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DICER1 is essential for the generation of mature miRNAs and other short noncoding RNAs. Several lines of investigation implicate DICER1 as a tumor suppressor. Reduced DICER1 levels and changes in miRNA abundance have been associated with aggressive tumor phenotypes. The global effects of reduced DICER1 on mRNA transcript abundance in tumor cells remain largely unknown. We used short hairpin RNA to stably knock down DICER1 in endometrial cancer cell lines to begin to determine how reduced DICER1 activity contributes to tumor phenotypes. DICER1 knockdown did not affect cell proliferation but caused enhanced cell migration and growth in soft agar. miRNA and mRNA profiling in KLE cells revealed overall decreases in miRNA levels and changes in the relative abundance of many mRNAs. One of the most striking changes in mRNA levels was the upregulation of IFN-stimulated genes (ISG), the majority of which lack known miRNA target sequences. IFNβ, a key upstream regulator of the IFN response, was significantly increased in DICER1 knockdowns in the AN3CA, Ishikawa, and KLE endometrial cancer cell lines and in the normal endometrial cell line EM-E6/E7/TERT. IFNβ secreted in media from KLE and EM-E6/E7/TERT shDcr cells was sufficient to activate an IFN response in HT29 cells. The reduced miRNA processing in DICER1 knockdowns was associated with increases in pre-miRNAs in the cytoplasm. Our findings suggest that elevated pre-miRNA levels trigger the IFN response to double-stranded RNA. We thus report a novel effect of reduced DICER1 function in cancer cells. Mol Cancer Res; 10(3); 316–25. ©2012 AACR.

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Lung cancer accounts for approximately 14% of all newly diagnosed cancers and is the leading cause of cancer-related deaths. Chimeric RNA resulting from gene fusions (RNA fusions) and other RNA splicing errors are driver events and clinically addressable targets for non-small cell lung cancer (NSCLC). The reliable assessment of these RNA markers by next-generation sequencing requires integrated reagents, protocols, and interpretive software that can harmonize procedures and ensure consistent results across laboratories. We describe the development and verification of a system for targeted RNA sequencing for the analysis of challenging, low-input solid tumor biopsies that includes reagents for nucleic acid quantification and library preparation, run controls, and companion bioinformatics software. Assay development reconciled sequence discrepancies in public databases, created predictive formalin-fixed, paraffin-embedded RNA qualification metrics, and eliminated read misidentification attributable to index hopping events on the next-generation sequencing flow cell. The optimized and standardized system was analytically verified internally and in a multiphase study conducted at five independent laboratories. The results show accurate, reproducible, and sensitive detection of RNA fusions, alternative splicing events, and other expression markers of NSCLC. This comprehensive approach, combining sample quantification, quality control, library preparation, and interpretive bioinformatics software, may accelerate the routine implementation of targeted RNA sequencing of formalin-fixed, paraffin-embedded samples relevant to NSCLC.

Newborn screening is designed for presymptomatic identification of serious conditions with effective early interventions. Clinical laboratories must perform prospective pilot studies to ensure that the analytical performance and workflow for a given screening test are appropriate. We assessed the potential to screen newborns for fragile X syndrome, a monogenic neurodevelopmental disorder, by establishing a customized, high-throughput PCR and analysis software system designed to detect fragile X mental retardation 1 gene repeat expansions from dried blood spots (DBSs). Assay precision, accuracy, sensitivity, and specificity were characterized across the categorical range of repeat expansions. The assay consistently resolved genotypes within three CGG repeats of reference values up to at least 137 repeats and within six repeats for larger expansions up to 200 repeats. Accuracy testing results were concordant with reference results. Full and premutation alleles were detected from subnanogram DNA inputs eluted from DBSs and from mixtures with down to 1% relative abundance of the respective expansion. Analysis of 963 deidentified newborn DBS samples identified 957 normal and 6 premutation specimens, consistent with previously published prevalence estimates. These studies demonstrate that the assay system can support high-throughput newborn screening programs.

