131 results on '"Flemington EK"'
Search Results
2. High-density resolution of the Kaposi's sarcoma associated herpesvirus transcriptome identifies novel transcript isoforms generated by long-range transcription and alternative splicing.
- Author
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Shekhar R, O'Grady T, Keil N, Feswick A, Amador DAM, Tibbetts SA, Flemington EK, and Renne R
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- Humans, Transcription, Genetic, Gene Expression Regulation, Viral, Open Reading Frames genetics, High-Throughput Nucleotide Sequencing, Sarcoma, Kaposi virology, Sarcoma, Kaposi genetics, RNA, Viral genetics, RNA, Viral metabolism, Herpesvirus 8, Human genetics, Alternative Splicing, Transcriptome genetics
- Abstract
Kaposi's sarcoma-associated herpesvirus is the etiologic agent of Kaposi's sarcoma and two B-cell malignancies. Recent advancements in sequencing technologies have led to high resolution transcriptomes for several human herpesviruses that densely encode genes on both strands. However, for KSHV progress remained limited due to the overall low percentage of KSHV transcripts, even during lytic replication. To address this challenge, we have developed a target enrichment method to increase the KSHV-specific reads for both short- and long-read sequencing platforms. Furthermore, we combined this approach with the Transcriptome Resolution through Integration of Multi-platform Data (TRIMD) pipeline developed previously to annotate transcript structures. TRIMD first builds a scaffold based on long-read sequencing and validates each transcript feature with supporting evidence from Illumina RNA-Seq and deepCAGE sequencing data. Our stringent innovative approach identified 994 unique KSHV transcripts, thus providing the first high-density KSHV lytic transcriptome. We describe a plethora of novel coding and non-coding KSHV transcript isoforms with alternative untranslated regions, splice junctions and open-reading frames, thus providing deeper insights on gene expression regulation of KSHV. Interestingly, as described for Epstein-Barr virus, we identified transcription start sites that augment long-range transcription and may increase the number of latency-associated genes potentially expressed in KS tumors., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2024
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3. Correction: Transactivation of human endogenous retrovirus K (HERV-K) by KSHV promotes Kaposi's sarcoma development.
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Dai L, Del Valle L, Miley W, Whitby D, Ochoa AC, Flemington EK, and Qin Z
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- 2024
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4. Viral reprogramming of host transcription initiation.
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Ungerleider NA, Roberts C, O'Grady TM, Nguyen TT, Baddoo M, Wang J, Ishaq E, Concha M, Lam M, Bass J, Nguyen TD, Van Otterloo N, Wickramarachchige-Dona N, Wyczechowska D, Morales M, Ma T, Dong Y, and Flemington EK
- Subjects
- Humans, Host-Pathogen Interactions, Promoter Regions, Genetic, TATA Box, Transcription Factors metabolism, Transcription Initiation Site, Transcription, Genetic, Viral Proteins metabolism, Viral Proteins genetics, Herpesvirus 4, Human physiology, Transcription Initiation, Genetic, Virus Replication, Epstein-Barr Virus Infections metabolism, Epstein-Barr Virus Infections virology
- Abstract
Viruses are master remodelers of the host cell environment in support of infection and virus production. For example, viruses typically regulate cell gene expression through modulating canonical cell promoter activity. Here, we show that Epstein Barr virus (EBV) replication causes 'de novo' transcription initiation at 29674 new transcription start sites throughout the cell genome. De novo transcription initiation is facilitated in part by the unique properties of the viral pre-initiation complex (vPIC) that binds a TATT[T/A]AA, TATA box-like sequence and activates transcription with minimal support by additional transcription factors. Other de novo promoters are driven by the viral transcription factors, Zta and Rta and are influenced by directional proximity to existing canonical cell promoters, a configuration that fosters transcription through existing promoters and transcriptional interference. These studies reveal a new way that viruses interact with the host transcriptome to inhibit host gene expression and they shed light on primal features driving eukaryotic promoter function., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
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- 2024
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5. Profiling the activity of the para-caspase MALT1 in B-cell acute lymphoblastic leukemia for potential targeted therapeutic application.
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Safa FM, Rasmussen T, Fontan L, Xia M, Melnick A, Wiestner A, Lobelle-Rich P, Burger JA, Mouawad Y, Safah H, Flemington EK, and Saba NS
- Subjects
- Humans, Apoptosis, Caspases metabolism, Cell Line, Tumor, Gene Expression Profiling, Molecular Targeted Therapy, Precursor B-Cell Lymphoblastic Leukemia-Lymphoma metabolism, Precursor B-Cell Lymphoblastic Leukemia-Lymphoma drug therapy, Precursor B-Cell Lymphoblastic Leukemia-Lymphoma genetics, Precursor B-Cell Lymphoblastic Leukemia-Lymphoma pathology, Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein metabolism, Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein antagonists & inhibitors, Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein genetics, Neoplasm Proteins genetics, Neoplasm Proteins metabolism, Neoplasm Proteins antagonists & inhibitors
- Abstract
B-cell acute lymphoblastic leukemia (B-ALL) remains a hard-to-treat disease with a poor prognosis in adults. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a para-caspase required for B-cell receptor (BCR)-mediated NF-κB activation. Inhibition of MALT1 in preclinical models has proven efficacious in many B-cell malignancies including chronic lymphocytic leukemia, mantle cell lymphoma and diffuse large B-cell lymphoma. We sought to examine the role of MALT1 in B-ALL and determine the biological consequences of its inhibition. Targeting MALT1 with both Z-VRPR-fmk and MI-2 efficiently kills B-ALL cells independent of the cell-of-origin (pro, pre, mature) or the presence of the Philadelphia chromosome, and spares normal B cells. The mechanism of cell death was through apoptotic induction, mostly in cycling cells. The proteolytic activity of MALT1 can be studied by measuring its ability to cleave its substrates. Surprisingly, with the exception of mature B-ALL, we did not detect cleavage of MALT1 substrates at baseline, nor after proteasomal inhibition or following activation of pre-BCR. To explore the possibility of a distinct role for MALT1 in B-ALL, independent of signaling through BCR, we studied the changes in gene expression profiling following a 24-hour treatment with MI-2 in 12 B-ALL cell lines. Our transcriptome analysis revealed a strong inhibitory effect on MYC-regulated gene signatures, further confirmed by Myc protein downregulation, concomitant with an increase in the Myc degrader FBXW7. In conclusion, our evidence suggests a novel role for MALT1 in B-ALL through Myc regulation and provides support for clinical testing of MALT1 inhibitors in B-ALL.
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- 2024
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6. Alterations in gene expression and microbiome composition upon calcium-sensing receptor deletion in the mouse esophagus.
- Author
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Abdulnour-Nakhoul SM, Kolls JK, Flemington EK, Ungerleider NA, Nakhoul HN, Song K, and Nakhoul NL
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- Animals, Mice, Receptors, Calcium-Sensing genetics, Receptors, Calcium-Sensing metabolism, Esophagus metabolism, Inflammation, Gene Expression, Calcium metabolism, Microbiota
- Abstract
The calcium-sensing receptor (CaSR), a G protein-coupled receptor, regulates Ca
2+ concentration in plasma by regulating parathyroid hormone secretion. In other tissues, it is reported to play roles in cellular differentiation and migration and in secretion and absorption. We reported previously that CaSR can be conditionally deleted in the mouse esophagus. This conditional knockout (KO) (Eso CaSR-/- ) model showed a significant reduction in the levels of adherens and tight junction proteins and had a marked buildup of bacteria on the luminal esophageal surface. To further examine the role of CaSR, we used RNA sequencing to determine gene expression profiles in esophageal epithelia of control andEso CaSR-/- mice RNA Seq data indicated upregulation of gene sets involved in DNA replication and cell cycle inEso CaSR-/- . This is accompanied by the downregulation of gene sets involved in the innate immune response and protein homeostasis including peptide elongation and protein trafficking. Ingenuity pathway analysis (IPA) demonstrated that these genes are mapped to important biological networks including calcium and Ras homologus A (RhoA) signaling pathways. To further explore the bacterial buildup inEso CaSR-/- esophageal tissue, 16S sequencing of the mucosal-associated bacterial microbiome was performed. Three bacterial species, g_Rodentibacter , s _Rodentibacter_unclassified , and s_Lactobacillus_hilgardi were significantly increased inEso CaSR-/- . Furthermore, metagenomic analysis of 16S sequences indicated that pathways related to oxidative phosphorylation and metabolism were downregulated inEso CaSR-/- tissues. These data demonstrate that CaSR impacts major pathways of cell proliferation, differentiation, cell cycle, and innate immune response in esophageal epithelium. The disruption of these pathways causes inflammation and significant modifications of the microbiome. NEW & NOTEWORTHY Calcium-sensing receptor (CaSR) plays a significant role in maintaining the barrier function of esophageal epithelium. Using RNA sequencing, we show that conditional deletion of CaSR from mouse esophagus causes upregulation of genes involved in DNA replication and cell cycle and downregulation of genes involved in the innate immune response, protein translation, and cellular protein synthesis. Pathway analysis shows disruption of signaling pathways of calcium and actin cytoskeleton. These changes caused inflammation and esophageal dysbiosis.- Published
- 2024
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7. Loss of feedback regulation between FAM3B and androgen receptor driving prostate cancer progression.
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Ma T, Jin L, Bai S, Liu Z, Wang S, Shen B, Cho Y, Cao S, Sun MJS, Fazli L, Zhang D, Wedderburn C, Zhang DY, Mugon G, Ungerleider N, Baddoo M, Zhang K, Schiavone LH, Burkhardt BR, Fan J, You Z, Flemington EK, Dong X, and Dong Y
- Subjects
- Male, Humans, Feedback, Transcriptome, Oncogene Proteins, Fusion genetics, Transcriptional Regulator ERG genetics, Transcriptional Regulator ERG metabolism, Neoplasm Proteins genetics, Cytokines genetics, Receptors, Androgen genetics, Prostatic Neoplasms genetics, Prostatic Neoplasms metabolism
- Abstract
Background: Although the fusion of the transmembrane serine protease 2 gene (TMPRSS2) with the erythroblast transformation-specific-related gene (ERG), or TMPRSS2-ERG, occurs frequently in prostate cancer, its impact on clinical outcomes remains controversial. Roughly half of TMPRSS2-ERG fusions occur through intrachromosomal deletion of interstitial genes and the remainder via insertional chromosomal rearrangements. Because prostate cancers with deletion-derived TMPRSS2-ERG fusions are more aggressive than those with insertional fusions, we investigated the impact of interstitial gene loss on prostate cancer progression., Methods: We conducted an unbiased analysis of transcriptome data from large collections of prostate cancer samples and employed diverse in vitro and in vivo models combined with genetic approaches to characterize the interstitial gene loss that imposes the most important impact on clinical outcome., Results: This analysis identified FAM3B as the top-ranked interstitial gene whose loss is associated with a poor prognosis. The association between FAM3B loss and poor clinical outcome extended to fusion-negative prostate cancers where FAM3B downregulation occurred through epigenetic imprinting. Importantly, FAM3B loss drives disease progression in prostate cancer. FAM3B acts as an intermediator of a self-governing androgen receptor feedback loop. Specifically, androgen receptor upregulates FAM3B expression by binding to an intronic enhancer to induce an enhancer RNA and facilitate enhancer-promoter looping. FAM3B, in turn, attenuates androgen receptor signaling., Conclusion: Loss of FAM3B in prostate cancer, whether through the TMPRSS2-ERG translocation or epigenetic imprinting, causes an exit from this autoregulatory loop to unleash androgen receptor activity and prostate cancer progression. These findings establish FAM3B loss as a new driver of prostate cancer progression and support the utility of FAM3B loss as a biomarker to better define aggressive prostate cancer., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
- Published
- 2024
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8. Transcriptomic analysis identifies B-lymphocyte kinase as a therapeutic target for desmoplastic small round cell tumor cancer stem cell-like cells.
