Telomere Organization Research Articles

Shaping human telomeres: from shelterin and CST complexes to telomeric chromatin organization.

The regulation of telomere length in mammals is crucial for chromosome end-capping and thus for maintaining genome stability and cellular lifespan. This process requires coordination between telomeric protein complexes and the ribonucleoprotein telomerase, which extends the telomeric DNA. Telomeric proteins modulate telomere architecture, recruit telomerase to accessible telomeres and orchestrate the conversion of the newly synthesized telomeric single-stranded DNA tail into double-stranded DNA. Dysfunctional telomere maintenance leads to telomere shortening, which causes human diseases including bone marrow failure, premature ageing and cancer. Recent studies provide new insights into telomerase-related interactions (the 'telomere replisome') and reveal new challenges for future telomere structural biology endeavours owing to the dynamic nature of telomere architecture and the great number of structures that telomeres form. In this Review, we discuss recently determined structures of the shelterin and CTC1-STN1-TEN1 (CST) complexes, how they may participate in the regulation of telomere replication and chromosome end-capping, and how disease-causing mutations in their encoding genes may affect specific functions. Major outstanding questions in the field include how all of the telomere components assemble relative to each other and how the switching between different telomere structures is achieved.

Functional Significance of DNA in Animal Science

G-quadruplexes (G4s) are non-canonical structures that can be formed in DNA and RNA sequences which carry four short runs of guanines. They are distributed in the whole genome but are enriched in gene promoter regions, gene UTRs and chromosome telomeres. The entire spectrum of their functional functions is not yet completely explored, but there is clear evidence supporting their involvement in a variety of processes, including transcription control, replication, telomere organization, among others. There has been an increased interest in research for G4s over the last decade that has led to a deeper understanding of their function in many physiological and pathways. On the other hand, in plant species and animals of agricultural interest, these mechanisms are poorly studied. Here, we summarize the existing methods used to study G4s, analyze plant and farm animal studies, and address the benefits of a more rigorous inclusion of G4 research in the agricultural sciences.

Identification of Key mRNAs and lncRNAs Associated with the Effects of Anti-TWEAK on Osteosarcoma

Background: Osteosarcoma (OS) is one of the most common primary malignant bone tumors in teenagers. Emerging studies demonstrated TWEAK and Fn14 were involved in regulating cancer cell differentiation, proliferation, apoptosis, migration and invasion. Objective: The present study identified differently expressed mRNAs and lncRNAs after anti-TWEAK treatment in OS cells using GSE41828. Methods: We identified 922 up-regulated mRNAs, 863 downregulated mRNAs, 29 up-regulated lncRNAs, and 58 down-regulated lncRNAs after anti-TWEAK treatment in OS cells. By constructing PPI networks, we identified several key proteins involved in anti-TWEAK treatment in OS cells, including MYC, IL6, CD44, ITGAM, STAT1, CCL5, FN1, PTEN, SPP1, TOP2A, and NCAM1. By constructing lncRNAs coexpression networks, we identified several key lncRNAs, including LINC00623, LINC00944, PSMB8-AS1, LOC101929787. Result: Bioinformatics analysis revealed DEGs after anti-TWEAK treatment in OS were involved in regulating type I interferon signaling pathway, immune response related pathways, telomere organization, chromatin silencing at rDNA, and DNA replication. Bioinformatics analysis revealed differently expressed lncRNAs after antiTWEAK treatment in OS were related to telomere organization, protein heterotetramerization, DNA replication, response to hypoxia, TNF signaling pathway, PI3K-Akt signaling pathway, Focal adhesion, Apoptosis, NF-kappa B signaling pathway, MAPK signaling pathway, FoxO signaling pathway. Conclusion: : This study provided useful information for understanding the mechanisms of TWEAK underlying OS progression and identifying novel therapeutic markers for OS.