Key steps in understanding a biological process include identifying genes that are involved and determining how they are regulated. We developed a novel method for identifying transcription factors (TFs) involved in a specific process and used it to map regulation of the key virulence factor of a deadly fungus—its capsule. The map, built from expression profiles of 41 TF mutants, includes 20 TFs not previously known to regulate virulence attributes. It also reveals a hierarchy comprising executive, midlevel, and “foreman” TFs. When grouped by temporal expression pattern, these TFs explain much of the transcriptional dynamics of capsule induction. Phenotypic analysis of TF deletion mutants revealed complex relationships among virulence factors and virulence in mice. These resources and analyses provide the first integrated, systems-level view of capsule regulation and biosynthesis. Our methods dramatically improve the efficiency with which transcriptional networks can be analyzed, making genomic approaches accessible to laboratories focused on specific physiological processes.

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench ("wet bench") and data analyses conducted using bioinformatics pipelines ("dry bench"). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible "tag" PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen that kills over 600,000 people annually. Here, we report integrated computational and experimental investigations of the role and mechanisms of transcriptional regulation in cryptococcal infection. Major cryptococcal virulence traits include melanin production and the development of a large polysaccharide capsule upon host entry; shed capsule polysaccharides also impair host defenses. We found that both transcription and translation are required for capsule growth and that Usv101 is a master regulator of pathogenesis, regulating melanin production, capsule growth, and capsule shedding. It does this by directly regulating genes encoding glycoactive enzymes and genes encoding three other transcription factors that are essential for capsule growth: GAT201, RIM101, and SP1. Murine infection with cryptococci lacking Usv101 significantly alters the kinetics and pathogenesis of disease, with extended survival and, unexpectedly, death by pneumonia rather than meningitis. Our approaches and findings will inform studies of other pathogenic microbes., IMPORTANCE Cryptococcus neoformans causes fatal meningitis in immunocompromised individuals, mainly HIV positive, killing over 600,000 each year. A unique feature of this yeast, which makes it particularly virulent, is its polysaccharide capsule; this structure impedes host efforts to combat infection. Capsule size and structure respond to environmental conditions, such as those encountered in an infected host. We have combined computational and experimental tools to elucidate capsule regulation, which we show primarily occurs at the transcriptional level. We also demonstrate that loss of a novel transcription factor alters virulence factor expression and host cell interactions, changing the lethal condition from meningitis to pneumonia with an exacerbated host response. We further demonstrate the relevant targets of regulation and kinetically map key regulatory and host interactions. Our work elucidates mechanisms of capsule regulation, provides methods and resources to the research community, and demonstrates an altered pathogenic outcome that resembles some human conditions.

DICER1 is essential for the generation of mature miRNAs and other short noncoding RNAs. Several lines of investigation implicate DICER1 as a tumor suppressor. Reduced DICER1 levels and changes in miRNA abundance have been associated with aggressive tumor phenotypes. The global effects of reduced DICER1 on mRNA transcript abundance in tumor cells remain largely unknown. We used short hairpin RNA to stably knock down DICER1 in endometrial cancer cell lines to begin to determine how reduced DICER1 activity contributes to tumor phenotypes. DICER1 knockdown did not affect cell proliferation but caused enhanced cell migration and growth in soft agar. miRNA and mRNA profiling in KLE cells revealed overall decreases in miRNA levels and changes in the relative abundance of many mRNAs. One of the most striking changes in mRNA levels was the upregulation of IFN-stimulated genes (ISG), the majority of which lack known miRNA target sequences. IFNβ, a key upstream regulator of the IFN response, was significantly increased in DICER1 knockdowns in the AN3CA, Ishikawa, and KLE endometrial cancer cell lines and in the normal endometrial cell line EM-E6/E7/TERT. IFNβ secreted in media from KLE and EM-E6/E7/TERT shDcr cells was sufficient to activate an IFN response in HT29 cells. The reduced miRNA processing in DICER1 knockdowns was associated with increases in pre-miRNAs in the cytoplasm. Our findings suggest that elevated pre-miRNA levels trigger the IFN response to double-stranded RNA. We thus report a novel effect of reduced DICER1 function in cancer cells. Mol Cancer Res; 10(3); 316–25. ©2012 AACR.

