[Show abstract][Hide abstract] ABSTRACT: Cell fate acquisition is heavily influenced by direct interactions between master regulators and tissue-specific enhancers. However, it remains unclear how lineage-specifying transcription factors, which are often expressed in both progenitor and mature cell populations, influence cell differentiation. Using in vivo mouse liver development as a model, we identified thousands of enhancers that are bound by the master regulators HNF4A and FOXA2 in a differentiation-dependent manner, subject to chromatin remodeling, and associated with differentially expressed target genes. Enhancers exclusively occupied in the embryo were found to be responsive to developmentally regulated TEAD2 and coactivator YAP1. Our data suggest that Hippo signaling may affect hepatocyte differentiation by influencing HNF4A and FOXA2 interactions with temporal enhancers. In summary, transcription factor-enhancer interactions are not only tissue specific but also differentiation dependent, which is an important consideration for researchers studying cancer biology or mammalian development and/or using transformed cell lines.
[Show abstract][Hide abstract] ABSTRACT: Extraordinary advancements in sequencing technology have made what was once a decade-long multi-institutional endeavor into a methodology with the potential for practical use in a clinical setting. We therefore set out to examine the clinical value of next-generation sequencing by enrolling patients with incurable or ambiguous tumors into the Personalized OncoGenomics initiative at the British Columbia Cancer Agency whereby whole genome and transcriptome analyses of tumor/normal tissue pairs are completed with the ultimate goal of directing therapeutics. First, we established that the sequencing, analysis, and communication with oncologists could be completed in less than 5 weeks. Second, we found that cancer diagnostics is an area that can greatly benefit from the comprehensiveness of a whole genome analysis. Here, we present a scenario in which a metastasized sphenoid mass, which was initially thought of as an undifferentiated squamous cell carcinoma, was rediagnosed as an SMARCB1-negative rhabdoid tumor based on the newly acquired finding of homozygous SMARCB1 deletion. The new diagnosis led to a change in chemotherapy and a complete nodal response in the patient. This study also provides additional insight into the mutational landscape of an adult SMARCB1-negative tumor that has not been explored at a whole genome and transcriptome level.
[Show abstract][Hide abstract] ABSTRACT: Cellular barcoding offers a powerful approach to characterize the growth and differentiation activity of large numbers of cotransplanted stem cells. Here, we describe a lentiviral genomic-barcoding and analysis strategy and its use to compare the clonal outputs of transplants of purified mouse and human basal mammary epithelial cells. We found that both sources of transplanted cells produced many bilineage mammary epithelial clones in primary recipients, although primary clones containing only one detectable mammary lineage were also common. Interestingly, regardless of the species of origin, many clones evident in secondary recipients were not detected in the primary hosts, and others that were changed from appearing luminal-restricted to appearing bilineage. This barcoding methodology has thus revealed conservation between mice and humans of a previously unknown diversity in the growth and differentiation activities of their basal mammary epithelial cells stimulated to grow in transplanted hosts.
[Show abstract][Hide abstract] ABSTRACT: Tumor recurrence is a leading cause of cancer mortality. Therapies for recurrent disease may fail, at least in part, because the genomic alterations driving the growth of recurrences are distinct from those in the initial tumor. To explore this hypothesis, we sequenced the exomes of 23 initial low-grade gliomas and recurrent tumors resected from the same patients. In 43% of cases, at least half of the mutations in the initial tumor were undetected at recurrence, including driver mutations in TP53, ATRX, SMARCA4, and BRAF, suggesting recurrent tumors are often seeded by cells derived from the initial tumor at a very early stage of their evolution. Notably, tumors from 6 of 10 patients treated with the chemotherapeutic drug temozolomide (TMZ) followed an alternative evolutionary path to high-grade glioma. At recurrence, these tumors were hypermutated and harbored driver mutations in the RB and AKT-mTOR pathways that bore the signature of TMZ-induced mutagenesis.
[Show abstract][Hide abstract] ABSTRACT: Chimeric transcripts, including partial and internal tandem duplications (PTDs, ITDs) and gene fusions, are important in the detection, prognosis, and treatment of human cancers.
