Middha, Sumit

ABSTRACT Our understanding of adenovirus (Ad) biology is largely extrapolated from human species C Ad5. Most humans are immune to Ad5, so lower-seroprevalence viruses like human Ad6 and Ad26 are being tested as therapeutic vectors. Ad6 and Ad26 differ at the DNA level by 34%. To better understand how this might impact their biology, we examined the life cycle of the two viruses in human lung cells in vitro . Both viruses infected A549 cells with similar efficiencies, executed DNA replication with identical kinetics within 12 h, and began killing cells within 72 h. While Ad6-infected cells remained adherent until death, Ad26-infected cells detached within 12 h of infection but remained viable. Next-generation sequencing (NGS) of mRNA from infected cells demonstrated that viral transcripts constituted 1% of cellular mRNAs within 6 h and 8 to 16% within 12 h. Quantitative PCR and NGS revealed the activation of key early genes at 6 h and transition to late gene activation by 12 h by both viruses. There were marked differences in the balance of E1A and E1B activation by the two viruses and in the expression of E3 immune evasion mRNAs. Ad6 was markedly more effective at suppressing major histocompatibility complex class I (MHC I) display on the cell surface and in evading TRAIL-mediated apoptosis than was Ad26. These data demonstrate shared as well as divergent life cycles in these genetically distant human adenoviruses. An understanding of these differences expands the knowledge of alternative Ad species and may inform the selection of related Ads for therapeutic development. IMPORTANCE A burgeoning number of adenoviruses (Ads) are being harnessed as therapeutics, yet the biology of these viruses is generally extrapolated from Ad2 and Ad5. Here, we are the first to compare the transcriptional programs of two genetically distant Ads by mRNA next-generation sequencing (NGS). Species C Ad6 and Ad26 are being pursued as lower-seroprevalence Ad vectors but differ at the DNA level by 34%. Head-to-head comparison in human lung cells by NGS revealed that the two viruses generally conform to our general understanding of the Ad transcriptional program. However, fine mapping revealed subtle and strong differences in how these two viruses execute these programs, including differences in the balance of E1A and E1B mRNAs and in E3 immune evasion genes. This suggests that not all adenoviruses behave like Ad2 and Ad5 and that they may have unique strategies to infect cells and evade the immune system.

Abstract Purpose: Discriminant markers for pancreatic cancer detection are needed. We sought to identify and validate methylated DNA markers for pancreatic cancer using next-generation sequencing unbiased by known targets. Experimental Design: At a referral center, we conducted four sequential case–control studies: discovery, technical validation, biologic validation, and clinical piloting. Candidate markers were identified using variance-inflated logistic regression on reduced-representation bisulfite DNA sequencing results from matched pancreatic cancers, benign pancreas, and normal colon tissues. Markers were validated technically on replicate discovery study DNA and biologically on independent, matched, blinded tissues by methylation-specific PCR. Clinical testing of six methylation candidates and mutant KRAS was performed on secretin-stimulated pancreatic juice samples from 61 patients with pancreatic cancer, 22 with chronic pancreatitis, and 19 with normal pancreas on endoscopic ultrasound. Areas under receiver-operating characteristics curves (AUC) for markers were calculated. Results: Sequencing identified >500 differentially hyper-methylated regions. On independent tissues, AUC on 19 selected markers ranged between 0.73 and 0.97. Pancreatic juice AUC values for CD1D, KCNK12, CLEC11A, NDRG4, IKZF1, PKRCB, and KRAS were 0.92*, 0.88, 0.85, 0.85, 0.84, 0.83, and 0.75, respectively, for pancreatic cancer compared with normal pancreas and 0.92*, 0.73, 0.76, 0.85*, 0.73, 0.77, and 0.62 for pancreatic cancer compared with chronic pancreatitis (*, P = 0.001 vs. KRAS). Conclusions: We identified and validated novel DNA methylation markers strongly associated with pancreatic cancer. On pilot testing in pancreatic juice, best markers (especially CD1D) highly discriminated pancreatic cases from controls. Clin Cancer Res; 21(19); 4473–81. ©2015 AACR.

Abstract Background: Fusion genes, transcripts, and proteins are key drivers of hematopoietic oncogenesis and can serve as effective therapeutic targets. Among peripheral T-cell lymphomas (PTCLs), little is known about fusion genes except for those involving ALK. Discovery of fusion genes may provide therapeutic targets for PTCLs, which have poor outcomes with conventional therapy. We have developed methods for detecting candidate fusions using (1) mate-pair DNA sequencing (MPseq), an approach that spans the entire genome at a fraction of the cost of traditional whole-genome sequencing, and (2) RNA sequencing (RNAseq). Each technique has strengths, limitations, and false discovery rates. Here, we integrated results of both methods to discover and orthogonally validate expressed fusion genes in PTCLs. Methods: DNA and RNA were extracted from 49 frozen, OCT-embedded clinical PTCL specimens, including 25 anaplastic large cell lymphomas; 16 PTCLs, not otherwise specified; and 8 other PTCLs. MPseq and RNAseq were performed using Illumina library protocols and a HiSeq 2000. Detection of fusion genes and transcripts followed our published methods (Blood 2012;120:2280 and Nucleic Acids Res 2011;39:e100, respectively). Functional annotation of genes involved in fusions was performed using DAVID. Results: Of 2818 candidate chromosomal rearrangements identified by MPseq and 90 candidate fusion transcripts identified by RNAseq, 45 events were present in both datasets (mean/case, 0.9; range, 0-7). Fusions were inter-chromosomal in 16 and intra-chromosomal in 29. Annotation clusters with the highest enrichment scores related to SH2 domain-containing proteins and tyrosine kinases (including FER and MAP2K3). Seven genes were recurrently involved in fusions, including known fusion partners such as ALK and previously unreported fusion partners such as PPP1R8, a phosphatase inhibitor important for the activity of Polycomb group proteins such as EZH2. Detection of candidate events only by MPseq could be explained in part by breakpoints mapping to non-genic regions (79% of events). Detection only by RNAseq could be explained in part by unannotated transcripts spanning neighboring named genes (median intra-chromosomal distance, 0.09 MB vs. 12.7 MB for intra-chromosomal candidates identified by MPseq; p<0.0001, Wilcoxon test). Conclusions: Integrated DNA/RNA sequencing is an effective, clinically feasible tool for simultaneous discovery and orthogonal validation of expressed fusion genes. This approach yielded fusions in PTCLs involving genes with key functions in lymphocytes, including novel and recurrent fusions, some of which might be targetable with compounds currently in clinical trial. Candidate intra-chromosomal fusion transcripts not identified by MPseq may represent unrecognized isoforms or the results of novel read-through mechanisms, and merit further study. Citation Format: Andrew L. Feldman, George Vasmatzis, Yan W. Asmann, Sarah H. Johnson, Bruce W. Eckloff, Sumit Middha, Jaime I. Davila, Paul J. Kurtin, Brian K. Link, Stephen M. Ansell, James R. Cerhan. Integrated DNA/RNA sequencing for discovery and orthogonal validation of expressed fusion genes in peripheral T-cell lymphomas. [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 5585. doi:10.1158/1538-7445.AM2014-5585

Background— Long-QT syndrome (LQTS) may result in syncope, seizures, or sudden cardiac arrest. Although 16 LQTS-susceptibility genes have been discovered, 20% to 25% of LQTS remains genetically elusive. Methods and Results— We performed whole-exome sequencing child–parent trio analysis followed by recessive and sporadic inheritance modeling and disease-network candidate analysis gene ranking to identify a novel underlying genetic mechanism for LQTS. Subsequent mutational analysis of the candidate gene was performed with polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing on a cohort of 33 additional unrelated patients with genetically elusive LQTS. After whole-exome sequencing and variant filtration, a homozygous p.D18fs*13 TRDN -encoded triadin frameshift mutation was discovered in a 10-year-old female patient with LQTS with a QTc of 500 milliseconds who experienced recurrent exertion-induced syncope/cardiac arrest beginning at 1 year of age. Subsequent mutational analysis of TRDN revealed either homozygous or compound heterozygous frameshift mutations in 4 of 33 unrelated cases of LQTS (12%). All 5 TRDN-null patients displayed extensive T-wave inversions in precordial leads V 1 through V 4 , with either persistent or transient QT prolongation and severe disease expression of exercise-induced cardiac arrest in early childhood (≤3 years of age) and required aggressive therapy. The overall yield of TRDN mutations was significantly greater in patients ≤10 years of age (5 of 10, 50%) compared with older patients (0 of 24, 0%; P =0.0009). Conclusions— We identified TRDN as a novel underlying genetic basis for recessively inherited LQTS. All TRDN-null patients had strikingly similar phenotypes. Given the recurrent nature of potential lethal arrhythmias, patients fitting this phenotypic profile should undergo cardiac TRDN genetic testing.