Introduction: HER2-positive breast cancers comprise 20-30% by subtype and represent the second poorest prognosis. In addition to HER2 signaling activation via protein overexpression or gene amplification (measured by IHC or ISH), somatic mutations in the ERBB2 (HER2) gene have been identified in 2-4% of breast cancers and reported to drive HER2 pathway signaling and tumorigenesis. Irreversible HER2 tyrosine kinase inhibitors such as neratinib have been shown to potently inhibit breast tumors harboring somatic HER2 activating mutations and are under active clinical investigation. A targeted molecular characterization strategy is thus needed to support clinical trial research. Here, we present an integrated NGS system that identifies multiple classes of ERBB2aberrations, including single nucleotide variants, insertion/deletion mutations, and copy number variations (CNVs). Methods: Genomic DNA was isolated from FFPE human tumor tissues or FFPE-embedded engineered cell lines expressing ERBB2 mutations. Pre-analytical sample QC was performed by a novel qPCR assay that quantifies amplifiable DNA templates suitable for library preparation. NGS libraries were prepared using the QuantideX® NGS ERBB2 Mutations assay, with two-pool PCR-based enrichment to detect multiple types of ERBB2 genomic alterations. Libraries were sequenced (Illumina MiSeq®) and analyzed using QuantideX® Reporter software, directly incorporating the pre-analytical QC data from each DNA sample to improve variant call accuracy. Results: The QuantideX® NGS ERBB2 Mutations system targets 28% of ERBB2 coding bases covering exons 8 and 17 - 24, including codons 713-989 of the tyrosine kinase domain. The assay also detects focal, whole-arm and polysomy-induced HER2 amplification using sentinel flanking amplicons, and amplicons covering CEN17 and copy number-neutral chromosomal regions identified in the TCGA breast cancer cohort. The assay was analytically validated in a CLIA-laboratory which included an analysis of 6 mixtures and titrations of synthesized DNA bearing 31 ERBB2 mutations, 17 engineered cell lines and FFPE cell-line blocks, and 12 surgically-resected FFPE carcinomas. A multi-day/operator study revealed 100% sensitivity and PPV for detection of ERBB2 SNVs, indels and complex variants down to 5% mutation positive in wild-type DNA. The NGS system was also used to profile ERBB2 variants in 473 FFPE human tumor tissues, including 280 breast carcinomas. Mutation-positive samples were confirmed by an orthogonal NGS method. A subset of 46 FFPE tissue samples were assessed for CNVs by NGS and compared with matched droplet digital PCR (ddPCR) analysis. A correlation of R2 > 0.91 between NGS and ddPCR underscores the potential of the assay to reveal both ERBB2 mutations and copy number changes. Conclusions: The QuantideX® NGS ERBB2system integrates optimized wet- and dry-bench elements to achieve broad and accurate detection of ERBB2 mutations and copy number variants from challenging FFPE specimens. The technology has the potential to advance breast cancer clinical research and aid in the selection of patients for clinical trials, particularly those that assess HER2-directed therapies such as afatinib, lapatinib, and neratinib. Citation Format: Zhu H, Zeigler R, Bridger S, Larson JL, Kincaid A, Cardwell R, Haynes BC, Andruss BF, Latham GJ. A fit-for-purpose NGS system that reports ERBB2 (HER2) mutations and copy number variants for clinical trials research and drug development [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-03-10.

Cryptococcus neoformans , a basidiomycete yeast and opportunistic pathogen, expends significant biosynthetic effort on construction of a polysaccharide capsule with a radius that may be many times that of the cell. Beyond posing a stimulating challenge in terms of defining biosynthetic pathways, the capsule is required for this yeast to cause fatal disease. This combination has focused the attention of researchers on this system. Here we briefly review two aspects of the rapidly advancing field of capsule synthesis: the extensive variation that occurs in capsule polymers and the regulation of capsule biosynthesis.

