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 melanoma. 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.

Figure 1. Transgenic zebrafish melanoma and neural crest gene expression

a, Transgenic zebrafish expressing BRAF^V600E under the melanocyte specific mitf promoter develop pigmentation abnormalities, and melanoma when crossed with p53^-/- fish. Gross embryonic development is largely normal. b, Gene expression analysis reveals a unique gene signature at 72hpf in the BRAF^V600E;p53^-/- strain (left). Gene set enrichment analysis (GSEA) reveals an enrichment between the embryonic gene signature and the adult melanomas which form 4-12 months later (middle and right; see Methods for full GSEA methods). Embryo heatmap columns represent average of 3 clutches (log₂ scale, range -2 to +2 fold); adult heatmap columns represent individual fish (log₂ scale, range -10 to +10 fold). c, Sagittal section of WT and BRAF^V600E;p53^-/- adults reveal homogeneous crestin expression (blue staining) only within the dorsal melanoma, whereas it is absent in normal adult tissues.

Figure 2. A chemical genetic screen to identify suppressors of neural crest development

a, A chemical genetic screen to identify suppressors of the crestin⁺ lineage during embryogenesis identified NSC210627, a compound which completely abrogates expression by ISH (a, left and middle). The Discoverygate chemoinformatic algorithm revealed structural similarity between NSC210627 and brequinar (see Supplemental Figure 5), an inhibitor of dihydroorotate dehydrogenase (DHODH). Leflunomide, a structurally distinct DHODH inhibitor, phenocopies the crestin phenotype of NSC210627 (a, right). b-d, Leflunomide caused an absence of multiple neural crest derivatives, including pigmented melanocytes (b), mitf-GFP⁺ melanocyte progenitors (c), and mbp-mCherry⁺ glial cells (d). e, Leflunomide or A771726 (see Supplemental Figure 9a) significantly reduced the number of multipotent daughter cells that could be subcloned from individual primary neural crest stem cell colonies (Values shown are mean +/-SD of n=3 replicates; *, p<0.05 compared to control, t-test).

Figure 3. DHODH inhibition modulates transcriptional elongation

a, The hypomorphic spt5^m806 mutant has only a mild pigment defect on its own (top). Treatment with low-dose leflunomide (3uM) leads to an almost complete absence of neural crest derived melanocytes in the mutant line. See Supplemental Figure 11 for dose-response quantification of this effect. b, Metagene analysis of RNA pol II occupancy in A375 human melanoma cells after treatment with leflunomide. Pol II occupancy at the promoter region is unaffected, but diminished at the 3′ end of the genes. Inset shows a higher magnification of the 3′ region of the genes. c, Representative examples of myc target genes which demonstrate defects in transcriptional elongation after leflunomide treatment, along with a non-affected gene. For Npm1, the TR in DMSO=5.04, and in LEF=8.10. For Ccnd1, the TR in DMSO=3.47, and in LEF=4.67.

Figure 4. DHODH blockade suppresses melanoma growth in concert with BRAF^V600E inhibition

a, Leflunomide causes a dose-dependent decrease in melanoma proliferation as measured by CellTiterGlo assay in 3 BRAF^V600E melanoma cells lines tested (A375, RPMI7951, Hs.294T). b, c Leflunomide cooperates with the BRAF^V600E inhibitor PLX4720 in inhibiting melanoma cell proliferation in the A375 (b) and Hs.294T (c) cell lines as well as the other tested lines (See Supplemental Figure 15). d, After subcutaneous transplantation of A375 cells (3×10⁵) into nude mice, both leflunomide and PLX4720 impair tumor progression, with the combination showing a nearly complete abrogation of tumor growth and in 2/5 animals complete tumor regression. (*p=0.036 DMSO vs. PLX; **p=0.006 DMSO vs. LEF; ***p=0.006 PLX or LEF vs. PLX/LEF; PLX vs. LEF: p=NS, ANOVA followed by Tukey post-hoc analysis). Values shown are mean +/- SEM of n=3-5 replicates, as shown.