We describe Barnacle, a production-grade analysis tool that detects such chimeras in de novo assemblies of RNA-seq data, and supports prioritizing them for review and validation by reporting the relative coverage of co-occurring chimeric and wild-type transcripts. We demonstrate applications in large-scale disease studies, by identifying PTDs in MLL, ITDs in FLT3, and reciprocal fusions between PML and RARA, in two deeply sequenced acute myeloid leukemia (AML) RNA-seq datasets.
Our analyses of real and simulated data sets show that, with appropriate filter settings, Barnacle makes highly specific predictions for three types of chimeric transcripts that are important in a range of cancers: PTDs, ITDs, and fusions. High specificity makes manual review and validation efficient, which is necessary in large-scale disease studies. Characterizing an extended range of chimera types will help generate insights into progression, treatment, and outcomes for complex diseases.
[Show abstract][Hide abstract] ABSTRACT: Genetic changes underlying clear cell renal cell carcinoma (ccRCC) include alterations in genes controlling cellular oxygen sensing (for example, VHL) and the maintenance of chromatin states (for example, PBRM1). We surveyed more than 400 tumours using different genomic platforms and identified 19 significantly mutated genes. The PI(3)K/AKT pathway was recurrently mutated, suggesting this pathway as a potential therapeutic target. Widespread DNA hypomethylation was associated with mutation of the H3K36 methyltransferase SETD2, and integrative analysis suggested that mutations involving the SWI/SNF chromatin remodelling complex (PBRM1, ARID1A, SMARCA4) could have far-reaching effects on other pathways. Aggressive cancers demonstrated evidence of a metabolic shift, involving downregulation of genes involved in the TCA cycle, decreased AMPK and PTEN protein levels, upregulation of the pentose phosphate pathway and the glutamine transporter genes, increased acetyl-CoA carboxylase protein, and altered promoter methylation of miR-21 (also known as MIR21) and GRB10. Remodelling cellular metabolism thus constitutes a recurrent pattern in ccRCC that correlates with tumour stage and severity and offers new views on the opportunities for disease treatment.
[Show abstract][Hide abstract] ABSTRACT: High-grade serous ovarian cancer (HGSC) is characterized by poor outcome, often attributed to emergence of treatment-resistant sub-clones. We sought to measure the degree of genomic diversity within primary, untreated HGSC to examine the natural state of tumor evolution prior to therapy. We performed exome sequencing, copy number analysis, targeted amplicon deep sequencing and gene expression profiling on thirty-one spatially and temporally separated HGSC tumor specimens (six patients) including ovarian masses, distant metastases, and fallopian tube lesions. We found widespread intra-tumoral variation in mutation, copy number, and gene expression profiles, with key driver alterations in genes present in only a subset of samples (e.g. PIK3CA, CTNNB1, NF1). On average, only 51.5% of mutations were present in every sample of a given case (range: 10.2%-91.4%), with TP53 as the only somatic mutation consistently present in all samples. Complex segmental aneuploidies, such as whole genome doubling, were present in a subset of samples from the same individual, with divergent copy number changes segregating independently of point mutation acquisition. Reconstruction of evolutionary histories showed one patient with mixed HGSC and endometrioid histology with common etiologic origin in the fallopian tube and subsequent selection of different driver mutations in the histologically distinct samples. In this patient, we observed mixed cell populations in the early fallopian tube lesion, indicating diversity arises at early stages of tumorigenesis. Our results reveal that HGSC exhibit highly individual evolutionary trajectories and diverse genomic tapestries prior to therapy, exposing an essential biological characteristic to inform future design of personalized therapeutic solutions and investigation of drug resistance mechanisms.