Introduction: Mutation analysis of circulating tumor DNA (ctDNA) in blood-based liquid biopsies provides a minimally invasive approach to detect and monitor disease. Existing next-generation sequencing (NGS) liquid biopsy techniques have laborious and/or inefficient workflows, heuristic error-correction algorithms, and variable performance with clinical tumor-plasma samples. We describe a method that combines a kittable and efficient wet-bench workflow with accurate dry-bench analytics to reduce costs and turnaround time, and is relevant to clinical research and patient testing. Methods: We developed a comprehensive, targeted NGS technology to enable the ultra-sensitive detection of variants from liquid biopsy samples. Input DNA molecules from Seraseq™ ctDNA v2 reference materials (SeraCare) and >50 matched FFPE and plasma samples were uniquely tagged with a random molecular barcode (MBC), amplified in an efficient PCR protocol, and sequenced using a targeted panel covering >500 somatic mutation hotspots. Sequence-ready libraries were prepared from input DNA within 9 hours. Data were analyzed with a robust bioinformatics pipeline tailored to the library chemistry to correct for multiple background errors. Variants were identified with a site-specific model, which effectively eliminated recurring non-biological aberrations that remained despite MBC-facilitated error-suppression. We verified variant calls in plasma by Droplet Digital™ PCR (Bio-Rad). To evaluate the limit of detection, healthy control and mutation-positive plasma admixtures were prepared and sequenced. Results: Input template molecules were efficiently recovered. The median number of unique MBCs across all amplicons was >90% of expectation based on input DNA quantities for both the tumor-associated plasma and Seraseq™ ctDNA v2 material. SNVs and indels were recognized with >90% sensitivity and PPV at 0.5% allele frequency (AF) in the Seraseq™ ctDNA mutation mixes. In both neat plasma samples and plasma admixtures, we correctly identified tumor mutations down to ~0.1% AF while maintaining a low false-positive rate (sensitivity and PPV remained high for AF ≥0.1%). Reference ctDNA material and tumor-associated plasma had analogous template amplification and variant frequency rates. Conclusions: A fast, efficient, and sensitive NGS panel approach was developed and evaluated, and it demonstrated the reliable and specific detection of rare variants in liquid biopsy specimens. The method is able to distinguish a low frequency ctDNA signal from the overwhelming background noise in plasma cell-free DNA using a streamlined workflow and purpose-built bioinformatics pipeline. This technology may provide an easy-to-use, high-performance, and adaptable NGS diagnostic framework for disease detection and therapeutic intervention monitoring. Citation Format: Jessica L. Larson, Liangjing Chen, Lando Ringel, Blake Printy, Farol L. Tomson, Yves Konigshofer, Sarah Statt, Joseph K. Kaplan, Shobha Gokul, Jeffrey Shelton, Gary J. Latham, Brian C. Haynes. A comprehensive, targeted next-generation sequencing method that rapidly and accurately detects circulating tumor DNA variants at 0.1% frequency in plasma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5574.

Introduction: Reliable assessment of cancer-associated RNA markers in lung cancer produced by gene-fusions or exon skipping events by next-generation sequencing requires integrated reagents, protocols, and interpretive software that can harmonize procedures and ensure consistent results across laboratories. We evaluated a comprehensive system for targeted RNA-Seq that includes reagents for nucleic acid quantification, library prep, run controls, and companion bioinformatics software. The reproducibility of this system was evaluated in a multi-phase study design at 5 independent laboratories. Methods: Total nucleic acid (TNA) was isolated from formalin-fixed, paraffin-embedded (FFPE) residual non-small cell lung cancer (NSCLC) tumor biopsies and cell-lines (RT-112, H596, HCC78). These TNA isolates were used to prepare a set of 30 test samples, including a dilution series to assess assay sensitivity. A non-template control and kit positive control were also included. The sample set was evaluated using the QuantideX® NGS RNA Lung Cancer Kit RUO (Asuragen) and sequenced on the MiSeq® system (Illumina) at Asuragen and 4 independent laboratories. Analyses were conducted using QuantideX® NGS Reporter RUO (Asuragen), a software suite that includes a FASTQ processing pipeline and incorporates pre-analytical QC information into the fusion-caller algorithm and reporting tool. Results: Laboratories were trained on the assay workflow and companion bioinformatics software in less than two days followed by independent library preparation and sequencing workflows. A total of 266 sample libraries were evaluated from inputs down to Conclusions: The accuracy of this novel targeted NGS assay for RNA fusions and splice variants in NSCLC was demonstrated in a multi-site laboratory evaluation using clinically-relevant specimens and low inputs of TNA. The ability of the panel to detect both common and rare gene fusion transcripts and exon skipping events within an integrated wet- and dry-bench workflow provides a foundation for the reliable detection of oncogenic RNA fusions and aberrant splicing events that can respond to current and emerging targeted therapies. This study highlighted the ease of implementation and consistent performance that can be achieved in different laboratories when the process from sample-to-report is highly integrated. Citation Format: Gary J. Latham, Richard Blidner, Brian C. Haynes, Shobha Gokul, Maria L. Aguirre, Stephen Hyter, Ziyan Y. Pessetto, Maria Curtis, Dan Su, Tom Halsey, Victor Weigman, Patrick Hurban, Andrew K. Godwin, Leon C. van Kempen. Accurate and reproducible detection of fusions and exon skipping events in NSCLC-derived samples using a comprehensive, targeted RNA-Seq system across multiple laboratories [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1764. doi:10.1158/1538-7445.AM2017-1764

