Activating B-RAF(V600E) (also known as BRAF) kinase mutations occur in ~7% of human malignancies and ~60% of melanomas1. Early clinical experience with a novel class I RAF-selective inhibitor, PLX4032, demonstrated an unprecedented 80% anti-tumour response rate among patients with B-RAF(V600E)-positive melanomas, but acquired drug resistance frequently develops after initial responses2. Hypotheses for mechanisms of acquired resistance to B-RAF inhibition include secondary mutations in B-RAF(V600E), MAPK reactivation, and activation of alternative survival pathways3–5. Here we show that acquired resistance to PLX4032 develops by mutually exclusive PDGFRβ (also known as PDGFRB) upregulation or N-RAS (also known as NRAS) mutations but not through secondary mutations in B-RAF(V600E). We used PLX4032-resistant sub-lines artificially derived from B-RAF(V600E)-positive melanoma cell lines and validated key findings in PLX4032-resistant tumours and tumour-matched, short-term cultures from clinical trial patients. Induction of PDGFRβ RNA, protein and tyrosine phosphorylation emerged as a dominant feature of acquired PLX4032 resistance in a subset of melanoma sub-lines, patient-derived biopsies and short-term cultures. PDGFRβ-upregulated tumour cells have low activated RAS levels and, when treated with PLX4032, do not reactivate the MAPK pathway significantly. In another subset, high levels of activated N-RAS resulting from mutations lead to significant MAPK pathway reactivation upon PLX4032 treatment. Knockdown of PDGFRβ or N-RAS reduced growth of the respective PLX4032-resistantsubsets. Overexpression of PDGFRβ or N-RAS(Q61K) conferred PLX4032 resistance to PLX4032-sensitive parental cell lines. Importantly, MAPK reactivation predicts MEK inhibitor sensitivity. Thus, melanomas escape B-RAF(V600E) targeting not through secondary B-RAF(V600E) mutations but via receptor tyrosine kinase (RTK)-mediated activation of alternative survival pathway(s) or activated RAS-mediated reactivation of the MAPK pathway, suggesting additional therapeutic strategies.
Cytosine DNA methylation is important in regulating gene expression and in silencing transposons and other repetitive sequences 1, 2 . Recent genomic studies in Arabidopsis have revealed that many endogenous genes are methylated either within their promoters or within their transcribed regions, and that gene methylation is highly correlated with transcription levels 3-5 . However, plants have different types of methylation controlled by different genetic pathways, and detailed information on the methylation status of each cytosine in any given genome is lacking. To this end, we generated a map at single base pair resolution of methylated cytosines for Arabidopsis, by combining bisulfite treatment of genomic DNA with ultra-high-throughput sequencing using the Illumina 1G Genome Analyzer and Solexa sequencing technology 6 . This approach, termed BS-Seq, unlike previous microarray-based methods, allows one to sensitively measure cytosine methylation on a genomewide scale within specific sequence contexts. We describe methylation on previously inaccessible components of the genome along with an analysis of the DNA methylation sequence composition and distribution. We also describe the effect of various DNA methylation mutants on genome-wide methylation patterns, and demonstrate that our newly developed library construction and computational methods can be applied to large genomes such as mouse.To generate a DNA methylation map at one nucleotide resolution across the genome, we adapted the Illumina 1G Genome Analyzer using Solexa sequencing technology (Illumina GA) for shotgun sequencing of bisulfite-treated Arabidopsis genomic DNA. Sodium bisulfite converts unmethylated cytosines to uracils, but 5-methylcytosines remain unconverted. Hence, Author Information. 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. Correspondence and requests for materials should be addressed to S.E.J. (jacobsen@ucla.edu) or M.P. (matteop@mcdb.ucla.edu). 6 These authors contributed equally to this work. 7 Present address: Department of Plant Biology, University of Georgia, Athens, Georgia 30602, USA. Author Contributions. S.J.C. developed computational methods for mapping and basecalling. S.F. designed and created DNA libraries and performed all molecular biology experiments. S.F., Z.C., B.M., and S.F.N. sequenced libraries. M.P., S.J.C., S.F., and S.E.J. analyzed data. S.E.J. and M.P. designed and directed the study. X.Z., C.D.H., and S.P. assisted in the design of experiments. S.F. and S.J.C. wrote the manuscript. HHMI Author ManuscriptHHMI Author Manuscript HHMI Author Manuscript after polymerase chain reaction amplification, unmethylated cytosines appear as thymines and methylated cytosines appear as cytosines 7 . We created genomic DNA libraries after bisulfite conversion and produced ~3.8 billion nucleotides of high quality sequence which successfully mapped to the...
Schizophrenia is a devastating neurodevelopmental disorder whose genetic influences remain elusive. We hypothesize that individually rare structural variants contribute to the illness. Microdeletions and microduplications >100 kilobases were identified by microarray comparative genomic hybridization of genomic DNA from 150 individuals with schizophrenia and 268 ancestry-matched controls. All variants were validated by high-resolution platforms. Novel deletions and duplications of genes were present in 5% of controls versus 15% of cases and 20% of young-onset cases, both highly significant differences. The association was independently replicated in patients with childhood-onset schizophrenia as compared with their parents. Mutations in cases disrupted genes disproportionately from signaling networks controlling neurodevelopment, including neuregulin and glutamate pathways. These results suggest that multiple, individually rare mutations altering genes in neurodevelopmental pathways contribute to schizophrenia.
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