DHODH modulates transcriptional elongation in the
neural crest and melanoma
Richard Mark White1,2, Jennifer Cech1, Sutheera Ratanasirintrawoot1, Charles Y. Lin3,4, Peter B. Rahl3, Christopher J. Burke1,
Erin Langdon1, Matthew L. Tomlinson5, Jack Mosher6, Charles Kaufman1,2, Frank Chen7, Hannah K. Long8, Martin Kramer9,
Sumon Datta1, Donna Neuberg10, Scott Granter11, Richard A. Young3,4, Sean Morrison6, Grant N. Wheeler5& Leonard I. Zon1
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
fish embryos to identify the initiating transcriptional events that
occur on activation of human BRAF(V600E) (which encodes an
Zebrafish embryos that are transgenic for mitfa:BRAF(V600E) and
lack p53 (also known astp53) have a gene signature that isenriched
for markers of multipotent neural crest cells, and neural crest pro-
genitors from these embryos fail to terminally differentiate. To
determine whether these early transcriptional events are important
to identify small-molecule suppressors of the neural crest lineage,
which were then tested for their effects on melanoma. One class of
for example leflunomide, led to an almost complete abrogation of
neural crest development inzebrafish and toa reduction inthe 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
decrease in melanoma growth bothinvitro and inmouse xenograft
ways in neural crest cells that have a direct bearing on melanoma
In melanoma, it is unknown to what extent BRAF(V600E) muta-
tions depend on transcriptional programs that are present in the
developmental lineage of tumour initiation. These programs may be
therapeutic targets when combined with BRAF(V600E) inhibition.
We have used zebrafish embryos to identify small-molecule suppres-
zebrafish expressing human BRAF(V600E) under the melanocyte-
specific mitfa promoter, Tg(mitfa:BRAF(V600E)), develop melanoma
at 4–12 months of age when crossed with p532/2mutant zebrafish,
drives the expression of BRAF(V600E) from 16h after fertilization
(a time point that overlaps with the expression of embryonic neural
crest markerssuch assox10), eventsthat occur early inembryogenesis
are analogous to those that occur at tumour initiation. To gain
insight into these initiating events, we compared the gene expression
profiles of Tg(mitfa:BRAF(V600E));p532/2embryos with those of
ment analysis (Fig. 1b). This approach uncovered a signature of 123
overlapping genes, whichis enriched formarkersof embryonic neural
crest progenitors (crestin, sox10 and ednrb (also known as ednrb1))
sets). The overlapping gene signature is similar to the signature of a
multipotent neural crest progenitor, suggesting that the melanomas
have adopted this cell fate.
We analysed alterations in embryonic neural crest development by
using in situ hybridization. At 24h post fertilization, Tg(mitfa:BRAF
(V600E));p532/2embryos show an abnormal expansion in the num-
tail and dorsal epidermis only in Tg(mitfa:BRAF(V600E));p532/2
embryos but not in embryos with either single mutation (Supplemen-
tary Fig. 2a). The gene encoding Crestin is zebrafish specific2and is
normally downregulated after the terminal differentiation of neural
crest progenitors3. Our finding therefore suggests that activated
BRAF(V600E) promotes the maintenance of multipotency in neural
crest progenitors, which become expanded during tumorigenesis. In
adult Tg(mitfa:BRAF(V600E));p532/2melanomas, almost all tumour
cells, but no normal cells, were positive for crestin (Fig. 1c). Only 10–
15% of the melanoma cells were pigmented (Supplementary Fig. 2b),
which is consistent with the concept that adult zebrafish melanomas
retain a progenitor-like state. A human melanoma tissue array yielded
positive for the neural crest progenitor gene ednrb, but only 9 of 70
(12.9%) were positive for the melanocyte lineage marker dct
melanomas express the neural crest marker sox10 (ref. 4). These data
to the maintenance of a neural crest progenitor phenotype5.