Introduction: Ovarian cancer is a leading cause of cancer related death in women. A comprehensive genomic characterization of platinum-sensitive tumors is required to further refine the definition of molecular subtypes and identify targeted therapies for this patient population. To this end we have performed a large-scale genomic and transcriptomic characterization of 348 primary FFPE tissues from a cohort of platinum-sensitive HGSOC patients collected from multiple clinical sites. Methods: Macrodissection of FFPE resected tumor slides or sectioned blocks was performed to enrich for tumor content. RNA and DNA were each isolated from 2x 5µm sections of FFPE material. RNA expression and gene fusions were profiled by whole transcriptome RNA-Seq. DNA variants were analyzed by the AmpliSeq™ Cancer Hotspot Panel (Thermo Fisher). A subset of tumor and matched germline (PBL) specimens (N=181) were assessed for TP53 mutations by the QuantideX® NGS TP53 Assay (Asuragen, Inc.). CNV analysis of FFPE tumor DNA was performed using the OncoScan® FFPE Assay Kit (Affymetrix) and microsatellite instability was characterized by comparing matched tumor and PBL specimens with capillary electrophoresis using the Bethesda panel. Germline BRCA1/2 mutation status was determined by profiling PBL specimens with a custom AmpliSeq™ NGS panel. Results: The spectrum of DNA mutations and CNVs was consistent with other HGSOC cohorts with mutations in TP53 present in the majority of specimens (87% for specimens with full exon coverage of TP53). Germline mutations in BRCA1 and BRCA2 were identified at 10.3% and 6.8% respectively. Analysis of recurrent whole chromosomal arm gain and loss displayed a striking agreement with the TCGA HGSOC cohort. Unsupervised analysis of the RNA-Seq expression data through non-negative matrix factorization revealed 4 distinct transcriptional subtypes, corresponding to the 4 established CLOVAR subtypes: differentiated, immunoreactive, mesenchymal and proliferative. Patients classified as mesenchymal had the poorest prognosis. Further integrative analysis identified additional associations between the CLOVAR subtypes and other molecular indications. Conclusions: Through integrative genomic analyses on this challenging set of clinical specimens we have reproduced and refined the molecular subtypes of platinum-sensitive HGSOC and highlighted alterations that may lead to improved diagnostic and precision medicine strategies. In contrast to previous large-scale molecular characterization efforts such as TCGA where fresh-frozen tissues were collected under highly controlled settings, this study is based on molecular profiling of FFPE tissues collected at dozens of clinical sites. Thus, our study serves as a model for future molecular characterization efforts of FFPE specimens collected in real-world clinical settings. Citation Format: Brian C. Haynes, Marie E. Fahey, Darcy Myers, Diane Ilsley, Gary J. Latham, Elizabeth B. Somers, Nicholas C. Nicolaides, Charles Schweizer, Daniel J. O’Shannessy. Comprehensive genomic characterization of a large cohort of platinum-sensitive, high-grade serous ovarian cancer (HGSOC) FFPE specimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1743. doi:10.1158/1538-7445.AM2017-1743

