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See detailCis-perturbation of cancer drivers by the HTLV-1/BLV proviruses is an early determinant of leukemogenesis
Rosewick, Nicolas; Durkin, Keith ULg; Artesi, Maria ULg et al

in Nature Communications (2017), 8

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See detailInvestigating non-coding viral transcripts in Bovine Leukemia Virus induced leukemia
Hahaut, Vincent ULg; Artesi, Maria ULg; Durkin, Keith ULg et al

Poster (2017, March 08)

Bovine Leukemia Virus (BLV) is a deltaretrovirus closely related to the Human T-cell leukemia virus-1 (HTLV-1). The natural host of BLV is cattle and much like the case of HTLV-1 in humans, about ~5% of ... [more ▼]

Bovine Leukemia Virus (BLV) is a deltaretrovirus closely related to the Human T-cell leukemia virus-1 (HTLV-1). The natural host of BLV is cattle and much like the case of HTLV-1 in humans, about ~5% of infected individuals develop leukemia/lymphoma following a long period of asymptomatic infection (~7 years in cattle, several decades in human). Experimental infection of sheep with BLV results in a reduced latency period (2 years on average), making for an attractive cancer model. A further advantage of the BLV system is that it is possible to infect sheep via injection of a cloned provirus, facilitating the mutation of specific parts of the viral genome to examine the function of viral products in vivo. Like HTLV-1, the BLV mRNAs/proteins are transcribed from the viral 5’ long terminal repeat (LTR), a region rich in regulatory elements. It was previously believed that the BLV provirus was transcriptionally silent in tumors, however we identified a cluster of five abundantly expressed non-canonical RNA polymerase III dependent microRNAs (miRNAs) encoded by BLV (Rosewick et al., PNAS 2013). In addition, using RNA sequencing we recently discovered viral antisense transcripts originating in the 3' Long Terminal Repeat (LTR) of the BLV provirus (Durkin et al., Retrovirology 2016) . While 5'LTR dependent transcription is absent in malignant cells, both the viral miRNAs and the antisense transcripts are expressed in all BLV induced leukemic and pre-leukemic samples examined to date, pointing to a vital role in the life cycle of the virus and a critical function in cellular transformation. [less ▲]

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See detailImproved high throughput DNA-seq based mapping of HTLV-1 integration sites: a tool to define response to treatment, ATL prognosis and guide therapeutic strategies
Marçais, Ambroise; Artesi, Maria ULg; Durkin, Keith ULg et al

Conference (2017)

Background Adult T-cell leukemia/lymphoma (ATL) is an aggressive CD4+ T-cell malignancy caused by HTLV-1 infection and associated with extremely poor prognosis. In France, treatment strategies mainly ... [more ▼]

