Evolution in action: highlighting a role for the Agouti gene in development?

crest stem cells treated with DHODH inhibitor produced fewer and smaller colonies in a colony formation assay, with no effect on differentiation or survival, nor biased toward particular neural crest lineages. This suggests that DHODH inhibition specifically interferes with self-renewal of NCSCs. The morphological phenotype resulting from DHODH inhibition during embryogenesis was noted to be similar to that of zebrafish with mutations in genes encoding transcriptional elongation factors spt5 or spt6 (Keegan et al., 2002), and this similarity was borne out by comparison of drug treatmentversus-mutant gene expression profiles. Demonstration that spt5 heterozygous embryos, as well as a hypomorphic spt5 allele, are sensitized to low doses of Leflunomide relative to wild-type siblings indicated a genetic interaction between DHODH and spt5. Together these experiments suggested that Leflunomide works at the level of transcriptional elongation, which was further supported by quantitative RT-PCR comparison between 5¢ and 3¢ ends of target transcripts in cultured melanoma cells. However, the definitive test was chromatin immunoprecipitation-sequencing (ChIP-seq) with RNA polymerase II, showing greater occupancy by transcription complexes near promoters versus within the body of signature genes after drug treatment, with no effect on initiation. This genome-wide approach also identified among the genes enriched in the transcriptionally-stalled subset numerous targets of c-Myc, shown recently to be integral to regulated transcriptional pausing (Rahl et al., 2010). So what effect does a compound that blocks transcriptional elongation in neural crest progenitor cells have on melanoma? White and co-workers show that the Leflunomide derivative A771726 reduces the proliferation of melanoma cells in culture, a result also produced by shRNA versus DHODH itself. When combined, the BRAF inhibitor PLX4720 (Tsai et al., 2008) and A771726 showed a cooperative effect. A771726 has a weak effect on nonmelanoma cell lines (that have wildtype BRAF) on which PLX4720 has no effect. Finally, the authors examine the effects of Leflunomide and the Leflunomide ⁄ PLX4720 combination in A375 melanoma cell xenografts into nude mice: while Leflunomide was as effective as PLX4720 on its own in suppressing tumor growth, the combination was yet more effective, with further reduced tumor growth and 40% of animals showing complete tumor regression. These intriguing findings of White and colleagues add to data highlighting the newfound importance of elongation factors in development and in cancer. This includes evidence that CHD7, the chromatin-remodeling factor associated with CHARGE syndrome (which has a significant neural crest component), acts as a global regulator of transcription by RNA pol II (Srinivasan et al., 2008). The reasons that the neural crest appears to be particularly sensitive to DHODH inhibition and dysregulation of elongation will be an interesting avenue of future investigation. The identification of a class of compounds including Leflunomide (a medication already approved to treat rheumatoid arthritis) as a potential therapy against melanoma shows that when properly designed and executed, small molecule screens can be ‘fishing expeditions’ of the successful variety.