The Journal of Pathology 06/2013; · 7.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are recently discovered small RNA molecules that regulate developmental processes, such as proliferation, differentiation, and apoptosis; however, the identity of miRNAs and their functions during liver development are largely unknown. Here we investigated the miRNA and gene expression profiles for embryonic day (E)8.5 endoderm, E14.5 Dlk1+ liver cells (hepatoblasts), and adult liver by employing Illumina sequencing. We found that miRNAs were abundantly expressed at all three stages. Using K-means clustering analysis, 13 miRNA clusters with distinct temporal expression patterns were identified. mir302b, an endoderm-enriched miRNA, was identified as an miRNA whose predicted targets are expressed highly in E14.5 hepatoblasts but low in the endoderm. We validated the expression of mir302b in the endoderm by whole-mount in situ hybridization. Interestingly, mir20a, the most highly expressed miRNA in the endoderm library, was also predicted to regulate some of the same targets as mir302b. We found that through targeting Tgfbr2, mir302b and mir20a are able to regulate transforming growth factor beta (TGFβ) signal transduction. Moreover, mir302b can repress liver markers in an embryonic stem cell differentiation model. Collectively, we uncovered dynamic patterns of individual miRNAs during liver development, as well as miRNA networks that could be essential for the specification and differentiation of liver progenitors. (HEPATOLOGY 2013)
[Show abstract][Hide abstract] ABSTRACT: White spruce (Picea glauca) is a dominant conifer of the boreal forests of North America, and providing genomics resources for this commercially valuable tree will help improve forest management and conservation efforts. Sequencing and assembling the large and highly repetitive spruce genome though pushes the boundaries of the current technology. Here, we describe a whole-genome shotgun sequencing strategy using two Illumina sequencing platforms and an assembly approach using the ABySS software. We report a 20.8 giga base pairs draft genome in 4.9 million scaffolds, with a scaffold N50 of 20 356 bp. We demonstrate how recent improvements in the sequencing technology, especially increasing read lengths and paired end reads from longer fragments have a major impact on the assembly contiguity. We also note that scalable bioinformatics tools are instrumental in providing rapid draft assemblies. AVAILABILITY: The Picea glauca genome sequencing and assembly data are available through NCBI (Accession#: ALWZ0100000000 PID: PRJNA83435). http://www.ncbi.nlm.nih.gov/bioproject/83435. CONTACT: email@example.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) show differential expression across breast cancer subtypes, and have both oncogenic and tumour-suppressive roles. Here we report the miRNA expression profiles of 1,302 breast tumours with matching detailed clinical annotation, long-term follow-up and genomic and messenger RNA expression data. This provides a comprehensive overview of the quantity, distribution and variation of the miRNA population and provides information on the extent to which genomic, transcriptional and post-transcriptional events contribute to miRNA expression architecture, suggesting an important role for post-transcriptional regulation. The key clinical parameters and cellular pathways related to the miRNA landscape are characterized, revealing context-dependent interactions, for example with regards to cell adhesion and Wnt signalling. Notably, only prognostic miRNA signatures derived from breast tumours devoid of somatic copy-number aberrations (CNA-devoid) are consistently prognostic across several other subtypes and can be validated in external cohorts. We then use a data-driven approach to seek the effects of miRNAs associated with differential co-expression of mRNAs, and find that miRNAs act as modulators of mRNA-mRNA interactions rather than as on-off molecular switches. We demonstrate such an important modulatory role for miRNAs in the biology of CNA-devoid breast cancers, a common subtype in which the immune response is prominent. These findings represent a new framework for studying the biology of miRNAs in human breast cancer.
[Show abstract][Hide abstract] ABSTRACT: Parathyroid carcinoma is a rare endocrine malignancy with an estimated incidence of less than 1 per million population. Excessive secretion of parathyroid hormone, extremely high serum calcium level and the deleterious effects of hypercalcemia are the clinical manifestations of the disease. Up to 60% of patients develop multiple disease recurrences and although long-term survival is possible with palliative surgery, permanent remission is rarely achieved. Molecular drivers of sporadic parathyroid carcinoma have remained largely unknown. Previous studies, mostly based on familial cases of the disease, suggested potential roles for the tumor suppressor MEN1 and proto-oncogene RET in benign parathyroid tumorigenesis while the tumor suppressor HRPT2 and proto-oncogene CCND1 may also act as drivers in parathyroid cancer. Here, we report the complete genomic analysis of a sporadic and recurring parathyroid carcinoma. Mutational landscapes of the primary and recurrent tumor specimens were analyzed using high throughput sequencing technologies. Such molecular profiling allowed for identification of somatic mutations never previously identified in this malignancy. These included single nucleotide point mutations in well characterized cancer genes such as mTOR, MLL2, CDKN2C and PIK3CA. Comparison of acquired mutations in patient-matched primary and recurrent tumors revealed loss of PIK3CA activating mutation during the evolution of the tumor from the primary to the recurrence. Structural variations leading to gene fusions and regions of copy loss and gain were identified at a single-base resolution. Loss of short arm of chromosome 1 along with somatic mis-sense and truncating mutations in CDKN2C and THRAP3, respectively, provide new evidence for the potential role of these genes as tumor suppressors in parathyroid cancer. The key somatic mutations indentified in this study can serve as novel diagnostic markers as well as therapeutic targets.