- Author
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Magrath JW, Flinchum DA, Hartono AB, Sampath SS, O'Grady TM, Baddoo M, Haoyang L, Xu X, Flemington EK, and Lee SB
- Abstract
Desmoplastic small round cell tumor (DSRCT) is an aggressive pediatric cancer caused by the EWSR1-WT1 fusion oncoprotein. The tumor is refractory to treatment with a 5-year survival rate of only 15-25%, necessitating the development of novel therapeutics, especially those able to target chemoresistant subpopulations. Novel in vitro cancer stem cell-like (CSC-like) culture conditions increase the expression of stemness markers (SOX2, NANOG) and reduce DSRCT cell line susceptibility to chemotherapy while maintaining the ability of DSRCT cells to form xenografts. To gain insights into this chemoresistant model, RNA-seq was performed to elucidate transcriptional alterations between DSRCT cells grown in CSC-like spheres and normal 2-dimensional adherent state. Commonly upregulated and downregulated genes were identified and utilized in pathway analysis revealing upregulation of pathways related to chromatin assembly and disassembly and downregulation of pathways including cell junction assembly and extracellular matrix organization. Alterations in chromatin assembly suggest a role for epigenetics in the DSRCT CSC-like state, which was further investigated with ATAC-seq, identifying over 10,000 differentially accessible peaks, including 4444 sphere accessible peaks and 6,120 adherent accessible peaks. Accessible regions were associated with higher gene expression, including increased accessibility of the CSC marker SOX2 in CSC-like culture conditions. These analyses were further utilized to identify potential CSC therapeutic targets, leading to the identification of B-lymphocyte kinase (BLK) as a CSC-enriched, EWSR1-WT1-regulated, druggable target. BLK inhibition and knockdown reduced CSC-like properties, including abrogation of tumorsphere formation and stemness marker expression. Importantly, BLK knockdown reduced DSRCT CSC-like cell chemoresistance, making its inhibition a promising target for future combination therapy., (© 2024. The Author(s).)
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- 2024
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9. SpliceTools, a suite of downstream RNA splicing analysis tools to investigate mechanisms and impact of alternative splicing.
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Flemington EK, Flemington SA, O'Grady TM, Baddoo M, Nguyen T, Dong Y, and Ungerleider NA
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- Sulfonamides, Transcriptome genetics, Sequence Analysis, RNA methods, Alternative Splicing genetics, RNA Splicing
- Abstract
As a fundamental aspect of normal cell signaling and disease states, there is great interest in determining alternative splicing (AS) changes in physiologic, pathologic, and pharmacologic settings. High throughput RNA sequencing and specialized software to detect AS has greatly enhanced our ability to determine transcriptome-wide splicing changes. Despite the richness of this data, deriving meaning from sometimes thousands of AS events is a substantial bottleneck for most investigators. We present SpliceTools, a suite of data processing modules that arms investigators with the ability to quickly produce summary statistics, mechanistic insights, and functional significance of AS changes through command line or through an online user interface. Utilizing RNA-seq datasets for 186 RNA binding protein knockdowns, nonsense mediated RNA decay inhibition, and pharmacologic splicing inhibition, we illustrate the utility of SpliceTools to distinguish splicing disruption from regulated transcript isoform changes, we show the broad transcriptome footprint of the pharmacologic splicing inhibitor, indisulam, we illustrate the utility in uncovering mechanistic underpinnings of splicing inhibition, we identify predicted neo-epitopes in pharmacologic splicing inhibition, and we show the impact of splicing alterations induced by indisulam on cell cycle progression. Together, SpliceTools puts rapid and easy downstream analysis at the fingertips of any investigator studying AS., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2023
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10. Reversal of splicing infidelity is a pre-activation step in B cell differentiation.
- Author
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O'Grady TM, Baddoo M, Flemington SA, Ishaq EY, Ungerleider NA, and Flemington EK
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- Humans, Herpesvirus 4, Human genetics, RNA, Messenger genetics, Cell Differentiation genetics, Alternative Splicing, Epstein-Barr Virus Infections
- Abstract
Introduction: B cell activation and differentiation is central to the adaptive immune response. Changes in exon usage can have major impacts on cellular signaling and differentiation but have not been systematically explored in differentiating B cells., Methods: We analyzed exon usage and intron retention in RNA-Seq data from subsets of human B cells at various stages of differentiation, and in an in vitro laboratory model of B cell activation and differentiation (Epstein Barr virus infection)., Results: Blood naïve B cells were found to have an unusual splicing profile, with unannotated splicing events in over 30% of expressed genes. Splicing changed substantially upon naïve B cell entry into secondary lymphoid tissue and before activation, involving significant increases in exon commitment and reductions in intron retention. These changes preferentially involved short introns with weak splice sites and were likely mediated by an overall increase in splicing efficiency induced by the lymphoid environment. The majority of transcripts affected by splicing changes showed restoration of encoded conserved protein domains and/or reduced targeting to the nonsense-mediated decay pathway. Affected genes were enriched in functionally important immune cell activation pathways such as antigen-mediated signaling, cell cycle control and mRNA processing and splicing., Discussion: Functional observations from donor B cell subsets in progressive states of differentiation and from timecourse experiments using the in vitro model suggest that these widespread changes in mRNA splicing play a role in preparing naïve B cells for the decisive step of antigen-mediated activation and differentiation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 O’Grady, Baddoo, Flemington, Ishaq, Ungerleider and Flemington.)
- Published
- 2022
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11. Establishment and characterization of a new mantle cell lymphoma cell line with a NOTCH2 mutation, Arbo.
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Safa F, Rasmussen T, Lobelle-Rich P, Collier S, Milligan N, Schmeig J, Schmid J, Wiewiorowski C, Totaro D, Brown TC, Satyavarapu I, Badoo M, Ungerleider N, Flemington EK, Safah H, and Saba NS
- Abstract
Cell lines represent an essential tool used in preclinical research. Most hematologic malignancies have a wide array of cell lines representing their respective molecular and pathologic spectra. In mantle cell lymphoma (MCL), cell lines become specifically valuable in view of the heterogeneity of this disease. Unfortunately, the number of MCL cell lines that are available for the research community remains small, with only nine cell lines available for purchase through the American Type Culture Collection (ATCC). We have established a novel blastoid MCL cell line, isolated from the malignant pleural effusion of a 69-year-old male with refractory MCL. Arbo was fully characterized with cytogenetics, immunophenotyping, whole exome sequencing and drug sensitivity assays. One of the most notable mutations identified in Arbo (but not in normal tissue) was the missense mutation NOTCH2 R2400*, which has been proposed as a clinically significant mutation in MCL seen in 5% of cases. NOTCH2 R2400* results in a truncated Notch2 protein, leading to a more stable and active protein. Using pharmacologic inhibition of Notch2, we showed a dependence of Arbo on NOTCH2 signaling, as well as a link between CD23 expression on Arbo and NOTCH2 activity. Arbo represents a NOTCH2 mutated model that is useful in MCL as well as other lymphomas with such mutation. We plan to deposit Arbo at the ATCC to be available for the research community., Competing Interests: The authors declare they have no conflicts of interest., (© 2022 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)
- Published
- 2022
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12. A Polymorphism in the Epstein-Barr Virus EBER2 Noncoding RNA Drives In Vivo Expansion of Latently Infected B Cells.
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Wang Y, Ungerleider N, Hoffman BA, Kara M, Farrell PJ, Flemington EK, Lee N, and Tibbetts SA
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- Animals, Herpesvirus 4, Human physiology, Humans, Mice, Nucleotides, Polymorphism, Genetic, RNA, Untranslated genetics, RNA, Untranslated metabolism, RNA, Viral, Virus Latency genetics, Epstein-Barr Virus Infections genetics, Gammaherpesvirinae genetics, Herpesvirus 8, Human genetics, Rhadinovirus genetics
- Abstract
The oncogenic gammaherpesviruses, including human Epstein-Barr virus (EBV), human Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, γHV68, MuHV-4), are associated with numerous malignancies, including B cell lymphomas and nasopharyngeal carcinoma. These viruses employ numerous molecular strategies to colonize the host, including the expression of noncoding RNAs (ncRNAs). As the first viral ncRNAs identified, EBV-encoded RNA 1 and 2 (EBER1 and EBER2, respectively) have been investigated extensively for decades; however, their specific in vivo functions remain largely unknown. In work here, we used chimeric MHV68 viruses in an in vivo complementation system to test whether EBV EBER2 contributes to acute and/or chronic phases of infection. Expression of EBER2 derived from EBV strain B95-8 resulted in a significant expansion of latently infected B cells in vivo , which was accompanied by a decrease in virus-infected plasma cells. EBV strains typically carry one of two variants of EBER2, which differ primarily by a 5-nucleotide core polymorphism identified initially in the EBV strain M81. Strikingly, mutation of the 5 nucleotides that define this core polymorphism resulted in the loss of the infected B cell expansion and restored plasma cell infection. This work reveals that the B95-8 variant of EBER2 promotes the expansion of the latently infected B cell pool in vivo and may do so in part through inhibition of terminal differentiation. These findings provide new insight into mechanisms by which viral ncRNAs promote in vivo colonization and further and provide further evidence of the inherent tumorigenic risks associated with gammaherpesvirus manipulation of B cell differentiation. IMPORTANCE The oncogenic gammaherpesviruses, including human Epstein-Barr virus (EBV), human Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68, employ numerous strategies to colonize the host, including expression of noncoding RNAs (ncRNAs). As the first viral ncRNAs ever identified, EBV-encoded RNA 1 and 2 (EBER1 and EBER2) have been investigated extensively for decades; however, their specific in vivo functions remain largely unknown. Work here reveals that an EBV EBER2 variant highly associated with B cell lymphoma promoted a significantly increased expansion of the infected B cell pool in vivo , which coincided with altered B cell differentiation. Mutation of the 5 nucleotides that define this EBER2 variant resulted in the loss of B cell expansion and normal B cell differentiation. These findings provide new insight into the mechanisms by which EBV manipulates B cells in vivo to retain infected cells in the high-risk B cell differentiation pathway where they are poised for tumorigenesis.
- Published
- 2022
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13. Ebola virus delta peptide is an enterotoxin.
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Melnik LI, Guha S, Ghimire J, Smither AR, Beddingfield BJ, Hoffmann AR, Sun L, Ungerleider NA, Baddoo MC, Flemington EK, Gallaher WR, Wimley WC, and Garry RF
- Subjects
- Animals, Diarrhea virology, Female, Gastroenteritis pathology, Male, Mice, Mice, Inbred BALB C, Ebolavirus metabolism, Enterotoxins toxicity, Gastroenteritis virology, Hemorrhagic Fever, Ebola pathology, Viral Envelope Proteins toxicity
- Abstract
During the 2013-2016 West African (WA) Ebola virus (EBOV) outbreak, severe gastrointestinal symptoms were common in patients and associated with poor outcome. Delta peptide is a conserved product of post-translational processing of the abundant EBOV soluble glycoprotein (sGP). The murine ligated ileal loop model was used to demonstrate that delta peptide is a potent enterotoxin. Dramatic intestinal fluid accumulation follows injection of biologically relevant amounts of delta peptide into ileal loops, along with gross alteration of villous architecture and loss of goblet cells. Transcriptomic analyses show that delta peptide triggers damage response and cell survival pathways and downregulates expression of transporters and exchangers. Induction of diarrhea by delta peptide occurs via cellular damage and regulation of genes that encode proteins involved in fluid secretion. While distinct differences exist between the ileal loop murine model and EBOV infection in humans, these results suggest that delta peptide may contribute to EBOV-induced gastrointestinal pathology., Competing Interests: Declaration of interests Dr. Garry is co-founder of Zalgen Labs, a biotechnology company that develops countermeasures for emerging viruses., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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14. SON drives oncogenic RNA splicing in glioblastoma by regulating PTBP1/PTBP2 switching and RBFOX2 activity.