The pathogenic fungus Cryptococcus neoformans synthesizes a complex family of glycosylinositolphosphoceramide (GIPC) structures. These glycosphingolipids (GSLs) consist of mannosylinositolphosphoceramide (MIPC) extended by β1-6-linked galactose, a unique structure that has to date only been identified in basidiomycetes. Further extension by up to five mannose residues and a branching xylose has been described. In this study, we identified and determined the gene structure of the enzyme Ggt1, which catalyzes the transfer of a galactose residue to MIPC. Deletion of the gene in C. neoformans resulted in complete loss of GIPCs containing galactose, a phenotype that could be restored by the episomal expression of Ggt1 in the deletion mutant. The entire annotated open reading frame, encoding a C-terminal GT31 galactosyltransferase domain and a large N-terminal domain of unknown function, was required for complementation. Notably, this gene does not encode a predicted signal sequence or transmembrane domain. The demonstration that Ggt1 is responsible for the transfer of a galactose residue to a GSL thus raises questions regarding the topology of this biosynthetic pathway and the function of the N-terminal domain. Phylogenetic analysis of the GGT1 gene shows conservation in hetero- and homobasidiomycetes but no homologs in ascomycetes or outside of the fungal kingdom.

A critical step in understanding how a genome functions is determining which transcription factors (TFs) regulate each gene. Accordingly, extensive effort has been devoted to mapping TF networks. In Saccharomyces cerevisiae, protein–DNA interactions have been identified for most TFs by ChIP-chip, and expression profiling has been done on strains deleted for most TFs. These studies revealed that there is little overlap between the genes whose promoters are bound by a TF and those whose expression changes when the TF is deleted, leaving us without a definitive TF network for any eukaryote and without an efficient method for mapping functional TF networks. This paper describes NetProphet, a novel algorithm that improves the efficiency of network mapping from gene expression data. NetProphet exploits a fundamental observation about the nature of TF networks: The response to disrupting or overexpressing a TF is strongest on its direct targets and dissipates rapidly as it propagates through the network. Using S. cerevisiae data, we show that NetProphet can predict thousands of direct, functional regulatory interactions, using only gene expression data. The targets that NetProphet predicts for a TF are at least as likely to have sites matching the TF's binding specificity as the targets implicated by ChIP. Unlike most ChIP targets, the NetProphet targets also show evidence of functional regulation. This suggests a surprising conclusion: The best way to begin mapping direct, functional TF-promoter interactions may not be by measuring binding. We also show that NetProphet yields new insights into the functions of several yeast TFs, including a well-studied TF, Cbf1, and a completely unstudied TF, Eds1.

