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1.  Outlier Kinase Expression by RNA Sequencing as Targets for Precision Therapy 
Cancer discovery  2013;3(3):280-293.
Protein kinases represent the most effective class of therapeutic targets in cancer; therefore determination of kinase aberrations is a major focus of cancer genomic studies. Here, we analyzed transcriptome sequencing data from a compendium of 482 cancer and benign samples from 25 different tissue types, and defined distinct ‘outlier kinases’ in individual breast and pancreatic cancer samples, based on highest levels of absolute and differential expression. Frequent outlier kinases in breast cancer included therapeutic targets like ERBB2 and FGFR4, distinct from MET, AKT2, and PLK2 in pancreatic cancer. Outlier kinases imparted sample-specific dependencies in various cell lines as tested by siRNA knockdown and/or pharmacologic inhibition. Outlier expression of polo-like kinases was observed in a subset of KRAS-dependent pancreatic cancer cell lines, and conferred increased sensitivity to the pan-PLK inhibitor BI 6727. Our results suggest that outlier kinases represent effective precision therapeutic targets that are readily identifiable through RNA-sequencing of tumors.
PMCID: PMC3597439  PMID: 23384775
Pancreatic Cancer; RNA-Seq; Kinases; Outlier Expression; Personalized Medicine
2.  Identification of Recurrent NAB2-STAT6 Gene Fusions in Solitary Fibrous Tumor by Integrative Sequencing 
Nature genetics  2013;45(2):180-185.
A 44-year old woman with recurrent solitary fibrous tumor (SFT)/hemangiopericytoma was enrolled in a clinical sequencing program including whole exome and transcriptome sequencing. A gene fusion of the transcriptional repressor NAB2 with the transcriptional activator STAT6 was detected. Transcriptome sequencing of 27 additional SFTs all revealed the presence of a NAB2-STAT6 gene fusion. Using RT-PCR and sequencing, we detected this fusion in 51 of 51 SFTs, indicating high levels of recurrence. Expression of NAB2-STAT6 fusion proteins was confirmed in SFT, and the predicted fusion products harbor the early growth response (EGR)-binding domain of NAB2 fused to the activation domain of STAT6. Overexpression of the NAB2-STAT6 gene fusion induced proliferation in cultured cells and activated EGR-responsive genes. These studies establish NAB2-STAT6 as the defining driver mutation of SFT and provide an example of how neoplasia can be initiated by converting a transcriptional repressor of mitogenic pathways into a transcriptional activator.
PMCID: PMC3654808  PMID: 23313952
3.  Expressed Pseudogenes in the Transcriptional Landscape of Human Cancers 
Cell  2012;149(7):1622-1634.
Pseudogene transcripts can provide a novel tier of gene regulation through generation of endogenous siRNAs or miRNA-binding sites. Characterization of pseudogene expression, however, has remained confined to anecdotal observations due to analytical challenges posed by the extremely close sequence similarity with their counterpart coding genes. Here, we describe a systematic analysis of pseudogene “transcription” from an RNA-Seq resource of 293 samples, representing 13 cancer and normal tissue types, and observe a surprisingly prevalent, genome-wide expression of pseudogenes that could be categorized as ubiquitously expressed or lineage and/or cancer specific. Further, we explore disease subtype specificity and functions of selected expressed pseudogenes. Taken together, we provide evidence that transcribed pseudogenes are a significant contributor to the transcriptional landscape of cells and are positioned to play significant roles in cellular differentiation and cancer progression, especially in light of the recently described ceRNA networks. Our work provides a transcriptome resource that enables high-throughput analyses of pseudogene expression.
PMCID: PMC3597446  PMID: 22726445
4.  The Mutational Landscape of Lethal Castrate Resistant Prostate Cancer 
Nature  2012;487(7406):239-243.
Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains/losses, including ETS gene fusions, PTEN loss and androgen receptor (AR) amplification, that drive prostate cancer development and progression to lethal, metastatic castrate resistant prostate cancer (CRPC)1. As less is known about the role of mutations2–4, here we sequenced the exomes of 50 lethal, heavily-pretreated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment naïve, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPC (2.00/Mb) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1, which define a subtype of ETS fusionnegative prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in ~1/3 of CRPCs (commonly through TMPRSS2:ERG fusions), is also deregulated through mutation. Further, we identified recurrent mutations in multiple chromatin/histone modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with AR, which is required for AR-mediated signaling. We also identified novel recurrent mutations in the AR collaborating factor FOXA1, which is mutated in 5 of 147 (3.4%) prostate cancers (both untreated localized prostate cancer and CRPC), and showed that mutated FOXA1 represses androgen signaling and increases tumour growth. Proteins that physically interact with AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX, and ASXL1 were found to be mutated in CRPC. In summary, we describe the mutational landscape of a heavily treated metastatic cancer, identify novel mechanisms of AR signaling deregulated in prostate cancer, and prioritize candidates for future study.
PMCID: PMC3396711  PMID: 22722839
5.  Personalized Oncology Through Integrative High-Throughput Sequencing: A Pilot Study 
Science translational medicine  2011;3(111):111ra121.
Individual cancers harbor a set of genetic aberrations that can be informative for identifying rational therapies currently available or in clinical trials. We implemented a pilot study to explore the practical challenges of applying high-throughput sequencing in clinical oncology. We enrolled patients with advanced or refractory cancer who were eligible for clinical trials. For each patient, we performed whole-genome sequencing of the tumor, targeted whole-exome sequencing of tumor and normal DNA, and transcriptome sequencing (RNA-Seq) of the tumor to identify potentially informative mutations in a clinically relevant time frame of 3 to 4 weeks. With this approach, we detected several classes of cancer mutations including structural rearrangements, copy number alterations, point mutations, and gene expression alterations. A multidisciplinary Sequencing Tumor Board (STB) deliberated on the clinical interpretation of the sequencing results obtained. We tested our sequencing strategy on human prostate cancer xenografts. Next, we enrolled two patients into the clinical protocol and were able to review the results at our STB within 24 days of biopsy. The first patient had metastatic colorectal cancer in which we identified somatic point mutations in NRAS, TP53, AURKA, FAS, and MYH11, plus amplification and overexpression of cyclin-dependent kinase 8 (CDK8). The second patient had malignant melanoma, in which we identified a somatic point mutation in HRAS and a structural rearrangement affecting CDKN2C. The STB identified the CDK8 amplification and Ras mutation as providing a rationale for clinical trials with CDK inhibitors or MEK (mitogenactivated or extracellular signal–regulated protein kinase kinase) and PI3K (phosphatidylinositol 3-kinase) inhibitors, respectively. Integrative high-throughput sequencing of patients with advanced cancer generates a comprehensive, individual mutational landscape to facilitate biomarker-driven clinical trials in oncology.
PMCID: PMC3476478  PMID: 22133722
6.  Functionally Recurrent Rearrangements of the MAST Kinase and Notch Gene Families in Breast Cancer 
Nature Medicine  2011;17(12):1646-1651.
Breast cancer is a heterogeneous disease, exhibiting a wide range of molecular aberrations and clinical outcomes. Here we employed paired-end transcriptome sequencing to explore the landscape of gene fusions in a panel of breast cancer cell lines and tissues. We observed that individual breast cancers harbor an array of expressed gene fusions. We identified two classes of recurrent gene rearrangements involving microtubule associated serine-threonine kinase (MAST) and Notch family genes. Both MAST and Notch family gene fusions exerted significant phenotypic effects in breast epithelial cells. Breast cancer lines harboring Notch gene rearrangements are uniquely sensitive to inhibition of Notch signaling, and over-expression of MAST1 or MAST2 gene fusions had a proliferative effect both in vitro and in vivo. These findings illustrate that recurrent gene rearrangements play significant roles in subsets of carcinomas and suggest that transcriptome sequencing may serve to identify patients with rare, actionable gene fusions.
PMCID: PMC3233654  PMID: 22101766
7.  Detection of Somatic Copy Number Alterations in Cancer Using Targeted Exome Capture Sequencing12 
Neoplasia (New York, N.Y.)  2011;13(11):1019-1025.
The research community at large is expending considerable resources to sequence the coding region of the genomes of tumors and other human diseases using targeted exome capture (i.e., “whole exome sequencing”). The primary goal of targeted exome sequencing is to identify nonsynonymous mutations that potentially have functional consequences. Here, we demonstrate that whole-exome sequencing data can also be analyzed for comprehensively monitoring somatic copy number alterations (CNAs) by benchmarking the technique against conventional array CGH. A series of 17 matched tumor and normal tissues from patients with metastatic castrate-resistant prostate cancer was used for this assessment. We show that targeted exome sequencing reliably identifies CNAs that are common in advanced prostate cancer, such as androgen receptor (AR) gain and PTEN loss. Taken together, these data suggest that targeted exome sequencing data can be effectively leveraged for the detection of somatic CNAs in cancer.
PMCID: PMC3223606  PMID: 22131877
8.  Marek's Disease Virus (MDV) Encodes an Interleukin-8 Homolog (vIL-8): Characterization of the vIL-8 Protein and a vIL-8 Deletion Mutant MDV† 
Journal of Virology  2001;75(11):5159-5173.
Chemokines induce chemotaxis, cell migration, and inflammatory responses. We report the identification of an interleukin-8 (IL-8) homolog, termed vIL-8, encoded within the genome of Marek's disease virus (MDV). The 134-amino-acid vIL-8 shares closest homology to mammalian and avian IL-8, molecules representing the prototype CXC chemokine. The gene for vIL-8 consists of three exons which map to the BamHI-L fragment within the repeats flanking the unique long region of the MDV genome. A 0.7-kb transcript encoding vIL-8 was detected in an n-butyrate-treated, MDV-transformed T-lymphoblastoid cell line, MSB-1. This induction is essentially abolished by cycloheximide and herpesvirus DNA polymerase inhibitor phosphonoacetate, indicating that vIL-8 is expressed with true late (γ2) kinetics. Baculovirus-expressed vIL-8 was found to be secreted into the medium and shown to be functional as a chemoattractant for chicken peripheral blood mononuclear cells but not for heterophils. To characterize the function of vIL-8 with respect to MDV infection in vivo, a recombinant MDV was constructed with a deletion of all three exons and a soluble-modified green fluorescent protein (smGFP) expression cassette inserted at the site of deletion. In two in vivo experiments, the vIL-8 deletion mutant (RB1BvIL-8ΔsmGFP) showed a decreased level of lytic infection in comparison to its parent virus, an equal-passage-level parent virus, and to another recombinant MDV containing the insertion of a GFP expression cassette at the nonessential US2 gene. RB1BvIL-8ΔsmGFP retained oncogenicity, albeit at a greatly reduced level. Nonetheless, we have been able to establish a lymphoblastoid cell line from an RB1BvIL-8ΔsmGFP-induced ovarian lymphoma (MDCC-UA20) and verify the presence of a latent MDV genome lacking vIL-8. Taken together, these data describe the identification and characterization of a chemokine homolog encoded within the MDV genome that is dispensable for transformation but may affect the level of MDV in vivo lytic infection.
PMCID: PMC114921  PMID: 11333897
9.  Kaposi’s Sarcoma-Associated Herpesvirus Encodes a bZIP Protein with Homology to BZLF1 of Epstein-Barr Virus 
Journal of Virology  1999;73(3):1909-1917.
Kaposi’s sarcoma-associated herpesvirus (KSHV) is a recently discovered human gamma herpesvirus strongly implicated in AIDS-related neoplasms. We report here the identification in the KSHV genome of a gene for a protein designated K-bZIP and belonging to the basic-leucine zipper (bZIP) family of transcription factors. K-bZIP shows significant homology to BZLF1, which plays a key role in the replication and reactivation of Epstein-Barr virus. K-bZIP is a homodimerizing protein of 237 amino acids with a prototypic bZIP domain at the C terminus. The N-terminal portion of K-bZIP is derived from the K8 open reading frame which, through in-frame splicing, adjoins the ZIP domain. This structure was revealed by rapid analysis of cDNA ends, followed by cloning of the entire cDNA. A 1.35-kb transcript encoding K-bZIP was detected in BCBL-1 cells treated with 12-O-tetradecanoylphorbol-13-acetate. The synthesis of this transcript was blocked by the protein synthesis inhibitor cycloheximide but not by the viral DNA synthesis inhibitor phosphonoacetate, a result which classifies it as an early lytic gene. RNase protection analysis further mapped the major transcription start site for the 1.35-kb K-bZIP mRNA and identified two other splice variants which encode proteins with the N-terminal portion of K-bZIP but lacking the C-terminal ZIP domain. Full-length K-bZIP forms dimers with itself, and the C terminus encompassing the ZIP domain is required for this process. Our studies set the stage for understanding the role of K-bZIP in the replication and reactivation of the KSHV genome.
PMCID: PMC104432  PMID: 9971770
10.  Identification of Targetable FGFR Gene Fusions in Diverse Cancers 
Cancer discovery  2013;3(6):636-647.
Through a prospective clinical sequencing program for advanced cancers, four index cases were identified which harbor gene rearrangements of FGFR2 including patients with cholangiocarcinoma, breast cancer, and prostate cancer. After extending our assessment of FGFR rearrangements across multiple tumor cohorts, we identified additional FGFR gene fusions with intact kinase domains in lung squamous cell cancer, bladder cancer, thyroid cancer, oral cancer, glioblastoma, and head and neck squamous cell cancer. All FGFR fusion partners tested exhibit oligomerization capability, suggesting a shared mode of kinase activation. Overexpression of FGFR fusion proteins induced cell proliferation. Two bladder cancer cell lines that harbor FGFR3 fusion proteins exhibited enhanced susceptibility to pharmacologic inhibition in vitro and in vivo. Due to the combinatorial possibilities of FGFR family fusion to a variety of oligomerization partners, clinical sequencing efforts which incorporate transcriptome analysis for gene fusions are poised to identify rare, targetable FGFR fusions across diverse cancer types.
PMCID: PMC3694764  PMID: 23558953
MI-ONCOSEQ; integrative clinical sequencing; FGFR fusions; driver mutations; therapeutic targets

Results 1-10 (10)