Mechanisms Of Genomic Instability Research Articles

Exploring Novel Genome Instability-associated lncRNAs and their Potential Function in Pan-Renal Cell Carcinoma.

Genomic instability can drive clonal evolution, continuous modification of tumor genomes, and tumor genomic heterogeneity. The molecular mechanism of genomic instability still needs further investigation. This study aims to identify novel genome instabilityassociated lncRNAs (GI-lncRNAs) and investigate the role of genome instability in pan-Renal cell carcinoma (RCC). A mutator hypothesis was employed, combining the TCGA database of somatic mutation (SM) information, to identify GI-lncRNAs. Subsequently, a training cohort (n = 442) and a testing cohort (n = 439) were formed by randomly dividing all RCC patients. Based on the training cohort dataset, a multivariate Cox regression analysis lncRNAs risk model was created. Further validations were performed in the testing cohort, TCGA cohort, and different RCC subtypes. To confirm the relative expression levels of lncRNAs in HK-2, 786-O, and 769-P cells, qPCR was carried out. Functional pathway enrichment analyses were performed for further investigation. A total of 170 novel GI-lncRNAs were identified. The lncRNA prognostic risk model was constructed based on LINC00460, AC073218.1, AC010789.1, and COLCA1. This risk model successfully differentiated patients into distinct risk groups with significantly different clinical outcomes. The model was further validated in multiple independent patient cohorts. Additionally, functional and pathway enrichment analyses revealed that GI-lncRNAs play a crucial role in GI. Furthermore, the assessments of immune response, drug sensitivity, and cancer stemness revealed a significant relationship between GI-lncRNAs and tumor microenvironment infiltration, mutational burden, microsatellite instability, and drug resistance. In this study, we discovered four novel GI-lncRNAs and developed a novel signature that effectively predicted clinical outcomes in pan-RCC. The findings provide valuable insights for pan-RCC immunotherapy and shed light on potential underlying mechanisms.

Inherent Genome Instability Underlies Trisomy 21-Associated Myeloid Malignancies

Background Children with Down syndrome (DS) are highly predisposed to pediatric leukemia, particularly to acute myeloid leukemia (AML) with a ~400-fold increased risk before age 5. DS-associated AML is preceded by a unique transient myeloproliferative disorder (TMD) in infancy, which is a pre-leukemic state driven by somatic mutations in exon 2 of GATA1 leading to the expression of a short ‘ GATA1s‘ isoform. Acquisition of secondary mutations in GATA1s-mutant clones causes fulminant AML. It remains a key question as to why children with DS are particularly susceptible to the genetic aberrations promoting TMD and AML so early in life. Objective DS is a condition of aneuploidy, i.e., trisomy 21 (T21). Several critical hematopoietic genes, e.g. RUNX1 and ERG, are located on chromosome 21 (chr21) whose dosage increase is linked to malignancy. In addition, aneuploidy per se exerts various detrimental impacts on cellular physiology, one of them being elevated genome instability. We aim to determine whether genome instability is an inherent property of T21 hematopoietic cells as a contributing factor to TMD and AML in DS. Results We investigated the impacts of T21 on genome stability in primary leukocytes and in a TMD disease model based on human induced pluripotent stem cells (iPSCs). We asked whether chromosomal aberrations are increased in primary leukocytes from individuals with T21 (n=14) compared to euploid/disomy 21 individuals (n=4) by assaying copy number variations (CNVs) using single-cell genomic sequencing. Leukocytes with CNVs were rare in euploid individuals (2.8%; total 178 cells). By contrast, we found that leukocytes from individuals with T21 showed an approximately 2-fold increase in the frequency of single-cells carrying CNVs other than constitutional T21 (6.5%; total 815 cells), particularly the cells with large CNVs (> 10 megabases, chi-square test p=0.011; Fig. 1A-B). We did not detect mutations in the exon 2 of GATA1 in bulk leukocytes, indicating that the background of T21 alone is associated with more frequent chromosomal aberrations. We then examined the effects of GATA1s mutations on genome integrity in an iPSC-based model of TMD. We derived hematopoietic progenitor cells (HPCs) from isogenic euploid and T21 iPSCs harboring CRISPR/Cas9-induced GATA1s mutations. We found that GATA1s mutations in T21 HPCs further augmented the accumulation of CNVs (chi-square test p=0.0007; Fig. 1C). Tetrasomy 21 was also detected in HPCs, recapitulating a frequent karyotypic aberration in DS-AML (Fig. 1D). On the cellular level, GATA1s caused myeloid skewing independently of chr21 status but significantly promoted progenitor cell maintenance only in the T21 context. Transcriptome analysis in HPCs identified altered signatures associated with genome integrity, including downregulation of mitotic spindle and apoptosis gene sets in T21- GATA1s HPCs as well as further upregulation of the chr21 gene DYRK1A, which has been shown to regulate pathways of DNA double-strand break (DSB) repair. We found that increased expression of DYRK1A in hematopoietic cells diminished homologous recombination, thereby impeding error-free repair and disrupting DSB repair pathway choices as a mechanism of genome instability in T21 and associated TMD and AML. Conclusions We conclude that hematopoietic cells carrying T21 display inherent genome instability, and that the acquisition of GATA1s further amplifies genome instability while promoting myeloid skewing and aberrant maintenance of the progenitor state. The inherently unstable nature of T21 blood cells is a likely contributor to the predisposition to leukemia in DS.

