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.

DHODH modulates transcriptional elongation in the neural crest and melanoma

Nature volume 471, pages 518–522 (2011)Cite this article

Subjects

Abstract

Melanoma is a tumour of transformed melanocytes, which are originally derived from the embryonic neural crest. It is unknown to what extent the programs that regulate neural crest development interact with mutations in the BRAF oncogene, which is the most commonly mutated gene in human melanoma1. We have used zebrafish embryos to identify the initiating transcriptional events that occur on activation of human BRAF(V600E) (which encodes an amino acid substitution mutant of BRAF) in the neural crest lineage. Zebrafish embryos that are transgenic for mitfa:BRAF(V600E) and lack p53 (also known as tp53) have a gene signature that is enriched for markers of multipotent neural crest cells, and neural crest progenitors from these embryos fail to terminally differentiate. To determine whether these early transcriptional events are important for melanoma pathogenesis, we performed a chemical genetic screen to identify small-molecule suppressors of the neural crest lineage, which were then tested for their effects on melanoma. One class of compound, inhibitors of dihydroorotate dehydrogenase (DHODH), for example leflunomide, led to an almost complete abrogation of neural crest development in zebrafish and to a reduction in the self-renewal of mammalian neural crest stem cells. Leflunomide exerts these effects by inhibiting the transcriptional elongation of genes that are required for neural crest development and melanoma growth. When used alone or in combination with a specific inhibitor of the BRAF(V600E) oncogene, DHODH inhibition led to a marked decrease in melanoma growth both in vitro and in mouse xenograft studies. Taken together, these studies highlight developmental pathways in neural crest cells that have a direct bearing on melanoma formation.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: Transgenic zebrafish melanoma and neural crest gene expression.
Figure 2: A chemical genetic screen to identify suppressors of neural crest development.
Figure 3: DHODH inhibition modulates transcriptional elongation.
Figure 4: Melanoma growth is suppressed by DHODH blockade in concert with BRAF(V600E) inhibition.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

The microarray data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus database under accession numbers GSE24526, GSE24527, GSE24528 and GSE24529.

References

  1. Davies, H. et al. Mutations of the BRAF gene in human cancer. Nature 417, 949–954 (2002)

    Article  ADS  CAS  Google Scholar 

  2. Rubinstein, A. L., Lee, D., Luo, R., Henion, P. D. & Halpern, M. E. Genes dependent on zebrafish cyclops function identified by AFLP differential gene expression screen. Genesis 26, 86–97 (2000)

    Article  CAS  Google Scholar 

  3. Luo, R., An, M., Arduini, B. L. & Henion, P. D. Specific pan-neural crest expression of zebrafish Crestin throughout embryonic development. Dev. Dyn. 220, 169–174 (2001)

    Article  CAS  Google Scholar 

  4. Bakos, R. M. et al. Nestin and SOX9 and SOX10 transcription factors are coexpressed in melanoma. Exp. Dermatol. 19, e89–e94 (2010)

    Article  Google Scholar 

  5. Boiko, A. D. et al. Human melanoma-initiating cells express neural crest nerve growth factor receptor CD271. Nature 466, 133–137 (2010)

    Article  ADS  CAS  Google Scholar 

  6. DiscoveryGate. 〈https://www.discoverygate.com/〉 (2008)

  7. McLean, J. E., Neidhardt, E. A., Grossman, T. H. & Hedstrom, L. Multiple inhibitor analysis of the brequinar and leflunomide binding sites on human dihydroorotate dehydrogenase. Biochemistry 40, 2194–2200 (2001)

    Article  CAS  Google Scholar 

  8. Kaplan, M. J. Leflunomide Aventis Pharma. Curr. Opin. Investig. Drugs 2, 222–230 (2001)

    CAS  PubMed  Google Scholar 

  9. Bixby, S., Kruger, G. M., Mosher, J. T., Joseph, N. M. & Morrison, S. J. Cell-intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity. Neuron 35, 643–656 (2002)

    Article  CAS  Google Scholar 

  10. Molofsky, A. V. et al. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 425, 962–967 (2003)

    Article  ADS  CAS  Google Scholar 

  11. Löffler, M., Jockel, J., Schuster, G. & Becker, C. Dihydroorotat-ubiquinone oxidoreductase links mitochondria in the biosynthesis of pyrimidine nucleotides. Mol. Cell. Biochem. 174, 125–129 (1997)

