011).

    Article  Google Scholar 

  16. Blum, R. et al. Genome-wide identification of enhancers in skeletal muscle: the role of MyoD1. Genes Dev. 26, 2763–2779 (2012).

    Article  CAS  Google Scholar 

  17. Chen, X. et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133, 1106–1117 (2008).

    Article  CAS  Google Scholar 

  18. Davicioni, E. et al. Molecular classification of rhabdomyosarcoma—genotypic and phenotypic determinants of diagnosis: a report from the Children's Oncology Group. Am. J. Pathol. 174, 550–564 (2009).

    Article  CAS  Google Scholar 

  19. Scrable, H.J. et al. Rhabdomyosarcoma-associated locus and MYOD1 are syntenic but separate loci on the short arm of human chromosome 11. Proc. Natl. Acad. Sci. USA 87, 2182–2186 (1990).

    Article  CAS  Google Scholar 

  20. Stratton, M.R. et al. Detection of point mutations in N-ras and K-ras genes of human embryonal rhabdomyosarcomas using oligonucleotide probes and the polymerase chain reaction. Cancer Res. 49, 6324–6327 (1989).

    CAS  PubMed  Google Scholar 

  21. Calhabeu, F. et al. Alveolar rhabdomyosarcoma-associated proteins PAX3/FOXO1A and PAX7/FOXO1A suppress the transcriptional activity of MyoD-target genes in muscle stem cells. Oncogene 32, 651–662 (2013).

    Article  CAS  Google Scholar 

  22. Cao, L. et al. Genome-wide identification of PAX3-FKHR binding sites in rhabdomyosarcoma reveals candidate target genes important for development and cancer. Cancer Res. 70, 6497–6508 (2010).

    Article  CAS  Google Scholar 

  23. Ahn, E.H. et al. Identification of target genes of PAX3-FOXO1 in alveolar rhabdomyosarcoma. Oncol. Rep. 30, 968–978 (2013).

    Article  CAS  Google Scholar 

  24. Davis, R.L., Weintraub, H. & Lassar, A.B. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51, 987–1000 (1987).

    Article  CAS  Google Scholar 

  25. Anand, G. et al. Rhabdomyosarcomas do not contain mutations in the DNA binding domains of myogenic transcription factors. J. Clin. Invest. 93, 5–9 (1994).

    Article  CAS  Google Scholar 

  26. Zhu, S. et al. Activated ALK collaborates with MYCN in neuroblastoma pathogenesis. Cancer Cell 21, 362–373 (2012).

    Article  CAS  Google Scholar 

  27. Berry, T. et al. The ALKF1174L mutation potentiates the oncogenic activity of MYCN in neuroblastoma. Cancer Cell 22, 117–130 (2012).

    Article  CAS  Google Scholar 

  28. Akagi, T., Sasai, K. & Hanafusa, H. Refractory nature of normal human diploid fibroblasts with respect to oncogene-mediated transformation. Proc. Natl. Acad. Sci. USA 100, 13567–13572 (2003).

    Article  CAS  Google Scholar 

  29. Irizarry, R.A. et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249–264 (2003).

    Article  Google Scholar 

  30. Gentleman, R.C. et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5, R80 (2004).

    Article  Google Scholar 

  31. Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010).

    Article  Google Scholar 

  32. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    Article  Google Scholar 

  33. Ross-Innes, C.S. et al. Differential oestrogen receptor binding is associated with clinical outcome in breast cancer. Nature 481, 389–393 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank H. Hosoi (Kyoto Prefectural University of Medicine) and S. Tanaka and M. Tsuda (both at Hokkaido University) for providing cell lines. We also thank T. Akagi and K. Sasai (both at KAN Research Institute, Inc.) for providing the pcx4bleo plasmid. We are also grateful to J. Zhao for technical assistance with microarray analysis, to L. Borsu for assistance with Sequenom analyses and to E. de Stanchina and R. Tieu for the xenograft studies. This work was supported by a generous donation from M.B. Zuckerman (M.L.), by NIH P01 CA047179 (S.S.), by NCI P50 CA140146 (M.L., N.D.S. and S.S.), by a National Center for Research Resources (NCRR) Clinical Translational Science Center grant (M.P. and N.D.S.) and by the Virginia and Daniel K. Ludwig Trust for Cancer Research (J.A.F.). S.K. was supported in part by the Yasuda Medical Foundation and the HIROKO International Academic Exchange Foundation. F.G.B. is supported by the Intramural Research Program of the National Cancer Institute. The Memorial Sloan Kettering Cancer Center Sequenom facility was supported by the Anbinder Fund, and the Genomics and Bioinformatics Cores are supported by Cancer Center Core grant NCI P30 CA008748.