Pediatric Burkitt Leukemia with Underlying MSH2, MUTYH and POT1 Germline Variants: The Complexities and Relevance of Understanding Cancer Predisposition

Monoallelic germline variants in POT1 can lead to POT1-tumor predisposition (POT1-TPD), an autosomal dominant disorder leading to a spectrum of neoplasia, predominantly, melanoma, glioma, angiosarcoma and chronic lymphocytic leukemia (CLL). Recently, germline POT1 variants have been described to be associated with familial clonal hematopoiesis syndrome.The POT1 protein forms a part of shelterin complex, which has been implicated in DNA damage protection, telomere length regulation and chromosomal stability. The proposed mechanism of oncogenesis by POT1 variants is presumed to be secondary to increased telomere length causing genomic instability and cellular immortality. While POT1-TPD can cause CLL in adulthood, childhood leukemias have not been commonly observed. Here, we present a case of a previously healthy 12-year-old male diagnosed with Burkitt leukemia. He presented with leukocytosis and tumor lysis syndrome. Bone marrow evaluation revealed hypercellular marrow involved by Burkitt lymphoma, comprising over 90% marrow cellularity. Flow cytometry showed an abnormal lambda monotypic B-lymphoid population positive for CD10, CD19, CD20, CD22 and CD45. FISH studies demonstrated low-level MYC rearrangement (9%); negative for BCL2 and BCL6 rearrangement. EBV staining was negative. Acute leukemia NGS panel (63 genes) revealed a Tier 1 somatic variant in PHF6. RNA sequencing did not detect additional fusions. His chemotherapy course was complicated by significant toxicities including prolonged count recovery, severe peripheral neuropathy, neurotoxicity, kidney injury and several infectious complications. Given extensive family history of cancers on the maternal side including colorectal, duodenal, brain and skin cancers, a germline cancer predisposition NGS panel was sent from cultured skin fibroblast and identified two heterozygous pathogenic variants in MSH2 (c.942+3A>T) and MUTYH (c.1187G>A) and a heterozygous likely pathogenic variant in POT1 (c.233T>C). To delineate potential roles of these germline variants in oncogenesis, additional testing was performed. Immunohistochemical staining of bone marrow revealed intact nuclear expression of MLH1, PMS2, MSH2,and MSH6,suggesting microsatellite stability. Extended tumor profiling through whole exome sequencing did not identify additional somatic variants or copy loss of heterozygosity in MSH2, MUTYH and POT1 but additional somatic loss of function variants were seen in CCND3, CASP8, ARID1, and PTPRD.To assess potential functional consequences of the POT1 germline variant on DNA double-strand break (DSB) damage repair, the patient lymphocytes were exposed to 2 Gy ionizing radiation and mobilization and kinetics of DNA DSB repair was assessed at 1h and 24h post-irradiation (IR) using phosphorylation (p) of ATM, SMC1 and H2AX (gamma), and cell death and apoptosis was also assessed at 24h post-irradiation by flow cytometry. There was normal mobilization and repair of DNA DSBs in T, B and NK cells although there was a subset of CD19dim B cells that showed constitutive DNA DSBs at1h with partial repair at 24h. It is not evident that this defect can be attributed to the germline defects as they are also observed in several other clinical contexts, including post-chemotherapy. Telomere length assay from peripheral blood demonstrated telomere length at 50 th percentile in both lymphocytes and granulocytes. Children in families with adult-onset cancer predisposition syndromes may present with cancers but the potential contribution of these germline variants to these childhood cancers is unknown. Our molecular and functional studies on both germline and tumor tissues did not suggest direct contribution of these germline variants on lymphomagenesis, however there might be other mechanism of inactivation not identified from this study. This case highlights the complexities of understanding genetic susceptibility in childhood cancers. We propose a multifactorial oligogenic inheritance of cancer susceptibility in this patient through several mechanisms of genomic instability.

DNA Damage Atlas: an atlas of DNA damage and repair.