Background Adult T-cell leukemia/lymphoma (ATL) is an aggressive CD4+ T-cell malignancy caused by HTLV-1 infection and associated with extremely poor prognosis. In France, treatment strategies mainly include chemotherapy, antiviral therapy and allogeneic stem cell transplantation, based on clinical subtype and therapeutic responses. Response to treatment is evaluated based on consensus criteria defined in 2009 (Tsukasaki K, Hermine O et al, JCO 2009). Although immuno-phenotyping, TCRγ rearrangement and HTLV-1 proviral load (PVL) quantification are often assayed, complete clinical remission (CR) is currently defined by morphological and cytological criteria i.e. complete blood cell counts (CBC) and the presence of abnormal lymphocytes. Given the extremely poor prognosis and high rates of early relapse, a revision of the response criteria is required, calling for improved tools that integrate specific aspects of the pathophysiology of ATL to better estimate response to treatment. Methods We retrospectively analyzed longitudinal PBMC samples from 6 ATL patients diagnosed with a leukemic subtype (5 acute and 1 chronic-aggressive) which all achieved CR upon therapy, yet relapsed after a median time of 15,9 months (range 2,4-70,7). CR was assessed by morphological and cytological criteria. An improved high throughput sequencing (HTS) based method was utilized to map and quantify the abundance of HTLV-1 genomic integration sites (IS), overcoming some of the limitations of previously published protocols. The dynamic range was increased by assaying both the 5’LTR and 3’LTRs, allowing better determination of clone abundance and revealing 5’ deletions. An enrichment step limited PCR duplicates. The addition of off-the-shelf Illumina primers simplified library multiplexing and reduced the costs to the point where the protocol could be applied to a clinical setting. Results HTS- mapping of HTLV-1 IS at diagnosis revealed in all cases a unique IS that constituted 92-99% of proviral genomes, with PVLs of 33-510%. All patients were treated and achieved CR which was characterized by normalized CBC, <5% abnormal lymphocytes and the absence of measurable tumors for >4 weeks. For 3/6 patients, the clone frequency distribution of HTLV-1 infected cells at CR was composed of multiple low abundance clones, of which the unique presumed malignant IS contributed to less than 2% of proviral genomes. In contrast, clonality analysis of the remaining 3/6 patients revealed that the relative abundance of the malignant clone detected at diagnosis remained dominant at CR (36-83% of PVL), despite clinical response criteria typical of CR and a 3-20-fold decrease in PVLs. These patients relapsed after 2.4, 2.9 and 3.4 months respectively with a dominant malignant clone >95% while patients with a polyclonal architecture showed significantly longer CR (28, 59 and 71 months). Conclusions Our observations highlight the great heterogeneity within an identical CR group, underlining the need for revisiting response criteria for ATL. Our results call for the use of this improved HTS-based method to measure HTLV-1 clonality as a tool to better estimate response to treatment, predict relapse and guide therapeutic choices in the course of treatment. [less ▲]

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See detailIdentification and characterization of novel bovine leukemia virus (BLV) antisense transcripts in leukemic and pre-leukemic clones
Durkin, Keith ULg; Rosewick, Nicolas; Artesi, Maria ULg et al

Conference (2016, May 21)

The deltaretrovirus Bovine Leukemia Virus (BLV) is closely related to the Human T-cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells, produces a lifelong ... [more ▼]

The deltaretrovirus Bovine Leukemia Virus (BLV) is closely related to the Human T-cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells, produces a lifelong infection. Most infected animals remain asymptomatic but following a protracted latency period about ~5% develop an aggressive leukemia/lymphoma, mirroring the disease trajectory of HTLV-1. Like the case in HTLV-1 the 5’LTR BLV provirus is transcriptionally silent in tumors, however the provirus is not entirely quiescent, constitutively express the BLV microRNAs in tumors. Using RNA-seq, we found that in addition to microRNAs, the BLV provirus also constitutively expresses two antisense transcripts in all BLV infected samples examined. The first transcript (AS1) has alternate potential polyadenylation sites generating a short transcript of ~600bp (AS1-S) and a less abundant longer transcript of ~2200bp (AS1-L). Alternative splicing also creates a second transcript of ~400bp (AS2) utilizing the first exon of AS1. Production of AS transcripts from the 3’LTR was supported by reporter assays demonstrating that the BLV LTR has substantial and Tax-independent antisense promoter activity. BLV AS transcripts predominantly localize in the nucleus. Examination of protein coding potential showed AS2 to be non-coding, while the AS1-S/L transcripts coding potential is ambiguous, with a small potential open reading frame (ORF) of 264bp present. The AS1-L transcript overlaps the BLV microRNAs transcribed in the sense direction. Using high throughput sequencing of RNA-ligase-mediated (RLM) 5' RACE products, we show that the perfect complementary between the transcripts leads to RNA-induced silencing complex (RISC) mediated cleavage of AS1-L. Furthermore, experiments using BLV proviruses where the microRNAs were removed or inverted point to additional transcriptional interactions between the two viral RNA species. Knock down of AS1-S/L using locked nucleic acids (LNAs) showed no obvious effect on the cells phenotype. While a detailed elucidation of the BLV antisense transcripts function remains in the future, the constitutive expression in all samples examined, points to a vital role for the transcripts in the life cycle and oncogenic potential of BLV. [less ▲]

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See detailImproving the methodology for high throughput mapping of proviral integration sites
Artesi, Maria ULg; Durkin, Keith ULg; Rosewick, Nicolas et al

Conference (2016)

Bovine Leukemia Virus (BLV) and Human T-cell leukemia virus-1 (HTLV-1) are closely related delta-retroviruses provoking a polyclonal expansion of infected B- and T-cells respectively, with monoclonal ... [more ▼]

Bovine Leukemia Virus (BLV) and Human T-cell leukemia virus-1 (HTLV-1) are closely related delta-retroviruses provoking a polyclonal expansion of infected B- and T-cells respectively, with monoclonal leukemia/lymphoma developing in about ~5% of infected individuals. To date, the molecular mechanisms leading to cellular transformation remain unclear. Both proviruses are largely transcriptionally silent in tumors and their integration sites in the host genome appear very variable. Mapping proviral insertion sites using high throughput sequencing techniques has provided insights into the evolution of BLV/HTLV-1 infections and the expansion of transformed clones in delta-retrovirus induced leukemia/lymphoma. The methods currently used have a number of limitations such as the utilisation of custom sequencing primers, relatively high sequencing costs, no examination of the 5’LTR host flanking region and a limited dynamic range for determining clone abundance. We have developed an alternative high-throughput sequencing protocol for tracking proviral integration sites and determining clonal abundance in BLV and HTLV-1 infected individuals. We implemented the use of off-the-shelf Illumina primers for the addition of adapters and indexes, which facilitates library multiplexing and avoids the need for custom sequencing primers. Our method reduces the amount of sequencing of PCR duplicates by reducing the number of PCR cycles via an enrichment of BLV- and HTLV-1-carrying DNA fragments. Moreover, in addition to the proviral 3’LTR, our approach also assays the 5’LTR, providing additional information on the frequency of 5’-end deletions in proviruses and increasing the dynamic range of the assay. We have tested the approach on over 100 BLV and HTLV-1 samples, representing both tumors and asymptomatic stages. Our approach allowed for a more accurate determination of clone abundance in tumors and by assaying the provirus 5’ end, identified clones overlooked with previously published methods. Finally, by facilitating greater multiplexing of libraries we have reduced the cost to a level where the technique may be attractive in a clinical setting. [less ▲]

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See detailCharacterization of novel Bovine Leukemia Virus (BLV) antisense transcripts by deep sequencing reveals constitutive expression in tumors and transcriptional interaction with viral microRNAs.
Durkin, Keith ULg; Rosewick, Nicolas; Artesi, Maria ULg et al

in Retrovirology (2016), 13(1), 33

BACKGROUND: Bovine Leukemia Virus (BLV) is a deltaretrovirus closely related to the Human T cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells, producing a ... [more ▼]

BACKGROUND: Bovine Leukemia Virus (BLV) is a deltaretrovirus closely related to the Human T cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells, producing a lifelong infection. Most infected animals remain asymptomatic but following a protracted latency period about 5 % develop an aggressive leukemia/lymphoma, mirroring the disease trajectory of HTLV-1. The mechanisms by which these viruses provoke cellular transformation remain opaque. In both viruses little or no transcription is observed from the 5'LTR in tumors, however the proviruses are not transcriptionally silent. In the case of BLV a cluster of RNA polymerase III transcribed microRNAs are highly expressed, while the HTLV-1 antisense transcript HBZ is consistently found in all tumors examined. RESULTS: Here, using RNA-seq, we demonstrate that the BLV provirus also constitutively expresses antisense transcripts in all leukemic and asymptomatic samples examined. The first transcript (AS1) can be alternately polyadenylated, generating a transcript of ~600 bp (AS1-S) and a less abundant transcript of ~2200 bp (AS1-L). Alternative splicing creates a second transcript of ~400 bp (AS2). The coding potential of AS1-S/L is ambiguous, with a small open reading frame of 264 bp, however the transcripts are primarily retained in the nucleus, hinting at a lncRNA-like role. The AS1-L transcript overlaps the BLV microRNAs and using high throughput sequencing of RNA-ligase-mediated (RLM) 5'RACE, we show that the RNA-induced silencing complex (RISC) cleaves AS1-L. Furthermore, experiments using altered BLV proviruses with the microRNAs either deleted or inverted point to additional transcriptional interference between the two viral RNA species. CONCLUSIONS: The identification of novel viral antisense transcripts shows the BLV provirus to be far from silent in tumors. Furthermore, the consistent expression of these transcripts in both leukemic and nonmalignant clones points to a vital role in the life cycle of the virus and its tumorigenic potential. Additionally, the cleavage of the AS1-L transcript by the BLV encoded microRNAs and the transcriptional interference between the two viral RNA species suggest a shared role in the regulation of BLV. [less ▲]

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See detailHTLV-1/BLV antisense-RNA dependent host gene perturbation in pre-leukemic and leukemic clones
Rosewick, Nicolas; Durkin, Keith ULg; Marçais, Ambroise et al

in Retrovirology (2015, August 28), 12(1),

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See detailImproving the methodology for the detection of proviral integration sites in the host genome via high throughput sequencing.
Durkin, Keith ULg; Artesi, Maria ULg; Rosewick, Nicolas et al

in Retrovirology (2015, August 28), 12(1),

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See detailImproving proviral integration site detection with high throughput sequencing
Durkin, Keith ULg; Artesi, Maria ULg; Rosewick, Nicolas et al

Poster (2015, May)

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See detailExploring the Deltaretrovirus Tumor Transcriptome: Lessons from RNA-Seq
Rosewick, Nicolas; Durkin, Keith ULg; Thys, Wannes et al

Conference (2014, June)

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See detailIdentification of two noncoding antisense transcripts in BLV and their interaction with the BLV encoded miRNAs
Durkin, Keith ULg; Rosewick, Nicolas; Momont, Mélanie et al

Conference (2014, June)

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See detailIdentification of two antisense transcripts in BLV and their interaction with BLV-encoded microRNAs.
Durkin, Keith ULg; Rosewick, Nicolas; Momont, Mélanie et al

Poster (2014, February)

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See detailElucidating the role of Bovine Leukemia Virus encoded micro-RNAs
Durkin, Keith ULg; Rosewick, Nicolas; Momont, Momont et al

in Retrovirology (2014, January 07), 11(1), 62

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See detailDeep sequencing reveals abundant non-canonical retroviral microRNAs in B-cell leukemia/lymphoma
Durkin, Keith ULg

Scientific conference (2013, January 28)

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See detailDeep sequencing reveals abundant non-canonical retroviral microRNAs in B-cell leukemia/lymphoma
Rosewick, Nicolas; Momont, Mélanie ULg; Durkin, Keith ULg et al

in Proceedings of the National Academy of Sciences of the United States of America (2013)

Viral tumor models have significantly contributed to our understanding of oncogenic mechanisms. How transforming delta-retroviruses induce malignancy however remains poorly understood, especially as viral ... [more ▼]

Viral tumor models have significantly contributed to our understanding of oncogenic mechanisms. How transforming delta-retroviruses induce malignancy however remains poorly understood, especially as viral mRNA/protein are tightly silenced in tumors. Here, using deep sequencing of broad windows of small RNA sizes in the Bovine Leukemia Virus ovine model of leukemia/lymphoma, we provide in vivo evidence of the production of non-canonical Pol IIItranscribed viral microRNAs in leukemic B-cells in the complete absence of Pol II 5’ LTR-driven transcriptional activity. Processed from a cluster of five independent self-sufficient transcriptional units located in a proviral region dispensable for in vivo infectivity, BLV microRNAs represent ~ 40 % of all microRNAs in both experimental and natural malignancy. They are subject to strong purifying selection and associate with Argonautes, consistent with a critical function in silencing of important cellular and/or viral targets. BLV microRNAs are strongly expressed in preleukemic and malignant cells in which structural and regulatory gene expression is repressed, suggesting a key role in tumor onset and progression. Understanding how Pol III-dependent microRNAs subvert cellular and viral pathways will contribute in deciphering the intricate perturbations that underlie malignant transformation. [less ▲]

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See detailDetection of copy number variants in the horse genome and examination of their association with recurrent laryngeal neuropathy
Dupuis, Marie-Capucine; Zhang, Zhiyan ULg; Durkin, Keith ULg et al

in Animal Genetics (2013)

We used the data from a recently performed genome-wide association study using the Illumina Equine SNP50 beadchip for the detection of copy number variants (CNVs) and examined their association with ... [more ▼]

We used the data from a recently performed genome-wide association study using the Illumina Equine SNP50 beadchip for the detection of copy number variants (CNVs) and examined their association with recurrent laryngeal neuropathy (RLN), an important equine upper airway disease compromising performance. A total of 2797 CNVs were detected for 477 horses, covering 229 kb and seven SNPs on average. Overlapping CNVs were merged to define 478 CNV regions (CNVRs). CNVRs, particularly deletions, were shown to be significantly depleted in genes. Fifty-two of the 67 common CNVRs (frequency ! 1%) were validated by association mapping, Mendelian inheritance, and/or Mendelian inconsistencies. None of the 67 common CNVRs were significantly associated with RLN when accounting for multiple testing. However, a duplication on chromosome 10 was detected in 10 cases (representing three breeds) and two unphenotyped parents but in none of the controls. The duplication was embedded in an 8-Mb haplotype shared across breeds. [less ▲]

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See detailSerial translocation via circular intermediates underlies color-sidedness in cattle.
Durkin, Keith ULg

Scientific conference (2012, May 04)

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See detailSerial translocation by means of circular intermediates underlies colour sidedness in cattle.
Durkin, Keith ULg; Coppieters, Wouter ULg; Drogemuller, Cord et al

in Nature (2012), 482(7383), 81-4

Colour sidedness is a dominantly inherited phenotype of cattle characterized by the polarization of pigmented sectors on the flanks, snout and ear tips. It is also referred to as 'lineback' or 'witrik ... [more ▼]

Colour sidedness is a dominantly inherited phenotype of cattle characterized by the polarization of pigmented sectors on the flanks, snout and ear tips. It is also referred to as 'lineback' or 'witrik' (which means white back), as colour-sided animals typically display a white band along their spine. Colour sidedness is documented at least since the Middle Ages and is presently segregating in several cattle breeds around the globe, including in Belgian blue and brown Swiss. Here we report that colour sidedness is determined by a first allele on chromosome 29 (Cs(29)), which results from the translocation of a 492-kilobase chromosome 6 segment encompassing KIT to chromosome 29, and a second allele on chromosome 6 (Cs(6)), derived from the first by repatriation of fused 575-kilobase chromosome 6 and 29 sequences to the KIT locus. We provide evidence that both translocation events involved circular intermediates. This is the first example, to our knowledge, of a phenotype determined by homologous yet non-syntenic alleles that result from a novel copy-number-variant-generating mechanism. [less ▲]

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See detailThe bovine genomic DNA sequence data reveal three IGHV subgroups, only one of which is functionally expressed
Niku, Mikael; Liljavirta, Jenni; Durkin, Keith ULg et al

in Developmental & Comparative Immunology (2012), 37

A comprehensive analysis of cattle shotgun sequencing data reveals 36 immunoglobulin heavy chain variable genes. The previously described bovine subgroup IGHV1 contains 10 functional genes with a ... [more ▼]

A comprehensive analysis of cattle shotgun sequencing data reveals 36 immunoglobulin heavy chain variable genes. The previously described bovine subgroup IGHV1 contains 10 functional genes with a conserved promoter including the consensus octamer and several other transcription factor binding sites, intact exons and matching cDNA sequences. Subgroups IGHV2 and IGHV3 consist entirely of pseudogenes. Thus, the bovine germline IGHV repertoire is very limited. The IGHV genes are distributed in mammalian clans I and II, while no clan III genes were detected. Clan-specific PCR of genomic DNA from cattle, sheep, Eurasian elk, white-tailed deer, pig and dolphin indicates highly dynamic evolution of IGHV gene usage within Cetartiodactyla. The bovine germline IGHV repertoire was probably generated by recent duplications of an IGHV1-IGHV2 homology unit. Immunoglobulin heavy chain genes are largely incorrectly assembled in the current cattle genome versions Btau_4.2 and UMD_3.1. FISH experiments confirm an IGHV locus close to terminus of BTA21. [less ▲]

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