Exploration of Prognostic Biomarkers among Replication Factor C Family in the Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the common cancers with a high incidence and mortality. The human replication factor C (RFC) family contains 5 subunits that play an important role in DNA replication and DNA damage repair. RFCs are abnormally expressed in a variety of cancers; some of them are differentially expressed in HCC tissues and related to tumor growth. However, the expression, prognostic value, and effect targets of the whole RFC family in HCC are still unclear. To address these issues, we performed a multidimensional analysis of RFCs in HCC patients by Oncomine, UALCAN, GEPIA, Human protein atlas, Kaplan-Meier plotter, cBioPortal, GeneMANIA, String, and LinkedOmics. mRNA expression of RFCs was significantly increased in HCC tissues. There was a significant correlation between the expression of RFC2/3/4/5 and tumor stage of HCC patients. Besides, high mRNA expression of RFC2/4 was associated with worse overall survival (OS). Moreover, genetic alterations of RFCs were associated with worse OS in HCC patients. We found that genes co-expressed with RFC2/4 were mainly involved in biological processes, such as chromosome segregation, mitotic cell cycle phase transition, and telomere organization and they activated the cell cycle and spliceosome pathways. The gene set is mainly enriched in cancer-related kinases AURKA, ATR, CDK1, PLK1, and CHEK1. E2F family members were the key transcription factors for RFCs. Our results suggest that differentially expressed RFC2 and RFC4 are potential prognostic biomarkers in HCC and may act on E2F transcription factors and some kinase targets to dysregulate the cell cycle pathway. These efforts may provide new research directions for prognostic biomarkers and therapeutic targets in HCC.

Dynamics of TRF1 organizing a single human telomere.

Chromosome stability is primarily determined by telomere length. TRF1 is the core subunit of shelterin that plays a critical role in telomere organization and replication. However, the dynamics of TRF1 in scenarios of telomere-processing activities remain elusive. Using single-molecule magnetic tweezers, we here investigated the dynamics of TRF1 upon organizing a human telomere and the protein-DNA interactions at a moving telomeric fork. We first developed a method to obtain telomeres from human cells for directly measuring the telomere length by single-molecule force spectroscopy. Next, we examined the compaction and decompaction of a telomere by TRF1 dimers. TRF1 dissociates from a compacted telomere with heterogenous loops in ∼20 s. We also found a negative correlation between the number of telomeric loops and loop sizes. We further characterized the dynamics of TRF1 at a telomeric DNA fork. With binding energies of 11 kBT, TRF1 can modulate the forward and backward steps of DNA fork movements by 2–9 s at a critical force of F1/2, temporarily maintaining the telomeric fork open. Our results shed light on the mechanisms of how TRF1 organizes human telomeres and facilitates the efficient replication of telomeric DNA. Our work will help future research on the chemical biology of telomeres and shelterin-targeted drug discovery.

Identification and characterization of critical genes associated with tamoxifen resistance in breast cancer.

BackgroundTamoxifen resistance in breast cancer is an unsolved problem in clinical practice. The aim of this study was to determine the potential mechanisms of tamoxifen resistance through bioinformatics analysis.MethodsGene expression profiles of tamoxifen-resistant MCF-7/TR and MCF-7 cells were acquired from the Gene Expression Omnibus dataset GSE26459, and differentially expressed genes (DEGs) were detected with R software. We conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses using Database for Annotation, Visualization and Integrated Discovery. A protein–protein interaction (PPI) network was generated, and we analyzed hub genes in the network with the Search Tool for the Retrieval of Interacting Genes database. Finally, we used siRNAs to silence the target genes and conducted the MTS assay.ResultsWe identified 865 DEGs, 399 of which were upregulated. GO analysis indicated that most genes are related to telomere organization, extracellular exosomes, and binding-related items for protein heterodimerization. PPI network construction revealed that the top 10 hub genes—ACLY, HSPD1, PFAS, GART, TXN, HSPH1, HSPE1, IRAS, TRAP1, and ATIC—might be associated with tamoxifen resistance. Consistently, RT-qPCR analysis indicated that the expression of these 10 genes was increased in MCF-7/TR cells comparing with MCF-7 cells. Four hub genes (TXN, HSPD1, HSPH1 and ATIC) were related to overall survival in patients who accepted tamoxifen. In addition, knockdown of HSPH1 by siRNA may lead to reduced growth of MCF-7/TR cell with a trend close to significance (P = 0.07), indicating that upregulation of HSPH1 may play a role in tamoxifen resistance.ConclusionThis study revealed a number of critical hub genes that might serve as therapeutic targets in breast cancer resistant to tamoxifen and provided potential directions for uncovering the mechanisms of tamoxifen resistance.