DNA damage and its improper repair are the major source of genomic alterations responsible for many human diseases, particularly cancer. To aid researchers in understanding the underlying mechanisms of genome instability, a number of genome-wide profiling approaches have been developed to monitor DNA damage and repair events. The rapid accumulation of published datasets underscores the critical necessity of a comprehensive database to curate sequencing data on DNA damage and repair intermediates. Here, we present DNA Damage Atlas (DDA, http://www.bioinformaticspa.com/DDA/), the first large-scale repository of DNA damage and repair information. Currently, DDA comprises 6,030 samples from 262 datasets by 59 technologies, covering 16 species, 10 types of damage and 135 treatments. Data collected in DDA was processed through a standardized workflow, including quality checks, hotspots identification and a series of feature characterization for the hotspots. Notably, DDA encompasses analyses of highly repetitive regions, ribosomal DNA and telomere. DDA offers a user-friendly interface that facilitates browsing, searching, genome browser visualization, hotspots comparison and data downloading, enabling convenient and thorough exploration for datasets of interest. In summary, DDA will stand as a valuable resource for research in genome instability and its association with diseases.

A mutator-derived prognostic eRNA signature provides insight into the pathogenesis of breast cancer

Abundant evidence suggests that enhancer RNA (eRNA) is closely related to tumorigenesis, and the role of eRNA transcription in promoting genomic instability in cancers is gradually unveiled. However, research on the evaluation of the prognostic value and molecular mechanisms of genomic instability associated eRNAs in breast cancer is long overdue.Here, we integratively analyzed eRNA expression and somatic mutation profiles in breast cancer genome. We identified genomic instability associated eRNAs and developed a prognostic signature based on these eRNAs with the area under the curve (AUC) around 0.8 at 9-year survival. We further found the prognostic value of this signature is independent of common clinical factors and is better than TP53 status. Higher expression of genomic instability associated genes in the high-risk group was observed, suggesting that this eRNA signature may serve as an indicator of genomic instability in breast cancer.We found prognostic eRNA co-expressed genes are mainly enriched in Gene set ‘Breast Cancer 8P12–P11 Amplicon’, Gene set ‘Metabolism of lipids’ and GO process ‘Ubiquitin protein ligase binding’. Furthermore, 11 eRNA-signature regulated genes are identified by assessing promoter-enhancer interaction. Among these genes, F11R, BHLHE40, and NECTIN4 are previously reported oncogenes and EGOT is a tumor suppressor gene, indicating the direct roles of eRNAs in tumorigenesis.

The genomic landscape and prognostic significance of 11.q.13 amplification in breast, head & neck, and lung cancer.

e18037 Background: The 11.q.13 (11q) chromosomal band is amplified in many cancers and is associated with poor outcomes in Head & Neck (HN) cancer. The 11q amplicon contains several putative oncogenes, but it’s unclear which amplified genes drive clinical outcomes. We compared the genomic landscape and prognostic significance of 11q amplification in Breast (BC), HN, and non-small cell lung cancer (NSCLC) tumors and identify genes whose expression may be driving clinical outcomes. Methods: DNA (592 genes or WES)/RNA (WTS) sequencing was performed for BC (N = 6,967), HN (N = 2,337), and NSCLC (N = 13,433) tumors submitted to Caris Life Sciences (Phoenix, AZ). 11q amplification (CNA) was defined as tumors with concurrent copy number amplification with copies of FGF3, FGF4, FGF19, and CCND1. Non-amplified tumors (copy number normal, CNN) were defined as a < 4 copies for all four genes. Her2/Neu (+: ≥3+ and > 10%) and HR (ER or PR+: ≥1+ and ≥1%) expression was tested by IHC. tests were applied as appropriate, with P-values adjusted for multiple comparisons. Real-world overall survival (OS) data was obtained from insurance claims, and Kaplan-Meier estimates were calculated for a molecularly defined subset of tumors. 11q genes that were two-fold over expressed in CNA vs CNN were further investigated. The hazard ratio (HR) was calculated for subgroups stratified by median expression of 11q genes. The genes with the five largest HRs (worse OS for CNA, p < .05) were identified as potential driver genes. Results: 11q CNA was observed in 10% of BC, 12% of HN, and 3% of NSCLC. 11q CNA rates were enriched in HR+ BC (HR+/HER2-: 17%, HR+/HER2+: 19%) as compared to HR- tumors (HR-/HER2+: 6%, TNBC: 3%, p < .05). TP53 mutations were less frequent in CNA BC (32 vs 53% CNN) but more frequent in CNA HN (91 vs 52%) and NSCLC (89 vs 66%, p < .05 all). In NSCLC, CNA tumors had a lower rate of EGFR (4.3 vs 12%), KRAS (6.7 vs 28%) and STK11 (5.7 vs 13%) mutations ( p < .05). Over 34 gene were amplified as compared to CNN ( > 3% increase, p < .05), including FGFR3 (11 vs 0.2%) and PDCD1 (10 vs 0). MEN1 (5 vs 0%) was the only 11q gene amplified. CNA was associated with worse OS in HR-/HER2+ (HR 6.3 [2.6-15.5], p = .001), while no difference in OS was observed in BC (HR 1.0 [.9-1.1], p = .62). Compared to CNN, CNA had worse OS in HN (HR 1.5 [1.3-1.7], p < .001) and NSCLC (HR 1.3 [1.2-1.4], p < .001). Stratification by 11q gene expression identified DHCR7, GAL, ANO1, MRPL21, and FADD as the strongest predictors of OS in HN tumors, while GAL, MYEOV, MRPL21, OVOL1, and DHCR7 were strongest in NSCLC (HR: 1.3-1.2). Conclusions: 11q CNA shows differential association with key driver mutations across cancer types that provides new insight to patient subpopulations harboring these alterations. Co-amplification of non-11q genes in NSCLC suggests a unique mechanism of genome instability. The role of DHCR7 and GAL as predictors of clinical outcomes in HN and NSCLC CNA tumors warrants further investigation.