Introduction: The promise of precision medicine relies on the identification of DNA and RNA markers that can individualize patient management. Methods such as next-generation sequencing (NGS) can deduce DNA or RNA sequences, but both types of nucleic acid have not been efficiently and effectively combined into a single NGS workflow. We describe a comprehensive methodology for targeted clinical NGS that reports DNA and RNA variants, provides a streamlined workflow, and accommodates low-input total nucleic acid (TNA) from challenging clinical specimens. Methods: Sample QC was performed using a novel qPCR assay that quantifies discrete populations of amplifiable DNA and RNA from TNA material. PCR-based target enrichment was performed using QuantideX® NGS reagents (Asuragen) and sequenced on the MiSeq® System (Illumina). Bioinformatic analyses were conducted using QuantideX® Reporter (Asuragen), a software suite that directly incorporates pre-analytical QC information into the variant calling. Results: Targeted DNA- and RNA-seq panels were developed that query 54 lung cancer DNA targets and 135 RNA targets, including >100 gene fusions and mRNA expression markers associated with clinical actionability. Gene-specific primers were formulated as multiplex PCR or RT-PCR reactions to independently interrogate DNA or RNA variants, respectively, from TNA or combined into a 189-plex RT-PCR to report multi-categorical nucleic acid variants. Integration of the bioinformatics pipeline and variant caller with wet-lab QC results enhanced mutation call sensitivity at The NGS panels were assessed with cell-line and synthetic controls and a residual clinical cohort of 97 NSCLC FFPE specimens. Mutations were accurately detected from as few as 5-10 DNA templates and down to Conclusions: QuantideX targeted NGS chemistries can unify the analysis of DNA and RNA markers associated with lung cancer and report SNVs, indels, fusions, and aberrantly expressed mRNA transcripts from a single NGS run. This novel technology offers reliable, accurate and comprehensive molecular characterizations of challenging tumor specimens by integrating wet-bench and dry-bench methods and improving the efficacy of routine laboratory testing. Citation Format: Gary J. Latham, Julie Krosting, Michael Dodge, Robert Zeigler, Liangjing Chen, Jason Plyler, Shobha Gokul, Junya Fujimoto, Vassiliki Papadimitrakopoulou, Ignacio Wistuba, Richard Blidner, Brian Haynes. A unified and streamlined targeted sequencing system for the quantification of DNA mutations and RNA expression markers in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1389.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes serious human disease in immunocompromised populations. Its polysaccharide capsule is a key virulence factor which is regulated in response to growth conditions, becoming enlarged in the context of infection. We used microarray analysis of cells stimulated to form capsule over a range of growth conditions to identify a transcriptional signature associated with capsule enlargement. The signature contains 880 genes, is enriched for genes encoding known capsule regulators, and includes many uncharacterized sequences. One uncharacterized sequence encodes a novel regulator of capsule and of fungal virulence. This factor is a homolog of the yeast protein Ada2, a member of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex that regulates transcription of stress response genes via histone acetylation. Consistent with this homology, the C. neoformans null mutant exhibits reduced histone H3 lysine 9 acetylation. It is also defective in response to a variety of stress conditions, demonstrating phenotypes that overlap with, but are not identical to, those of other fungi with altered SAGA complexes. The mutant also exhibits significant defects in sexual development and virulence. To establish the role of Ada2 in the broader network of capsule regulation we performed RNA-Seq on strains lacking either Ada2 or one of two other capsule regulators: Cir1 and Nrg1. Analysis of the results suggested that Ada2 functions downstream of both Cir1 and Nrg1 via components of the high osmolarity glycerol (HOG) pathway. To identify direct targets of Ada2, we performed ChIP-Seq analysis of histone acetylation in the Ada2 null mutant. These studies supported the role of Ada2 in the direct regulation of capsule and mating responses and suggested that it may also play a direct role in regulating capsule-independent antiphagocytic virulence factors. These results validate our experimental approach to dissecting capsule regulation and provide multiple targets for future investigation., Author Summary Cryptococcus neoformans is a fungal pathogen that causes serious disease in immunocompromised individuals, killing over 600,000 people per year worldwide. A major factor in the ability of this microbe to cause disease is an extensive polysaccharide capsule that surrounds the cell and interferes with the host immune response to infection. This capsule expands dramatically in certain growth conditions, including those found in the mammalian host. We grew cells in multiple conditions and assessed gene expression and capsule size. This allowed us to identify a ‘transcriptional signature’ of genes whose expression correlates with capsule size; we speculated that a subset of these genes acts in capsule regulation. To test this hypothesis, we characterized one previously unstudied gene in this signature and found it to be a novel regulator of capsule expansion, fungal virulence, and mating. This gene encodes cryptococcal Ada2, a well-conserved protein that regulates genes involved in stress response and development. We used phenotypic analysis, RNA sequencing, and chromatin-immunoprecipitation sequencing (ChIP-Seq) to situate Ada2 in the complex network of genes that regulate capsule and other cryptococcal virulence factors. This approach, which yielded insights into the regulation of a critical fungal virulence factor, is applicable to similar questions in other pathogens.

DICER1 is required for generation of mature microRNAs (miRNAs) and other short noncoding RNAs. Reduced DICER1 levels are associated with adverse outcomes in a variety of cancers and multiple lines of evidence suggest DICER1 is a tumor suppressor. The effects of reduced DICER1 on mRNA transcript abundance in tumor cells remain largely unknown. To determine how reduced DICER1 levels contribute to tumor phenotypes we used shRNA to stably knock down DICER1 in endometrial cancer cell lines for extended periods (up to 25 passages). Reduced DICER1 levels had no effect on cell proliferation but were associated with enhanced cell migration and increased growth in soft agar. MiRNA profiling for the KLE cancer cell line DICER1 knockdowns (two different DICER1 shRNA constructs) revealed that miRNA levels were decreased overall. Not surprisingly, many mRNA transcripts were upregulated in the DICER1 knockdowns. A striking number of interferon stimulated genes (ISGs) showed upregulation. Upregulation of six ISG transcripts was validated by qRT-PCR in three independent knockdowns of DICER1 in KLE cells. IFNα, a key upstream regulator of the interferon response, was significantly increased in DICER1 knockdowns (KLE and additional endometrial cancer cell lines). IFNα protein secreted in media of DICER1 knockdown cells activated an interferon response in HT29 colon carcinoma cells. The majority of ISGs with increased transcript levels lacked miRNA target sequences and as such it seemed unlikely that the increase in expression levels was secondary to reduced miRNA levels. We tested the hypothesis that defects in miRNA processing could trigger an interferon response. We showed that DICER1 knockdowns in which miRNA processing is reduced have increased levels of pre-microRNAs in the cytoplasm. Pre-microRNAs are double-stranded molecules and long enough (60-100 nt) to activate the dsRNA sensors in the cytoplasm, such as Mda5/IFIH1, that typically sense viral dsRNA (> 30 nt) and stimulate the interferon response. Knockdown of DROSHA, necessary for processing of pri-miRNAs to pre-miRNAs, did not result in accumulation of pre-miRNAs and did not cause an interferon response. Our findings suggest elevated pre-microRNA levels trigger the interferon response to double stranded RNA (incompletely processed miRNA and other species). Rescue experiments (restoring DICER1 function) are currently underway to shed additional light on how reduced double-stranded RNA processing might elicit an interferon response. Work is ongoing to determine the relationship between the observed interferon response and the migration and soft agar phenotypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2312. doi:1538-7445.AM2012-2312

DICER1 is required for the generation of mature microRNAs (22-24 nt miRNAs) and other short noncoding RNAs. Several lines of evidence point to DICER1's role as a tumor suppressor. In particular, reduced DICER1 levels are associated with adverse outcomes in a variety of human and mouse cancers. To date the effects of reduced DICER1 on mRNA transcript abundance in tumor cells remain largely unknown. Furthermore, DICER1 may have non-canonical (unrelated to miRNA activity) functions in tumorigenesis. In model organisms, small RNAs initiate transcriptional silencing. DICER1 may have a similar role in transcriptional silencing and DNA methylation in mammalian cells. To begin to determine how reduced DICER1 levels contribute to tumor phenotypes we used shRNA to stably knock down DICER1 in endometrial cancer cell lines for extended periods (up to 25 passages). Reduced DICER1 levels (measured by Western blot) had no effect on cell proliferation but were associated with enhanced cell migration. We performed microRNA, mRNA, and DNA methylation profiling experiments in the KLE endometrial cancer cell line. MiRNA levels were decreased overall and the fold reductions for individual miRNAs were highly correlated in two different DICER1 shRNA knockdowns. RNAseq analysis (Illumina) revealed a 4-fold increase in the TARBP2 (TRBP) and MOV-10 transcripts. TARBP2 and MOV-10 are integral components of the DICER1 complex and the observed upregulation of their transcripts could reflect selection to stabilize DICER1 in the knockdowns. Furthermore, miR-103 and miR-107, which target DICER1 mRNA, were downregulated in the knockdown cells, possibility reflecting selection to maintain DICER1 activity. Consistent with decreases in miRNA levels, many mRNA transcripts were upregulated with DICER1 knockdown (average 1.1 fold increase across 8077 informative transcripts). Most striking was the number of interferon responsive genes that showed upregulation. Among the 8077 informative transcripts, 28 were classified as interferon responsive, and for those the average and median fold increase was 3.1 and 2.1 respectively (range 0.9 – 12.9). The majority of those interferon responsive genes with increased transcript levels lack miRNA target sequences. Previous work has shown that shRNAs do not induce a general antiviral interferon response. We are currently performing studies to confirm that the effect observed is DICER1 dependent. Technical and biologic validations for those interferon responsive genes that show upregulation based on RNAseq are underway. Increases in IFI6 and IFI44L transcript levels have been confirmed by qPCR. We hypothesize a transcription-based mechanism of DICER1 control of interferon responsive transcripts and are attempting to determine if there is such an effect and whether it is a direct or indirect effect. 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 1173. doi:10.1158/1538-7445.AM2011-1173