Repeat Elements Research Articles

De Novo Assembly and Annotation of the Siganus fuscescens (Houttuyn, 1782) Genome: Marking a Pioneering Advance for the Siganidae Family.

This study presents the first draft genome of Siganus fuscescens, and thereby establishes the first whole-genome sequence for a species in the Siganidae family. Leveraging both long and short read sequencing technologies, i.e., Oxford Nanopore and Illumina sequencing, we successfully assembled a mitogenome spanning 16.494 Kb and a first haploid genome encompassing 498Mb. The assembled genome accounted for a 99.6% of the estimated genome size and was organized into 164 contigs with an N50 of 7.2Mb. This genome assembly showed a GC content of 42.9% and a high Benchmarking Universal Single-Copy Orthologue (BUSCO) completeness score of 99.5% using actinopterygii_odb10 lineage, thereby meeting stringent quality standards. In addition to its structural aspects, our study also examined the functional genomics of this species, including the intricate capacity to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFAs) and secrete venom. Notably, our analyses revealed various repeats elements, which collectively constituted 17.43% of the genome. Moreover, annotation of 28,351 genes uncovered both shared genetic signatures and those that are unique to S. fuscescens. Our assembled genome also displayed a moderate prevalence of gene duplication compared to other fish species, which suggests that this species has a distinctive evolutionary trajectory and potentially unique functional constraints. Taken altogether, this genomic resource establishes a robust foundation for future research on the biology, evolution, and the aquaculture potential of S. fuscescens.

Species-specific variation in mitochondrial genome tandem repeat polymorphisms in hares (Lepus spp., Lagomorpha, Leporidae) provides insight into their evolution

The non-coding regions of the mitochondrial DNAs (mtDNAs) of hares, rabbits, and pikas (Lagomorpha) contain short (∼20 bp) and long (130–160 bp) tandem repeats, absent in related mammalian orders. In the presented study, we provide in-depth analysis for mountain hare (Lepus timidus) and brown hare (L. europaeus) mtDNA non-coding regions, together with a species- and population-level analysis of tandem repeat variation. Mountain hare short tandem repeats (SRs) as well as other analyzed hare species consist of two conserved 10 bp motifs, with only brown hares exhibiting a single, more variable motif. Long tandem repeats (LRs) also differ in sequence and copy number between species. Mountain hares have four to seven LRs, median value five, while brown hares exhibit five to nine LRs, median value six. Interestingly, introgressed mountain hare mtDNA in brown hares obtained an intermediate LR length distribution, with median copy number being the same as with conspecific brown hare mtDNA. In contrast, transfer of brown hare mtDNA into cultured mtDNA-less mountain hare cells maintained the original LR number, whereas the reciprocal transfer caused copy number instability, suggesting that cellular environment rather than the nuclear genomic background plays a role in the LR maintenance. Due to their dynamic nature and separation from other known conserved sequence elements on the non-coding region of hare mitochondrial genomes, the tandem repeat elements likely to represent signatures of ancient genetic rearrangements. clarifying the nature and dynamics of these rearrangements may shed light on the possible role of NCR repeated elements in mitochondria and in species evolution.

Energy absorption behaviour and shape change effects of 3D printed polyurethane hexagonal honeycombs

Honeycombs have been used in a wide range of repeatable energy-absorbing elements due to their light weight and high energy absorption capacity. In the engineering environment, honeycombs will produce a shape change effect and rebound under multiple loading and unloading, so it is of great importance to investigate the energy-absorbing properties of honeycombs under the shape change effect for the application of honeycombs. In this study, the deformation process and energy absorption properties of 3D printed honeycombs under multiple loading were obtained through multiple quasi-static compression tests. On this basis, the coupling between shape change effects and honeycomb energy absorption is revealed. It is shown that the number of compressions does not affect the four-stage force-displacement curve characteristics of the honeycombs, but only the energy absorption efficiency of the honeycombs. There is an inverse relationship between the resilience of honeycombs and the energy absorption capacity of honeycombs, and the change of honeycomb stiffness under repeated compression is the direct cause of this relationship. The research results provide a reference for the application of honeycombs in the field of repetitive energy absorption.