Telomeres and telomerase in oncogenesis.

Telomeres are located at the ends of chromosomes and protect them from degradation. Suppressing the activity of telomerase, a telomere-synthesizing enzyme, and maintaining short telomeres is a protective mechanism against cancer in humans. In most human somatic cells, the expression of telomerase reverse transcriptase (TERT) is repressed and telomerase activity is inhibited. This leads to the progressive shortening of telomeres and inhibition of cell growth in a process called replicative senescence. Most types of primary cancer exhibit telomerase activation, which allows uncontrolled cell proliferation. Previous research indicates that TERT activation also affects cancer development through activities other than the canonical function of mediating telomere elongation. Recent studies have improved the understanding of the structure and function of telomeres and telomerase as well as key mechanisms underlying the activation of TERT and its role in oncogenesis. These advances led to a search for drugs that inhibit telomerase as a target for cancer therapy. The present review article summarizes the organization and function of telomeres, their role in carcinogenesis, and advances in telomerase-targeted therapy.

Karyotype and putative chromosomal inversion suggested by integration of cytogenetic and molecular data of the fungus-farming ant Mycetomoellerius iheringi Emery, 1888.

Comparative cytogenetic analyses are being increasingly used to collect information on species evolution, for example, diversification of closely related lineages and identification of morphologically indistinguishable species or lineages. Here, we have described the karyotype of the fungus-farming ant Mycetomoellerius iheringi Emery, 1888 and investigated its evolutionary relationships on the basis of molecular and cytogenetic data. The M. iheringi karyotype consists of 2n = 20 chromosomes (2K = 18M + 2SM). We also demonstrated that this species has the classical insect TTAGG telomere organization. Phylogenetic reconstruction showed that M. iheringi is phylogenetically closer to M. cirratus Mayhé-Nunes & Brandão, 2005 and M. kempfi Fowler, 1982. We compared M. iheringi with other congeneric species such as M. holmgreni Wheeler, 1925 and inferred that M. iheringi probably underwent a major pericentric inversion in one of its largest chromosomes, making it submetacentric. We discussed our results in the light of the phylogenetic relationships and chromosomal evolution.

Abstract P4-05-09: Molecular differences between high and low tumor mutational burden (TMB) across breast cancer (BC) subtypes

Abstract Background. High-TMB (HTMB) is an emerging promising agnostic biomarker for predicting benefit from immune-checkpoint inhibitors, independently of tumor type. At ASCO 2019, the TAPUR trial reported an interesting 21% ORR in heavily pretreated metastatic BC patients with very HTMB [vHTMB, ≥9 mutations/megabase (Muts/Mb)]. We aimed to define the differential gene expression and methylation landscape between low and high TMB in each BC subtype. Methods. In TCGA, we identified 848 patients with WES data available for TMB estimation. [ER+/HER2- (LumA by PAM50)) n = 364; ER+/HER2- (LumB by PAM50), n = 147; HER2+, n = 158; and TN, n = 179]. High TMB was defined according to two different cut-offs: ≥9 (vHTMB) and≥5 Muts/Mb (HTMB). The second arbitrary cut-off was used to define a larger group allowing to better characterize the different molecular landscapes associated with high and low TMB in each BC subtype. The HTMB group was compared with an equal number of tumors with low TMB. We assessed the differential RNA expression and methylation of single genes and pathways (defined using Gene Ontology - GO). “Common” genes and pathways were defined as recurrently associated with TMB (p<0.05) in all subtypes and with a combined p value ≤0.00001. Results. The overall prevalence of vHTMB (≥9) was 4.5%, with no substantial differences across subtypes (4.4%, LumA; 4.8% LumB; 5.7% HER2+; 3.3% TN). The prevalence of HTMB (≥5) was 13.7%, but it was different across BC subtypes (p=8.0E-07) (8.2%, LumA; 12.9% LumB; 13.3% HER2+; 25.7% TN). We found more “common” genes down-regulated (n=70) than up-regulated (n=3) in HTMB group. Two of these three genes (HSPE1 and FEZF1.AS1) have been associated with poor prognosis in BC. When we considered the “common” pathways, only 3 were up-regulated in HTMB, all implicated in post-transcriptional repression of gene expression (gene silencing by miRNA and mRNA binding involved in post-transcriptional gene silencing). Conversely, 66 were significantly down-regulated (including transcription coregulator and coactivator activity, protein serine/threonine kinase activity and ubiquitin-protein transferase activity and binding). Some genes and pathways were associated with TMB only in a specific BC subtype (p ≤0.00001). For instance, 16 pathways were down-regulated in the HTMB group of TNBC. These inlcuded 12 pathways implicated in immune response. Conversely, in LumB, 11 pathways were up-regulated in HTMB group and implicated in immune response. Intrestingly, these pathways were all significantly down-regulated in the HTMB group of LumA and TN. In HTMB group, we found 7 and 4 “common” genes hypermetylated and hypomethylated, respectively. Four pathways were commonly hypermethylated (chromatin silencing at rDNA, telomere organization and positive regulation of G1/S transition of mitotic cell cycle) and five were hypomethilated (including mitotic sister chromatid segregation). Considering private alterations, in TNBC, 23 of 27 significant pathways were hypermetylated in HTMB group including double-strand break repair via nonhomologous end joining, epigenetic negative regulation of gene expression, and regulation of gene silencing by miRNA. Conclusions. Very-high TMB which is considered potentially druggable (≥9 Muts/Mb) is rare in BC, and equally frequent in all subtypes. Instead, HTMB (≥5 Muts/Mb) is more frequent in TNBC. BCs with HTMB had a different molecular landscape. Overall, several genes are recurrently down-regulated in HTMB group, and this is at least partly due to miRNA regulated post-trascriptional silencing, which might rapresents a new mechanism of immune escape. The positive association between TMB and immune genes in LumB, as well as the negative association in TN and LumA, suggest that immune editing and surveillance might be dependent on the molecular context. Citation Format: Luca Licata, Barbara Galbardi, Balázs Győrffy, Thomas Karn, Lorenzo Sica, Alessia Rognone, Patrizia Zucchinelli, Daniela Aldrighetti, Stefania Zambelli, Luca Gianni, Giampaolo Bianchini. Molecular differences between high and low tumor mutational burden (TMB) across breast cancer (BC) subtypes [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-09.

Potential Molecular Mechanisms of Chaihu-Shugan-San in Treatment of Breast Cancer Based on Network Pharmacology.

Breast cancer is one of the most common cancers endangering women's health all over the world. Traditional Chinese medicine is increasingly recognized as a possible complementary and alternative therapy for breast cancer. Chaihu-Shugan-San is a traditional Chinese medicine prescription, which is extensively used in clinical practice. Its therapeutic effect on breast cancer has attracted extensive attention, but its mechanism of action is still unclear. In this study, we explored the molecular mechanism of Chaihu-Shugan-San in the treatment of breast cancer by network pharmacology. The results showed that 157 active ingredients and 8074 potential drug targets were obtained in the TCMSP database according to the screening conditions. 2384 disease targets were collected in the TTD, OMIM, DrugBank, GeneCards disease database. We applied the Bisogenet plug-in in Cytoscape 3.7.1 to obtain 451 core targets. The biological process of gene ontology (GO) involves the mRNA catabolic process, RNA catabolic process, telomere organization, nucleobase-containing compound catabolic process, heterocycle catabolic process, and so on. In cellular component, cytosolic part, focal adhesion, cell-substrate adherens junction, and cell-substrate junction are highly correlated with breast cancer. In the molecular function category, most proteins were addressed to ubiquitin-like protein ligase binding, protein domain specific binding, and Nop56p-associated pre-rRNA complex. Besides, the results of the KEGG pathway analysis showed that the pathways mainly involved in apoptosis, cell cycle, transcriptional dysregulation, endocrine resistance, and viral infection. In conclusion, the treatment of breast cancer by Chaihu-Shugan-San is the result of multicomponent, multitarget, and multipathway interaction. This study provides a certain theoretical basis for the treatment of breast cancer by Chaihu-Shugan-San and has certain reference value for the development and application of new drugs.

Comparative FISH mapping of ribosomal DNA clusters and TTAGG telomeric sequences to holokinetic chromosomes of eight species of the insect order Psocoptera.

Repetitive DNAs are the main components of eukaryotic genome. We mapped the 18S rDNA and TTAGG telomeric probe sequences by FISH to meiotic chromosomes of eight species of the order Psocoptera considered a basal taxon of Paraneoptera: Valenzuela burmeisteri (Brauer, 1876), Stenopsocus lachlani Kolbe, 1960, Graphopsocus cruciatus (Linnaeus, 1768), Peripsocus phaeopterus (Stephens, 1836), Philotarsus picicornis (Fabricius, 1793), Amphigerontia bifasciata (Latreille, 1799), Psococerastis gibbosa (Sulzer, 1766), and Metylophorus nebulosus (Stephens, 1836). These species belong to five distantly related families of the largest psocid suborder Psocomorpha: Caeciliusidae, Stenopsocidae, Peripsocidae, Philotarsidae, and Psocidae. We show that all the examined species share a similar location of 18S rDNA on a medium-sized pair of autosomes. This is the first study of rDNA clusters in the order Psocoptera using FISH. We also demonstrate that these species have the classical insect (TTAGG)n telomere organization. Our results provide a foundation for further cytogenetic characterization and chromosome evolution studies in Psocoptera.

Comprehensive Analysis of Therapy-Related Messenger RNAs and Long Noncoding RNAs as Novel Biomarkers for Advanced Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common types of human cancers. However, the mechanisms underlying CRC progression remained elusive. This study identified differently expressed messenger RNAs (mRNAs), long noncoding RNAs (lncRNAs), and small nucleolar RNAs (snoRNAs) between pre-therapeutic biopsies and post-therapeutic resections of locally advanced CRC by analyzing a public dataset, GSE94104. We identified 427 dysregulated mRNAs, 4 dysregulated lncRNAs, and 19 dysregulated snoRNAs between pre- and post-therapeutic locally advanced CRC samples. By constructing a protein–protein interaction network and co-expressing networks, we identified 10 key mRNAs, 4 key lncRNAs, and 7 key snoRNAs. Bioinformatics analysis showed therapy-related mRNAs were associated with nucleosome assembly, chromatin silencing at recombinant DNA, negative regulation of gene expression, and DNA replication. Therapy-related lncRNAs were associated with cell adhesion, extracellular matrix organization, angiogenesis, and sister chromatid cohesion. In addition, therapy-related snoRNAs were associated with DNA replication, nucleosome assembly, and telomere organization. We thought this study provided useful information for identifying novel biomarkers for CRC.

3D Telomeric Fingerprint of Advanced Cutaneous T-Cell Lymphoma

Introduction Cutaneous T-cell lymphomas (CTCLs) are characterized by dermal and epidermal infiltration of skin homing clonal CD4+ memory T-cells. Little is known about the oncogenic events driving either the progression of skin-limited disease such as Mycosis Fungoides (MF) to a leukemic form or Sézary Syndrome (SS), and there are no histologic means to predict evolution. Genetic instability is a hallmark of malignancy progression and telomere remodeling has been shown to play a role in the progression of hematological malignancies. Thus, the aim of this study is to characterize the three-dimensional (3D) telomeric organization in early and advanced CTCLs. Methods We performed 3D telomeric quantitative fluorescent in situ hybridization (3D Telo-Q-FISH) of 5mm skin tissue slides of 10 patients with MF and SS and of CD4+ lymphocytes of 3 healthy controls (Figure 1). Using the program TeloView (Vermolen et al., 2005), the proportion of telomeres of low intensity (TLI) (<5000 u), the nuclear volume and the total number of telomeric signals per cell were calculated. Patients were stratified based on CD30 expression (CD30 high, n=3 versus CD30 low, n=7) and clinical stage (early stages I-IIA, n=6 versus advanced stages IIB-IV, n=4). Results TLI represented 27% of telomeres in CTCL cells compared to 16% in control lymphocytes (p<0.0001). The highest proportion of TLIs was found in tumors CD30 high (34%), compared to 22% in tumors CD30 low (p<0.0001). Similar findings were observed when stratifying for advanced and early clinical stages (30% vs 24%, p<0.0001). Nuclear volume and total number of telomeric signals increased in CTCL cells expressing CD30 (both p<0.0001), and in CTCL cells associated with advanced clinical stages (p=0.0021 and p=0.001, respectively). However, as expected, nuclear volume and total number of telomeric signals were higher in control cells compared to CTCL cells (both p<0.0001). Conclusion We report clear evidence that CTCLs with either CD30 expression or advanced clinical stage are associated with loss of telomeres and telomeric signal intensity. These very small telomeres, termed 't-stumps', are a hallmark feature in many tumor cells. Analysis of the nuclear volume and total number of telomeric signals suggest that CTCL cells undergo in a first step telomere shortening and loss compared to healthy controls. In a second step further telomeric shortening associated with chromosomal rearrangements and bridge-breakage-fusion cycles may be involved in the progression of CTCL. Disclosures Pehr: Merck: Consultancy, Other: Clinical research on Alzheimers Drug; Eli Lilly: Consultancy, Other: Clinical research on Alzheimers Drug; galderma: Consultancy, Other: tactupump Forte; Actelion: Consultancy; valeant: Consultancy; CeraVe: Consultancy; Janssen-Cilag: Other: Train the trainer.

<p>TCDD-Inducible Poly-ADP-Ribose Polymerase (TIPARP), A Novel Therapeutic Target Of Breast Cancer</p>

BackgroundTIPARP (TCDD-inducible poly-ADP-ribose polymerase), a mono-ADP-ribosyltransferase and a transcriptional repressor of aryl hydrocarbon receptor (AHR), was one of the potential therapeutic targets for human cancers identified by CRISPR–Cas9 screens recently. Studies about TIPARP on cancers are scarce till now, most of which just focus on expressions, while the functions have not been widely reported yet. Moreover, the TIPARP prognostic significance and therapeutic value of breast cancer is also uncertain.MethodsThe present study was performed to comprehensively analyze the expression pattern, prognostic effect, potential therapeutic function of TIPARP in breast cancer by pooling all currently available databases online including Oncomine, UALCAN, bc-GenExMiner, Kaplan–Meier Plotter, COSMIC, UCSC Xena, STRING, DAVID and Comparative Toxicogenomics Database. Further, we also performed several cell biology experiments including RT-qPCR, Western blot and CCK-8 in cellular and clinical sample levels to confirm the conclusions from bioinformatics analysis.ResultsTIPARP was expressed lower in tumor tissues comparing with normal tissues. Meanwhile, several clinical parameters of breast cancer patients were correlated with TIPARP expression. Further, higher TIPARP expression was related to preferable survival. Moreover, the mutations and DNA methylation of TIPARP might contribute to TIPARP dysregulation in breast cancer. Interactors with TIPARP were significantly enriched in telomere maintenance, telomere organization and mainly participated in pathways in cancer. Finally, several common drugs including metformin were observed to up-regulate the expression of TIPARP.ConclusionTIPARP might act as a preferable prognostic marker of breast cancer through multiple biological processes such as DNA methylation, mutation as well as pathway related to telomere and so on. TIPARP could be considered as a potential therapeutic target for breast cancer. However, large-scale and comprehensive research is needed to clarify our results.

Distinct Nuclear Organization of Telomeresand Centromeres in Monoclonal Gammopathyof Undetermined Significance and Multiple Myeloma.

Both multiple myeloma (MM) and its precursor state of monoclonal gammopathy of undetermined significance (MGUS) are characterized by an infiltration of plasma cells into the bone marrow, but the mechanisms underlying the disease progression remain poorly understood. Previous research has indicated that 3D nuclear telomeric and centromeric organization may represent important structural indicators for numerous malignancies. Here we corroborate with previously noted differences in the 3D telomeric architecture and report that modifications in the nuclear distribution of centromeres may serve as a novel structural marker with potential to distinguish MM from MGUS. Our findings improve the current characterization of the two disease stages, providing two structural indicators that may become altered in the progression of MGUS to MM.

PB1694 KEVETRIN DAMPENS MYC EXPRESSION AND CELLULAR METABOLISM IN ACUTE MYELOID LEUKEMIA

Background:Tumor protein p53 (TP53) is the most frequently mutated gene in cancer and it regulates a number of genes involved in DNA repair, cell cycle, apoptosis and angiogenesis. Kevetrin is a small compound that showed activity against TP53 wt and mutant solid tumors. We explored, for the first time, the consequences of kevetrin exposure in AML cell lines.Aims:In the present study we addressed the response of AML cell lines to kevetrin treatment and investigated gene expression changes in TP53 mutant and wt models, in order to better understand the molecular and biological consequences of kevetrin exposure.Methods:TP53‐wt MOLM‐13 and TP53‐mut KASUMI‐1 were treated with kevetrin [concentration range 15‐60 μg/ml]. After 24 and 48 h MTS and Annexin‐V were performed. Gene expression profiling (GEP) was performed on cells treated for 6 and 48 h with the highest kevetrin dose (60 μg/ml or no drug control) using Human Transcriptome Array 2.0 (Thermo Scientific). Data quality control and normalization were carried out by Expression Console software. Supervised analysis was performed with Transcriptome Analysis Console software. Functional annotation clustering was performed using David Bioinformatics Resources 6.8. Gene set enrichment analysis (GSEA) was performed with GSEA software.Results:The TP53‐wt cell line showed a sensibility only at the highest drug concentration with a decreased cell viability and a significant increase in Annexin V+ cells (54.95 ± 5.63 % vs 12.53 ± 6.15 % of the CTRL). KASUMI‐1 cell line showed a significant dose‐ and time‐dependent cell growth inhibition and apoptosis increase with 79.70 ± 4.57 % of Annexin V+ cells at 60 μg/ml (vs 13.18 ± 0.80 % of the CTRL). Short‐term (6 h) kevetrin treatment induced few alterations of the overall transcriptional program, with a differential expression of FOXK2 and STAT5A, which were 2‐fold downregulated in KASUMI‐1. Moreover, metallothionein 1 and 2 were upregulated by kevetrin exposure in both cell lines. After prolonged kevetrin treatment (48 h) we found 1024 upregulated and 1563 downregulated genes in MOLM‐13 cell line, whereas KASUMI‐1 showed increased expression of 325 and decreased levels of 1535 genes. Kevetrin exposure targeted also a common core transcriptional program in the selected models, including 162 upregulated and 812 downregulated genes. Enrichment analysis of the core transcripts showed that upregulated genes were mainly involved in transcription, nucleosome assembly and telomere organization, apoptosis, autophagy, NF‐kB pathway and MAPK activity. Downregulated genes were mostly involved in cell cycle, DNA repair, biosynthetic processes, bioenergetics, translation, telomere maintenance and splicing. Among the most relevant genes we found critical regulators of myelopoiesis and leukemogenesis, such as MYC, MYB, BCL11A and EZH1, leukemia‐related genes involved in unfolded protein response (JUN), signaling, glycolysis (HK2), DNA methylation (IDH2) and the tumor suppressor gene TP53. GSEA of microarray data showed that kevetrin‐treated cells shared the downregulation of glycolysis, DNA repair, unfolded protein response and MYC target gene sets (Figure).Summary/Conclusion:Our results show kevetrin alters several key genes and cellular metabolism. Along with cellular data, this study could provide a rationale for an experimental trial in AML patients, especially those carrying TP53 mutation who actually have very few therapeutic options.image

Cytotoxicity and Transcriptomic Analyses of Biogenic Palladium Nanoparticles in Human Ovarian Cancer Cells (SKOV3).