Anoctamin‐1 (Ano1) is a widely expressed protein responsible for endogenous Ca 2+ ‐activated Cl 2 currents. Ano1 is overexpressed in cancer. Differential expression of transcriptional variants is also found in other diseases. However, the mechanisms underlying regulation of Ano1 are unknown. This study identifies the Ano1 promoter and defines a mechanism for regulating its expression. Next‐generation RNA sequencing (RNA‐seq) analysis in human gastric muscle found a new exon upstream of the reported exon 1 and identified a promoter proximal to this new exon. Reporter assays in human embryonic kidney 293 cells showed a 6.7 ± 2.1fold increase in activity over empty vector. Treatment with a known regulator of Ano1 expression, IL‐4, increased promoter activity by 1.6 ± 0.02‐fold over untreated cells. The promoter region contained putative binding sites for multiple transcription factors including signal transducer and activator of transcription 6 (STAT6), a downstream effector of IL‐4. Chromatin immunoprecipitation (ChIP) experiments on T84 cells, which endogenously express Ano1, showed a 2.1 ± 0.12‐fold increase in binding of STAT6 to P0 after IL‐4 treatment. These results were confirmed by mutagenesis, expression, and RNA interference techniques. This work allows deeper understanding of the regulation of Ano1 in physiology and as a potential therapeutic target in a variety of diseases—Mazzone, A., Gibbons, S. J., Bernard, C. E., Nowsheen, S., Middha, S., Almada, L. L., Ordog, T., Kendrick, M. L., Reid Lombardo, KM., Shen, K. R., Galietta, L. J. V., Fernandez‐Zapico, M. E., and Farrugia, G., Identification and characterization of a novel promoter for the human ANO1 gene regulated by the transcription factor signal transducer and activator of transcription 6 (STAT6). FASEB J. 29, 152–163 (2015). www.fasebj.org

Abstract Colorectal cancer (CRC) is estimated to be the third most common cancer in both men and women in the US in 2012, accounting for over 50,000 deaths. Familial CRC accounts for approximately 30% of all CRC, yet only 5% of familial CRCs can be attributed to mutations in known hereditary colon cancer (HCC) genes. Many families with a strong family history of CRC do not have mutations in known CRC genes. Finding the genes underpinning CRC in these families may improve screening for disease and allow the development of more targeted therapeutics. We completed targeted sequencing of over 2,000 genes and miRNAs in 1,500 cases of CRC from the Colon Cancer Family Registry (CCFR) in an effort to identify rare variants associated with CRC risk and to identify mutations in known CRC genes missed by conventional sequencing. Several categories of genes were included: 18 known HCC genes; 18 candidate HCC genes; 185 genes in previously reported CRC linkage peaks; 1,423 miRNAs; and 363 genes identified through whole exome sequencing of 40 affected cases in 16 families with hereditary CRC (n=2,007 total genes sequenced). Genomic DNA samples were indexed, then libraries were made using Agilent's SureSelect Targeted Enrichment kit. Samples were pooled (96 samples/pool), then each pool of samples was run on five lanes on an Illumina HiSeq2000 (average of 19.2 samples/lane). GenomeGPS, an in-house developed workflow, was used for read alignment, variant calling, and annotation. In early analyses of the first 161 cases, the mean number of mapped reads exceeded 20 million per sample and over 90% of the target region exceeded 40x coverage. A total of 26,484 SNPs were identified and passed quality filtering. Sequencing identified known mutations in MMR genes in positive control cases as well as mutations in HCC genes in previously untested cases. Analysis is currently on-going on the remaining samples and we will report results on the full set of 1500 cases at the meeting. Citation Format: Melissa S. DeRycke, Shanaka R. Gunawardena, Sumit Middha, Shannon K. McDonnell, Shaun M. Riska, Zachary C. Fogarty, Bruce W. Eckloff, Daniel J. Schaid, Ellen L. Goode, Stephen N. Thibodeau. Targeted sequencing to identify rare variants in colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2548. doi:10.1158/1538-7445.AM2013-2548