ACMGA: a reference-free multiple-genome alignment pipeline for plant species

BackgroundThe short-read whole-genome sequencing (WGS) approach has been widely applied to investigate the genomic variation in the natural populations of many plant species. With the rapid advancements in long-read sequencing and genome assembly technologies, high-quality genome sequences are available for a group of varieties for many plant species. These genome sequences are expected to help researchers comprehensively investigate any type of genomic variants that are missed by the WGS technology. However, multiple genome alignment (MGA) tools designed by the human genome research community might be unsuitable for plant genomes.ResultsTo fill this gap, we developed the AnchorWave-Cactus Multiple Genome Alignment (ACMGA) pipeline, which improved the alignment of repeat elements and could identify long (> 50 bp) deletions or insertions (INDELs). We conducted MGA using ACMGA and Cactus for 8 Arabidopsis (Arabidopsis thaliana) and 26 Maize (Zea mays) de novo assembled genome sequences and compared them with the previously published short-read variant calling results. MGA identified more single nucleotide variants (SNVs) and long INDELs than did previously published WGS variant callings. Additionally, ACMGA detected significantly more SNVs and long INDELs in repetitive regions and the whole genome than did Cactus. Compared with the results of Cactus, the results of ACMGA were more similar to the previously published variants called using short-read. These two MGA pipelines identified numerous multi-allelic variants that were missed by the WGS variant calling pipeline.ConclusionsAligning denovo assembled genome sequences could identify more SNVs and INDELs than mapping short-read. ACMGA combines the advantages of AnchorWave and Cactus and offers a practical solution for plant MGA by integrating global alignment, a 2-piece-affine-gap cost strategy, and the progressive MGA algorithm.

A high-resolution map of functional miR-181 response elements in the thymus reveals the role of coding sequence targeting and an alternative seed match.

MicroRNAs (miRNAs) are critical post-transcriptional regulators in many biological processes. They act by guiding RNA-induced silencing complexes to miRNA response elements (MREs) in target mRNAs, inducing translational inhibition and/or mRNA degradation. Functional MREs are expected to predominantly occur in the 3' untranslated region and involve perfect base-pairing of the miRNA seed. Here, we generate a high-resolution map of miR-181a/b-1 (miR-181) MREs to define the targeting rules of miR-181 in developing murine Tcells. By combining a multi-omics approach with computational high-resolution analyses, we uncover novel miR-181 targets and demonstrate that miR-181 acts predominantly through RNA destabilization. Importantly, we discover an alternative seed match and identify a distinct set of targets with repeat elements in the coding sequence which are targeted by miR-181 and mediate translational inhibition. In conclusion, deep profiling of MREs in primary cells is critical to expand physiologically relevant targetomes and establish context-dependent miRNA targeting rules.

The chromosome-level genome and functional database accelerate research about biosynthesis of secondary metabolites in Rosa roxburghii

Rosa roxburghii Tratt, a valuable plant in China with long history, is famous for its fruit. It possesses various secondary metabolites, such as L-ascorbic acid (vitamin C), alkaloids and poly saccharides, which make it a high nutritional and medicinal value. Here we characterized the chromosome-level genome sequence of R. roxburghii, comprising seven pseudo-chromosomes with a total size of 531 Mb and a heterozygosity of 0.25%. We also annotated 45,226 coding gene loci after masking repeat elements. Orthologs for 90.1% of the Complete Single-Copy BUSCOs were found in the R. roxburghii annotation. By aligning with protein sequences from public platform, we annotated 85.89% genes from R. roxburghii. Comparative genomic analysis revealed that R. roxburghii diverged from Rosa chinensis approximately 5.58 to 13.17 million years ago, and no whole-genome duplication event occurred after the divergence from eudicots. To fully utilize this genomic resource, we constructed a genomic database RroFGD with various analysis tools. Otherwise, 69 enzyme genes involved in L-ascorbate biosynthesis were identified and a key enzyme in the biosynthesis of vitamin C, GDH (L-Gal-1-dehydrogenase), is used as an example to introduce the functions of the database. This genome and database will facilitate the future investigations into gene function and molecular breeding in R. roxburghii.

Specific DNMT3C flanking sequence preferences facilitate methylation of young murine retrotransposons

The DNA methyltransferase DNMT3C appeared as a duplication of the DNMT3B gene in muroids and is required for silencing of young retrotransposons in the male germline. Using specialized assay systems, we investigate the flanking sequence preferences of DNMT3C and observe characteristic preferences for cytosine at the -2 and -1 flank that are unique among DNMT3 enzymes. We identify two amino acids in the catalytic domain of DNMT3C (C543 and V547) that are responsible for the DNMT3C-specific flanking sequence preferences and evolutionary conserved in muroids. Reanalysis of published data shows that DNMT3C flanking preferences are consistent with genome-wide methylation patterns in mouse ES cells only expressing DNMT3C. Strikingly, we show that CpG sites with the preferred flanking sequences of DNMT3C are enriched in murine retrotransposons that were previously identified as DNMT3C targets. Finally, we demonstrate experimentally that DNMT3C has elevated methylation activity on substrates derived from these biological targets. Our data show that DNMT3C flanking sequence preferences match the sequences of young murine retrotransposons which facilitates their methylation. By this, our data provide mechanistic insights into the molecular co-evolution of repeat elements and (epi)genetic defense systems dedicated to maintain genomic stability in mammals.

Characterization of the Plastid Genomes of Four Caroxylon Thunb. Species from Kazakhstan.

The family Chenopodiaceae Vent. (Amaranthaceae s.l.) is known for its taxonomic complexity, comprising species of significant economic and ecological importance. Despite its significance, the availability of plastid genome data for this family remains limited. This study involved assembling and characterizing the complete plastid genomes of four Caroxylon Thunb. species within the tribe Salsoleae s.l., utilizing next-generation sequencing technology. We compared genome features, nucleotide diversity, and repeat sequences and conducted a phylogenetic analysis of ten Salsoleae s.l. species. The size of the plastid genome varied among four Caroxylon species, ranging from 150,777 bp (C. nitrarium) to 151,307 bp (C. orientale). Each studied plastid genome encoded 133 genes, including 114 unique genes. This set of genes includes 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Eight divergent regions (accD, atpF, matK, ndhF-ndhG, petB, rpl20-rpl22, rpoC2, and ycf3) were identified in ten Salsoleae s.l. plastid genomes, which could be potential DNA-barcoding markers. Additionally, 1106 repeat elements were detected, consisting of 814 simple sequence repeats, 92 tandem repeats, 88 forward repeats, 111 palindromic repeats, and one reverse repeat. The phylogenetic analysis provided robust support for the relationships within Caroxylon species. These data represent a valuable resource for future phylogenetic studies within the genus.

Exploring the extrachromosomal plasmid rDNA of Naegleria fowleri AY27 genotype II: A human brain-eating amoeba via high-throughput sequencing

Naegleria fowleri, also known as brain-earing amoeba, causes severe and rapidly fatal CNS infection in humans called primary amebic meningoencephalitis (PAM). The DNA from the N. fowleri clinical isolate was sequenced for circular extrachromosomal ribosomal DNA (CERE - rDNA). The CERE contains 18 S, 5.8 S, and 28 S ribosomal subunits separated by internal transcribed spacers, 5 open reading frames (ORFs), and mostly repeat elements comprising 7268 bp out of 15,786 bp (46%). A wide variety of variations and recombination events were observed. Finally, the ORFs that comprised only 4 hypothetical proteins were modeled and screened against Zinc drug-like compounds. Two compounds [ZINC77564275 (ethyl 2-(((4-isopropyl-4 H-1,2,4-triazol-3-yl) methyl) (methyl)amino) oxazole-4-carboxylate) and ZINC15022129 (5-(2-methoxyphenoxy)-[2,2’-bipyrimidine]-4,6(1 H,5 H)-dione)] were finalized as potential druggable compounds based on ADME toxicity analysis. We propose that the compounds showing the least toxicity would be potential drug candidates after laboratory experimental validation is performed.

Deciphering novel TCF4-driven mechanisms underlying a common triplet repeat expansion-mediated disease.

Fuchs endothelial corneal dystrophy (FECD) is an age-related cause of vision loss, and the most common repeat expansion-mediated disease in humans characterised to date. Up to 80% of European FECD cases have been attributed to expansion of a non-coding CTG repeat element (termed CTG18.1) located within the ubiquitously expressed transcription factor encoding gene, TCF4. The non-coding nature of the repeat and the transcriptomic complexity of TCF4 have made it extremely challenging to experimentally decipher the molecular mechanisms underlying this disease. Here we comprehensively describe CTG18.1 expansion-driven molecular components of disease within primary patient-derived corneal endothelial cells (CECs), generated from a large cohort of individuals with CTG18.1-expanded (Exp+) and CTG 18.1-independent (Exp-) FECD. We employ long-read, short-read, and spatial transcriptomic techniques to interrogate expansion-specific transcriptomic biomarkers. Interrogation of long-read sequencing and alternative splicing analysis of short-read transcriptomic data together reveals the global extent of altered splicing occurring within Exp+ FECD, and unique transcripts associated with CTG18.1-expansions. Similarly, differential gene expression analysis highlights the total transcriptomic consequences of Exp+ FECD within CECs. Furthermore, differential exon usage, pathway enrichment and spatial transcriptomics reveal TCF4 isoform ratio skewing solely in Exp+ FECD with potential downstream functional consequences. Lastly, exome data from 134 Exp- FECD cases identified rare (minor allele frequency <0.005) and potentially deleterious (CADD>15) TCF4 variants in 7/134 FECD Exp- cases, suggesting that TCF4 variants independent of CTG18.1 may increase FECD risk. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity and TCF4 isoform-specific dysregulation, both underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease.

Shared Features Underlying Compact Genomes and Extreme Habitat Use in Chironomid Midges.

Nonbiting midges (family Chironomidae) are found throughout the world in a diverse array of aquatic and terrestrial habitats, can often tolerate harsh conditions such as hypoxia or desiccation, and have consistently compact genomes. Yet we know little about the shared molecular basis for these attributes and how they have evolved across the family. Here, we address these questions by first creating high-quality, annotated reference assemblies for Tanytarsus gracilentus (subfamily Chironominae, tribe Tanytarsini) and Parochlus steinenii (subfamily Podonominae). Using these and other publicly available assemblies, we created a time-calibrated phylogenomic tree for family Chironomidae with outgroups from order Diptera. We used this phylogeny to test for features associated with compact genomes, as well as examining patterns of gene family evolution and positive selection that may underlie chironomid habitat tolerances. Our results suggest that compact genomes evolved in the common ancestor of Chironomidae and Ceratopogonidae and that this occurred mainly through reductions in noncoding regions (introns, intergenic sequences, and repeat elements). Significantly expanded gene families in Chironomidae included biological processes that may relate to tolerance of stressful environments, such as temperature homeostasis, carbohydrate transport, melanization defense response, and trehalose transport. We identified several positively selected genes in Chironomidae, notably sulfonylurea receptor, CREB-binding protein, and protein kinase D. Our results improve our understanding of the evolution of small genomes and extreme habitat use in this widely distributed group.

Chromosome-level genome assembly of the yellow-cheek carp Elopichthys bambusa

Yellow-cheek carp (Elopichthys bambusa) is a typical large and ferocious carnivorous fish endemic to East Asia, with high growth rate, nutritional value and economic value. In this study, a chromosome-level genome of yellow-cheek carp was generated by combining PacBio reads, Illumina reads and Hi-C data. The genome size is 827.63 Mb with a scaffold N50 size of 33.65 Mb, and 99.51% (823.61 Mb) of the assembled sequences were anchored to 24 pseudo-chromosomes. The genome is predicted to contain 24,153 protein-coding genes, with 95.54% having functional annotations. Repeat elements account for approximately 55.17% of the genomic landscape. The completeness of yellow-cheek carp genome assembly is highlighted by a BUSCO score of 98.4%. This genome will help us understand the genetic diversity of yellow-cheek carp and facilitate its conservation planning.

Toward understanding the role of genomic repeat elements in neurodegenerative diseases.

Neurodegenerative diseases cause great medical and economic burdens for both patients and society; however, the complex molecular mechanisms thereof are not yet well understood. With the development of high-coverage sequencing technology, researchers have started to notice that genomic repeat regions, previously neglected in search of disease culprits, are active contributors to multiple neurodegenerative diseases. In this review, we describe the association between repeat element variants and multiple degenerative diseases through genome-wide association studies and targeted sequencing. We discuss the identification of disease-relevant repeat element variants, further powered by the advancement of long-read sequencing technologies and their related tools, and summarize recent findings in the molecular mechanisms of repeat element variants in brain degeneration, such as those causing transcriptional silencing or RNA-mediated gain of toxic function. Furthermore, we describe how in silico predictions using innovative computational models, such as deep learning language models, could enhance and accelerate our understanding of the functional impact of repeat element variants. Finally, we discuss future directions to advance current findings for a better understanding of neurodegenerative diseases and the clinical applications of genomic repeat elements.

Architecture of the Far East in the first third of the XX century

The architecture of the Enlightenment is considered as a derivative of the architecture of the Far Eastern frontier, which consists of garrison and railway buildings. The article analyzes the projects of Far Eastern schools and colleges first identified in archival funds. To explain the phenomenon of “Russian style” schools, the author puts forward a version of the existence of a corporate “railroad Russian style” uniting a number of buildings from 1900 to 1926. Repeated elements speak of a plan, which was realized during a quarter of a century. The railway schools of the CER can be considered together with the Far Eastern railway stations as an integral part of a single ensemble.

The landscape and predicted roles of structural variants in Fusarium graminearum genomes.

Structural rearrangements, such as inversions, translocations, duplications, and large insertions and deletions, are large-scale genomic variants that can play an important role in shaping phenotypic variation and in genome adaptation and evolution. We used chromosomal-level assemblies from eight Fusarium graminearum isolates to study structural variants and their role in fungal evolution. We generated the assemblies of four of these genomes after Oxford Nanopore sequencing. A total of 87 inversions, 159 translocations, 245 duplications, 58,489 insertions, and 34,102 deletions were detected. Regions of high recombination rate are associated with structural rearrangements, and a significant proportion of inversions, translocations, and duplications overlap with the repeat content of the genome, suggesting recombination and repeat elements are major factors in the origin of structural rearrangements in F. graminearum. Large insertions and deletions introduce presence-absence polymorphisms for many genes, including secondary metabolite biosynthesis cluster genes and predicted effectors genes. Translocation events were found to be shuffling predicted effector-rich regions of the genomes and are likely contributing to the gain and loss of effectors facilitated by recombination. Breakpoints of some structural rearrangements fall within coding sequences and are likely altering the protein products. Structural rearrangements in F. graminearum thus have an important role to play in shaping pathogen-host interactions and broader evolution through genome reorganization, the introduction of presence-absence polymorphisms, and changing protein products and gene regulation.

A chromosome-level genome assembly of East Asia endemic minnow Zacco platypus

Zacco platypus is an endemic colorful freshwater minnow that is intensively distributed in East Asia. In this study, two adult female individuals collected from Haihe River basin were used for karyotypic study and genome sequencing, respectively. The karyotype formula of Z. platypus is 2N = 48 = 18 M + 24SM/ST + 6 T. We used PacBio long-read sequencing and Hi-C technology to assemble a chromosome-level genome of Z. platypus. As a result, an 814.87 Mb genome was assembled with the PacBio long reads. Subsequently, 98.64% assembled sequences were anchored into 24 chromosomes based on the Hi-C data. The chromosome-level assembly contained 54 scaffolds with a N50 length of 32.32 Mb. Repeat elements accounted for 52.35% in genome, and 24,779 protein-coding genes were predicted, with 92.11% were functionally annotated with the public databases. BUSCO analysis yielded a completeness score of 96.5%. This high-quality genome assembly provides valuable resources for future functional genomic research, comparative genomics, and evolutionary studies of genus Zacco.

Abstract 988: Genome-wide repeat landscapes in cancer and cell-free DNA

Abstract Introduction: Repeat sequences comprise &amp;gt;50% of the human genome and structural and epigenetic changes in these regions are implicated in cancer. However, no systematic analysis of the compendium of repeat sequences has ever been performed in human cancer or cell-free DNA (cfDNA), largely due to the inability to identify and quantify repeat sequences genome-wide. We describe here the first comprehensive analysis of genome-wide repeat landscapes in cancer and demonstrate their utility in cfDNA liquid biopsies. Methods: We developed ARTEMIS (Analysis of RepeaT EleMents in dISease) an alignment-free, genome-wide approach for analyzing repeat landscapes in short read sequencing. This approach uses a de novo search of short sequences (kmers) in the telomere to telomere (chm13) reference genome to identify 1.2 billion 24-mers uniquely defining 1280 individual repeat types occurring genome-wide across 57 subfamilies and 6 families. We analyzed ARTEMIS kmers in whole genome sequences of 525 matched tumor/normal pairs from breast, colorectal, liver, lung, ovarian, cervical, prostate, thyroid, head and neck, gastric, and bladder cancers in the Pan Cancer Analysis of Whole Genomes (PCAWG), and in low coverage (1-2x) whole genome sequences of 1450 cfDNA samples from individuals with and without 8 types of cancer. Results: Analysis of ARTEMIS kmer repeat landscapes in 525 PCAWG tumors identified changes in all 1280 repeat element types, including 820 novel elements not previously known to be altered in cancer. A median of 807 repeat elements (range 246-1280) were altered in each tumor compared to its matched normal. The majority of changes were in repeat elements not previously described as altered in tumorigenesis and were most frequently found within Satellites, LINEs and SINEs, though changes were also observed in LTRs, Transposable Elements, and RNA Elements. A cross-validated cfDNA model using repeat landscapes (ARTEMIS) and fragmentation features (DELFI) detected individuals in a diagnostic cohort (n=287) across all stages of lung cancer with high performance (AUC 0.91, 95% CI 0.88-0.95) and was externally validated in a separate population (n=513). The locked model generated scores that correlated with circulating tumor mutant allele fractions for patients (n=19) undergoing targeted lung cancer therapy (r=0.80, p&amp;lt;2.2e-16), and stratified progression-free survival (p&amp;lt;0.001). ARTEMIS repeat landscape analyses of cfDNA also detected liver cancer in a high-risk cohort (n=208) of individuals with cirrhosis or viral hepatitis (AUC 0.91, 95% CI 0.87-0.95), and identified tissue of origin among seven tumor types (n=423). Conclusions: ARTEMIS reveals genome-wide repeat landscapes in human cancer, including in 820 novel elements not previously known to be altered in tumorigenesis. These repeat landscapes that can now be described are evaluable in the circulation and provide an avenue for noninvasive detection and characterization of cancer. Citation Format: Akshaya Annapragada, Noushin Niknafs, James R. White, Daniel C. Bruhm, Christopher Cherry, Jamie E. Medina, Vilmos Adleff, Carolyn Hruban, Dimitrios Mathios, Zachariah H. Foda, Jillian Phallen, Robert B. Scharpf, Victor E. Velculescu. Genome-wide repeat landscapes in cancer and cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 988.

Abstract 7480: Human LINE-1 ORF1p retrotransposition independent function in pancreatic cancer

Abstract The aberrant expression of repeat elements is a common feature found in both mouse models and human pancreatic ductal adenocarcinoma (PDAC). The majority of these are non-coding RNAs that have shown the ability to stimulate a viral-like innate immune response. The long interspersed nuclear element 1 (LINE-1) retrotransposon is one of the few protein coding repeat elements in the human genome. LINE-1 has two open reading frames, ORF1 and ORF2. The former (ORF1p) encodes a ribonucleotide protein and the latter (ORF2p) a reverse transcriptase and endonuclease. Although nuclear ORF1p and ORF2p is required for retrotransposition, ORF1p is primarily localized in the cytoplasm, which suggests the possibility of ORF1p function independent of LINE-1 retrotransposition. Here, we show that ORF1p has a function to shield repeat RNAs from activating pathogen recognition receptor (PRR) mediated antiviral response in PDAC cells. LINE-1 ORF1p-targeting shRNA significantly inhibits patient-derived PDAC tumor growth both in vitro and in vivo. Total RNA sequencing (RNA-seq) reveals distinct transcriptional profiles with ORF1p deficiency. Gene set enrichment analysis (GSEA) identified high enrichment of innate immune response-related pathways including IL6-JAK-STAT3 signaling, inflammatory response, interferon response, and apoptosis in ORF1 knockdown PDAC cell lines. These immune related pathways are suppressed by deletion of RIG-I and MAVS, two components of viral RNA sensing. Mechanistically, we demonstrated that ORF1p is associated with repeat RNAs including LINE-1 RNA (L1HS) in processing bodies (PBs), which are cytoplasmic membraneless ribonucleoprotein (RNP) granules. In the PBs, ORF1p interacts with MOV10 RNA helicase, an essential enzyme for dsRNA unwinding. Using MOV10 truncated mutants, we found that the ORF1p-MOV10 interaction is important in attenuation of PRR sensing and apoptosis upon repeat RNA exposure. Finally, we evalulated the activation of the innate immune response in the tumor microenvironment by ORF1p depletion. These results suggest that ORF1p loss induces a desmoplastic reaction driven by Acta2+ myofibroblastic CAFs in response to inflammatory cytokines. Collectively, our results demonstrate a critical and previously uncharacterized role of ORF1p in evasion of endogenous repeat RNA-triggered antiviral immune response in PDAC cells with implications in the tumor microenvironment. Citation Format: Eunae You, Bidish Patel, Alexandra Rojas, Natalie Ho, Siyu Sun, Michael Raabe, Yuhui Song, Ildiko Phillips, Katherine Xu, Peter Richieri, Linda Nieman, Benjamin Greenbaum, David Ting. Human LINE-1 ORF1p retrotransposition independent function in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7480.

Abstract 6551: Comprehensive genomic and transcriptomic analyses capture the effects of epigenetic therapy priming on immune checkpoint blockade response in non-small cell lung cancer