Author information

Authors and Affiliations

Authors

Contributions

S.K. performed myogenic differentiation studies, individual ChIP assays and in vivo studies. N.S. collected and analyzed clinical data and performed Sequenom genotyping studies with assistance from D.L. and A.M. N.A. obtained whole-exome and whole-transcriptome sequencing data with assistance from A.V., and M.P. and N.D.S. processed and analyzed the data. S.K. obtained expression microarray data with assistance from A.V., and L.-X.Q. analyzed the data. T.I. generated ChIP-seq data with assistance from A.V., and the data were processed by M.P. and N.D.S. and analyzed by C.K.Y.N. and J.S.R.-F. L.W. generated and interpreted the Affymetrix OncoScan array data. R.S., J.B., S.S., P.M., L.H.W., F.G.B., S.D. and J.A.F. provided rhabdomyosarcoma samples or cell lines, including clinical or pathological data. S.K., N.S. and M.L. drafted, edited and wrote the manuscript. M.L. led the project and manuscript preparation.

Corresponding author

Correspondence to Marc Ladanyi.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Identification of MYOD1 L122R in RMS.

IGV views of exome and RNA-seq reads at MYOD1 L122 in embryonal RMS case RMS1. A mutation (c.365T>G) was seen in the tumor exome and tumor transcriptome but not in the normal exome; the read counts for the reference and variant alleles in these three sequencing runs were, respectively, 35T:40G, 284T:389G and 143T:0G.

Supplementary Figure 2 Sequenom mass spectrometry–based genotyping data showing MYOD1 L122R point mutation in ten ERMS cases.

In human tumors with admixed non-neoplastic cells, heterozygous mutant (M) peaks are typically smaller than the wild-type (WT) allele peaks. The cases with LOH at MYOD1 by Affymetrix Oncoscan array analysis (see text for details) are grouped by the boxes. The latter data were not available in case RMS8, but the fact that the wild-type allele is less abundant than the mutant allele in this tumor biopsy is also suggestive of loss of the wild-type allele in this tumor.

Supplementary Figure 3 Introduction of MYOD1 L122R and PIK3CA H1047R into C2C12 mouse myoblast cells.

(a) Protein blot analysis of MYOD1 and MYC transfectants. The expression levels of MYOD1 (top panel), MYC (second from the top panel) and PI3 kinase p110α (third from the top panel) were evaluated. GAPDH is shown as a loading control (bottom panel). This confirmed MYOD1 protein expression and MYC protein expression in the respective transfectants. (b) Phase-contrast microscopy of C2C12 mouse cells transfected with GFP, wild-type MYOD1, MYOD1 L122R or MYC expression constructs, cultured in normal medium (not differentiation medium) (top panel; 200×magnification; scale bar, 100 μm). (c) Phase-contrast microscopy of C2C12 cells transfected with the indicated expression plasmids cultured in normal medium (not differentiation medium) are shown (200× magnification; scale bar, 100 μm). Arrowheads indicate multinucleated myotubes.

Supplementary Figure 4 Assessment of whole-genome copy number, LOH and PTEN deletion status in MYOD1-mutated samples using the Affymetrix OncoScan FFPE assay kit.

(a) The results of 9 of 10 mutant cases are summarized (sample RMS8 was inadequate for this analysis). MYOD1 is located on chromosome 11p15.1. LOH through uniparental disomy (UPD) was identified at this region in 44% (4/9) of the mutant cases. PTEN deletions were identified in 22% (2/9) of the mutant cases. Neither of these cases harbored PIK3CA mutations. (b) Representative data from case RMS5, depicting UPD at 11p12-15 and homozygous loss of PTEN exon 1.

Supplementary Figure 5 Global gene expression changes associated with MYOD1 L122R.