We proposed that chemical suppressors of neural crest progenitors
would be useful for treating melanoma. We screened 2,000 chemicals
genesis. Most chemicals (90%) had a minimal effect or were toxic
strongly abrogated the expression of crestin (Fig. 2a, centre and left).
The chemoinformatic algorithm DiscoveryGate6revealed similarity
of DHODH7. NSC210627 inhibited DHODH activity in vitro (Sup-
distinct from NSC210627 (ref. 8), phenocopied NSC210627 (Fig. 2a,
right) and was used for further studies because of its availability.
zebrafish embryos were devoid of pigmented melanocytes at 36–48h
post fertilization (Fig. 2b) and iridophores at 72h post fertilization
1Stem Cell Program and Hematology/Oncology, Children’s Hospital Boston, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.5School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.6Center for Stem Cell Biology,
9Genzyme Corporation, Cambridge, Massachusetts 02142, USA.10Department of Biostatistics and Computational Biology, Dana Farber Cancer Institute, Boston, Massachusetts 02115, USA.
11Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA.
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(Supplementary Fig. 7a). dhodh inhibition led to a loss of ventral
melanocytes in stage 38 Xenopus embryos (Supplementary Fig. 7b).
Leflunomide treatment led to an almost complete loss of melanocyte
progenitors at 24h post fertilization (Fig. 2c), a reduction in the num-
cartilage at 72h post fertilization (data not shown). Leflunomide
crest progenitors and melanocytes, respectively, while leaving other
lineages such as blood and notochord less affected (Supplementary
Fig. 8). Microarray analysis of leflunomide-treated embryos showed
downregulation of 49% of the genes that were upregulated in the 123-
genemelanomasignature,and morethanhalfoftheseare neural crest
related (see Supplementary Table 2 for the complete list).
The loss of several types of neural crest derivative suggested that
self-renewing neural crest stem cells in primary stem cell colonies, to
2765.35% (leflunomide) and 3566.16% (A771726) of control num-
and Supplementary Fig. 9a). Colony size was also reduced compared
with controls (by 18% and 24%, respectively; P,0.02, Student’s
t-test), but there was no effect on the differentiation or survival of
that DHODH inhibitors negatively regulate the self-renewal of neural
crest stem cells and have an affect on these cells in multiple species.
DHODH catalyses the fourth step in the synthesis of pyrimidine
nucleotides (NTPs)11. We noted striking morphological similarity
between leflunomide-treated embryos and spt5/spt6 mutants12, sug-
gesting that leflunomide acts to suppress transcriptional elongation.
In the spt5sk8null mutant, we found a lack of both crestin expression
and pigmented melanocytes (similar to leflunomide-treated embryos)
profiles of spt5sk8mutants and leflunomide-treated embryos13were
nearly identical; of 223 genes downregulated after leflunomide treat-
ment, 183 were similarly downregulated in spt5sk8mutants (Sup-
plementary Table 3 and Supplementary Fig. 10b). These downregu-
lated genes include neural crest genes (crestin, sox10 and mitfa) and
action of Dhodh with spt5 by incubating the hypomorphic spt5m806
mutant (which has only mild melanocyte defects)14in low concentra-
tions of leflunomide (3–5mM) and then analysing the number of pig-
mented melanocytes. Enhanced sensitivity to leflunomide was shown
by spt5m806embryos (Fig. 3a and Supplementary Fig. 11); at 3mM
leflunomide, 99% of mutant embryos had few or no melanocytes,
compared with 0% of wild-type embryos (P50.000018, Kruskal–
Wallis test; Supplementary Fig. 11b). These data confirm that
DHODH inhibition affects transcriptional elongation, which is con-
pools in vitro leads to defects in elongation15.
We assessed whether leflunomide specifically caused defects in the
transcriptional elongation of genes required for neural crest develop-
plementary Fig. 10c and Supplementary Table 4). Leflunomide caused
cant downregulation of 39 transcripts of mitfa (for 59 transcripts
3.7561.19-fold increase versus 0.3960.07-fold increase for 39 tran-
increase versus 39 transcripts 0.7460.07-fold increase; P,0.05).
Leflunomide did not have a similar effect on control genes such as the
gene encoding b-actin (59 transcripts 1.0360.07-fold increase versus 39
In the presence of leflunomide, transcription is initiated normally, but
the transcripts accumulate and do not undergo productive elongation.
To confirm thatthismechanismisconserved inhuman melanoma,
we performed chromatin immunoprecipitation using an antibody
Figure 1 | Transgeniczebrafishmelanomaandneuralcrestgeneexpression.
a, Transgenic zebrafish expressing BRAF(V600E) under the control of the
promoter of the melanocyte-specific gene mitfa, Tg(mitfa:BRAF(V600E)),
fish. Their gross embryonic development is largely normal. hpf, hours post
(left). Gene set enrichment analysis uncovered an enrichment between the
embryonic gene signature and the adult melanomas that form 4–12 months
later (centre and right; see the Supplementary Information for full protocol
scale, range 22-fold to 12-fold increase); adult heat-map columns represent
individual fish (log2scale, range 210-fold to 110-fold increase). c, In situ
hybridization of sagittal sections of WT and Tg(mitfa:BRAF(V600E));p532/2
adults reveal homogeneous crestin expression (blue) only within the dorsal
melanoma; it is absent from normal adult tissues.
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specific for RNA polymerase II (RNA Pol II), followed by sequencing
(ChIP-seq). Transcriptional elongation was measured using the
travelling ratio (TR)16, in which the ratio of RNA Pol II density in
In the human melanoma cell lines A375 and Malme-3M, leflunomide
treatment caused a significant inhibition of transcriptional elongation
(measured as an increase in the TR), particularly for genes with a TR
that was initially low (,7.5). For example, in A375 cells, the TR
increased by .1.3 fold at 21.3% of loci; in Malme-3M cells, this
occurred for 36.3% of loci (Supplementary Table 5). Examination of
RNA Pol II occupancy using metagene analysis at a variety of fold-
change cutoffs (Fig. 3b (A375), Supplementary Fig. 12 (Malme-3M)
and Supplementary Table 5) revealed no defect in transcription ini-
tiation but a decrease in elongation that was pronounced at the 39 end
of genes such as NPM1 and CCND1 (Fig. 3c). Ingenuity Pathway
Analysisonthe lociaffected inbothcelllinesrevealedastrongenrich-
ment for Myc targets and pathway members17(Supplementary Fig.
13a, b). Myc, in addition to its requirement for neural crest develop-
ment18, was recently shown to be a potent regulator of transcriptional
pause release in embryonic stem cells16. Our data suggest that the
mechanism by which Myc target genes are regulated at the transcrip-
tional elongation level operates in neural-crest-derived melanoma as
well. Taken together, the genetic and biochemical data demonstrate
that leflunomide acts to modulate transcriptional elongation in both
neural crest development and human melanoma.
we tested its effects on melanoma growth. A771726 caused a dose-
dependentdecreasein theproliferation of human melanoma cell lines
(A375, Hs 294T and RPMI-7951; Fig. 4a). Similarly, a short hairpin
RNA directed against DHODH led to a 57.7% decrease in the pro-
by ChIP-PCR (Supplementary Fig. 14). Microarray analysis of the
A375 cell line treated with leflunomide revealed downregulation of
genes that are required for neural crest development (such as
SNAI2) and members of the NOTCH pathway (for example, HES6
and JAG1), which is consistent with the effects of leflunomide on
zebrafish embryos (Supplementary Table 6).
Pyrimidine NTP production is regulated at the level of carbamoyl-
phosphate synthetase(CAD)19, theenzymethatisdirectlyupstreamof
DHODH. CAD is phosphorylated by the mitogen-activated protein
kinase ERK220, a protein that would be activated in melanoma as a
result of the BRAF(V600E) mutation. We reasoned that combined
blockade of BRAF(V600E) and DHODH would suppress melanoma
the BRAF(V600E) inhibitor PLX4720 (ref. 21) together with A771726
(Fig. 4b, c and Supplementary Fig. 15a, b), and we found that the
combination led to a cooperative suppression of melanoma growth.
Transcription start site
Distance from promoter
Mean rank normalized
Figure 3 | DHODHinhibitionmodulatestranscriptionalelongation. a,The
hypomorphic spt5m806homozygous mutant (top right) has only a mild
animals. Treatment with a low dose of leflunomide (3mM) leads to an almost
complete absence of neural-crest-derived melanocytes in the mutant line. See
Supplementary Fig. 11 for dose–response quantification of this effect.
after treatment with leflunomide. The metagene plot allows visualization of all
of the genes that are occupied by RNA Pol II, corrected for individual gene
lengths. Genome-wide RNA Pol II occupancy at the promoter region is
magnification of the 39 region of the genes. c, Representative examples of Myc
elongation after treatment with leflunomide. A gene that is minimally affected
(DGAT) is also depicted. ForNPM1, theTR is 5.04 after DMSO treatmentand
8.10 after leflunomide treatment. For CCND1, the TR is 3.47 after DMSO
DMSO treatment and 5.34 after leflunomide treatment.
NSC210627 40 μm Lefunomide 6.5 μm
Neural crest stem cell self-renewal
Lefunomide 5 μm
Lefunomide 40 μm
Multipotent daughter cells
(% of control group value)
Figure 2 | A chemical genetic screen to identify suppressors of neural crest
development. a, A search for chemical suppressors of the crestin1lineage
during embryogenesis identified NSC210627, a compound that completely
the dorsum and in ventrally migrating neural crest cells), as shown by in situ
hybridization (a, left and centre). DMSO is used as a control. The
DiscoveryGate chemoinformatic algorithm revealed structural similarity
between NSC210627 and brequinar (Supplementary Fig. 5), an inhibitor of
the crestin phenotype induced by treatment with NSC210627 (a, right).
b–d, Treatment with leflunomide caused an absence of multiple neural crest
derivatives, including pigmented melanocytes (b); melanocyte progenitors,
control of the mitfa promoter (c); and glial cells, which were visualized by
expressing the fluorescent protein mCherry under the control of the myelin
basic protein (mbp) promoter (d). e, Treatment with leflunomide, or A771726
(Supplementary Fig. 9a), significantly reduced the number of multipotent
daughter cells that could be subcloned from individual primary neural crest
compared with control, Student’s t-test.
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PLX4720 had no effect in non-melanoma cell lines (Supplementary
Fig. 15c, BRAFWT). A771726 demonstrated some antiproliferative
activity in non-melanoma cell lines but was less potent in these cells
than in melanoma cells (Supplementary Fig. 15d).
We examined the in vivo effects of leflunomide and PLX4720 by
Supplementary Fig. 16). At 12 days post treatment, tumours in mice
that had been treated with dimethylsulphoxide (DMSO) as a control
had grown 7.461.3 fold. By contrast, tumours in PLX4720-treated
had grown 4.760.12 fold. The combination of PLX4720 and lefluno-
mide led to an enhanced abrogation of tumour growth, with only
complete tumour regression (PLX4720 and leflunomide versus
PLX4720 alone or leflunomide alone, P,0.001, analysis of variance
followed by Tukey’s post hoc test). Therefore, we have found that an
inhibitor of embryonic neural crest development, leflunomide, blocks
in vivo tumour growth in combination with the BRAF(V600E) inhi-
bitor PLX4720 when used at clinically meaningful doses.
Our data suggest that inhibition of DHODH abrogates the tran-
scriptional elongation of genes that are required for both neural crest
development and melanoma growth, including Myc target genes and
mitfa. Although DHODH inhibition would be expected to lead to
ubiquitous defects, human mutations in DHODH cause Miller’s syn-
drome22, a craniofacial disorder that is similar to syndromes with
defects in neural crest development. Our data support recent findings
that elongation factors are important for both neural crest develop-
ment23and cancer growth24. Developmental regulators of transcrip-
tional elongation have recently been identified to be important in
haematopoiesis25, and identification of such factors in the neural crest
awaits further study.
the identification of molecules that require in vivo contexts for the
expression of relevant phenotypes26. Inhibition of DHODH may be a
unique in vivo mechanism for modulating transcriptional elongation.
Leflunomide is a well-tolerated anti-arthritis drug in humans27, and
effective in combination with a BRAF(V600E) inhibitor. This com-
bination therapy may help to overcome resistance to BRAF(V600E)
inhibitors28. As an increasing number of genomic changes are iden-
tifiedin cancerous cells,the challenge is to target thesein concertwith
lineage-specific factors to achieve therapeutic synergy. Our approach
generalized to other cell types, with direct relevance to human cancer.
Microarray analysis was performed on four groups of embryos at 72h post fertiliza-
tion: wild type, Tg(mitfa:BRAF(V600E)), p532/2, and Tg(mitfa:BRAF(V600E));
p532/2double mutants. Array analysis was also performed for adult
tional signature of the melanomas was used in gene set enrichment analysis to
identify genes that were significantly enriched in the Tg(mitfa:BRAF
(V600E));p532/2embryos. The 123 genes that make up this signature, which is
regulated in bothBRAF(V600E);p532/2embryos andtumours. Insitu hybridiza-
tion was used to examine the expression of crestin (a pan neural crest marker)
and that of other neural crest genes in embryos (at 24–72h post fertilization) and
adult tumours. Chemical screening was performed to identify suppressors of the
crestin1lineage by treating wild-type embryos from 50% epiboly to 24h post
fertilization with various chemicals, followed by robot-controlled in situ hybrid-
leflunomide. The latter was used for further studies owing to its more widespread
availability. The effect of leflunomide on embryonic neural crest development in
zebrafish was assessed by scoring embryos for melanocytes, iridophores and glial
main text as neural crest stem cells). The effects of leflunomide on transcriptional
elongation in the neural crest were tested using the spt5m806allele and measuring
pigmentation in response to 3–5 mM leflunomide. Elongation in melanoma cells
the TR. Leflunomide was tested for anti-melanoma effects on human melanoma
cell lines in the presence or absence of the BRAF(V600E) inhibitor PLX4720. In
vitro proliferation assays were performed using the CellTiter-Glo system
daily intraperitoneal doses of PLX4720 alone, leflunomide alone or both, and the
tumour growth rate was measured on days 4, 7 and 12.
Received 26 March 2010; accepted 31 January 2011.
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Hs 294T melanoma
A375 xenografts in nude mice
Days after start of treatment
Tumour volume (fold increase)
Viable cells (%)120
DMSO DMSO PLX4720
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Supplementary Information is linked to the online version of the paper at
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,
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
M.L.T.was aBiotechnologyand BiologicalSciencesResearchCouncil/PfizerIndustrial
CASE award recipient.
Author 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. TheXenopus 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
performedbyM.K.atGenzyme. Theratneuralcrest workwas performedbyJ.M.inthe
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
microarray analysis was performed by S.G. All authors discussed the results and
commented on the manuscript.
Author Information 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. Reprints and permissions
information is available at www.nature.com/reprints. The authors declare competing
financial interests: details accompany the full-text HTML version of the paper at
www.nature.com/nature. Readers are welcome to comment on the online version of
this article at www.nature.com/nature. Correspondence and requests for materials
should be addressed to L.I.Z. (email@example.com).
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