The Journal of Pathology 04/2013; · 7.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
The mountain pine beetle, Dendroctonus ponderosae Hopkins, is the most serious insect pest of western North American pine forests. A recent outbreak has destroyed more than 15 million hectares of pine forests with major environmental impacts on forest health, and economic impacts on the forest industry. The outbreak has in part been driven by climate change and will contribute to increased carbon emissions through decaying forests.
We developed a mountain pine beetle genome sequence resource to better understand the unique aspects of this beetle’s biology. A draft de novo genome sequence was assembled from paired-end, short-read sequences from an individual field-collected male pupa and scaffolded using mate-paired, short-read genomic sequences from pooled field-collected pupae, paired-end short-insert whole transcriptome shotgun sequencing reads of mRNA from adult beetle tissues, and paired-end Sanger EST sequences from various life stages. We describe the cytochrome P450, glutathione-S- transferase, and plant cell wall degrading enzyme gene families important to mountain pine beetle survival in their harsh and nutrient-poor host environment, and examine genome-wide SNP variation. A horizontally transferred bacterial sucrose-6-phosphate hydrolase was evident in the genome and its tissue-specific transcription suggests a functional role for the species.
Despite Coleoptera being the largest insect order with over 400,000 described species, including many agricultural and forest pest species, this is only the second genome
sequence reported in Coleoptera and will provide an important resource for the Curculionoidea and other insects.
[Show abstract][Hide abstract] ABSTRACT: Diffuse large B-cell lymphoma (DLBCL) accounts for 30% to 40% of newly diagnosed lymphomas and has an overall cure rate of approximately 60%. Previously, we observed FOXO1 mutations in NHL patient samples. To explore the effects of FOXO1 mutations, we assessed FOXO1 status in 279 DLBCL patient samples and 22 DLBCL-derived cell lines. FOXO1 mutations were found in 8.6% (24/279) of DLBCL cases. 92.3% (24/26) of mutations were in the first exon, 46.2% (12/26) were recurrent mutations affecting the N-terminal region and another 38.5% (10/26) affected the Forkhead DNA binding domain. Recurrent mutations in the N-terminal region resulted in diminished T24 phosphorylation, loss of interaction with 14-3-3, and nuclear retention. FOXO1 mutation was associated with decreased overall survival in patients treated with R-CHOP (P = 0.037), independent of cell-of-origin and the Revised International Prognostic Index. This association was particularly evident (P = 0.003) in patients in the low-risk R-IPI categories. The independent relationship of mutations in FOXO1 to survival, transcending the prognostic influence of the R-IPI and COO, indicates that FOXO1 mutation is a novel prognostic factor that plays an important role in DLBCL pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: Neuroblastoma is a malignancy of the developing sympathetic nervous system that often presents with widespread metastatic disease, resulting in survival rates of less than 50%. To determine the spectrum of somatic mutation in high-risk neuroblastoma, we studied 240 affected individuals (cases) using a combination of whole-exome, genome and transcriptome sequencing as part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. Here we report a low median exonic mutation frequency of 0.60 per Mb (0.48 nonsilent) and notably few recurrently mutated genes in these tumors. Genes with significant somatic mutation frequencies included ALK (9.2% of cases), PTPN11 (2.9%), ATRX (2.5%, and an additional 7.1% had focal deletions), MYCN (1.7%, causing a recurrent p.Pro44Leu alteration) and NRAS (0.83%). Rare, potentially pathogenic germline variants were significantly enriched in ALK, CHEK2, PINK1 and BARD1. The relative paucity of recurrent somatic mutations in neuroblastoma challenges current therapeutic strategies that rely on frequently altered oncogenic drivers.