DHODH modulates transcriptional elongation in the neural crest and melanoma

Stem Cell Program and Hematology/Oncology, Children's Hospital Boston, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Nature (Impact Factor: 41.46). 03/2011; 471(7339):518-22. DOI: 10.1038/nature09882
Source: PubMed


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.

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    • "Miller syndrome or postaxial acrofacial dysostosis (MIM 263750), characterised by both craniofacial and limb anomalies, is another example of a clinically recognisable acrofacial dysostosis in which recessive mutations in DHODH result in reduced de novo pyrimidine synthesis [Rainger et al., 2012]. Interestingly, inhibition of DHODH in zebrafish embryos leads to complete abrogation of neural crest development similar to Treacle [White et al., 2011]. The involvement of PHAX in PRS with or without CCA merits further work particularly in the absence of any human cases with intragenic mutations. "
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    ABSTRACT: Pierre Robin sequence (PRS) is an etiologically distinct subgroup of cleft palate. We aimed to define the critical genomic interval from five different 5q22-5q31 deletions associated with PRS or PRS-associated features and assess each gene within the region as a candidate for the PRS component of the phenotype. Clinical array-based comparative genome hybridisation (aCGH) data were used to define a 2.08 Mb minimum region of overlap among four de novo deletions and one mother-son inherited deletion associated with at least one component of PRS. Commonly associated anomalies were talipes equinovarus (TEV), finger contractures and crumpled ear helices. Expression analysis of the orthologous genes within the PRS critical region in embryonic mice showed that the strongest candidate genes were FBN2 and PHAX. Targeted aCGH of the critical region and sequencing of these genes in a cohort of 25 PRS patients revealed no plausible disease-causing mutations. In conclusion, deletion of ∼2 Mb on 5q23 region causes a clinically recognisable subtype of PRS. Haploinsufficiency for FBN2 accounts for the digital and auricular features. A possible critical region for TEV is distinct and telomeric to the PRS region. The molecular basis of PRS in these cases remains undetermined but haploinsufficiency for PHAX is a plausible mechanism.
    European Journal of Medical Genetics 10/2014; 57(10). DOI:10.1016/j.ejmg.2014.08.007 · 1.47 Impact Factor
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    • "White et al. [14] reported that leflunomide inhibits transcriptional elongation of the genes involved in self-renewal of neural progenitor cells through inhibition of DHO-DHase activity. These investigators further demonstrated that leflunomide at low doses cooperates with PLX4720, a B-Raf kinase inhibitor, to effectively inhibit melanoma cell proliferation and tumor growth [14]. Our early studies using a lymphadenopathy and autoimmune disease model in MRL/MpJ-lpr/lpr mice and a tumor xenograft model demonstrated that the immunosuppressive and antitumor activities of leflunomide are largely independent of the pyrimidine nucleotide synthesis pathway [4] [5] since uridine co-administration with leflunomide normalized pyrimidine nucleotide levels in tumor tissues but did not antagonize the antitumor activity of leflunomide in two xenograft models [5]. "
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    ABSTRACT: Leflunomide is a novel immunomodulatory drug prescribed for treating rheumatoid arthritis. It inhibits the activity of protein tyrosine kinases and dihydroorotate dehydrogenase, a rate-limiting enzyme in the pyrimidine nucleotide synthesis pathway. Here, we report that A77 1726, the active metabolite of leflunomide, inhibited the phosphorylation of ribosomal protein S6 and two other substrates of S6K1, insulin receptor substrate-1 and carbamoyl phosphate synthetase 2, in an A375 melanoma cell line. A77 1726 increased the phosphorylation of AKT, p70 S6 (S6K1), ERK1/2, and MEK through the feedback activation of the IGF-1 receptor–mediated signaling pathway. Invitro kinase assay revealed that leflunomide and A77 1726 inhibited S6K1 activity with IC50 values of approximately 55 and 80 μM, respectively. Exogenous uridine partially blocked A77 1726–induced inhibition of A375 cell proliferation. S6K1 knockdown led to the inhibition of A375 cell proliferation but did not potentiate the antiproliferative effect of A77 1726. A77 1726 stimulated bromodeoxyuridine incorporation in A375 cells but arrested the cell cycle in the S phase, which was reversed by addition of exogenous uridine or by MAP kinase pathway inhibitors but not by rapamycin and LY294002 (a phosphoinositide 3-kinase inhibitor). These observations suggest that A77 1726 accelerates cell cycle entry into the S phase through MAP kinase activation and that pyrimidine nucleotide depletion halts the completion of the cell cycle. Our study identified a novel molecular target of A77 1726 and showed that the inhibition of S6K1 activity was in part responsible for its antiproliferative activity. Our study also provides a novel mechanistic insight into A77 1726–induced cell cycle arrest in the S phase.
    Neoplasia (New York, N.Y.) 10/2014; 16(10):824–834. DOI:10.1016/j.neo.2014.08.006 · 4.25 Impact Factor
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    • "Animal models of a disease can be used as tools for therapeutic discovery of compounds that suppress the disease phenotype, regardless of the specific molecular targets. In the last few years, it has become apparent that zebrafish is a powerful model organism amenable to high-throughput drug screening in vivo at embryonic and larval stages (North et al., 2007; White et al., 2011; Zon and Peterson, 2005). Thus, this model organism is becoming valuable in the preclinical pipeline to bridge the gap between in vitro assays and more costly screens in mammals (Giacomotto and Ségalat, 2010; Lieschke and Currie, 2007). "
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    ABSTRACT: Motor neuron disorders (MNDs) are a clinically heterogeneous group of neurological diseases characterized by progressive degeneration of motor neurons, and share some common pathological pathways. Despite remarkable advances in our understanding of these diseases, no curative treatment for MNDs exists. To better understand the pathogenesis of MNDs and to help develop new treatments, the establishment of animal models that can be studied efficiently and thoroughly is paramount. The zebrafish (Danio rerio) is increasingly becoming a valuable model for studying human diseases and in screening for potential therapeutics. In this Review, we highlight recent progress in using zebrafish to study the pathology of the most common MNDs: spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP). These studies indicate the power of zebrafish as a model to study the consequences of disease-related genes, because zebrafish homologues of human genes have conserved functions with respect to the aetiology of MNDs. Zebrafish also complement other animal models for the study of pathological mechanisms of MNDs and are particularly advantageous for the screening of compounds with therapeutic potential. We present an overview of their potential usefulness in MND drug discovery, which is just beginning and holds much promise for future therapeutic development.
    Disease Models and Mechanisms 07/2014; 7(7):799-809. DOI:10.1242/dmm.015719 · 4.97 Impact Factor
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