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Kim JH, Jeong K, Li J, Murphy JM, Vukadin L, Stone JK, Richard A, Tran J, Gillespie GY, Flemington EK, Sobol RW, Lim SS, and Ahn EE
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- Animals, Brain Neoplasms metabolism, Brain Neoplasms mortality, Brain Neoplasms pathology, Cell Cycle genetics, Cell Line, Tumor, Cell Proliferation, DNA-Binding Proteins antagonists & inhibitors, DNA-Binding Proteins metabolism, Exons, Gene Expression Regulation, Neoplastic, Glioblastoma metabolism, Glioblastoma mortality, Glioblastoma pathology, Heterogeneous-Nuclear Ribonucleoprotein Group A-B genetics, Heterogeneous-Nuclear Ribonucleoprotein Group A-B metabolism, Heterogeneous-Nuclear Ribonucleoproteins metabolism, Heterografts, Humans, Introns, Mice, Minor Histocompatibility Antigens metabolism, Nerve Tissue Proteins metabolism, Neuroglia metabolism, Neuroglia pathology, Neurons metabolism, Neurons pathology, Polypyrimidine Tract-Binding Protein metabolism, RNA Splicing Factors metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Repressor Proteins metabolism, Signal Transduction, Spheroids, Cellular metabolism, Spheroids, Cellular pathology, Survival Analysis, Brain Neoplasms genetics, DNA-Binding Proteins genetics, Glioblastoma genetics, Heterogeneous-Nuclear Ribonucleoproteins genetics, Minor Histocompatibility Antigens genetics, Nerve Tissue Proteins genetics, Polypyrimidine Tract-Binding Protein genetics, RNA Splicing, RNA Splicing Factors genetics, Repressor Proteins genetics
- Abstract
While dysregulation of RNA splicing has been recognized as an emerging target for cancer therapy, the functional significance of RNA splicing and individual splicing factors in brain tumors is poorly understood. Here, we identify SON as a master regulator that activates PTBP1-mediated oncogenic splicing while suppressing RBFOX2-mediated non-oncogenic neuronal splicing in glioblastoma multiforme (GBM). SON is overexpressed in GBM patients and SON knockdown causes failure in intron removal from the PTBP1 transcript, resulting in PTBP1 downregulation and inhibition of its downstream oncogenic splicing. Furthermore, SON forms a complex with hnRNP A2B1 and antagonizes RBFOX2, which leads to skipping of RBFOX2-targeted cassette exons, including the PTBP2 neuronal exon. SON knockdown inhibits proliferation and clonogenicity of GBM cells in vitro and significantly suppresses tumor growth in orthotopic xenografts in vivo. Collectively, our study reveals that SON-mediated RNA splicing is a GBM vulnerability, implicating SON as a potential therapeutic target in brain tumors., (© 2021. The Author(s).)
- Published
- 2021
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15. SON inhibits megakaryocytic differentiation via repressing RUNX1 and the megakaryocytic gene expression program in acute megakaryoblastic leukemia.
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Vukadin L, Kim JH, Park EY, Stone JK, Ungerleider N, Baddoo MC, Kong HK, Richard A, Tran J, Giannini H, Flemington EK, Lim SS, and Ahn EE
- Subjects
- Cell Differentiation, Down Syndrome genetics, Gene Expression, Genetic Predisposition to Disease, Humans, Leukemia, Megakaryoblastic, Acute genetics, Leukemia, Megakaryoblastic, Acute pathology, Transfection, Core Binding Factor Alpha 2 Subunit metabolism, DNA-Binding Proteins metabolism, Leukemia, Megakaryoblastic, Acute metabolism, Megakaryocytes metabolism, Minor Histocompatibility Antigens metabolism
- Abstract
A high incidence of acute megakaryoblastic leukemia (AMKL) in Down syndrome patients implies that chromosome 21 genes have a pivotal role in AMKL development, but the functional contribution of individual genes remains elusive. Here, we report that SON, a chromosome 21-encoded DNA- and RNA-binding protein, inhibits megakaryocytic differentiation by suppressing RUNX1 and the megakaryocytic gene expression program. As megakaryocytic progenitors differentiate, SON expression is drastically reduced, with mature megakaryocytes having the lowest levels. In contrast, AMKL cells express an aberrantly high level of SON, and knockdown of SON induced the onset of megakaryocytic differentiation in AMKL cell lines. Genome-wide transcriptome analyses revealed that SON knockdown turns on the expression of pro-megakaryocytic genes while reducing erythroid gene expression. Mechanistically, SON represses RUNX1 expression by directly binding to the proximal promoter and two enhancer regions, the known +23 kb enhancer and the novel +139 kb enhancer, at the RUNX1 locus to suppress H3K4 methylation. In addition, SON represses the expression of the AP-1 complex subunits JUN, JUNB, and FOSB which are required for late megakaryocytic gene expression. Our findings define SON as a negative regulator of RUNX1 and megakaryocytic differentiation, implicating SON overexpression in impaired differentiation during AMKL development., (© 2020. The Author(s), under exclusive licence to Springer Nature America, Inc. part of Springer Nature.)
- Published
- 2021
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16. Transcriptome analysis reveals sexual disparities in gene expression in rat brain microvessels.
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Chandra PK, Cikic S, Baddoo MC, Rutkai I, Guidry JJ, Flemington EK, Katakam PV, and Busija DW
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- Animals, Female, Male, Rats, Rats, Sprague-Dawley, Sex Characteristics, Brain physiopathology, Gene Expression Profiling methods, Gene Expression Regulation genetics, Microvessels physiopathology, Proteomics methods
- Abstract
Sex is an important determinant of brain microvessels (MVs) function and susceptibility to cerebrovascular and neurological diseases, but underlying mechanisms are unclear. Using high throughput RNA sequencing analysis, we examined differentially expressed (DE) genes in brain MVs from young, male, and female rats. Bioinformatics analysis of the 23,786 identified genes indicates that 298 (1.2%) genes were DE using False Discovery Rate criteria (FDR; p < 0.05), of which 119 (40%) and 179 (60%) genes were abundantly expressed in male and female MVs, respectively. Nucleic acid binding, enzyme modulator, and transcription factor were the top three DE genes, which were more highly expressed in male than female MVs. Synthesis of glycosylphosphatidylinositol (GPI), biosynthesis of GPI-anchored proteins, steroid and cholesterol synthesis, were the top three significantly enriched canonical pathways in male MVs. In contrast, respiratory chain, ribosome, and 3 ́-UTR-mediated translational regulation were the top three enriched canonical pathways in female MVs. Different gene functions of MVs were validated by proteomic analysis and western blotting. Our novel findings reveal major sex disparities in gene expression and canonical pathways of MVs and these differences provide a foundation to study the underlying mechanisms and consequences of sex-dependent differences in cerebrovascular and other neurological diseases.
- Published
- 2021
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17. EBV miRNAs are potent effectors of tumor cell transcriptome remodeling in promoting immune escape.
- Author
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Ungerleider N, Bullard W, Kara M, Wang X, Roberts C, Renne R, Tibbetts S, and Flemington EK
- Subjects
- Epstein-Barr Virus Infections metabolism, Epstein-Barr Virus Infections pathology, Epstein-Barr Virus Infections virology, Herpesvirus 4, Human genetics, Humans, RNA-Induced Silencing Complex genetics, Epstein-Barr Virus Infections immunology, Gene Expression Regulation, Viral, Herpesvirus 4, Human immunology, MicroRNAs genetics, RNA, Viral genetics, RNA-Induced Silencing Complex metabolism, Transcriptome
- Abstract
The Epstein Barr virus (EBV) contributes to the tumor phenotype through a limited set of primarily non-coding viral RNAs, including 31 mature miRNAs. Here we investigated the impact of EBV miRNAs on remodeling the tumor cell transcriptome. Strikingly, EBV miRNAs displayed exceptionally abundant expression in primary EBV-associated Burkitt's Lymphomas (BLs) and Gastric Carcinomas (GCs). To investigate viral miRNA targeting, we used the high-resolution approach, CLASH in GC and BL cell models. Affinity constant calculations of targeting efficacies for CLASH hits showed that viral miRNAs bind their targets more effectively than their host counterparts, as did Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) miRNAs. Using public BL and GC RNA-seq datasets, we found that high EBV miRNA targeting efficacies translates to enhanced reduction of target expression. Pathway analysis of high efficacy EBV miRNA targets showed enrichment for innate and adaptive immune responses. Inhibition of the immune response by EBV miRNAs was functionally validated in vivo through the finding of inverse correlations between EBV miRNAs and immune cell infiltration and T-cell diversity in BL and GC datasets. Together, this study demonstrates that EBV miRNAs are potent effectors of the tumor transcriptome that play a role in suppressing host immune response., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2021
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18. Increased transcription and high translation efficiency lead to accumulation of androgen receptor splice variant after androgen deprivation therapy.
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Ma T, Bai S, Qi Y, Zhan Y, Ungerleider N, Zhang DY, Neklesa T, Corey E, Dehm SM, Zhang K, Flemington EK, and Dong Y
- Subjects
- Humans, Male, RNA, Messenger genetics, Androgen Antagonists pharmacology, Androgens deficiency, Protein Biosynthesis, RNA Splicing, Receptors, Androgen genetics, Transcription, Genetic
- Abstract
Upregulation of androgen receptor splice variants (AR-Vs), especially AR-V7, is associated with castration resistance of prostate cancer. At the RNA level, AR-V7 upregulation is generally coupled with increased full-length AR (AR-FL); consequently, AR-V7 and AR-Vs collectively constitute a minority of the AR population. However, Western blotting showed that the relative abundance of AR-V proteins is much higher in many castration-resistant prostate cancers (CRPCs). To address the mechanism underlying this discrepancy, we analyzed RNA-seq data from ~350 CRPC samples and found a positive correlation between all canonical and alternative AR splicing. This indicates that increased alternative splicing is not at the expense of canonical splicing. Instead, androgen deprivation releases AR-FL from repressing the transcription of the AR gene to induce coordinated increase of AR-FL and AR-V mRNAs. At the protein level, however, androgen deprivation induces AR-FL, but not AR-V, degradation. Moreover, AR-V7 is translated much faster than AR-FL. Thus, androgen-deprivation-induced AR-gene transcription and AR-FL protein decay, together with efficient AR-V7 translation, explain the discrepancy between the relative AR-V mRNA and protein abundances in many CRPCs, highlighting the inevitability of AR-V induction after endocrine therapy., (Copyright © 2021 Elsevier B.V. All rights reserved.)
- Published
- 2021
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19. Transcriptional signatures of Zika virus infection in astrocytes.
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Schouest B, Peterson TA, Szeltner DM, Scheef EA, Baddoo M, Ungerleider N, Flemington EK, MacLean AG, and Maness NJ
- Subjects
- Animals, Cells, Cultured, Macaca mulatta, Zika Virus, Astrocytes virology, Brain virology, Transcriptome, Zika Virus Infection virology
- Abstract
Astrocytes are an early and important target of Zika virus (ZIKV) infection in the developing brain, but the impacts of infection on astrocyte function remain controversial. Given that nonhuman primate (NHP) models of ZIKV infection replicate aspects of neurologic disease seen in human infections, we cultured primary astrocytes from the brain tissue of infant rhesus macaques and then infected the cells with Asian or African lineage ZIKV to identify transcriptional patterns associated with infection in these cells. The African lineage virus appeared to have greater infectivity and promote stronger antiviral signaling, but infection by either strain ultimately produced typical virus response patterns. Both viruses induced hypoxic stress, but the Asian lineage strain additionally had an effect on metabolic and lipid biosynthesis pathways. Together, these findings describe an NHP astrocyte model that may be used to assess transcriptional signatures following ZIKV infection.
- Published
- 2021
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20. Data of relative mRNA and protein abundances of androgen receptor splice variants in castration-resistant prostate cancer.
- Author
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Ma T, Ungerleider N, Zhang DY, Corey E, Flemington EK, and Dong Y
- Abstract
These data include secondary analysis of publicly available RNA-seq data from castration-resistant prostate cancer (CRPC) patients as well as RT-qPCR and Western blotting analyses of patient-derived xenograft models and a CRPC cell line. We applied Spearman correlation analysis to assess the relationship between canonical androgen receptor (AR) splicing and alternative AR splicing. We also assessed the ratio of AR splice variants (AR-Vs) to the full-length AR (AR-FL) at the RNA and protein levels by absolute RT-qPCR and Western blotting, respectively. These data are critical for studying the mechanisms underlying upregulated expression of AR-Vs after AR-directed therapies and the importance of AR-Vs to castration-resistant progression of prostate cancer. Data presented here are related to the research article by Ma et al., "Increased transcription and high translation efficiency lead to accumulation of androgen receptor splice variant after androgen deprivation therapy", Cancer Lett. In Press [1]., Competing Interests: This work was supported by the National Institutes of Health [grant numbers R01CA188609, R01AI106676, R01CA243793]. The Richard M Lucas Foundation supported the development of the LuCaP models. The authors declare that they have no known competing financial interests or personal relationships that have or could be perceived to have influenced the work reported in this article., (© 2021 The Authors.)