Abstract Introduction: Emergence of resistance to immune checkpoint blockade (ICB) mandates the development of strategies for ICB sensitization. Here we employed a multi-omic approach to understand the effects of epigenetic priming in re-shaping the tumor microenvironment, together with genomic drivers of therapeutic response of epigenetic therapy followed by ICB in non-small cell lung cancer (NSCLC). Methods: We performed whole exome sequencing (WES) on 39 baseline tumors and bulk RNA sequencing (RNAseq) on 18 serial tumors across 42 patients with metastatic immunotherapy-naïve NSCLC, who received azacitidine and entinostat followed by nivolumab (NCT01928576). WES was utilized to assess co-mutations, mutation signatures and genome-wide structural changes. RNAseq was utilized for gene and gene set enrichment analysis (GSEA) and repeat element analysis. Response after nivolumab initiation was assessed using RECIST 1.1 criteria. Patients who were progression-free at 6 months or alive at 2 years after initial treatment were defined as exceptional responders. Results: We found an enrichment in inactivating mutations in homologous recombination genes in radiographic responders (Fisher’s exact, p=0.0007). Higher germline and somatic HLA class II diversity (Mann-Whitney U-test, p=0.05 and p=0.03 respectively), and an enrichment of smoking and DNA damage mutational signatures were noted in radiographic responders (Mann-Whitney U-test, p=0.03 and p=0.15 respectively). Radiographically responding tumors harbored a higher number of mutations in genomic regions that were haploid or present in multiple copies (persistent tumor mutation burden; Mann-Whitney U-test, p=0.018). GSEA revealed an upregulation of inflammatory response gene sets post-epigenetic therapy in patients attaining an exceptional response. Antigen presentation, interferon-alpha, and interferon-gamma related gene sets were upregulated in exceptional responders post epigenetic priming (FDR-adjusted p=0.005, p=0.009, and p=0.011 respectively). Additionally, we observed an enrichment in interferon-alpha, cancer testis antigens, and interferon-gamma related gene sets in patients with a longer progression-free survival (FDR-adjusted p=0.007, p=0.011, and p=0.013 respectively). Repeat element analysis showed an increase in long terminal repeats on-therapy in exceptional responders, suggesting induction of repeat element expression with epigenetic therapy. A higher expression of MAGE family tumor associated antigens (MAGEA10, MAGEB1, MAGEB2) was noted post-epigenetic therapy in exceptional responders. Conclusions: Genomic and transcriptomic analyses reveal the potential of epigenetic-priming to re-shape the tumor microenvironment and highlight patients that could benefit from combination epigenetic therapy with immunotherapy. Citation Format: Blair V. Landon, Kristen A. Marrone, Michael J. Topper, Akshaya Annapragada, Hua-Ling Tsai, Archana Balan, Noushin Niknafs, Christopher Cherry, James R. White, Gavin Pereira, Vilmos Adleff, Chen Hu, Joanne Riemer, Margaret Fitzpatrick, Patrick Forde, Christine L. Hann, Ronan J. Kelly, David S. Ettinger, Benjamin Levy, Jorge Nieva, James Herman, Victor E. Velculescu, Stephen Baylin, Julie Brahmer, Valsamo Anagnostou. Comprehensive genomic and transcriptomic analyses capture the effects of epigenetic therapy priming on immune checkpoint blockade response in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6551.

Genome-wide repeat landscapes in cancer and cell-free DNA.

Genetic changes in repetitive sequences are a hallmark of cancer and other diseases, but characterizing these has been challenging using standard sequencing approaches. We developed a de novo kmer finding approach, called ARTEMIS (Analysis of RepeaT EleMents in dISease), to identify repeat elements from whole-genome sequencing. Using this method, we analyzed 1.2 billion kmers in 2837 tissue and plasma samples from 1975 patients, including those with lung, breast, colorectal, ovarian, liver, gastric, head and neck, bladder, cervical, thyroid, or prostate cancer. We identified tumor-specific changes in these patients in 1280 repeat element types from the LINE, SINE, LTR, transposable element, and human satellite families. These included changes to known repeats and 820 elements that were not previously known to be altered in human cancer. Repeat elements were enriched in regions of driver genes, and their representation was altered by structural changes and epigenetic states. Machine learning analyses of genome-wide repeat landscapes and fragmentation profiles in cfDNA detected patients with early-stage lung or liver cancer in cross-validated and externally validated cohorts. In addition, these repeat landscapes could be used to noninvasively identify the tissue of origin of tumors. These analyses reveal widespread changes in repeat landscapes of human cancers and provide an approach for their detection and characterization that could benefit early detection and disease monitoring of patients with cancer.