(a) Venn diagrams of the number of genes that were upregulated (left panel) or downregulated (right panel) by more than 2-fold in C2C12 mouse myoblast cells transfected with wild-type MYOD1, MYOD1 L122R or MYC compared to parental C2C12 cells. Overlaps among both upregulated and downregulated genes were greater between MYOD1 L122R and MYC than between the former and wild-type MYOD1 (81/347 versus 30/270, P < 0.001; 200/435 versus 18/182, P < 0.001). (b) Venn diagrams of genes that were more than 2-fold upregulated (left panel) or downregulated (right panel) in BJ human fibroblast cells transfected with wild-type MYOD1, MYOD1 L122R or MYC compared to parental BJ cells. Overlaps among downregulated genes were greater between MYOD1 L122R and MYC than between the former and wild-type MYOD1 (503/1,104 versus 266/718, P < 0.001), but upregulated genes were not significantly different (277/1,080 versus 213/777, P = 0.425). (c) Heat maps of hierarchical clustering of the expression microarray data from parental C2C12 cells and cells transfected with wild-type MYOD1, MYOD1 L122R or MYC. The analysis was performed on 693 statistically significant genes with a fold change of at least 2 in one of three comparisons, as described in the Online Methods. Levels of expression are indicated on a color scale where blue represents the upregulated genes and yellow represents the downregulated genes. Similarities or differences in the gene expression patterns of each cell line are represented in the horizontal dendrogram. (d) Heat maps of hierarchical clustering of the expression microarray data from parental BJ cells and cells transfected with wild-type MYOD1, MYOD1 L122R or MYC. The analysis was performed on 1,372 statistically significant genes (from a filtered list of 3,850 genes) with a fold change of at least 2 in one of three comparisons, as described in the Online Methods.

Supplementary Figure 6 Gene set enrichment analysis on the gene list from MYOD1 L122R–transfected C2C12 cells.

(a) GSEA plots for the top seven transcription factor binding motifs enriched among genes changed by the transfection of MYOD1 L122R in C2C12 cells. The genes from the microarray analysis are ranked according to their differential expression scores in MYOD1 L122R–transfected C2C12 cells from highest to lowest along the x axis. The over-representation of genes encoding DNA-binding motifs (represented by the black lines) at the top of the ranked gene list suggests that there is a correlation between genes encoding this binding motif and MYOD1 L122R–regulated genes. The green line represents the running enrichment score. (b) Table of the significant (FDR q value < 0.1) gene sets associated with the MYOD1 L122R–regulated gene signature. Sets are ranked in order of their normalized enrichment score (NES). The transcription factors associated with these DNA-binding motifs are shown.

Supplementary Figure 7 Cotransfection of wild-type MYOD1 and MYOD1 L122R into C2C12 cells.

Quantitative RT-PCR (qPCR) was performed to compare the expression levels of genes related to myogenic differentiation in C2C12 transfected with wild-type MYOD1, both wild-type MYOD1 and MYOD1 L122R, or MYOD1 L122R. Data were normalized to the expression levels of GAPDH and are expressed as the fold increase relative to C2C12 cells transfected with wild-type MYOD1.

Supplementary Figure 8 Histone modifications associated with wild-type MYOD1 and MYOD1 L122R.

(a) Individual ChIP assays were used to assess the deposition of the indicated histone marks at the enhancer regions of myotube-specific genes that are highly expressed in C2C12 cells upon transfection with wild-type MYOD1. DNA enrichment was measured by quantitative PCR (qPCR). ChIP enrichment is calculated as a percentage of input and is shown as the ratio to control. Error bars represent s.d. from three independent experiments. †P < 0.01, control versus MYOD1; *P < 0.01, MYOD1 versus MYOD1 L122R; N.S., not significant, control versus MYOD1 or MYOD1 versus MYOD1 L122R.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 1, 2 and 4–8, and Supplementary Note (PDF 1720 kb)

Supplementary Table 3

Complete lists of variant calls from next-generation sequencing. (XLSX 13963 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kohsaka, S., Shukla, N., Ameur, N. et al. A recurrent neomorphic mutation in MYOD1 defines a clinically aggressive subset of embryonal rhabdomyosarcoma associated with PI3K-AKT pathway mutations. Nat Genet 46, 595–600 (2014). https://doi.org/10.1038/ng.2969

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng.2969

This article is cited by

Search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer