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The use of routine monitoring of donor-derived cell-free DNA (dd-cfDNA) after kidney transplant may allow clinicians to identify subclinical allograft injury and intervene prior to development of clinically evident graft injury. To evaluate this, data from 1092 kidney transplant recipients monitored for dd-cfDNA over a three-year period was analyzed to assess the association of dd-cfDNA with histologic evidence of allograft rejection. Elevation of dd-cfDNA (0.5% or more) was significantly correlated with clinical and subclinical allograft rejection. dd-cfDNA values of 0.5% or more were associated with a nearly three-fold increase in risk development of de novo donor-specific antibodies (hazard ratio 2.71) and were determined to be elevated a median of 91 days (interquartile range of 30-125 days) ahead of donor specific antibody identification. Persistently elevated dd-cfDNA (more than one result above the 0.5% threshold) predicted over a 25% decline in the estimated glomerular filtration rate over three years (hazard ratio 1.97). Therefore, routine monitoring of dd-cfDNA allowed early identification of clinically important graft injury. Biomarker monitoring complemented histology and traditional laboratory surveillance strategies as a prognostic marker and risk-stratification tool post-transplant. Thus, persistently low dd-cfDNA levels may accurately identify allograft quiescence or absence of injury, paving the way for personalization of immunosuppression trials.

BACKGROUND: We previously reported that low 21 gene Recurrence Scores (RS) identify patients with ER-positive, lymph node-positive breast cancer who may not benefit from anthracycline-based adjuvant chemotherapy added to tamoxifen (SWOG-8814A NCI correlative science study; Albain et al. Lancet Oncol 2010). New exploratory and comprehensive quantitative analyses now permit whole transcriptome NGS on residual RNA extracted from FFPE blocks 12-18 years post-fixation. Herein, we report methodology details and feasibility results (see companion abstract, Albain et al., for clinical outcomes correlations). METHODS and RESULTS: Sequencing was carried out in Illumina HiSeq 2000 instruments, yielding 4.2 trillion data points. Messenger RNA expression was quantified using 3rd quartile normalization. Both Library (RNA) and Sequencing Standards showed high quality coverage as measured by median uniquely mapped reads over a 13 month window (168M and 182M, respectively, including duplicate reads). The median absolute deviation (MAD) of the relative log expression (RLE) of mapped reads for the Library and Sequencing Standards was 0.22 and 0.05, respectively. The Library Standard variation was greater than the Sequencing Standard, as library preparation was manual. Of 360 patient samples with sufficient RNA (≥ 100 ng total RNA), 354 (98.3%) were successfully sequenced and included in the final analysis data set. Average library yield was 39 ng/μL. Only 5 libraries failed yield requirements and one library failed expression quality metrics. The median insert length was 120 bp with the first and third quartiles 93 and 152 bp, respectively. After removal of duplicate reads, 82% of reads were uniquely mapped, and the median library size was 8.95M (number of unique mapped reads). Sequences with counts CONCLUSIONS: High quality whole transcriptome NGS is feasible from decades-old clinical trial FFPE specimens that have not been stored in any special fashion. Controlled laboratory, bioinformatics and biostatistics methods, with inclusion of appropriate process controls, ensure robustness and reliability of the NGS process. This in turn results in the discovery and validation of biologically and clinically relevant variations from prior landmark clinical trials. SUPPORT: NCI CA 180888, CA180819, CA180821, CA180820, CA180863; in part, Genomic Health, Inc. Citation Format: Cherbavaz DB, Hayes DF, Qu K, Crager MR, Barlow WR, Goddard AD, Beasley EM, Jeong J, Collin F, Liu M-L, Rae JM, Ravdin PM, Tripathy D, Gralow JR, Livingston RB, Osborne CK, Ingle JN, Pritchard KI, Davidson NE, Carey LA, Sing AP, Baehner FL, Hortobagyi GN, Shak S, Albain KS. Successful whole transcriptome analysis of 25-year-old breast tumor samples from the phase III trial SWOG-8814 by next generation sequencing (NGS): Standardized analytical methods for exploratory and validation studies. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-07-01.

Background: RNA biomarkers discovered by RT-PCR-based gene expression profiling of archival formalin-fixed paraffin-embedded (FFPE) tissue are the basis for very precise and sensitive clinical diagnostic tests, such as the 21 gene Oncotype DX® breast cancer assay. Both inherent limits of technical scalability and the small amounts of patient FFPE RNA available place practical constraints on the number of transcripts that can be interrogated by RT-PCR. We developed new methods for RNA profiling through massively parallel “next generation” sequencing (RNA-Seq) of archival FFPE specimens. We report here the technical performance of this methodology and compare the results to RT-PCR results obtained in one of the studies that were carried out to develop the 21 gene assay. Methods: RNA was extracted in 2002 from 136 invasive breast tumors that were formalin-fixed and paraffin-embedded between 1990 and 1997. RNA-Seq was carried out using minor modifications to methods we have reported previously (Sinicropi et al., Advances in Genome Biology and Technology Conference, p. 170, 2010 and p. 198, 2011). Briefly, 0.1 mg of total RNA was selectively depleted of ribosomal RNA and sequencing libraries were prepared using a modification of the ScriptSeq™ kit from Epicentre. The libraries were sequenced on an Illumina HiSeq 2000 instrument with multiplexing of two libraries per lane for 50 cycles in one direction. The resulting FASTQ sequences were mapped to version hg19 of the human genome using the Illumina CASAVA pipeline. The total number of sequences (reads) that uniquely mapped to all exons of each RefSeq entry was used for quantification of expression levels. Results: On average, there were 43 million reads per sample (range 31 - 58 million; SD=4.6 million) of which 69% uniquely mapped to the human genome. Ribosomal RNA was effectively removed and accounted for Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD03-09.

Aurora kinases play a key role in mitotic progression. Over-expression of Aurora kinases is found in several human cancers and correlated with histological malignancy and clinical outcomes. Therefore, Aurora kinase inhibitors should be useful in the treatment of cancers. Cell-based screening methods have an advantage over biochemical approaches because hits can be optimized to inhibit targets in the proper intracellular context. We developed a novel Aurora kinase inhibitor R763/AS703569 using an image-based phenotypic screen. The anti-proliferative effect was examined in a panel of tumor cell lines and primary cells. The efficacy was determined in a broad panel of xenograft models. R763/AS703569 inhibits Aurora kinases, along with a limited number of other kinases including FMS-related tyrosine kinase 3 (FLT3), and has potent anti-proliferative activity against many cell types accompanying unique phenotypic changes such as enlarged cell size, endoreduplication and apoptosis. The endoreduplication cycle induced by R763/AS703569 was irreversible even after the compound was withdrawn from the culture. Oral administration of R763/AS703569 demonstrated marked inhibition of tumor growth in xenograft models of pancreatic, breast, colon, ovarian, and lung tumors and leukemia. An acute myeloid leukemia cell line MV4-11, which carries a FLT3 internal tandem duplication mutation, is particularly sensitive to R763/AS703569 in vivo. R763/AS703569 is a potent inhibitor of Aurora kinases and exhibited significant anti-proliferative activity against a wide range of tumor cells both in vitro and in vivo. Inhibition of Aurora kinases has the potential to be a new addition to the treatment of cancers.

Hepatitis C virus (HCV) infection is one of the major causes of viral hepatitis, with a great propensity to induce chronicity (21). Liver inflammation can persist for decades in chronic HCV infection and eventually leads to cirrhosis, end-stage liver disease, and hepatocellular carcinoma. HCV infection is a significant health care problem: it is estimated that approximately 170 million individuals are chronically infected with HCV worldwide, with ∼30,000 cases of new infection each year in the United States alone (1, 2, 46). No vaccine is currently available to prevent HCV infection. The standard treatment for HCV infection, a combination of pegylated alpha interferon (IFN-α) and ribavirin (RBV), is limited by its suboptimal response rate in a significant patient population, side effects, and affordability (11). Thus, it is critical to discover highly effective, safer therapies to improve the clinical management of HCV infection. HCV is an enveloped RNA virus belonging to the family Flaviviridae (9). HCV clinical isolates display high heterogeneity in their genomic RNA and amino acid sequences, and they are classified into six genotypes and numerous subtypes (49). It is documented that infections by different genotypes may produce different clinical outcomes and may respond differently to IFN-α-based antiviral treatment (for a review, see reference 11). Significantly, patients infected with genotype 1 viruses, which account for approximately 70% of HCV infections in the United States, exhibit poor rates of response to the IFN-α-based treatment. An ideal antiviral should, therefore, be effective against the majority, if not all, of the HCV genotypes. Upon entering the host cell, HCV releases its 9.6-kb genomic RNA into the cytoplasm, where it directs the translation of a single polyprotein of about 3,000 amino acids. The giant polyprotein is cotranslationally processed by host and viral proteases into structural proteins (core, E1, and E2) and nonstructural proteins (P7, NS2, NS3, NS4a, NS4b, NS5a, and NS5b). The mature nonstructural proteins (except P7 and NS2) and host factors assemble into membrane-associated RNA replication complexes, where a vast quantity of progeny viral RNA molecules are amplified from the incoming HCV genomic RNA (14, 18, 35). Although all the steps in the HCV life cycle can be targeted for drug discovery against HCV, the viral nonstructural proteins, specifically NS3 and NS5b, which encode well-defined enzymatic activities crucial for viral replication, are the major targets for antiviral discovery (10, 53). However, the replication of HCV viral RNA by the viral replication complex is quickly becoming another focus for drug discovery with the development of the HCV replicon system. Until the establishment of HCV replicons, the analysis of HCV replication was hampered due to the lack of a robust HCV cell culture system (5, 38). The first-generation HCV replicons are human hepatoma Huh-7 cell lines carrying engineered genotype 1b subgenomic RNA with the following genome organization: HCV 5′ nontranslated region (5′ NTR)-neomycin phosphotransferase (NPT) gene (also referred to as the neomycin resistance [Neor] gene)-encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-HCV NS3-4a-4b-5a-5b-HCV 3′ NTR. Subsequent studies have shown that the efficiency of replicon establishment can be enhanced substantially by incorporating cell culture-adaptive mutations, especially those in NS3 and NS5a (5, 26, 37, 38). The HCV replicon system has been an effective tool for studying viral RNA replication and virus-host interactions. It also serves as an important cell-based system with which to evaluate antiviral drugs and to reveal drug resistance mechanisms (for a review, see reference 4). Moreover, the HCV replicon presents a unique drug-screening system, allowing for the screening of compounds inhibiting the viral enzymes as well as other targets of the HCV RNA replication process in a cellular environment. Such screens would perhaps facilitate the discovery of inhibitors that block the functions of NS4b and NS5a or interrupt virus-host interactions, discoveries that cannot be readily achieved with biochemical screens. Several efforts have already been made to screen small-molecule compound libraries against different versions of the HCV replicon system (17, 47, 50, 55). Here we describe the development of an HCV replicon assay for high-throughput screening and the characterization of one of the heterocyclic hits from screens of a 230,000-member chemical library. We show that the lead compound of this hit scaffold is effective at inhibiting HCV replicons of different genotypes and can enhance the inhibitory activity of IFN-α in the replicon model.

Ribosomal RNAs (rRNAs) are extremely abundant, often constituting 80% to 90% of total RNA. Since rRNA sequences are often not of interest in genomic RNA sequencing experiments, rRNAs can be removed from the sample before the library preparation step, in order to prevent the majority of the library and the majority of sequencing reads from being rRNA. Removal of rRNA can be especially challenging for low quality and formalin-fixed paraffin-embedded (FFPE) RNA samples due to the fragmented nature of these RNA molecules. The NEBNext rRNA Depletion Kit (Human/Mouse/Rat) depletes both cytoplasmic (5 S rRNA, 5.8 S rRNA, 18 S rRNA, and 28 S rRNA) and mitochondrial rRNA (12 S rRNA and 16 S rRNA) from total RNA preparations from human, mouse, and rat samples. Due to the high similarity among mammalian rRNA sequences, it is likely that rRNA depletion can also be achieved for other mammals but has not been empirically tested. This product is compatible with both intact and degraded RNA (e.g., FFPE RNA). The resulting rRNA-depleted RNA is suitable for RNA-seq, random-primed cDNA synthesis, or other downstream RNA analysis applications. Regardless of the quality or amount of input RNA, this method efficiently removes rRNA, while retaining non-coding and other non-poly(A) RNAs. The NEBNext rRNA Depletion Kit thus provides a more complete picture of the transcript repertoire than oligo d(T) poly(A) mRNA enrichment methods. © 2016 by John Wiley & Sons, Inc. Keywords: NGS; RNA depletion; sequencing; formalin-fixed paraffin-embedded; FFPE; RNA; rRNA; ribosomal RNA; library preparation; transcriptome; rRNA removal

A majority of the orthopoxviruses, including the variola virus that causes the dreaded smallpox disease, encode a highly conserved 28-kDa protein with a classic RING finger sequence motif (C(3)HC(4)) at their carboxyl-terminal domains. The RING domain of p28 has been shown to be a critical determinant of viral virulence for the ectromelia virus (mousepox virus) in a murine infection model (Senkevich, T. G., Koonin, E. V., and Buller, R. M. (1994) Virology 198, 118-128). Here, we demonstrate that the p28 proteins encoded by the ectromelia virus and the variola virus possess E3 ubiquitin ligase activity in biochemical assays as well as in cultured mammalian cells. Point mutations disrupting the RING finger domain of p28 completely abolish its E3 ligase activity. In addition, p28 functions cooperatively with Ubc4 and UbcH5c, the E2 conjugating enzymes involved in 26 S proteasome degradation of protein targets. Moreover, p28 catalyzes the formation of Lys-63-linked polyubiquitin chains in the presence of Ubc13/Uev1A, a heterodimeric E2 conjugating enzyme, indicating that p28 may regulate the biological activity of its cognate viral and/or host cell target(s) by Lys-63-linked ubiquitin multimers. We thus conclude that the poxvirus p28 virulence factor is a new member of the RING finger E3 ubiquitin ligase family and has a unique polyubiquitylation activity. We propose that the E3 ligase activity of the p28 virulence factor may be targeted for therapeutic intervention against infections by the variola virus and other poxviruses.

The ubiquitin system has been implicated in the pathogenesis of numerous disease states, including oncogenesis, inflammation, viral infection, CNS disorders and metabolic dysfunction. Ubiquitin conjugation and deconjugation to substrate proteins is carried out by multiple families of proteins, each with a defined role in the enzymatic cascade. This conjugation-deconjugation system parallels the kinase-phosphatase system in that both alter protein function by the addition and removal of post-translational modifiers. Our understanding of ubiquitin biology and strategies to interfere pharmacologically with the ubiquitin regulatory machinery is progressing rapidly. In light of increased interest in ubiquitin pathways as drug targets, we review the ubiquitin enzymatic cascades, highlighting therapeutic opportunities and enzymatic mechanisms. We also discuss the challenges of targeting this class of enzymes with small molecules, as well as current approaches and progress in drug discovery.

Fusion transcripts play an important role in a variety of human cancers. But bioinformatics algorithms that use RNA-Seq data to detect fusions tend to yield high false-positive rates due to both relatively short reads from next-generation sequencing (NGS) and the repetitive elements in human genome. The primary purpose of this chapter is to discuss the design strategy of the bioinformatics methods used for detection of fusion transcripts and to compare their strengths and weaknesses, or “fit for purpose,” on RNA-Seq data from non-fixed or formalin-fixed paraffin-embedded (FFPE) tumor tissues. A large number of archival FFPE tumor tissue samples are associated with mature medical records including disease outcome; these samples offer great potential for diagnostic and therapeutic target discovery. However, the chemical treatment by formalin causes RNA degradation and base deamination, which lead to low library complexity and mapping quality. It is important that bioinformatics tools are designed to address these challenges. Here we illustrate a framework to address them, using gFuse as the example. We present results by comparing the fusion transcripts discovered from analysis of RNA from fresh and FFPE MCF-7 breast cancer cells. We also describe the application of gFuse to RNA-Seq data generated from two independent breast cancer cohorts with clinical outcomes and identify candidate fusion transcripts relevant to disease progression.

Rare cancer types are not only understudied, but are typically represented by formalin-fixed paraffin-embedded (FFPE) (rather than freshly-frozen) specimens that are suboptimal for genomic analysis. Ameloblastoma is one such rare tumor type, thought to arise from ameloblasts, the cells that deposit enamel during tooth development. Though typically benign, ameloblastomas are locally destructive to the jaw and face, and new non-surgical interventions are needed. To discover novel driver mutations and therapeutic targets, we optimized methods and performed whole-transcriptome sequencing and/or targeted exon sequencing (TruSeq Cancer Panel) of 8 FFPE cases. Identified mutations were verified, and then evaluated on a larger, independent set of 22 FFPE cases by PCR and Sanger sequencing. From the analysis, we identified recurrent somatic mutations in three key developmental or signaling pathways, including Hedgehog, fibroblast growth factor, and MAP kinase pathways. Functional interrogation of a novel Hedgehog ...

The pyridoxal-P binding sites of the two isoforms of human glutamate decarboxylase (GAD65 and GAD67) were modeled by using PROBE (a recently developed algorithm for multiple sequence alignment and database searching) to align the primary sequence of GAD with pyridoxal-P binding proteins of known structure. GAD's cofactor binding site is particularly interesting because GAD activity in the brain is controlled in part by a regulated interconversion of the apo- and holoenzymes. PROBE identified six motifs shared by the two GADs and four proteins of known structure: bacterial ornithine decarboxylase, dialkylglycine decarboxylase, aspartate aminotransferase, and tyrosine phenol-lyase. Five of the motifs corresponded to the alpha/beta elements and loops that form most of the conserved fold of the pyridoxal-P binding cleft of the four enzymes of known structure; the sixth motif corresponded to a helical element of the small domain that closes when the substrate binds. Eight residues that interact with pyridoxal-P and a ninth residue that lies at the interface of the large and small domains were also identified. Eleven additional conserved residues were identified and their functions were evaluated by examining the proteins of known structure. The key residues that interact directly with pyridoxal-P were identical in ornithine decarboxylase and the two GADs, thus allowing us to make a specific structural prediction of the cofactor binding site of GAD. The strong conservation of the cofactor binding site in GAD indicates that the highly regulated transition between apo- and holoGAD is accomplished by modifications in this basic fold rather than through a novel folding pattern.

Here we report the discovery of oncogenic mutations in the Hedgehog and mitogen-activated protein kinase (MAPK) pathways in over 80% of ameloblastomas, locally destructive odontogenic tumors of the jaw, by genomic analysis of archival material. Mutations in SMO (encoding Smoothened, SMO) are common in ameloblastomas of the maxilla, whereas BRAF mutations are predominant in tumors of the mandible. We show that a frequently occurring SMO alteration encoding p.Leu412Phe is an activating mutation and that its effect on Hedgehog-pathway activity can be inhibited by arsenic trioxide (ATO), an anti-leukemia drug approved by the US Food and Drug Administration (FDA) that is currently in clinical trials for its Hedgehog-inhibitory activity. In a similar manner, ameloblastoma cells harboring an activating BRAF mutation encoding p.Val600Glu are sensitive to the BRAF inhibitor vemurafenib. Our findings establish a new paradigm for the diagnostic classification and treatment of ameloblastomas.

We present the technical details and relevant performance aspects of a dielectric resonator‐based electron paramagnetic resonance probe for stopped‐flow kinetic studies. The major benefits of this system are: (1) It incorporates a small, high sensitivity resonator system that is insensitive to stopped‐flow induced noisy transients. (2) The resonator system is cheap, robust, and easily assembled. (3) It contains a microwave coupling scheme that provides finesse in tuning and freedom from microphonics.

To cure or contain a disease is to selectively eliminate or restrain the disorder caused by the pathogen, the human body, or both, and at the same time to minimize damage to the healthy parts. The quest of drug discovery is to identify the causes of the phenotype of the disease, and to interfere with the abnormal gene or gene product in such a manner that the disease is cured. Selectivity for just the aberrant gene products remains the critical point throughout the modern drug discovery process.

Background: Analytically and clinically validated non-invasive blood tests that quantify breast cancer burden and clinical drug response/resistance are greatly needed. Many groups have successfully detected tumor markers in blood using a variety of technologies, including next generation sequencing (NGS). We performed a comprehensive NGS study on a small number of patients to evaluate the value of global versus individual markers for the quantitation of tumor-derived cell free DNA (cfDNA) in plasma. Methods: DNA isolated from formalin-fixed primary tumor, buffy coat cells, and plasma from 2 patients with metastatic breast cancer were characterized simultaneously for copy number aberrations (CNAs) and differentially methylated regions (DMRs) using whole genome bisulfite sequencing (WBGS), and targeted sequencing-based genotyping of 346 cancer-associated single nucleotide variations (SNVs). CNA and DMR regions were identified from log normalized, GC content corrected counts and DMR data using Poisson and binomial distribution theory and false discovery rate controlling methods. Percent tumor in cfDNA was estimated from the normalized ratio (plasma: primary tumor) of CNA or DMR compared to buffy coat, aggregating over genomic regions. Sample sets from 8 non-metastatic patients were also profiled using the targeted SNV panel in order to compare SNVs between samples and estimate percent tumor cfDNA. Results: WGBS detected tumor specific alterations in each primary tumor compared to buffy coat. By analyzing the genome using 100 Kb bins, we observed over 1000 bins with detectable CNA signal and, among 56 million CpG sites, over 30,000 DMRs. As expected, 5 or fewer informative somatic SNVs were detected in each patient. Analysis of these somatic changes in plasma revealed that the tumor fraction estimated from SNV detected in cfDNA varied widely between sites originally discovered in the patient’s primary tumor. In contrast, similar estimates of tumor fraction in cfDNA were obtained using CNA and DMR profiles within each patient; both methods yielded similar estimates of over 50% in one patient and less than 10% in the other. For the patient with high tumor fraction, both CNA and DMR profiles contained examples of individual large genomic regions that displayed additional clear aberrations in the plasma compared to the original tumor, such as a striking loss of a >25 Mb region of chromosome 4. Conclusions: Although individual somatic SNV in cfDNA can be detected in metastatic disease, calculated allelic fraction based on individual SNVs varies greatly within the same patient. Measuring and integrating CNA or DMR across the genome provided more consistent and reliable estimates of tumor DNA fraction in plasma, and also revealed alterations in plasma from patients with metastatic disease that were not prominent in the primary tumor. Citation Format: Ellen M Beasley, Richard D Abramson, Gregory E Alexander, David Chan, Kristen Bradley, Francois Collin, Michael Crager, Andrew Dei Rossi, Joseph Dorado, Adam Friedman, William J Gibb, Jennie Jeong, Col Jones, C J Ku, Yan Ma, John Morlan, Kunbin Qu, Aibing Rao, Aaron Scott, Haluk Tezcan. Global quantitative measures using next-generation sequencing for breast cancer presence outperform individual tumor markers in plasma [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-02-08.

Recent compelling evidence has lead to renewed interest in the role of antibodies and immune complexes in the pathogenesis of several autoimmune disorders, such as rheumatoid arthritis. These immune complexes, consisting of autoantibodies to self-antigens, can mediate inflammatory responses largely through binding and activating the immunoglobulin Fc receptors (FcRs). Using cell-based structure activity relationships with cultured human mast cells, we have identified the small molecule R406 [ N 4-(2,2-dimethyl-3-oxo-4 H -pyrid[1,4]oxazin-6-yl)-5-fluoro- N 2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine] as a potent inhibitor of immunoglobulin E (IgE)- and IgG-mediated activation of Fc receptor signaling (EC 50 for degranulation = 56–64 nM). Here we show that the primary target for R406 is the spleen tyrosine kinase (Syk), which plays a key role in the signaling of activating Fc receptors and the B-cell receptor (BCR). R406 inhibited phosphorylation of Syk substrate linker for activation of T cells in mast cells and B-cell linker protein/SLP65 in B cells. R406 bound to the ATP binding pocket of Syk and inhibited its kinase activity as an ATP-competitive inhibitor ( K i = 30 nM). Furthermore, R406 blocked Syk-dependent FcR-mediated activation of monocytes/macrophages and neutrophils and BCR-mediated activation of B lymphocytes. R406 was selective as assessed using a large panel of Syk-independent cell-based assays representing both specific and general signaling pathways. Consistent with Syk inhibition, oral administration of R406 to mice reduced immune complex-mediated inflammation in a reverse-passive Arthus reaction and two antibody-induced arthritis models. Finally, we report a first-inhuman study showing that R406 is orally bioavailable, achieving exposures capable of inhibiting Syk-dependent IgE-mediated basophil activation. Collectively, the results show R406 potential for modulating Syk activity in human disease.

Motivation: Although there are several databases storing protein–protein interactions, most such data still exist only in the scientific literature. They are scattered in scientific literature written in natural languages, defying data mining efforts. Much time and labor have to be spent on extracting protein pathways from literature. Our aim is to develop a robust and powerful methodology to mine protein–protein interactions from biomedical texts. Results: We present a novel and robust approach for extracting protein–protein interactions from literature. Our method uses a dynamic programming algorithm to compute distinguishing patterns by aligning relevant sentences and key verbs that describe protein interactions. A matching algorithm is designed to extract the interactions between proteins. Equipped only with a dictionary of protein names, our system achieves a recall rate of 80.0% and precision rate of 80.5%. Availability: The program is available on request from the authors.

We have studied the structure and function of Human Misshapen/NIK-related kinase (MINK-1) through a combination of computational methods and experimental approaches, including (1) fold recognition and sequence-structure alignment for each structural domain using the threading program PROSPECT, (2) gene expression and protein-protein interaction analysis of yeast homologs of human MINK-1 domains, and (3) yeast two-hybrid screening for proteins that interact with human MINK-1. Our structure prediction dissects MINK-1 into four domains: a conserved N-terminal kinase domain, followed by a coiled-coil region and a proline-rich region, and a C-terminal GCK domain. Gene expression and yeast two-hybrid analysis of yeast homologs of the MINK-1 domains suggest that MINK-1 may be involved in cell-cycle progression and cytoskeletal control. Consistent with these predicted functions, our in-house yeast two-hybrid screen for proteins that interact with human MINK-1 provides strong evidence that the coiled-coil and proline-rich domains of MINK-1 participate in the regulation of cytoskeletal organization, cell-cycle control and apoptosis. A homology model of the MINK-1 kinase domain was used to screen the NCI open compound database in DOCK, and chemical compounds with pharmaceutically acceptable properties were identified. Further medicinal chemistry compound structure optimization and kinase assays are underway.

Although there have been many research projects to extract protein pathways, most such information still exists only in the scientific literature, usually written in natural languages and defying data mining efforts. We present a novel and robust approach for extracting protein-protein interactions from the literature. Our method uses a dynamic programming algorithm to compute distinguishing patterns by aligning relevant sentences and key verbs that describe protein interactions. A matching algorithm is designed to extract the interactions between proteins. Equipped only with a protein name dictionary, our system achieves a recall rate of about 80.0% and a precision rate of about 80.5%.

Nat. Genet. 46, 722–725 (2014); published online 25 May 2014; corrected after print 12 November 2014 In the version of this article initially published, the introductory paragraph mistakenly stated that ameloblasts were “cells in the tooth roots of the upper (maxilla) and lower (mandible) jaw responsible for depositing enamel during tooth development (odontogenesis).

Rare cancer types are not only understudied, but are typically represented by formalin-fixed paraffin-embedded (FFPE) (rather than freshly-frozen) specimens that are suboptimal for genomic analysis. Ameloblastoma is one such rare tumor type, thought to arise from ameloblasts, the cells that deposit enamel during tooth development. Though typically benign, ameloblastomas are locally destructive to the jaw and face, and new non-surgical interventions are needed. To discover novel driver mutations and therapeutic targets, we optimized methods and performed whole-transcriptome sequencing and/or targeted exon sequencing (TruSeq Cancer Panel) of 8 FFPE cases. Identified mutations were verified, and then evaluated on a larger, independent set of 21 FFPE cases by PCR and Sanger sequencing. From the analysis, we identified recurrent somatic mutations in three key developmental or signaling pathways, including Hedgehog, fibroblast growth factor, and MAP kinase pathways. Functional interrogation of a novel Hedgehog pathway mutation confirmed increased basal pathway activity, and defined the response profile to various pharmacologic Hedgehog inhibitors. Together, our results define new ameloblastoma drivers and nominate new molecularly-directed therapies for this rare but disfiguring disease. More generally, our findings validate a robust approach for discovering driver mutations in rare cancers. Citation Format: Andrew C. McClary, Robert T. Sweeney, Jewison Biscocho, Benjamin R. Myers, Lila Neahring, Kevin A. Kwei, Kunbin Qu, Xue Gong, Tony Ng, Carol D. Jones, Sushama Varma, Justin I. Odegaard, Brian Rubin, Megan L. Troxell, Robert J. Pelham, James L. Zehnder, Philip A. Beachy, Jonathan R. Pollack, Robert B. West. Ameloblastoma driver mutations revealed by next-generation sequencing of formalin-fixed paraffin-embedded specimens. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3436. doi:10.1158/1538-7445.AM2014-3436

The kinetics of chemically induced folding and unfolding processes in spin-labeled yeast iso-1-cytochrome c were measured by stopped-flow electron paramagnetic resonance (EPR). Stopped-flow EPR, based on a new dielectric resonator structure [Sienkiewicz, A., Qu, K.,Scholes, C. P. (1994) Rev. Sci. Instrum. 65, 68-74], gives a new temporal component to probing nanosecond molecular tumbling motions that are modulated by macromolecular processes requiring time resolution of milliseconds to seconds. The stopped-flow EPR technique presented in this work is a kinetic technique that has not been previously used with such a time resolution on spin-labeled systems, and it has the potential for application to numerous spin-labeled sites in this and other proteins. The cysteine-specific spin-label, methanethiosulfonate spin-label (MTSSL), was attached to yeast iso-1-cytochrome c at the single naturally occurring cysteine102, and the emphasis for this work was on this disulfide-attached spin-labeled prototype. This probe has the advantage of reflecting the protein tertiary fold, as shown by recent, systematic site-directed spin labeling of T4 lysozyme [Mchaourab, H. S. Lietzow, M. A., Hideg, K.,Hubbell, W. L. (1996) Biochemistry 35, 7692-7704], and protein backbone dynamics, as also shown by model peptide studies [Todd, A. P.,Millhauser, G. L. (1991) Biochemistry 30, 5515-5523]. The C-terminal cytochrome c helix where the label is attached is thought to be critical in the initial steps of protein folding and unfolding. Stopped-flow EPR resolved the monoexponential, guanidinium-induced unfolding process at pH 6.5 with an approximately 20 ms time constant; this experiment required less than 150 microL of 80 microM spin-labeled protein. We observed an approximately 50-fold decrease of this unfolding time from the 1 s range to the 20 ms time range as the guanidinium denaturant concentration was increased from 0.6 to 2.0 M. The more complex refolding kinetics of our labeled cytochrome were studied by stopped-flow EPR at pH 5.0 and 6.5. The spin probe showed a fast kinetic process compatible with the time range over which hydrogen/deuterium amide protection indicates helix formation; this process was monoexponential at pH 5.0. At pH 6.5, there was evidence of an additional slower kinetic phase resolved by stopped-flow EPR and by heme-ligation-sensitive UV-Vis that indicated a slower folding where heme misligation may be involved. Since the disulfide-attached probe has reported folding and backbone dynamics in other systems, the implication is that our kinetic experiments were directly sensing events of the C-terminal helix formation and possibly the N- and C-terminal helical interaction. The cysteine-labeled protein was also studied under equilibrium conditions to characterize probe mobility and the effect of the probe on protein thermodynamics. The difference in spin probe mobility between folded and denatured protein was marked, and in the folded protein, the motion of the probe was anisotropically restricted. The motion of the attached nitroxide in the folded protein appears to be restricted about the carbon and sulfur bonds which tether it to the cysteine. The original point of cysteine sulfur attachment is approximately 11 A from the heme iron within the C-terminal helix near its interface with the N-terminal helix, but the low-temperature EPR spin probe line width showed that the probe lies more distant (15 A) from the heme iron. By all physical evidence, the protein labeled at cysteine102 folded, but the spin probe in this prototype system perturbed packing which lowered the thermal melting temperature, the free energy of folding, the guanidinium concentration at the midpoint of the unfolding transition, the m parameter of the denaturant, and the helical CD signature. This study prepares the way for study of protein folding/unfolding kinetics using EPR spectroscopy of spin-labels placed at specific cysteine-mutated sites within

11018 Background: While several recently discovered gene fusions already play an important role in personalized cancer treatment, many cancer gene fusions remain to be discovered. Next generation sequencing has enabled identification of many rare gene fusion events in fresh or frozen solid tumors. There is a need to detect gene fusions in transcriptomes of formalin-fixed paraffin-embedded (FFPE) tumor tissue, for which there is long-term clinical outcome data. We therefore sought to develop bioinformatics methods to detect fusion transcripts in FFPE tissue and to characterize their association with clinical outcomes. Methods: RNA sequencing libraries were created and sequenced from tumor biopsy tissues (Plos One 2012 7(7): e40092) of two ER+ breast cancer cohorts consisting of 136 and 77 patients, for which clinical outcomes were available. The fusion junctions were nominated by the RNA-seq aligner GSNAP and further filtered to consider discontinuous expression patterns at exon/intron levels. Results: A total of 108 candidate fusion transcripts were detected and RT-PCR assays confirmed 89% of the top ranking fusion transcript candidates. The majority (82%) of identified fusion gene partners are listed in the COSMIC database of known cancer sequence variations. Of note, several patients expressed multiple fusion transcripts that are significantly associated with tumor progression (P

We have used RNA-seq to profile and compare normal and cancerous human breast tissue. FFPE breast specimens from a total of 24 patients, 12 normal (N) and 12 tumor (T) specimens from surgical resections, were analyzed on an Illumina's GA IIx sequencer. Whole transcriptome RNA-Seq libraries were prepared after depletion of ribosomal RNA by a protocol developed at Genomic Health Inc. (GHI). The analysis was multiplexed across two flow cells using barcoding, with two specimens per sequencing lane (1 T and 1 closely age-matched N library from a different patient). FFPE tissue archive times ranged from 10 to 13 years and they were also closely matched within each lane. To evaluate reproducibility, triplicate libraries were created from 4 of the specimens and analyzed within and across flow cells. Libraries yielded, on average, 19 million 51 bp sequences. R2 values obtained from replicate libraries prepared from the same patient RNA were > 0.9 within and between flow cells. More than 80% of known genes in the human genome were detected in all patients. Several thousand intergenic transcripts were identified by an algorithm developed at GHI. A negative binomial model with tag-wise estimates of dispersion was applied to the known genes and intergenic regions. Inter-patient count variance is generally higher in the set of intergenic sequences than in the set of gene (RefSeq) sequences. Thousands of gene (RefSeq) and intergenic sequences were found to be differentially expressed between T and N tissues. We sought to build classifiers based on flow cell #1 data that could stratify T and N tissues when applied to flow cell #2 data. Sets of genes and intergenic regions were selected for analysis based on high inter-patient count variance. Support vector machine classifiers were trained and then applied to the data from flow cell #2, and also to another GHI tumor/normal RNA-Seq study. Either a set of 100 genes (RefSeq), or a set of 70 intergenic sequences accurately distinguished tumor and normal tissues. Our results offer further evidence of the potential of RNA-Seq for discovery of biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4859. doi:10.1158/1538-7445.AM2011-4859

Molecular analysis of archival tumor specimens has accelerated the discovery of clinically useful biomarkers. RNA-seq of RNA extracted from formalin fixed, paraffin-embedded (FFPE) specimens, a potentially valuable source of biomarkers, is hampered by the presence of highly abundant ribosomal RNA (rRNA), which is difficult to remove due to extensive RNA degradation. Here we report RNA-seq results from human FFPE samples. RNA was extracted from estrogen receptor positive (ER+) and negative (ER-) FFPE breast tumors 7-8 years after fixation and embedding. Sequencing libraries were prepared using a proprietary protocol with known strand direction. On average, 14 million reads were obtained from each lane of the Illumina GAII. After employing a proprietary rRNA depletion method, 18S and 28S rRNA are reduced to less than 5% of total mapped reads. Approximately 25 000 unique Ref-seq gene transcripts were detected in each library, demonstrating detection of most rare and intermediate transcripts as well as abundantly expressed transcripts. The relative abundance of the estrogen receptor gene ESR1 and co-expressed transcripts in the ESR1+ and ESR1- tumors correlated with expression differences measured by qPCR. Directional analysis indicated that 95% of the reads mapped to known genes had the correct direction, whereas remaining 5% with antisense direction, which possibly serve as regulatory functions. Among the uniquely mapped reads, ~ 25% were within known exons, 41% in introns, and 34% in intergenic regions. 'Valleys' and 'hills' were commonly observed across the three mapped regions. A proprietary region analysis method was developed to identify any region (not limited to known Ref-seqs) exhibiting differential expression. Using this method, we identified 34 regions that were highly differentially expressed between the ESR1+ and ESR1- tumor specimens. These regions represent exons, introns and intergenic areas. Our findings were confirmed by qPCR. These preliminary studies demonstrate the feasibility of using RNA-seq with FFPE tumor specimens. We anticipate that RNA-seq will provide a new perspective about transcriptional changes that underlie the phenotypes of individual tumors.

The activation of T cells, mediated by the T-cell receptor (TCR), activates a battery of specific membrane-associated, cytosolic and nuclear proteins. Identifying the signaling proteins downstream of TCR activation will help us to understand the regulation of immune responses and will contribute to developing therapeutic agents that target immune regulation. In an effort to identify novel signaling molecules specific for T-cell activation we undertook a large-scale dominant effector genetic screen using retroviral technology. We cloned and characterized 33 distinct genes from over 2,800 clones obtained in a screen of 7 × 108 Jurkat T cells on the basis of a reduction in TCR-activation-induced CD69 expression after expressing retrovirally derived cDNA libraries. We identified known signaling molecules such as Lck, ZAP70, Syk, PLCγ1 and SHP-1 (PTP1C) as truncation mutants with dominant-negative or constitutively active functions. We also discovered molecules not previously known to have functions in this pathway, including a novel protein with a RING domain (found in a class of ubiquitin ligases; we call this protein TRAC-1), transmembrane molecules (EDG1, IL-10Rα and integrin α2), cytoplasmic enzymes and adaptors (PAK2, A-Raf-1, TCPTP, Grb7, SH2-B and GG2-1), and cytoskeletal molecules (moesin and vimentin). Furthermore, using truncated Lck, PLCγ1, EDG1 and PAK2 mutants as examples, we showed that these dominant immune-regulatory molecules interfere with IL-2 production in human primary lymphocytes. This study identified important signal regulators in T-cell activation. It also demonstrated a highly efficient strategy for discovering many components of signal transduction pathways and validating them in physiological settings.