    Article  Google Scholar 

  12. Keegan, B. R. et al. The elongation factors Pandora/Spt6 and Foggy/Spt5 promote transcription in the zebrafish embryo. Development 129, 1623–1632 (2002)

    CAS  PubMed  Google Scholar 

  13. Krishnan, K., Salomonis, N. & Guo, S. Identification of Spt5 target genes in zebrafish development reveals its dual activity in vivo . PLoS ONE 3, e3621 (2008)

    Article  ADS  Google Scholar 

  14. Guo, S. et al. A regulator of transcriptional elongation controls vertebrate neuronal development. Nature 408, 366–369 (2000)

    Article  ADS  CAS  Google Scholar 

  15. Wada, T. et al. DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs. Genes Dev. 12, 343–356 (1998)

    Article  CAS  Google Scholar 

  16. Rahl, P. B. et al. c-Myc regulates transcriptional pause release. Cell 141, 432–445 (2010)

    Article  CAS  Google Scholar 

  17. Chen, Y. et al. Integration of genome and chromatin structure with gene expression profiles to predict c-MYC recognition site binding and function. PLOS Comput. Biol. 3, e63 (2007)

    Article  ADS  MathSciNet  Google Scholar 

  18. Hong, S. K., Tsang, M. & Dawid, I. B. The mych gene is required for neural crest survival during zebrafish development. PLoS ONE 3, e2029 (2008)

    Article  ADS  Google Scholar 

  19. Aoki, T., Morris, H. P. & Weber, G. Regulatory properties and behavior of activity of carbamoyl phosphate synthetase II (glutamine-hydrolyzing) in normal and proliferating tissues. J. Biol. Chem. 257, 432–438 (1982)

    CAS  PubMed  Google Scholar 

  20. Graves, L. M. et al. Regulation of carbamoyl phosphate synthetase by MAP kinase. Nature 403, 328–332 (2000)

    Article  ADS  CAS  Google Scholar 

  21. Tsai, J. et al. Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc. Natl Acad. Sci. USA 105, 3041–3046 (2008)

    Article  ADS  CAS  Google Scholar 

  22. Ng, S. B. et al. Exome sequencing identifies the cause of a mendelian disorder. Nature Genet. 42, 30–35 (2010)

    Article  CAS  Google Scholar 

  23. Nguyen, C. T., Langenbacher, A., Hsieh, M. & Chen, J. N. The PAF1 complex component Leo1 is essential for cardiac and neural crest development in zebrafish. Dev. Biol. 341, 167–175 (2010)

    Article  CAS  Google Scholar 

  24. Mueller, D. et al. Misguided transcriptional elongation causes mixed lineage leukemia. PLoS Biol. 7, e1000249 (2009)

    Article  Google Scholar 

  25. Bai, X. et al. TIF1γ controls erythroid cell fate by regulating transcription elongation. Cell 142, 133–143 (2010)

    Article  CAS  Google Scholar 

  26. Wheeler, G. N. & Brandli, A. W. Simple vertebrate models for chemical genetics and drug discovery screens: lessons from zebrafish and Xenopus . Dev. Dyn. 238, 1287–1308 (2009)

    Article  CAS  Google Scholar 

  27. Schiff, M. H., Strand, V., Oed, C. & Loew-Friedrich, I. Leflunomide: efficacy and safety in clinical trials for the treatment of rheumatoid arthritis. Drugs Today (Barc.) 36, 383–394 (2000)

    Article  CAS  Google Scholar 

  28. Flaherty, K. T. et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N. Engl. J. Med. 363, 809–819 (2010)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank G. Bollag and P. Lin for supplying PLX4720. The melanoma xenografts were performed with the assistance of T. Venezia-Bowman. In situ hybridization probes for crestin were supplied by P. Henion. We thank S. Lacadie, C. Ceol, Y. Houvras, T. Bowman, X. Bai and R. Field for discussions and/or comments on the manuscript. This work was supported by the Howard Hughes Medical Institute and the National Cancer Institute (National Institutes of Health) (L.I.Z.), Aid for Cancer Research, the American Society for Clinical Oncology and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (National Institutes of Health) (R.M.W.). M.L.T. was a Biotechnology and Biological Sciences Research Council/Pfizer Industrial CASE award recipient.