Genome wide hypomethylation and youth-associated DNA gap reduction promoting DNA damage and senescence-associated pathogenesis

Introduction: The United States currently faces two opioid crises, an evolved crisis currently manifesting as widespread abuse of illicit opioids, and a crisis in pain management largely manufactured by the Centers for Disease Control and Prevention 2016 Guideline. Our goal in this paper is to identify root causes, trace the trajectory of forces unleashed over time, and define potential solutions to these crises. Methods: Analytic review of the scientific, socioeconomic, and historical literature. Background: Age-associated epigenetic alteration is the underlying cause of DNA damage in aging cells. Two types of youth-associated DNA-protection epigenetic marks, global methylation, and youth-associated genomic stabilization DNA gap (youth-DNA-gap) reduce when cell ages. The epigenomic mark reduction promotes DNA damage and accelerates aging hallmarks. While DNA hypomethylation destabilizes DNA by several mechanisms, the DNA sequence around the youth-DNA-gap is hypermethylated. Therefore, the genomic instability mechanisms underlying DNA hypomethylation and youth-DNA-gap reduction are linked. Results: DNA gap prevents DNA damage by relieving the torsion forces caused by a twisted wave during DNA strand separation by transcription or replication. When the cells begin to age, hypomethylation and youth-DNA-gap reduction can occur as consequences of the efflux of intranuclear HMGB1. The methylated DNA gaps are formed by several proteins. Box A of HMGB1 possesses a molecular scissor role in producing youth-DNA-gaps. So the lack of a gap-producing role of HMGB1 results in a youth-DNA-gap reduction. The histone deacetylation role of SIRT1, an aging prevention protein, prevents DNA ends of youth-DNA-gaps from being recognized as pathologic DNA breaks. Youth-DNA-gaps are methylated and determined genome distribution by AGO4, an effector protein in RNA-directed DNA methylation. The lack of intranuclear HMGB1 promotes global hypomethylation due to two subsequent mechanisms. First is the loss of AGO4-methylating DNA. The other is the accumulation of DNA damage due to lacking HMGB1-produced DNA gap promoting DNA demethylation while undergoing DNA repair. DNA torsion due to youth-DNA-gap reduction increases DNA damage and, consequently, the DNA damage response (DDR). Persistent DDR promotes cellular senescence. Accumulating senescent cells leads to the deterioration of the structure and function of the human body. Rejuvenating DNA (RED) by adding DNA protection epigenetic marks using genomic stability molecule (GEM) such as box A of HMGB1 increases DNA durability, limits DNA damage, rejuvenates senescence cells, and improves organ structure and function deterioration due to aging. Conclusion: Reducing youth-associated epigenetic marks is degenerative diseases' primary molecular pathogenesis mechanism. REDGEM is a new therapeutic strategy inhibiting the upstream molecular aging process that will revolutionize the treatment of DNA damage or age-associated diseases and conditions.

Radiation-Induced Bystander Effect on the Genome of Bone Marrow Mesenchymal Stem Cells in Lung Cancer.

Aims: Radiation by-radiation effect (RIBE) can induce the genomic instability of bone marrow mesenchymal stem cells (BMSCs) adjacent to lung cancer, and this effect not only exists in the short-term, but also accompanies it in the long-term, but its specific mechanism is not clear. Our goal is to explore the similarities and differences in the mechanism of genomic damage in tumor-associated BMSCs induced by short-term and long-term RIBE, and to provide a theoretical basis for adjuvant drugs for protection against RIBE at different clinical time periods. Results: We found that both short- and long-term RIBE induced genomic instability. We could show a high expression of TGF-β1, TNF-α, and HIF-1α in tumor-associated BMSCs after short-term RIBE whereas only TNF-α and HIF-1α expression was increased in long-term RIBE. We further confirmed that genomic instability is associated with the activation of the HIF-1α pathway and that this is mediated by TNF-α and TGF-β1. In addition, we found differences in the mechanisms of genomic instability in the considered RIBE windows of analysis. In short-term RIBE, both TNF-α and TGF-β1 play a role, whereas only TNF-α plays a decisive role in long-term RIBE. In addition, there were differences in BMSC recruitment and genomic instability of different tissues with a more pronounced expression in tumor and bone marrow than compared to lung. Innovation and Conclusion: We could show dynamic changes in the expression of the cytokines TGF-β1 and TNF-α during short- and long-term RIBE. The differential expression of the two is the key to causing the genomic damage of tumor-associated BMSCs in the considered windows of analysis. Therefore, these results may serve as a guideline for the administration of radiation protection adjuvant drugs at different clinical stages. Antioxid. Redox Signal. 38, 747-767.

Theranostic Interpolation of Genomic Instability in Breast Cancer.

Breast cancer is a diverse disease caused by mutations in multiple genes accompanying epigenetic aberrations of hazardous genes and protein pathways, which distress tumor-suppressor genes and the expression of oncogenes. Alteration in any of the several physiological mechanisms such as cell cycle checkpoints, DNA repair machinery, mitotic checkpoints, and telomere maintenance results in genomic instability. Theranostic has the potential to foretell and estimate therapy response, contributing a valuable opportunity to modify the ongoing treatments and has developed new treatment strategies in a personalized manner. “Omics” technologies play a key role while studying genomic instability in breast cancer, and broadly include various aspects of proteomics, genomics, metabolomics, and tumor grading. Certain computational techniques have been designed to facilitate the early diagnosis of cancer and predict disease-specific therapies, which can produce many effective results. Several diverse tools are used to investigate genomic instability and underlying mechanisms. The current review aimed to explore the genomic landscape, tumor heterogeneity, and possible mechanisms of genomic instability involved in initiating breast cancer. We also discuss the implications of computational biology regarding mutational and pathway analyses, identification of prognostic markers, and the development of strategies for precision medicine. We also review different technologies required for the investigation of genomic instability in breast cancer cells, including recent therapeutic and preventive advances in breast cancer.

Integrated genome and transcriptome analyses reveal the mechanism of genome instability in ataxia with oculomotor apraxia 2

Mutations in the SETX gene, which encodes Senataxin, are associated with the progressive neurodegenerative diseases ataxia with oculomotor apraxia 2 (AOA2) and amyotrophic lateral sclerosis 4 (ALS4). To identify the causal defect in AOA2, patient-derived cells and SETX knockouts (human and mouse) were analyzed using integrated genomic and transcriptomic approaches. A genome-wide increase in chromosome instability (gains and losses) within genes and at chromosome fragile sites was observed, resulting in changes to gene-expression profiles. Transcription stress near promoters correlated with high GCskew and the accumulation of R-loops at promoter-proximal regions, which localized with chromosomal regions where gains and losses were observed. In the absence of Senataxin, the Cockayne syndrome protein CSB was required for the recruitment of the transcription-coupled repair endonucleases (XPG and XPF) and RAD52 recombination protein to target and resolve transcription bubbles containing R-loops, leading to genomic instability. These results show that transcription stress is an important contributor to SETX mutation-associated chromosome fragility and AOA2.

Mechanisms of Genome Instability in the Fragile X-Related Disorders.

The Fragile X-related disorders (FXDs), which include the intellectual disability fragile X syndrome (FXS), are disorders caused by expansion of a CGG-repeat tract in the 5′ UTR of the X-linked FMR1 gene. These disorders are named for FRAXA, the folate-sensitive fragile site that localizes with the CGG-repeat in individuals with FXS. Two pathological FMR1 allele size classes are distinguished. Premutation (PM) alleles have 54–200 repeats and confer the risk of fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X-associated primary ovarian insufficiency (FXPOI). PM alleles are prone to both somatic and germline expansion, with female PM carriers being at risk of having a child with >200+ repeats. Inheritance of such full mutation (FM) alleles causes FXS. Contractions of PM and FM alleles can also occur. As a result, many carriers are mosaic for different sized alleles, with the clinical presentation depending on the proportions of these alleles in affected tissues. Furthermore, it has become apparent that the chromosomal fragility of FXS individuals reflects an underlying problem that can lead to chromosomal numerical and structural abnormalities. Thus, large numbers of CGG-repeats in the FMR1 gene predisposes individuals to multiple forms of genome instability. This review will discuss our current understanding of these processes.

Genomic Instability Promotes the Progression of Clear Cell Renal Cell Carcinoma Through Influencing the Immune Microenvironment.

Background: Long non-coding RNAs (lncRNAs) are now under discussion as novel promising biomarkers for clear cell renal cell carcinoma (ccRCC). However, the role of genomic instability-associated lncRNA signatures in tumors has not been thoroughly uncovered. The purpose of our study is to probe the role of genomic instability-derived lncRNA signature (GILncSig) and to further investigate the mechanism of genomic instability-mediated ccRCC progression.Methods: The transcriptome data and somatic mutation profiles of ccRCC as well as clinical characteristics used in this study were obtained from The Cancer Genome Atlas database and Gene Expression Omnibus database. Lasso regression analysis was performed to construct the GILncSig. Gene set enrichment analysis (GSEA) was performed to elucidate the biological functions and relative pathways. CIBERSORT and EPIC algorithm were applied to calculate the proportion of immune cells in ccRCC. ESTIMATE algorithm was utilized to compute the immune microenvironment scores.Results: In total, 148 novel genomic instability-derived lncRNAs in ccRCC were identified. Immediately, on the basis of univariate cox analysis and lasso analysis, a GILncSig was appraised, through which the patients were allocated into High-Risk and Low-Risk groups with significantly different characteristics and prognoses. In addition, we confirmed that the somatic mutation count, tumor mutation burden, and the expression of UBQLN4, which were ascertainably associated with genomic instability, were significantly correlated with the GILncSig, indicating its reliability as a measurement of the genomic instability. Furthermore, the efficiency of GILncSig in prognostic aspects was better than the single mutation gene in ccRCC. In addition, MNX1-AS1 was defined to be a potential biomarker characterized by strong correlation with clinical features. Moreover, GSEA results indicated that the IL6/JAK/STAT3/SIGNALING pathway could be considered as a potential mechanism of genomic instability to influence tumor progression. Besides, the immune microenvironment showed significant differences between the GS-like group and the GU-like group, which was specifically manifested as high expression of CTLA4, GITR, TNFSF14, and regulatory T cells (Tregs) as well as low expression of endothelial cells (ECs) in the GU-like group. Finally, the prognostic value and clinical relevance of GILncSig were verified in GEO datasets and other urinary tumors in TCGA dataset.Conclusion: In conclusion, our study provided a new perspective for the role of lncRNAs in genomic instability and revealed that genomic instability may mediate tumor progression by affecting immunity. Besides, MNX1-AS1 played critical roles in promoting the progression of ccRCC, which may be a potential therapeutic target. What is more, the immune atlas of genomic instability was characterized by high expression of CTLA4, GITR, TNFSF14, and Tregs, and low expression of ECs.

R-loops and regulatory changes in chronologically ageing fission yeast cells drive non-random patterns of genome rearrangements.

Aberrant repair of DNA double-strand breaks can recombine distant chromosomal breakpoints. Chromosomal rearrangements compromise genome function and are a hallmark of ageing. Rearrangements are challenging to detect in non-dividing cell populations, because they reflect individually rare, heterogeneous events. The genomic distribution of de novo rearrangements in non-dividing cells, and their dynamics during ageing, remain therefore poorly characterized. Studies of genomic instability during ageing have focussed on mitochondrial DNA, small genetic variants, or proliferating cells. To characterize genome rearrangements during cellular ageing in non-dividing cells, we interrogated a single diagnostic measure, DNA breakpoint junctions, using Schizosaccharomyces pombe as a model system. Aberrant DNA junctions that accumulated with age were associated with microhomology sequences and R-loops. Global hotspots for age-associated breakpoint formation were evident near telomeric genes and linked to remote breakpoints elsewhere in the genome, including the mitochondrial chromosome. Formation of breakpoint junctions at global hotspots was inhibited by the Sir2 histone deacetylase and might be triggered by an age-dependent de-repression of chromatin silencing. An unexpected mechanism of genomic instability may cause more local hotspots: age-associated reduction in an RNA-binding protein triggering R-loops at target loci. This result suggests that biological processes other than transcription or replication can drive genome rearrangements. Notably, we detected similar signatures of genome rearrangements that accumulated in old brain cells of humans. These findings provide insights into the unique patterns and possible mechanisms of genome rearrangements in non-dividing cells, which can be promoted by ageing-related changes in gene-regulatory proteins.

Bone Marrow Microenvironment Induces Genomic Instability and Enables Clonal Evolution in Multiple Myeloma