- Published
- 2021
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21. Somatic mutations in the DNA repairome in prostate cancers in African Americans and Caucasians.
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Yadav S, Anbalagan M, Baddoo M, Chellamuthu VK, Mukhopadhyay S, Woods C, Jiang W, Moroz K, Flemington EK, and Makridakis N
- Subjects
- Aged, DNA, Neoplasm metabolism, Humans, Male, Middle Aged, Neoplasm Proteins metabolism, Prostatic Neoplasms metabolism, Black or African American, DNA Repair, DNA, Neoplasm genetics, Neoplasm Proteins genetics, Prostatic Neoplasms genetics, White People
- Abstract
Most hereditary tumors show aberrations in DNA repair genes or their regulators. In contrast, only a minority of sporadic tumors show alterations in these genes. As a result, genomic instability is currently considered an enhancer of tumorigenesis rather than an obligatory event in this process. However, tumor heterogeneity presents a significant technical challenge for most cancer genomics studies performed at less than 100× mean resolution depth. To address the importance of genomic instability in prostate carcinogenesis and tumor progression, we performed ultrahigh depth exome sequencing of 124 DNA damage repair/response (repairome) genes in 63 tumors and matched normal tissue samples in African Americans and Caucasians. The average sequence depth was 712-fold for DNA isolated from normal tissue and 368-fold for FFPE tumors. We identified 671 somatic mutations in tumors from African Americans and 762 somatic mutations in tumors in Caucasians. The most frequently mutated DNA repairome genes were EXO1, ATR, POLQ, NEIL3, ERCC6, BRCA2, BRCA1, XPC, JAG1, RPA1, POLE, ATM, and LIG1 in African American men, and POLQ, NEIL3, POLB, BRCA2, EXO1, ERCC6, ATR, RBBP8, BRCA1, ATM, JAG1, XPC, and POLE in Caucasians. We found that 89% of tumors had at least one mutation in nucleotide excision repair pathway genes in African Americans, whereas >40% of tumors had mutations in base excision repair pathway genes in Caucasians. We further identified a marginal increase in mutation rate in tumors in African Americans with increasing age. Tumors in Caucasians did not show a correlation with age, but a progressive increase in the mutation rate was observed at higher Gleason scores. Our data reveal significant differences in the molecular signatures in the DNA repairome in prostate cancer between African Americans and Caucasians. These data also have substantial implications regarding the well-known health disparities in prostate cancer, such as the higher mortality in African Americans than Caucasians.
- Published
- 2020
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22. SEER and Gene Expression Data Analysis Deciphers Racial Disparity Patterns in Prostate Cancer Mortality and the Public Health Implication.
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Zhang W, Dong Y, Sartor O, Flemington EK, and Zhang K
- Subjects
- Black or African American genetics, Age Factors, Aged, Aged, 80 and over, Cluster Analysis, Demography, Gene Expression Profiling, Humans, Incidence, Male, Middle Aged, Neoplasm Grading, Registries, Regression Analysis, Survival Analysis, White People genetics, Data Analysis, Gene Expression Regulation, Neoplastic, Prostatic Neoplasms genetics, Prostatic Neoplasms mortality, Public Health, Racial Groups genetics, SEER Program
- Abstract
A major racial disparity in prostate cancer (PCa) is that African American (AA) patients have a higher mortality rate than European American (EA) patients. We filtered the SEER 2009-2011 records and divided them into four groups regarding patient races and cancer grades. On such a partition, we performed a series of statistical analyses to further clarify the aforementioned disparity. Molecular evidence for a primary result of the epidemiological analysis was obtained from gene expression data. The results include: (1) Based on the registry-specific measures, a significant linear regression of total mortality rate (as well as PCa specific mortality rate) on the percentage of (Gleason pattern-based) high-grade cancers (PHG) is demonstrated in EAs (p < 0.01) but not in AAs; (2) PHG and its racial disparity are differentiated across ages and the groups defined by patient outcomes; (3) For patients with cancers in the same grade category, i.e. the high or low grade, the survival stratification between races is not significant in most geographical areas; and (4) The genes differentially expressed between AAs' and EAs' tumors of the same grade category are relatively rare. The perception that prostate tumors are more lethal in AAs than in EAs is reasonable regarding AAs' higher PHG, while high grade alone could not imply aggressiveness. However, this perception is questionable when the comparison is focused on cases within the same grade category. Supporting observations for this conclusion hold a remarkable implication for erasing racial disparity in PCa. That is, "Equal grade, equal outcomes" is not only a verifiable hypothesis but also an achievable public health goal.
- Published
- 2020
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23. Assessment of viral RNA in idiopathic pulmonary fibrosis using RNA-seq.
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Yin Q, Strong MJ, Zhuang Y, Flemington EK, Kaminski N, de Andrade JA, and Lasky JA
- Subjects
- Case-Control Studies, Humans, Idiopathic Pulmonary Fibrosis pathology, Lung pathology, Real-Time Polymerase Chain Reaction, Idiopathic Pulmonary Fibrosis virology, Lung virology, RNA, Viral analysis, RNA-Seq, Virus Diseases complications
- Abstract
Background: Numerous publications suggest an association between herpes virus infection and idiopathic pulmonary fibrosis (IPF). These reports have employed immunohistochemistry, in situ hybridization and/or PCR, which are susceptible to specificity artifacts., Methods: We investigated the possible association between IPF and viral RNA expression using next-generation sequencing, which has the potential to provide a high degree of both sensitivity and specificity. We quantified viral RNA expression for 740 viruses in 28 IPF patient lung biopsy samples and 20 controls. Key RNA-seq results were confirmed using Real-time RT-PCR for select viruses (EBV, HCV, herpesvirus saimiri and HERV-K)., Results: We identified sporadic low-level evidence of viral infections in our lung tissue specimens, but did not find a statistical difference for expression of any virus, including EBV, herpesvirus saimiri and HERV-K, between IPF and control lungs., Conclusions: To the best of our knowledge, this is the first publication that employs RNA-seq to assess whether viral infections are linked to the pathogenesis of IPF. Our results do not address the role of viral infection in acute exacerbations of IPF, however, this analysis patently did not support an association between herpes virus detection and IPF.
- Published
- 2020
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24. Screen technical noise in single cell RNA sequencing data.
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Bai YL, Baddoo M, Flemington EK, Nakhoul HN, and Liu YZ
- Subjects
- Cell Line, Genes, Essential, Humans, Software, RNA-Seq methods, Single-Cell Analysis methods
- Abstract
We proposed a data cleaning pipeline for single cell (SC) RNA-seq data, where we first screen genes (gene-wise screening) followed by screening cell libraries (library-wise screening). Gene-wise screening is based on the expectation that for a gene with a low technical noise, a gene's count in a library will tend to increase with the increase of library size, which was tested using negative binomial regression of gene count (as dependent variable) against library size (as independent variable). Library-wise screening is based on the expectation that across-library correlations for housekeeping (HK) genes is expected to be higher than the correlations for non-housekeeping (NHK) genes in those libraries with low technical noise. We removed those libraries, whose mean pairwise correlation for HK genes is NOT significantly higher than that for NHK genes. We successfully applied the pipeline to two large SC RNA-seq datasets. The pipeline was also developed into an R package., (Copyright © 2019. Published by Elsevier Inc.)
- Published
- 2020
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25. Circular RNAs add diversity to androgen receptor isoform repertoire in castration-resistant prostate cancer.
- Author
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Cao S, Ma T, Ungerleider N, Roberts C, Kobelski M, Jin L, Concha M, Wang X, Baddoo M, Nguyen HM, Corey E, Fazli L, Ledet E, Zhang R, Silberstein JL, Zhang W, Zhang K, Sartor O, Dong X, Flemington EK, and Dong Y
- Subjects
- Animals, Humans, Male, Mice, SCID, Protein Isoforms, Receptors, Androgen classification, Tumor Cells, Cultured, Xenograft Model Antitumor Assays, Gene Expression Regulation, Neoplastic, Prostatic Neoplasms, Castration-Resistant genetics, Prostatic Neoplasms, Castration-Resistant pathology, RNA, Circular genetics, Receptors, Androgen genetics
- Abstract
Deregulated expression of circular RNAs (circRNAs) is associated with various human diseases, including many types of cancer. Despite their growing links to cancer, there has been limited characterization of circRNAs in metastatic castration-resistant prostate cancer, the major cause of prostate cancer mortality. Here, through the analysis of an exome-capture RNA-seq dataset from 47 metastatic castration-resistant prostate cancer samples and ribodepletion and RNase R RNA-sequencing of patient-derived xenografts (PDXs) and cell models, we identified 13 circRNAs generated from the key prostate cancer driver gene-androgen receptor (AR). We validated and characterized the top four most abundant, clinically relevant AR circRNAs. Expression of these AR circRNAs was upregulated during castration-resistant progression of PDXs. The upregulation was not due to global increase of circRNA formation in these tumors. Instead, the levels of AR circRNAs correlated strongly with that of the linear AR transcripts (both AR and AR variants) in clinical samples and PDXs, indicating a transcriptional mechanism of regulation. In cultured cells, androgen suppressed the expression of these AR circRNAs and the linear AR transcripts, and the suppression was attenuated by an antiandrogen. Using nuclear/cytoplasmic fractionation and RNA in-situ hybridization assays, we demonstrated predominant cytoplasmic localization of these AR circRNAs, indicating likely cytoplasmic functions. Overall, this is the first comprehensive characterization of circRNAs arising from the AR gene. With greater resistance to exoribonuclease compared to the linear AR transcripts and detectability of AR circRNAs in patient plasma, these AR circRNAs may serve as surrogate circulating markers for AR/AR-variant expression and castration-resistant prostate cancer progression.
- Published
- 2019
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26. Genome-wide Transcript Structure Resolution Reveals Abundant Alternate Isoform Usage from Murine Gammaherpesvirus 68.
- Author
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O'Grady T, Feswick A, Hoffman BA, Wang Y, Medina EM, Kara M, van Dyk LF, Flemington EK, and Tibbetts SA
- Subjects
- Animals, Genome-Wide Association Study, Mice, NIH 3T3 Cells, Gammaherpesvirinae metabolism, Herpesviridae Infections metabolism, Open Reading Frames, RNA, Viral biosynthesis, Transcriptome
- Abstract
The gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, MuHV-4, γHV68), are etiologic agents of a wide range of lymphomas and non-hematological malignancies. These viruses possess large and highly dense dsDNA genomes that feature >80 bidirectionally positioned open reading frames (ORFs). The abundance of overlapping transcripts and extensive splicing throughout these genomes have until now prohibited high throughput-based resolution of transcript structures. Here, we integrate the capabilities of long-read sequencing with the accuracy of short-read platforms to globally resolve MHV68 transcript structures using the transcript resolution through integration of multi-platform data (TRIMD) pipeline. This approach reveals highly complex features, including: (1) pervasive overlapping transcript structures; (2) transcripts containing intra-gene or trans-gene splices that yield chimeric ORFs; (3) antisense and intergenic transcripts containing ORFs; and (4) noncoding transcripts. This work sheds light on the underappreciated complexity of gammaherpesvirus transcription and provides an extensively revised annotation of the MHV68 transcriptome., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
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27. A positive role of c-Myc in regulating androgen receptor and its splice variants in prostate cancer.
- Author
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Bai S, Cao S, Jin L, Kobelski M, Schouest B, Wang X, Ungerleider N, Baddoo M, Zhang W, Corey E, Vessella RL, Dong X, Zhang K, Yu X, Flemington EK, and Dong Y
- Subjects
- Adenocarcinoma pathology, Alternative Splicing genetics, Animals, Cells, Cultured, Gene Expression Regulation, Neoplastic, HEK293 Cells, Humans, Male, Mice, Mice, Inbred C57BL, Mice, SCID, Prostatic Neoplasms, Castration-Resistant pathology, Protein Isoforms genetics, Adenocarcinoma genetics, Prostatic Neoplasms, Castration-Resistant genetics, Proto-Oncogene Proteins c-myc physiology, Receptors, Androgen genetics
- Abstract
Increased expression of the full-length androgen receptor (AR-FL) and AR splice variants (AR-Vs) drives the progression of castration-resistant prostate cancer (CRPC). The levels of AR-FL and AR-V transcripts are often tightly correlated in individual CRPC samples, yet our understanding of how their expression is co-regulated is limited. Here, we report a role of c-Myc in accounting for coordinated AR-FL and AR-V expression. Analysis of gene-expression data from 159 metastatic CRPC samples and 2142 primary prostate tumors showed that the level of c-Myc is positively correlated with that of individual AR isoforms. A striking positive correlation also exists between the activity of the c-Myc pathway and the level of individual AR isoforms, between the level of c-Myc and the activity of the AR pathway, and between the activities of the two pathways. Moreover, the c-Myc signature is highly enriched in tumors expressing high levels of AR, as is the AR signature in c-Myc-high-expressing tumors. Using shRNA knockdown, we confirmed c-Myc regulation of expression and activity of AR-FL and AR-Vs in cell models and a patient-derived xenograft model. Mechanistically, c-Myc promotes the transcription of the AR gene and enhances the stability of the AR-FL and AR-V proteins without altering AR RNA splicing. Importantly, inhibiting c-Myc sensitizes enzalutamide-resistant cells to growth inhibition by enzalutamide. Overall, this study highlights a critical role of c-Myc in regulating the coordinated expression of AR-FL and AR-Vs that is commonly observed in CRPC and suggests the utility of targeting c-Myc as an adjuvant to AR-directed therapy.
- Published
- 2019
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28. Detection of Epstein-Barr Virus Infection in Non-Small Cell Lung Cancer.
- Author
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Kheir F, Zhao M, Strong MJ, Yu Y, Nanbo A, Flemington EK, Morris GF, Reiss K, Li L, and Lin Z
- Abstract
Previous investigations proposed a link between the Epstein-Barr virus (EBV) and lung cancer (LC), but the results are highly controversial largely due to the insufficient sample size and the inherent limitation of the traditional viral screening methods such as PCR. Unlike PCR, current next-generation sequencing (NGS) utilizes an unbiased method for the global assessment of all exogenous agents within a cancer sample with high sensitivity and specificity. In our current study, we aim to resolve this long-standing controversy by utilizing our unbiased NGS-based informatics approaches in conjunction with traditional molecular methods to investigate the role of EBV in a total of 1127 LC. In situ hybridization analysis of 110 LC and 10 normal lung samples detected EBV transcripts in 3 LC samples. Comprehensive virome analyses of RNA sequencing (RNA-seq) data sets from 1017 LC and 110 paired adjacent normal lung specimens revealed EBV transcripts in three lung squamous cell carcinoma and one lung adenocarcinoma samples. In the sample with the highest EBV coverage, transcripts from the BamHI A region accounted for the majority of EBV reads. Expression of EBNA-1, LMP-1 and LMP-2 was observed. A number of viral circular RNA candidates were also detected. Thus, we for the first time revealed a type II latency-like viral transcriptome in the setting of LC in vivo. The high-level expression of viral BamHI A transcripts in LC suggests a functional role of these transcripts, likely as long non-coding RNA. Analyses of cellular gene expression and stained tissue sections indicated an increased immune cell infiltration in the sample expressing high levels of EBV transcripts compared to samples expressing low EBV transcripts. Increased level of immune checkpoint blockade factors was also detected in the sample with higher levels of EBV transcripts, indicating an induced immune tolerance. Lastly, inhibition of immune pathways and activation of oncogenic pathways were detected in the sample with high EBV transcripts compared to the EBV-low LC indicating the direct regulation of cancer pathways by EBV. Taken together, our data support the notion that EBV likely plays a pathological role in a subset of LC.
- Published
- 2019
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29. Gammaherpesvirus RNAs Come Full Circle.
- Author
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Ungerleider NA, Tibbetts SA, Renne R, and Flemington EK
- Subjects
- Animals, Herpesviridae Infections virology, Humans, RNA, Circular, RNA, Untranslated genetics, Gammaherpesvirinae physiology, RNA genetics, RNA, Viral genetics, Virus Latency
- Abstract
After an adaptive immune response is mounted, gammaherpesviruses achieve persistence through the utilization of viral noncoding RNAs to craft a suitable host cell environment in an immunologically transparent manner. While gammaherpesvirus long noncoding RNAs (lncRNAs) and microRNAs have been recognized for some time and have been actively investigated, a recent spate of reports have now identified repertoires of the circular RNA (circRNA) class of noncoding RNAs in both the lymphocryptovirus and rhadinovirus genera of gammaherpesviruses. Despite the recent nature of these findings, the detection of circRNAs across viruses and viral gene expression programs, the conservation of some viral circRNAs, and their detection in the clinical setting already raises the spectrum of functional importance in gammaherpesvirus biology and associated malignancies. Here, we provide an overview of currently known gammaherpesvirus circular RNAs and discuss reported physical and contextual properties that may be germane to future functional studies. With the Epstein-Barr virus (EBV) circRNAome being the most extensively studied to date, our discussions will be weighted toward EBV circRNAs while also addressing circRNAs discovered in the rhesus macaque lymphocryptovirus (rLCV), the Kaposi's sarcoma herpesvirus (KSHV), and the murid gammaherpesvirus 68 (MHV68). We hope that this will help set the stage for future investigations into the functions and relevance of this new class of viral noncoding RNAs in infection and disease., (Copyright © 2019 Ungerleider et al.)
- Published
- 2019
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30. Comparative Analysis of Gammaherpesvirus Circular RNA Repertoires: Conserved and Unique Viral Circular RNAs.
- Author
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Ungerleider NA, Jain V, Wang Y, Maness NJ, Blair RV, Alvarez X, Midkiff C, Kolson D, Bai S, Roberts C, Moss WN, Wang X, Serfecz J, Seddon M, Lehman T, Ma T, Dong Y, Renne R, Tibbetts SA, and Flemington EK
- Subjects
- Animals, Cell Line, Gene Expression Regulation, Viral genetics, Herpesvirus 4, Human genetics, Herpesvirus 8, Human genetics, Humans, Lymphocryptovirus genetics, Macaca mulatta, Male, RNA, Circular, RNA, Viral genetics, Rhadinovirus genetics, Simian Immunodeficiency Virus genetics, Virus Latency genetics, Virus Replication genetics, Gammaherpesvirinae genetics, RNA genetics
- Abstract
Recent studies have identified circular RNAs (circRNAs) expressed from the Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) human DNA tumor viruses. To gain initial insights into the potential relevance of EBV circRNAs in virus biology and disease, we assessed the circRNAome of the interspecies homologue rhesus macaque lymphocryptovirus (rLCV) in a naturally occurring lymphoma from a simian immunodeficiency virus (SIV)-infected rhesus macaque. This analysis revealed rLCV orthologues of the latency-associated EBV circular RNAs circRPMS1_E4_E3a and circEBNA_U. Also identified in two samples displaying unusually high lytic gene expression was a novel rLCV circRNA that contains both conserved and rLCV-specific RPMS1 exons and whose backsplice junctions flank an rLCV lytic origin of replication (OriLyt). Analysis of a lytic infection model for the murid herpesvirus 68 (MHV68) rhadinovirus identified a cluster of circRNAs near an MHV68 lytic origin of replication, with the most abundant of these, circM11_ORF69, spanning the OriLyt. Lastly, analysis of KSHV latency and reactivation models revealed the latency associated circRNA originating from the vIRF4 gene as the predominant viral circRNA. Together, the results of this study broaden our appreciation for circRNA repertoires in the Lymphocryptovirus and Rhadinovirus genera of gammaherpesviruses and provide evolutionary support for viral circRNA functions in latency and viral replication. IMPORTANCE Infection with oncogenic gammaherpesviruses leads to long-term viral persistence through a dynamic interplay between the virus and the host immune system. Critical for remodeling of the host cell environment after the immune responses are viral noncoding RNAs that modulate host signaling pathways without attracting adaptive immune recognition. Despite the importance of noncoding RNAs in persistent infection, the circRNA class of noncoding RNAs has only recently been identified in gammaherpesviruses. Accordingly, their roles in virus infection and associated oncogenesis are unknown. Here we report evolutionary conservation of EBV-encoded circRNAs determined by assessing the circRNAome in rLCV-infected lymphomas from an SIV-infected rhesus macaque, and we report latent and lytic circRNAs from KSHV and MHV68. These experiments demonstrate utilization of the circular RNA class of RNAs across 4 members of the gammaherpesvirus subfamily, and they identify orthologues and potential homoplastic circRNAs, implying conserved circRNA functions in virus biology and associated malignancies., (Copyright © 2019 American Society for Microbiology.)
- Published
- 2019
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31. Gammaherpesvirus Readthrough Transcription Generates a Long Non-Coding RNA That Is Regulated by Antisense miRNAs and Correlates with Enhanced Lytic Replication In Vivo.
- Author
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Kara M, O'Grady T, Feldman ER, Feswick A, Wang Y, Flemington EK, and Tibbetts SA
- Abstract
Gammaherpesviruses, including the human pathogens Epstein⁻Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are oncogenic viruses that establish lifelong infections in hosts and are associated with the development of lymphoproliferative diseases and lymphomas. Recent studies have shown that the majority of the mammalian genome is transcribed and gives rise to numerous long non-coding RNAs (lncRNAs). Likewise, the large double-stranded DNA virus genomes of herpesviruses undergo pervasive transcription, including the expression of many as yet uncharacterized lncRNAs. Murine gammaperherpesvirus 68 (MHV68, MuHV-4, HV68) is a natural pathogen of rodents, and is genetically and pathogenically related to EBV and KSHV, providing a highly tractable model for studies of gammaherpesvirus biology and pathogenesis. Through the integrated use of parallel data sets from multiple sequencing platforms, we previously resolved transcripts throughout the MHV68 genome, including at least 144 novel transcript isoforms. Here, we sought to molecularly validate novel transcripts identified within the M3/M2 locus, which harbors genes that code for the chemokine binding protein M3, the latency B cell signaling protein M2, and 10 microRNAs (miRNAs). Using strand-specific northern blots, we validated the presence of M3-04, a 3.91 kb polyadenylated transcript that initiates at the M3 transcription start site and reads through the M3 open reading frame (ORF), the M3 poly(a) signal sequence, and the M2 ORF. This unexpected transcript was solely localized to the nucleus, strongly suggesting that it is not translated and instead may function as a lncRNA. Use of an MHV68 mutant lacking two M3-04 -antisense pre-miRNA stem loops resulted in highly increased expression of M3-04 and increased virus replication in the lungs of infected mice, demonstrating a key role for these RNAs in regulation of lytic infection. Together these findings suggest the possibility of a tripartite regulatory relationship between the lncRNA M3-04 , antisense miRNAs, and the latency gene M2 ., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.
- Published
- 2019
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32. Epigenetically Silenced Candidate Tumor Suppressor Genes in Prostate Cancer: Identified by Modeling Methylation Stratification and Applied to Progression Prediction.
- Author
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Zhang W, Flemington EK, Deng HW, and Zhang K
- Subjects
- Adenocarcinoma metabolism, CpG Islands, Gene Expression Profiling, Gene Expression Regulation, Neoplastic, Humans, Male, Promoter Regions, Genetic, Prostatic Neoplasms metabolism, Adenocarcinoma genetics, DNA Methylation, Gene Silencing, Genes, Tumor Suppressor, Prostatic Neoplasms genetics
- Abstract
Background: Recent studies have shown that epigenetic alterations, especially the hypermethylated promoters of tumor suppressor genes (TSGs), contribute to prostate cancer progression and metastasis. This article proposes a novel algorithm to identify epigenetically silenced TSGs (epi-TSGs) for prostate cancer., Methods: Our method is based on the perception that the promoter CpG island(s) of a typical epi-TSG has a stratified methylation profile over tumor samples. In other words, we assume that the methylation profile resembles the combination of a binary distribution of a driver mutation and a continuous distribution representing measurement noise and intratumor heterogeneity., Results: Applying the proposed algorithm and an existing method to The Cancer Genome Atlas prostate cancer data, we identify 57 candidate epi-TSGs. Over one third of these epi-TSGs have been reported to carry potential tumor suppression functions. The negative correlations between the expression levels and methylation levels of these genes are validated on external independent datasets. We further find that the expression profiling of these genes is a robust predictive signature for Gleason scores, with the AUC statistic ranging from 0.75 to 0.79. The identified signature also shows prediction strength for tumor progression stages, biochemical recurrences, and metastasis events., Conclusions: We propose a novel method for pinpointing candidate epi-TSGs in prostate cancer. The expression profiling of the identified epi-TSGs demonstrates significant prediction strength for tumor progression., Impact: The proposed epi-TSGs identification method can be adapted to other cancer types beyond prostate cancer. The identified clinically significant epi-TSGs would shed light on the carcinogenesis of prostate adenocarcinomas., (©2018 American Association for Cancer Research.)