Author information

Authors and Affiliations

  1. Stem Cell Program and Hematology/Oncology, Children’s Hospital Boston, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Richard Mark White, Jennifer Cech, Sutheera Ratanasirintrawoot, Christopher J. Burke, Erin Langdon, Charles Kaufman, Sumon Datta & Leonard I. Zon

  2. Department of Medical Oncology, Dana Farber Cancer Institute, Boston, 02115, Massachusetts, USA

    Richard Mark White & Charles Kaufman

  3. Whitehead Institute for Biomedical Research, Cambridge, 02142, Massachusetts, USA

    Charles Y. Lin, Peter B. Rahl & Richard A. Young

  4. Department of Biology, Massachusetts Institute of Technology, Cambridge, 02142, Massachusetts, USA

    Charles Y. Lin & Richard A. Young

  5. School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK

    Matthew L. Tomlinson & Grant N. Wheeler

  6. Center for Stem Cell Biology, University of Michigan, Howard Hughes Medical Institute, Ann Arbor, 48109, Michigan, USA

    Jack Mosher & Sean Morrison

  7. Harvard University, Cambridge, 02138, Massachusetts, USA

    Frank Chen

  8. Cambridge University, Cambridge CB2 1TN, UK

    Hannah K. Long

  9. Genzyme Corporation, Cambridge, 02142, Massachusetts, USA

    Martin Kramer

  10. Department of Biostatistics and Computational Biology, Dana Farber Cancer Institute, Boston, 02115, Massachusetts, USA

    Donna Neuberg

  11. Department of Pathology, Brigham and Women’s Hospital, Boston, 02115, Massachusetts, USA

    Scott Granter

Authors
  1. Richard Mark White
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jennifer Cech
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sutheera Ratanasirintrawoot
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Charles Y. Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter B. Rahl
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Christopher J. Burke
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Erin Langdon
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Matthew L. Tomlinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jack Mosher
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Charles Kaufman
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Frank Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Hannah K. Long
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Martin Kramer
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Sumon Datta
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Donna Neuberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Scott Granter
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Richard A. Young
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Sean Morrison
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Grant N. Wheeler
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Leonard I. Zon
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.M.W. and L.I.Z. planned the project. The chemical screen was performed by R.M.W., S.R., J.C., F.C., C.J.B., H.K.L. and S.D. The Xenopus work and initial identification of NSC210627 was performed by M.L.T. in the laboratory of G.N.W. The mbp:mCherry work was performed by R.M.W. and C.K. The human DHODH assay was performed by M.K. at Genzyme. The rat neural crest work was performed by J.M. in the laboratory of S.M. The ChIP-seq experiments and data analysis were performed by P.B.R. and C.Y.L. in the laboratory of R.A.Y. The ChIP-PCR assays were performed by P.B.R., R.M.W. and E.L. The zebrafish elongation and melanoma assays were performed by R.M.W. and J.C. Statistical analysis was performed by D.N. Human melanoma tissue microarray analysis was performed by S.G. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Leonard I. Zon.

Ethics declarations

Competing interests

L.I.Z. is a founder and stockholder of Fate Therapeutics and a scientific adviser for Stemgent.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-16 with legends, legends for Supplementary Tables 1-6 and Supplementary Methods with additional references. (PDF 1700 kb)

Supplementary Table 1

GSEA analysis showing genes enriched between BRAFV600E;p53‐/‐ embryos and tumors - see Supplementary Information file for full legend. (XLS 910 kb)

Supplementary Table 2

Neural crest genes affected by leflunomide - see Supplementary Information file for full legend. (XLS 134 kb)

Supplementary Table 3

GSEA analysis comparing leflunomide treated embryos to the spt5sk8 mutant line at 24hpf - see Supplementary Information file for full legend. (XLS 612 kb)

Supplementary Table 4

qRT‐PCR results analyzing 5’ or 3’ transcripts in 24hpf embryos treated with either leflunomide (6.5μM) or DMSO control - see Supplementary Information file for full legend. (XLS 12 kb)

Supplementary Table 5

ChIP‐seq results in the presence of leflunomide in A375 and MAMLE‐3M cell lines - see Supplementary Information file for full legend. (XLS 3373 kb)

Supplementary Table 6

Microarray analysis of A375 melanoma cells treated with leflunomide 25μM for 3 days - see Supplementary Information file for full legend. (XLS 534 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

White, R., Cech, J., Ratanasirintrawoot, S. et al. DHODH modulates transcriptional elongation in the neural crest and melanoma. Nature 471, 518–522 (2011). https://doi.org/10.1038/nature09882

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature09882

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing