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DHODH modulates transcriptional elongation in the neural crest and melanoma

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.

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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.

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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.

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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

Authors

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)

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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

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