- Published
- 2019
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33. Connivance, Complicity, or Collusion? The Role of Noncoding RNAs in Promoting Gammaherpesvirus Tumorigenesis.
- Author
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Bullard WL, Flemington EK, Renne R, and Tibbetts SA
- Subjects
- Animals, Herpesviridae Infections complications, Humans, Carcinogenesis genetics, Gammaherpesvirinae genetics, Herpesviridae Infections genetics, RNA, Untranslated, RNA, Viral
- Abstract
EBV and KSHV are etiologic agents of multiple types of lymphomas and carcinomas. The frequency of EBV
+ or KSHV+ malignancies arising in immunocompromised individuals reflects the intricate evolutionary balance established between these viruses and their immunocompetent hosts. However, the specific mechanisms by which these pathogens drive tumorigenesis remain poorly understood. In recent years an enormous array of cellular and viral noncoding RNAs (ncRNAs) have been discovered, and host ncRNAs have been revealed as contributory factors to every single cancer hallmark cellular process. As new evidence emerges that gammaherpesvirus ncRNAs also alter host processes and viral factors dysregulate host ncRNA expression, and as novel viral ncRNAs continue to be discovered, we examine the contribution of small, non-miRNA ncRNAs and long ncRNAs to gammaherpesvirus tumorigenesis., (Copyright © 2018 Elsevier Inc. All rights reserved.)- Published
- 2018
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34. The Epstein Barr virus circRNAome.
- Author
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Ungerleider N, Concha M, Lin Z, Roberts C, Wang X, Cao S, Baddoo M, Moss WN, Yu Y, Seddon M, Lehman T, Tibbetts S, Renne R, Dong Y, and Flemington EK
- Subjects
- Cell Line, Epstein-Barr Virus Infections genetics, Humans, RNA, Circular, RNA, Untranslated genetics, Gene Expression Regulation, Viral genetics, Herpesvirus 4, Human genetics, RNA genetics, RNA, Viral genetics, Virus Latency genetics
- Abstract
Our appreciation for the extent of Epstein Barr virus (EBV) transcriptome complexity continues to grow through findings of EBV encoded microRNAs, new long non-coding RNAs as well as the more recent discovery of over a hundred new polyadenylated lytic transcripts. Here we report an additional layer to the EBV transcriptome through the identification of a repertoire of latent and lytic viral circular RNAs. Utilizing RNase R-sequencing with cell models representing latency types I, II, and III, we identified EBV encoded circular RNAs expressed from the latency Cp promoter involving backsplicing from the W1 and W2 exons to the C1 exon, from the EBNA BamHI U fragment exon, and from the latency long non-coding RPMS1 locus. In addition, we identified circular RNAs expressed during reactivation including backsplicing from exon 8 to exon 2 of the LMP2 gene and a highly expressed circular RNA derived from intra-exonic backsplicing within the BHLF1 gene. While expression of most of these circular RNAs was found to depend on the EBV transcriptional program utilized and the transcription levels of the associated loci, expression of LMP2 exon 8 to exon 2 circular RNA was found to be cell model specific. Altogether we identified over 30 unique EBV circRNAs candidates and we validated and determined the structural features, expression profiles and nuclear/cytoplasmic distributions of several predominant and notable viral circRNAs. Further, we show that two of the EBV circular RNAs derived from the RPMS1 locus are detected in EBV positive clinical stomach cancer specimens. This study increases the known EBV latency and lytic transcriptome repertoires to include viral circular RNAs and it provides an essential foundation and resource for investigations into the functions and roles of this new class of EBV transcripts in EBV biology and diseases., Competing Interests: The authors, TL and MS, are currently under the employment of ReproCELL Incorporated. ReproCELL Incorporated provided funds, resources and equipment to conduct some of the studies and provided salary and benefits for TL and MS. This does not alter our adherence to all PLoS Pathogens policies on sharing data and materials.
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- 2018
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35. Transactivation of human endogenous retrovirus K (HERV-K) by KSHV promotes Kaposi's sarcoma development.
- Author
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Dai L, Del Valle L, Miley W, Whitby D, Ochoa AC, Flemington EK, and Qin Z
- Subjects
- Adult, Aged, Carcinogenesis genetics, Cell Line, Female, Human Umbilical Vein Endothelial Cells, Humans, Male, Middle Aged, Signal Transduction genetics, Transcription Factors genetics, Viral Proteins genetics, Young Adult, Endogenous Retroviruses genetics, Sarcoma, Kaposi genetics, Sarcoma, Kaposi virology, Transcriptional Activation genetics
- Abstract
Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several human cancers such as Kaposi's sarcoma (KS), which represents the most common AIDS-associated malignancy that lacks effective treatment options. Despite its clear role in AIDS malignancies, the fact that only a small set of KSHV-infected patients will eventually develop these tumors implies that additional co-factors are required for the development of KSHV-related cancers. In the current study, we demonstrate for the first time that KSHV de novo infection or viral latent proteins are able to transactivate human endogenous retrovirus K (HERV-K) through a variety of cellular signaling pathways and transcriptional factors. Moreover, we found that HERV-K transactivation, particularly activation of its encoded oncogenic NP9 protein, plays an important role in KSHV pathogenesis and tumorigenesis in vitro and in vivo. Our data provide innovative insights into the mechanisms of HERV-K transactivation contributing to viral oncogenesis, which may represent a promising target for KS treatment.
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- 2018
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36. Driver gene mutations based clustering of tumors: methods and applications.
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Zhang W, Flemington EK, and Zhang K
- Subjects
- Cluster Analysis, Humans, Mutation, Prognosis, DNA Mutational Analysis methods, Genes, Neoplasm, Genomics methods, Models, Genetic, Neoplasms genetics, Software
- Abstract
Motivation: Somatic mutations in proto-oncogenes and tumor suppressor genes constitute a major category of causal genetic abnormalities in tumor cells. The mutation spectra of thousands of tumors have been generated by The Cancer Genome Atlas (TCGA) and other whole genome (exome) sequencing projects. A promising approach to utilizing these resources for precision medicine is to identify genetic similarity-based sub-types within a cancer type and relate the pinpointed sub-types to the clinical outcomes and pathologic characteristics of patients., Results: We propose two novel methods, ccpwModel and xGeneModel, for mutation-based clustering of tumors. In the former, binary variables indicating the status of cancer driver genes in tumors and the genes' involvement in the core cancer pathways are treated as the features in the clustering process. In the latter, the functional similarities of putative cancer driver genes and their confidence scores as the 'true' driver genes are integrated with the mutation spectra to calculate the genetic distances between tumors. We apply both methods to the TCGA data of 16 cancer types. Promising results are obtained when these methods are compared to state-of-the-art approaches as to the associations between the determined tumor clusters and patient race (or survival time). We further extend the analysis to detect mutation-characterized transcriptomic prognostic signatures, which are directly relevant to the etiology of carcinogenesis., Availability and Implementation: R codes and example data for ccpwModel and xGeneModel can be obtained from http://webusers.xula.edu/kzhang/ISMB2018/ccpw_xGene_software.zip., Supplementary Information: Supplementary data are available at Bioinformatics online.
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- 2018
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37. Transferring knowledge of bacterial protein interaction networks to predict pathogen targeted human genes and immune signaling pathways: a case study on M. tuberculosis.
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Mei S, Flemington EK, and Zhang K
- Subjects
- Area Under Curve, Databases, Genetic, Drug Resistance, Bacterial genetics, Gene Ontology, Humans, Immune System metabolism, Immune System microbiology, Logistic Models, ROC Curve, Tuberculosis immunology, Tuberculosis microbiology, Tuberculosis pathology, Bacterial Proteins metabolism, Host-Pathogen Interactions genetics, Mycobacterium tuberculosis metabolism, Protein Interaction Maps genetics, Signal Transduction genetics, Tuberculosis genetics
- Abstract
Background: Bacterial invasive infection and host immune response is fundamental to the understanding of pathogen pathogenesis and the discovery of effective therapeutic drugs. However, there are very few experimental studies on the signaling cross-talks between bacteria and human host to date., Methods: In this work, taking M. tuberculosis H37Rv (MTB) that is co-evolving with its human host as an example, we propose a general computational framework that exploits the known bacterial pathogen protein interaction networks in STRING database to predict pathogen-host protein interactions and their signaling cross-talks. In this framework, significant interlogs are derived from the known pathogen protein interaction networks to train a predictive l
2 -regularized logistic regression model., Results: The computational results show that the proposed method achieves excellent performance of cross validation as well as low predicted positive rates on the less significant interlogs and non-interlogs, indicating a low risk of false discovery. We further conduct gene ontology (GO) and pathway enrichment analyses of the predicted pathogen-host protein interaction networks, which potentially provides insights into the machinery that M. tuberculosis H37Rv targets human genes and signaling pathways. In addition, we analyse the pathogen-host protein interactions related to drug resistance, inhibition of which potentially provides an alternative solution to M. tuberculosis H37Rv drug resistance., Conclusions: The proposed machine learning framework has been verified effective for predicting bacteria-host protein interactions via known bacterial protein interaction networks. For a vast majority of bacterial pathogens that lacks experimental studies of bacteria-host protein interactions, this framework is supposed to achieve a general-purpose applicability. The predicted protein interaction networks between M. tuberculosis H37Rv and Homo sapiens, provided in the Additional files, promise to gain applications in the two fields: (1) providing an alternative solution to drug resistance; (2) revealing the patterns that M. tuberculosis H37Rv genes target human immune signaling pathways.- Published
- 2018
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38. Maintenance of AP-2-Dependent Functional Activities of Nef Restricts Pathways of Immune Escape from CD8 T Lymphocyte Responses.
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Schouest B, Weiler AM, Janaka SK, Myers TA, Das A, Wilder SC, Furlott J, Baddoo M, Flemington EK, Rakasz EG, Evans DT, Friedrich TC, and Maness NJ
- Subjects
- Animals, Biological Evolution, Bone Marrow Stromal Antigen 2 immunology, Bone Marrow Stromal Antigen 2 metabolism, Epitopes, T-Lymphocyte genetics, Histocompatibility Antigens Class I immunology, Humans, Macaca virology, Membrane Glycoproteins, Membrane Proteins, Mutation, Neoplasm Proteins, RNA, Viral, Receptors, Cell Surface, Sequence Analysis, Simian Acquired Immunodeficiency Syndrome virology, Simian Immunodeficiency Virus genetics, Simian Immunodeficiency Virus immunology, Simian Immunodeficiency Virus pathogenicity, Viral Envelope Proteins immunology, Viral Regulatory and Accessory Proteins genetics, Virus Replication, nef Gene Products, Human Immunodeficiency Virus immunology, CD8-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes virology, Epitopes, T-Lymphocyte immunology, Immune Evasion immunology, Simian Acquired Immunodeficiency Syndrome immunology, Viral Regulatory and Accessory Proteins immunology
- Abstract
Nef-specific CD8
+ T lymphocytes (CD8TL) are linked to extraordinary control of primate lentiviral replication, but the mechanisms underlying their efficacy remain largely unknown. The immunodominant, Mamu-B*017:01+ -restricted Nef195-203 MW9 epitope in SIVmac239 partially overlaps a sorting motif important for interactions with host AP-2 proteins and, hence, downmodulation of several host proteins, including Tetherin (CD317/BST-2), CD28, CD4, SERINC3, and SERINC5. We reasoned that CD8TL-driven evolution in this epitope might compromise Nef's ability to modulate these important molecules. Here, we used deep sequencing of SIV from nine B*017:01+ macaques throughout infection with SIVmac239 to characterize the patterns of viral escape in this epitope and then assayed the impacts of these variants on Nef-mediated modulation of multiple host molecules. Acute variation in multiple Nef195-203 MW9 residues significantly compromised Nef's ability to downregulate surface Tetherin, CD4, and CD28 and reduced its ability to prevent SERINC5-mediated reduction in viral infectivity but did not impact downregulation of CD3 or major histocompatibility complex class I, suggesting the selective disruption of immunomodulatory pathways involving Nef AP-2 interactions. Together, our data illuminate a pattern of viral escape dictated by a selective balance to maintain AP-2-mediated downregulation while evading epitope-specific CD8TL responses. These data could shed light on mechanisms of both CD8TL-driven viral control generally and on Mamu-B*017:01-mediated viral control specifically. IMPORTANCE A rare subset of humans infected with HIV-1 and macaques infected with SIV can control the virus without aid of antiviral medications. A common feature of these individuals is the ability to mount unusually effective CD8 T lymphocyte responses against the virus. One of the most formidable aspects of HIV is its ability to evolve to evade immune responses, particularly CD8 T lymphocytes. We show that macaques that target a specific peptide in the SIV Nef protein are capable of better control of the virus and that, as the virus evolves to escape this response, it does so at a cost to specific functions performed by the Nef protein. Our results help show how the virus can be controlled by an immune response, which could help in designing effective vaccines., (Copyright © 2018 American Society for Microbiology.)- Published
- 2018
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39. In colonic ρ 0 (rho0) cells reduced mitochondrial function mediates transcriptomic alterations associated with cancer.