Multiple myeloma (MM) is characterized by ongoing DNA damage and genomic instability, with acquisition of mutations and structural changes during disease progression. Its growth and survival charactersitics are also affected by the bone marrow (BM) microenvironment, influencing its pathogenesis as well as development of drug resistance. However, it is unclear whether the BM microenvironment can affect the ability of the tumor cells to acquire new genomic changes. The susceptibility to genomic changes might come from an increase in the endogenous sources of DNA damage or an impairment or deregulation of the DNA Damage Response (DDR). We hypothesized that the microenvironment may also contribute to ongoing DNA damage and genomic instability in MM.To investigate the potential role of the microenvironment in genomic stability in MM, we evaluated change in frequency of spontaneous DNA damage in myeloma cells when co-cultured with various microenvironment cells (HS-5 stromal cell line and primary patient BM stromal cells [BMSC]). We observed that DNA breaks, already present at baseline as demonstrated by high levels of pH2AX, were significantly increased when MM cell lines were co-cultured with either HS5 cell or primary BMSCs (2-4 folds increase, p <0.05). Moreover, similar results were observed when MM cell lines were cultured in presence of stromal cell supernatant or IL6 alone, also suggesting a possible effect of the cytokine. Immunofluorescence experiments revealed that after co-culture with stromal cells pH2AX recruitment to sites of DNA DSBs was also increased.Since we have shown that elevated HR and nuclease (especially apurinic/apyrimidinic endonuclease) activities are important mechanisms of genomic instability in MM, we next evaluated the impact of microenvironment on nuclease and HR activities using functional assays. Interestingly, we found that MM-BMSC co-culture induced a significant increase in overall nuclease activity, with specific increase in the expression of APEX1, a major apurinic/apyrimidinic (AP) nuclease, leading to increase in AP acitivity as measured by a flourescent-based oliginucleotide assay. MM-BMSC co-culture also induced HR activity demonstrating the impact of BMSC on genomic instability.To more comprehensively evaluate effects of stromal cells on different DNA Damage Response pathways, we performed RNA-sequencing of 3 different MM cell lines cultured alone or in presence of HS-5. We observed that the network of genes involved in DNA Damage Response were significantly altered in MM in the presence of stromal cells. We further confirmed our results in publicly available microarray gene expression data in MM cell lines cultured alone or with HS-5. These results support the idea that microenvironment can impact DNA integrity and stability in MM cells.Finally, we cultured MM cell lines alone or in presence of BMSC from MM patients for 4 weeks and investigated genomic changes using cytoscan HD array to detect effects of co-culture on acquisition of new copy-number alterations. We observed that MM cell line growth in the presence of BMSCs provides a selective environment for clonal selection, demonstrating the role of stromal cells in clonal diversity in MM.In conclusion, our data suggest that BMSC enhance DNA damage and instability in MM. Understanding the mechanisms leading to genomic instability is important to develop new therapeutic strategies aimed at preventing MM evolution. DisclosuresAnderson:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder.

Mechanism of genome instability mediated by human DNA polymerase mu misincorporation

Pol μ is capable of performing gap-filling repair synthesis in the nonhomologous end joining (NHEJ) pathway. Together with DNA ligase, misincorporation of dGTP opposite the templating T by Pol μ results in a promutagenic T:G mispair, leading to genomic instability. Here, crystal structures and kinetics of Pol μ substituting dGTP for dATP on gapped DNA substrates containing templating T were determined and compared. Pol μ is highly mutagenic on a 2-nt gapped DNA substrate, with T:dGTP base pairing at the 3ʹ end of the gap. Two residues (Lys438 and Gln441) interact with T:dGTP and fine tune the active site microenvironments. The in-crystal misincorporation reaction of Pol μ revealed an unexpected second dGTP in the active site, suggesting its potential mutagenic role among human X family polymerases in NHEJ.

Integrated genomics and comprehensive validation reveal drivers of genomic evolution in esophageal adenocarcinoma

Esophageal adenocarcinoma (EAC) is associated with a marked genomic instability, which underlies disease progression and development of resistance to treatment. In this study, we used an integrated genomics approach to identify a genomic instability signature. Here we show that elevated expression of this signature correlates with poor survival in EAC as well as three other cancers. Knockout and overexpression screens establish the relevance of these genes to genomic instability. Indepth evaluation of three genes (TTK, TPX2 and RAD54B) confirms their role in genomic instability and tumor growth. Mutational signatures identified by whole genome sequencing and functional studies demonstrate that DNA damage and homologous recombination are common mechanisms of genomic instability induced by these genes. Our data suggest that the inhibitors of TTK and possibly other genes identified in this study have potential to inhibit/reduce growth and spontaneous as well as chemotherapy-induced genomic instability in EAC and possibly other cancers.

Aicardi-Goutières syndrome-associated gene SAMHD1 preserves genome integrity by preventing R-loop formation at transcription-replication conflict regions.

The comorbid association of autoimmune diseases with cancers has been a major obstacle to successful anti-cancer treatment. Cancer survival rate decreases significantly in patients with preexisting autoimmunity. However, to date, the molecular and cellular profiles of such comorbidities are poorly understood. We used Aicardi-Goutières syndrome (AGS) as a model autoimmune disease and explored the underlying mechanisms of genome instability in AGS-associated-gene-deficient patient cells. We found that R-loops are highly enriched at transcription-replication conflict regions of the genome in fibroblast of patients bearing SAMHD1 mutation, which is the AGS-associated-gene mutation most frequently reported with tumor and malignancies. In SAMHD1-depleted cells, R-loops accumulated with the concomitant activation of DNA damage responses. Removal of R-loops in SAMHD1 deficiency reduced cellular responses to genome instability. Furthermore, downregulation of SAMHD1 expression is associated with various types of cancer and poor survival rate. Our findings suggest that SAMHD1 functions as a tumor suppressor by resolving R-loops, and thus, SAMHD1 and R-loop may be novel diagnostic markers and targets for patient stratification in anti-cancer therapy.

Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq.

Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3' ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3' ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

Abstract 3538: Microsatellite instability (MSI) and homologous recombination deficiency (HRD) are mutually exclusive mechanisms of genomic instability

Abstract Background: Genomic instability refers to the tendency to accumulate genomic alterations. MSI-High (MSI-H) tumors are characterized by a mismatch repair deficiency triggering hypermutation, whereas HRD tumors display genomic lesions that result in genomic loss of heterozygosity (gLOH). These mechanistic differences have critical implications for treatment strategies, where immune checkpoint blockade is often efficacious in MSI-H tumors and PARP inhibition is linked to response in HRD tumors. We investigated the patterns of co-occurrence or mutual exclusivity between these two mechanisms of genomic instability. Methods: Comprehensive genomic profiling was performed on &amp;gt;130K tumors to coding exons and select introns of up to 465 genes. MSI was determined on up to 114 loci and high gLOH (≥14%) was used as a surrogate of HRD (PMID: 27908594). Results: In this cohort, HRD was most prevalent in triple-negative breast and fallopian tube cancers and absent in cancers such as skin neuroendocrine cancers. HRD was strongly associated with biallelic loss of homologous recombination (HR) pathway genes and not monoallelic loss of these genes. High MSI was most frequently found in endometrial and small intestine cancers, and absent in cancers such as fallopian tube cancers and GIST. MSI-H tumors and HRD tumors were mutually exclusive (6.5% of MSI-H tumors were HRD vs 25.7% of tumors that were not MSI-H; OR = 5.0; p&amp;lt;1e-108). Conclusions: We show that high MSI and HRD events are mutually exclusive across tumor types, suggesting that co-occurrence of these genomic instability mechanisms may be evolutionarily disadvantageous. High gLOH is associated with biallelic loss of HR pathway genes, supporting its use as a surrogate for HRD. The genomic differences between HRD and MSI-H may underlie differential susceptibility to targeted therapeutics. Gene% Biallelic loss in gLOH-H% Biallelic loss in gLOH-LowBiallelic loss p-value% Monoallelic loss in gLOH-H% Monoallelic loss in gLOH-LowMonoallelic loss p-valueATM2.45%1.54%1.24E-251.15%1.83%1.12E-17BARD10.25%0.03%5.15E-290.25%0.28%0.40BRCA15.15%0.23%&amp;lt;1.00E-1000.49%0.57%0.10BRCA25.32%0.76%&amp;lt;1.00E-1000.74%1.11%4.62E-09BRIP10.36%0.12%1.97E-160.41%0.49%0.07CDK120.49%0.31%3.95E-060.30%0.52%1.31E-07CHEK20.65%0.44%8.20E-060.54%0.86%1.92E-09FANCA0.51%0.25%1.36E-120.38%0.47%0.04FANCC0.22%0.07%2.03E-100.11%0.26%1.64E-07FANCG0.10%0.03%1.08E-050.08%0.12%0.04RAD51B0.39%0.15%5.36E-150.04%0.09%0.01RAD51C0.32%0.05%2.66E-320.10%0.12%0.41RAD51D0.18%0.02%2.52E-230.08%0.11%0.20 Citation Format: Dexter X. Jin, Ethan S. Sokol, Sally E. Trabucco, Garrett M. Frampton, Luciana Molinero. Microsatellite instability (MSI) and homologous recombination deficiency (HRD) are mutually exclusive mechanisms of genomic instability [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3538.

Integrin-Linked-Kinase Overexpression Is Implicated in Mechanisms of Genomic Instability in Human Colorectal Cancer.

Genomic instability is a hallmark of cancer cells contributing to tumor development and progression. Integrin-linked kinase (ILK) is a focal adhesion protein with well-established role in carcinogenesis. We have previously shown that ILK overexpression is critically implicated in human colorectal cancer (CRC) progression. In light of the recent findings that ILK regulates centrosomes and mitotic spindle formation, we aimed to determine its implication in mechanisms of genomic instability in human CRC. Association of ILK expression with markers of genomic instability (micronuclei formation, nucleus size, and intensity) was investigated in diploid human colon cancer cells HCT116 upon ectopic ILK overexpression, by immunofluorescence and in human CRC samples by Feulgen staining. We also evaluated the role of ILK in mitotic spindle formation, by immunofluorescence, in HCT116 cells upon inhibition and overexpression of ILK. Finally, we evaluated association of ILK overexpression with markers of DNA damage (p-H2AX, p-ATM/ATR) in human CRC tissue samples by immunohistochemistry and in ILK-overexpressing cells by immunofluorescence. We showed that ILK overexpression is associated with genomic instability markers in human colon cancer cells and tissues samples. Aberrant mitotic spindles were observed in cells treated with specific ILK inhibitor (QLT0267), while ILK-overexpressing cells failed to undergo nocodazole-induced mitotic arrest. ILK overexpression was also associated with markers of DNA damage in HCT116 cells and human CRC tissue samples. The above findings indicate that overexpression of ILK is implicated in mechanisms of genomic instability in CRC suggesting a novel role of this protein in cancer.