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Penrose HM, Heller S, Cable C, Nakhoul H, Ungerleider N, Baddoo M, Pursell ZF, Flemington EK, Crawford SE, and Savkovic SD
- Abstract
Background: Mitochondrial reprogramming has emerged as a hallmark of cancer pathobiology. Although it is believed this reprogramming is essential for cancer cells to thrive, how it supports cancer pathobiology is unclear. We previously generated colonic ρ0 (rho0) cells with reduced mitochondrial energy function and acquired their transcriptional signature. Here, we utilized a bioinformatics approach to identify their changes linked to cancer pathobiology., Methods: Human colon cancer HCT116 cells, control and ρ0, were used for qPCR. Bioinformatics analysis: GeneCards, Kaplan-Meier Survival, GENT, cBioPortal., Results: The colonic ρ0 transcriptome was linked with proliferation, DNA replication, survival, tumor morphology, and cancer. Among differentially expressed transcripts, 281 were regulators or biomarkers of human colon cancer especially those with inflammatory microsatellite instability (MSI). We identified and validated novel transcripts in ρ0 cells with altered expression in human colon cancer. Among them DGK1, HTR7, FLRT3, and ZBTB18 co-occurred with established regulators of human colon cancer pathobiology. Also, increased levels of DGKI, FLRT3, ZBTB18, and YPEL1 as well as decreased levels of HTR7, and CALML6 were linked to substantially poorer patient survival., Conclusion: We identified established and novel regulators in colon cancer pathobiology that are dependent on mitochondrial energy reprogramming and linked to poorer patient survival., Competing Interests: CONFLICTS OF INTERESTS Suzana D. Savkovic wishes to disclose ownership in Pegasus Biosolution, LLC. No other conflicts of interest, financial or otherwise, are declared by the authors.
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- 2017
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40. MALT1 Inhibition Is Efficacious in Both Naïve and Ibrutinib-Resistant Chronic Lymphocytic Leukemia.
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Saba NS, Wong DH, Tanios G, Iyer JR, Lobelle-Rich P, Dadashian EL, Liu D, Fontan L, Flemington EK, Nichols CM, Underbayev C, Safah H, Melnick A, Wiestner A, and Herman SEM
- Subjects
- Adenine analogs & derivatives, Apoptosis drug effects, B-Lymphocytes drug effects, B-Lymphocytes pathology, Cell Line, Tumor, Drug Screening Assays, Antitumor, Humans, Leukemia, Lymphocytic, Chronic, B-Cell pathology, Neoadjuvant Therapy, Piperidines, Protein Kinase Inhibitors therapeutic use, Treatment Outcome, Drug Resistance, Neoplasm drug effects, Enzyme Inhibitors therapeutic use, Leukemia, Lymphocytic, Chronic, B-Cell drug therapy, Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein antagonists & inhibitors, Pyrazoles therapeutic use, Pyrimidines therapeutic use
- Abstract
The clinical efficacy displayed by ibrutinib in chronic lymphocytic leukemia (CLL) has been challenged by the frequent emergence of resistant clones. The ibrutinib target, Bruton's tyrosine kinase (BTK), is essential for B-cell receptor signaling, and most resistant cases carry mutations in BTK or PLCG2 , a downstream effector target of BTK. Recent findings show that MI-2, a small molecule inhibitor of the para-caspase MALT1, is effective in preclinical models of another type of BCR pathway-dependent lymphoma. We therefore studied the activity of MI-2 against CLL and ibrutinib-resistant CLL. Treatment of CLL cells in vitro with MI-2 inhibited MALT1 proteolytic activity reduced BCR and NF-κB signaling, inhibited nuclear translocation of RelB and p50, and decreased Bcl-xL levels. MI-2 selectively induced dose and time-dependent apoptosis in CLL cells, sparing normal B lymphocytes. Furthermore, MI-2 abrogated survival signals provided by stromal cells and BCR cross-linking and was effective against CLL cells harboring features associated with poor outcomes, including 17p deletion and unmutated IGHV Notably, MI-2 was effective against CLL cells collected from patients harboring mutations conferring resistance to ibrutinib. Overall, our findings provide a preclinical rationale for the clinical development of MALT1 inhibitors in CLL, in particular for ibrutinib-resistant forms of this disease. Cancer Res; 77(24); 7038-48. ©2017 AACR ., (©2017 American Association for Cancer Research.)
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- 2017
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41. Induction of a novel isoform of the lncRNA HOTAIR in Claudin-low breast cancer cells attached to extracellular matrix.
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Li M, Li X, Zhuang Y, Flemington EK, Lin Z, and Shan B
- Subjects
- Breast Neoplasms metabolism, Cell Adhesion, Cell Culture Techniques methods, Cell Cycle Proteins, Epigenesis, Genetic, Extracellular Matrix metabolism, Female, Humans, Nuclear Proteins metabolism, Promoter Regions, Genetic, Transcription Factors metabolism, Breast Neoplasms genetics, Claudins metabolism, Extracellular Matrix genetics, Gene Expression Regulation, Neoplastic, RNA, Long Noncoding genetics
- Abstract
Elevated overexpression of the lncRNA HOTAIR mediates invasion and metastasis in breast cancer. In an apparent paradox, we observed low expression of HOTAIR in the invasive Claudin-low MDA-MB-231 and Hs578T cells in two-dimensional culture (2D). However, HOTAIR expression exhibited robust induction in laminin-rich extracellular matrix-based three-dimensional organotypic culture (lrECM 3D) over that in 2D culture. Induction of HOTAIR required intact ECM signaling, namely integrin α2 and SRC kinase activity. Moreover, invasive growth was suppressed by HOTAIR-specific siRNA. Induction of HOTAIR in lrECM 3D culture resulted from the activation of a novel isoform of HOTAIR (HOTAIR-N) whose transcription is started from the first intron of the HOXC11 gene. The HOTAIR-N promoter exhibited increased trimethylation of histone H3 lysine 4, a histone marker of active transcription, and binding of BRD4, a reader of transcriptionally active histone markers. Inhibition of BRD4 substantially reduced the expression of HOTAIR in lrECM 3D culture. In summary, our results indicate that HOTAIR expression is activated by BRD4 binding to a novel HOTAIR-N promoter in Claudin-low breast cancer cells that are attached to ECM. Induction of HOTAIR is required for invasive growth of Claudin-low breast cancer cells in lrECM 3D culture., (© 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)
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- 2017
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42. Racial disparities in patient survival and tumor mutation burden, and the association between tumor mutation burden and cancer incidence rate.
- Author
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Zhang W, Edwards A, Flemington EK, and Zhang K
- Subjects
- Genetic Association Studies, Humans, Incidence, Neoplasms mortality, Racial Groups, SEER Program, Survival Analysis, Mutation, Neoplasms ethnology, Neoplasms genetics
- Abstract
The causes underlying racial disparities in cancer are multifactorial. In addition to socioeconomic issues, biological factors may contribute to these inequities, especially in disease incidence and patient survival. To date, there have been few studies that relate the disparities in these aspects to genetic aberrations. In this work, we studied the impacts of race on the patient survival and tumor mutation burden using the data released by the Cancer Genome Atlas (TCGA). The potential relationship between mutation burden and disease incidence is further inferred by an integrative analysis of TCGA data and the data from the Surveillance, Epidemiology, and End Results (SEER) Program. The results show that disparities are present (p < 0.05) in patient survival of five cancers, such as head and neck squamous cell carcinoma. The numbers of tumor driver mutations are differentiated (p < 0.05) over the racial groups in five cancers, such as lung adenocarcinoma. By treating a specific cancer type and a racial group as an "experimental unit", driver mutation numbers demonstrate a significant (r = 0.46, p < 0.002) positive correlation with cancer incidence rates, especially when the five cancers with mutational disparities are exclusively focused (r = 0.88, p < 0.00002). These results enrich our understanding of racial disparities in cancer and carcinogenic process.
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- 2017
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43. Lipids, lipid metabolism and Kaposi's sarcoma-associated herpesvirus pathogenesis.
- Author
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Dai L, Lin Z, Jiang W, Flemington EK, and Qin Z
- Subjects
- Humans, Sarcoma, Kaposi metabolism, Sarcoma, Kaposi virology, Herpesvirus 8, Human pathogenicity, Lipid Metabolism physiology
- Abstract
Lipids are essential for mammalian cells to maintain many physiological functions. Emerging evidence has shown that cancer cells can develop specific alterations in lipid biosynthesis and metabolism to facilitate their survival and various malignant behaviors. To date, the precise role of cellular lipids and lipid metabolism in viral oncogenesis is still largely unclear with only a handful of literature covering this topic to implicate lipid metabolism in oncogenic virus associated pathogenesis. In this review, we focus on the role of lipid biosynthesis and metabolism in the pathogenesis of the Kaposi's sarcoma-associated herpesvirus, a common causative factor for cancers arising in the immunocompromised settings.
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- 2017
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44. Ribonucleotide reductase represents a novel therapeutic target in primary effusion lymphoma.
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Dai L, Lin Z, Qiao J, Chen Y, Flemington EK, and Qin Z
- Subjects
- Animals, Apoptosis drug effects, Cell Line, Tumor, Enzyme Inhibitors pharmacology, Humans, Lymphoma, Primary Effusion genetics, Lymphoma, Primary Effusion pathology, Male, Mice, Mice, Inbred NOD, Mice, SCID, Molecular Targeted Therapy, Signal Transduction, Transcriptome drug effects, Xenograft Model Antitumor Assays, Lymphoma, Primary Effusion drug therapy, Lymphoma, Primary Effusion enzymology, Pyridines pharmacology, Ribonucleotide Reductases antagonists & inhibitors, Ribonucleotide Reductases metabolism, Thiosemicarbazones pharmacology
- Abstract
Primary effusion lymphoma (PEL) is a highly aggressive B-cell malignancy that is closely associated with one of oncogenic viruses infection, Kaposi's sarcoma-associated herpesvirus. PEL prognosis is poor and patients barely survive >6 months even following active chemotherapy interventions. There is therefore an urgent need to discover more effective targets for PEL management. We recently found that the ribonucleotide reductase (RR) subunit M2 is potentially regulated by the key oncogenic hepatocyte growth factor/c-MET pathway in PEL. In this study, we set to investigate the role of RR in PEL pathogenesis and to evaluate its potential as a therapeutic target. We report that the RR inhibitor 3-AP actively induces PEL cell cycle arrest through inhibiting the activity of the nuclear factor-κB pathway. Using a xenograft model, we found that 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) effectively suppresses PEL progression in immunodeficient mice. Transcriptome analysis of 3-AP-treated PEL cell lines reveals altered cellular genes, most of whose roles in PEL have not yet been reported. Taken together, we propose that RR and its signaling pathway may serve as novel actionable targets for PEL management.
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- 2017
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45. A computational framework for distinguishing direct versus indirect interactions in human functional protein-protein interaction networks.
- Author
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Mei S, Flemington EK, and Zhang K
- Subjects
- Algorithms, Computational Biology, Computer Simulation, Databases, Protein, Gene Ontology, Humans, Logistic Models, Models, Biological, Signal Transduction, Protein Interaction Maps genetics
- Abstract
Recognition of indirect interactions is instrumental to in silico reconstruction of signaling pathways and sheds light on the exploration of unknown physical paths between two indirectly interacting genes. However, very limited computational methods have explicitly exploited the indirect interactions with experimental evidence thus far. In this work, we attempt to distinguish direct versus indirect interactions in human functional protein-protein interaction (PPI) networks via a predictive l
2 -regularized logistic regression model built on the experimental data. The l2 -regularized logistic regression method is adopted to counteract the potential homolog noise and reduce the computational complexity on large training data. Computational results show that the proposed model demonstrates promising performance even though the training data are highly skewed. From the 304 799 PPIs that are curated in several databases, the proposed method detects 23 131 indirect interactions, most of which have been verified by the breadth-first graph search algorithm to find dozens of physical paths between the interacting partners. Pathway enrichment analysis shows that most of the physical paths can be mapped onto more than one human signaling pathway, indicating that there do exist a series of biochemical signals between the two indirectly interacting genes. The interactome-scale computational results promise to provide useful cues to the following applications: (1) exploration of unknown physical PPIs or physical paths between two indirectly interacting genes; (2) amending or extending the existing signaling pathways; (3) recognition of the physical PPIs for druggable target discovery.- Published
- 2017
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46. Arsenic trioxide inhibits EBV reactivation and promotes cell death in EBV-positive lymphoma cells.
- Author
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Yin Q, Sides M, Parsons CH, Flemington EK, and Lasky JA
- Subjects
- Arsenic Trioxide, Cell Death, Cell Line, Tumor, Humans, Arsenicals metabolism, Cell Survival drug effects, Herpesvirus 4, Human physiology, Lymphocytes virology, Oxides metabolism, Virus Activation drug effects
- Abstract
Background: Epstein-Barr Virus (EBV) is associated with hematopoietic malignancies, such as Burkitt's lymphoma, post-transplantation lymphoproliferative disorder, and diffuse large B-cell lymphoma. The current approach for EBV-associated lymphoma involves chemotherapy to eradicate cancer cells, however, normal cells may be injured and organ dysfunction may occur with currently employed regimens. This research is focused on employing arsenic trioxide (ATO) as EBV-specific cancer therapy takes advantage of the fact the EBV resides within the malignant cells., Methods and Results: Our research reveals that low ATO inhibits EBV gene expression and genome replication. EBV spontaneous reactivation starts as early as 6 h after re-suspending EBV-positive Mutu cells in RPMI media in the absence of ATO, however this does not occur in Mutu cells cultured with ATO. ATO's inhibition of EBV spontaneous reactivation is dose dependent. The expression of the EBV immediate early gene Zta and early gene BMRF1 is blocked with low concentrations of ATO (0.5 nM - 2 nM) in EBV latency type I cells and EBV-infected PBMC cells. The combination of ATO and ganciclovir further diminishes EBV gene expression. ATO-mediated reduction of EBV gene expression can be rescued by co-treatment with the proteasome inhibitor MG132, indicating that ATO promotes ubiquitin conjugation and proteasomal degradation of EBV genes. Co-immunoprecipitation assays with antibodies against Zta pulls down more ubiquitin in ATO treated cell lysates. Furthermore, MG132 reverses the inhibitory effect of ATO on anti-IgM-, PMA- and TGF-β-mediated EBV reactivation. Thus, mechanistically ATO's inhibition of EBV gene expression occurs via the ubiquitin pathway. Moreover, ATO treatment results in increased cell death in EBV-positive cells compared to EBV-negative cells, as demonstrated by both MTT and trypan blue assays. ATO-induced cell death in EBV-positive cells is dose dependent. ATO and ganciclovir in combination further enhances cell death specifically in EBV-positive cells., Conclusion: ATO-mediated inhibition of EBV lytic gene expression results in cell death selectively in EBV-positive lymphocytes, suggesting that ATO may potentially serve as a drug to treat EBV-related lymphomas in the clinical setting.
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- 2017
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47. Interaction of Epstein-Barr virus genes with human gastric carcinoma transcriptome.
- Author
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Zhang R, Strong MJ, Baddoo M, Lin Z, Wang YP, Flemington EK, and Liu YZ
- Subjects
- Gene Expression Regulation, Neoplastic genetics, Gene Expression Regulation, Viral genetics, Herpesvirus 4, Human, Humans, Transcriptome genetics, Cell Transformation, Neoplastic genetics, Epstein-Barr Virus Infections complications, Epstein-Barr Virus Infections genetics, Genes, Viral genetics, Stomach Neoplasms genetics, Stomach Neoplasms virology
- Abstract
Gastric carcinoma (GC) is a leading cause of mortality. 10% of GC cases are related with EBV (Epstein-Barr virus) infection. The detailed mechanistic roles EBV genes play and especially the interaction between the viral genes and human genes in GC remain unclear. In this study, raw fastq data from 285 GC samples were downloaded from TCGA (The Cancer Genome Atlas), including 25 EBV positive (EBV+) GC samples and 260 EBV negative (EBV-) GC samples. RNA-seq based expression data were generated for both human genes (among all the samples) and for the EBV genes (among the 25 EBV+ samples). Bioinformatics analyses were performed to identify differentially expressed (DEx) human genes and DEx KEGG pathways in EBV+ vs. EBV- samples and co-expressed human gene modules and hub genes among the DEx genes. Within the EBV+ samples, analyses were conducted to find correlation between EBV gene expression and the human gene expression modules, between EBV gene expression and the human hub genes, and between EBV gene expression and the DEx human pathways. EBV genes LMP-1, BALF1 and BALF2 were found to have significant correlation with human hub genes, CNTD2 and VANGL2. EBV genes BALF4 and BALF5 were found to correlate with human pathways, including Jak-STAT signaling and Phosphatidylinositol Signaling System. Our study has revealed the coordinated expression patterns between EBV and human GC transcriptome and identified several key EBV genes that may play an important role in EBV+ GC pathogenesis through their interactions with human genes and pathways.
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- 2017
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48. A comprehensive approach to expression of L1 loci.
- Author
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Deininger P, Morales ME, White TB, Baddoo M, Hedges DJ, Servant G, Srivastav S, Smither ME, Concha M, DeHaro DL, Flemington EK, and Belancio VP
- Subjects
- Animals, Chromosome Mapping, Chromosomes, Human metabolism, DNA, Complementary genetics, DNA, Complementary metabolism, Genomic Instability, HeLa Cells, Humans, Mice, NIH 3T3 Cells, Nucleic Acid Amplification Techniques, Promoter Regions, Genetic, RNA, Messenger metabolism, Sequence Analysis, RNA, Chromosomes, Human chemistry, Genetic Loci, Genome, Human, Long Interspersed Nucleotide Elements, RNA, Messenger genetics, Transcription, Genetic
- Abstract
L1 elements represent the only currently active, autonomous retrotransposon in the human genome, and they make major contributions to human genetic instability. The vast majority of the 500 000 L1 elements in the genome are defective, and only a relatively few can contribute to the retrotransposition process. However, there is currently no comprehensive approach to identify the specific loci that are actively transcribed separate from the excess of L1-related sequences that are co-transcribed within genes. We have developed RNA-Seq procedures, as well as a 1200 bp 5΄ RACE product coupled with PACBio sequencing that can identify the specific L1 loci that contribute most of the L1-related RNA reads. At least 99% of L1-related sequences found in RNA do not arise from the L1 promoter, instead representing pieces of L1 incorporated in other cellular RNAs. In any given cell type a relatively few active L1 loci contribute to the 'authentic' L1 transcripts that arise from the L1 promoter, with significantly different loci seen expressed in different tissues., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)
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- 2017
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49. Significant Prognostic Features and Patterns of Somatic TP53 Mutations in Human Cancers.
- Author
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Zhang W, Edwards A, Flemington EK, and Zhang K
- Abstract
TP53 is the most frequently altered gene in human cancers. Numerous retrospective studies have related its mutation and abnormal p53 protein expression to poor patient survival. Nonetheless, the clinical significance of TP53 ( p53 ) status has been a controversial issue. In this work, we aimed to characterize TP53 somatic mutations in tumor cells across multiple cancer types, primarily focusing on several less investigated features of the mutation spectra, and determine their prognostic implications. We performed an integrative study on the clinically annotated genomic data released by The Cancer Genome Atlas. Standard statistical methods, such as the Cox proportional hazards model and logistic regression, were used. This study resulted in several novel findings. They include the following: (1) similar to previously reported cases in breast cancer, the mutations in exons 1 to 4 of TP53 were more lethal than those in exons 5 to 9 for the patients with lung adenocarcinomas; (2) TP53 mutants tended to be negatively selected in mammalian evolution, but the evolutionary conservation had various clinical implications for different cancers; (3) conserved correlation patterns (ie, consistent co-occurrence or consistent mutual exclusivity) between TP53 mutations and the alterations in several other cancer genes (ie, PIK3CA, PTEN, KRAS, APC, CDKN2A , and ATM ) were present in several cancers in which prognosis was associated with TP53 status and/or the mutational characteristics; (4) among TP53 -mutated tumors, the total mutation burden in other driver genes was a predictive signature ( P <.05, false discovery rate <0.11) for better patient survival outcome in several cancer types, including glioblastoma multiforme. Among these findings, the fourth is of special significance as it suggested the potential existence of epistatic interaction effects among the mutations in different cancer driver genes on clinical outcomes., Competing Interests: DECLARATION OF CONFLICTING INTERESTS: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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- 2017
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50. Interplay between Cytoplasmic and Nuclear Androgen Receptor Splice Variants Mediates Castration Resistance.
- Author
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Zhan Y, Zhang G, Wang X, Qi Y, Bai S, Li D, Ma T, Sartor O, Flemington EK, Zhang H, Lee P, and Dong Y
- Subjects
- Androgens metabolism, Cell Line, Tumor, HEK293 Cells, Humans, Male, Models, Biological, Prostatic Neoplasms, Castration-Resistant pathology, Protein Multimerization, Protein Transport, Receptors, Androgen metabolism, Transcriptional Activation genetics, Alternative Splicing genetics, Cell Nucleus metabolism, Prostatic Neoplasms, Castration-Resistant genetics, Receptors, Androgen genetics
- Abstract
Androgen receptor splice variants (AR-V) are implicated in resistance of prostate cancer to androgen-directed therapies. When expressed alone in cells, some AR-Vs (e.g., AR-V7) localize primarily to the nucleus, whereas others (e.g., AR-V1, AR-V4, and AR-V6) localize mainly to the cytoplasm. Significantly, the latter are often coexpressed with the nucleus-predominant AR-Vs and the full-length AR (AR-FL). An important question to be addressed is whether the cytoplasmic-localized AR-Vs play a role in castration-resistant prostate cancer (CRPC) through interaction with the nucleus-predominant AR-Vs and AR-FL. Here, it is demonstrated that AR-V1, -V4, and -V6 can dimerize with both AR-V7 and AR-FL. Consequently, AR-V7 and androgen-bound AR-FL induced nuclear localization of AR-V1, -V4, and -V6, and these variants, in turn, mitigated the ability of the antiandrogen enzalutamide to inhibit androgen-induced AR-FL nuclear localization. Interestingly, the impact of nuclear localization of AR-V4 and -V6 on AR transactivation differs from that of AR-V1. Nuclear localization leads to an increased ability of AR-V4 and -V6 to transactivate both canonical AR targets and AR-V-specific targets and to confer castration-resistant cell growth. However, although AR-V1, which lacks inherent transcriptional activity, appears to activate AR-FL in an androgen-independent manner, it significantly antagonizes AR-V7 transactivation. Together, these data demonstrate that the complex interactions among different AR-Vs and AR-FL play a significant role in castration-resistant disease., Implications: This study suggests important consequences for clinical castration resistance due to simultaneous expression of AR-FL and AR-Vs in patient tumors and suggests that dissecting these interactions should help develop effective strategies to disrupt AR-V signaling. Mol Cancer Res; 15(1); 59-68. ©2016 AACR., Competing Interests: None, (©2016 American Association for Cancer Research.)
- Published
- 2017
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