Ovarian cancer incidence continues to increase at an alarming rate. Although various therapeutic approaches exist for ovarian cancer, they have limitations, including undesired side effects. Therefore, nanoparticle (NP)-mediated therapy may be a viable, biocompatible, and suitable alternative. To the best of our knowledge, no comprehensive analysis has been undertaken on the cytotoxicity and cellular pathways involved in ovarian cancer cells, particularly SKOV3 cells. Here, we investigated the effect of palladium NPs (PdNPs) and the molecular mechanisms and cellular pathways involved in ovarian cancer. We assayed cell viability, proliferation, cytotoxicity, oxidative stress, DNA damage, and apoptosis and performed an RNA-Seq analysis. The results showed that PdNPs elicited concentration-dependent decreases in cell viability and proliferation and induced increasing cytotoxicity at increasing concentrations, as determined by leakage of lactate dehydrogenase, increased levels of reactive oxygen species and malondialdehyde, and decreased levels of antioxidants like glutathione and superoxide dismutase. Furthermore, our study revealed that PdNPs induce mitochondrial dysfunction by altering mitochondrial membrane potential, reducing adenosine triphosphate levels, inducing DNA damage, and activating caspase 3, all of which significantly induced apoptosis in SKOV3 cells following PdNPs treatment. Gene ontology (GO) term analysis of PdNPs-exposed SKOV3 cells showed various dysregulated pathways, particularly nucleosome assembly, telomere organization, and rDNA chromatin silencing. When genes downregulated by PdNPs were applied to GO term enrichment analysis, nucleosome assembly was the top-ranked biological pathway. We also provide evidence for an association between PdNPs exposure and multiple layers of epigenetic transcriptional control and establish a molecular basis for NP-mediated apoptosis. These findings provide a foundation, potential targets, and novel insights into the mechanism underlying toxicity and pathways in SKOV3 cells, and open new avenues to identify novel targets for ovarian cancer treatment.

3D Telomere Structure Analysis to DetectGenomic Instability and Cytogenetic Evolutionin Myelodysplastic Syndromes.

The disease course of myelodysplastic syndromes (MDS) features chromosome instability and clonal evolution, leading to the sequential acquisition of novel cytogenetic aberrations and the accumulation of these abnormalities in the bone marrow. Although clonal cytogenetic abnormalities can be detected by conventional cytogenetics in 50% of patients with MDS, such distinguishing patterns are lacking in the other 50%. Despite the increase in the prognostic value of some biomarkers, none of them is specific and able to discriminate between stable and unstable patients that subsequently progress to acute myeloid leukemia. This pilot study aimed to investigate the potential use of the 3D telomere profiling to detect genomic instability in MDS patients with or without clonal cytogenetic evolution. The comparison between different time points in patients with cytogenetic changes showed that in the CD34+ MDS cells, there was a significant decrease in the total number of telomeric signals, the average intensity of signals and the total intensity of telomeres. By contrast, the number of aggregates increased during cytogenetic evolution (p < 0.001). This pattern was observed only for MDS patients with cytogenetic evolution but was absent in patients without cytogenetic changes. In conclusion, we demonstrated that the 3D nuclear telomere organization was significantly altered during the MDS disease course, and may have contributed to cytogenetic clonal evolution.

Shelterin Components Modulate the Phase-Separation Propensity of Telomeres

Telomeres are nucleoprotein complexes that provide a mechanism to shelter the ends of chromosomes and are essential to maintain chromosome stability in Eukaryotes. In cells, individual telomeres are bound by the six-protein shelterin complex (TRF2-hRap1-Tin2-TRF1-TPP1-Pot1) and form distinct condensed globules of finite size. Interestingly, incells that maintain their telomeres via an alternative lengthening mechanism, multiple telomeric DNAs and shelterin proteins coalesce in membrane-less promyelocytic leukemia (PML) nuclear bodies. The mechanism controlling this dual nature of telomere organization is currently not understood. Here, we propose that shelterin proteins modulate the phase-separation propensity of telomeres, leading to either the condensation of single DNA chains or the coalescence of multiple ones. In particular, we use a combination of optical and fluorescence microscopy and single-molecule experiments to quantify the contribution of shelterin components to phase-separation and condensation of single DNA chains. Our experiments reveal that TRF2 promotes phase-separation of TRF2-DNA mixtures. Even though the N-terminal basic domain and the dimerization domain of TRF2 are sufficient to promote phase-separation, the C-terminal DNA-binding domain imparts specificity for telomeric DNA repeats. Moreover, addition of shelterin components, such as hRap1, provides a fine-tuning of the properties of the resulting dense and de-mixed phase.

Systematic identification of lincRNA-based prognostic biomarkers by integrating lincRNA expression and copy number variation in lung adenocarcinoma.

Copy number alterations (CNAs) of lincRNAs act as one of important mechanisms in disrupting lincRNA expression which may play critical roles during tumorigenesis in lung adenocarcinoma (LUAD). The copy number alterations of lincRNAs can mark the spectrum of cancer progression and may serve as biomarkers for prognosis in LUAD, however it is rarely studied. We analyzed RNASeq data for 488 LUAD patients from TCGA portal and 58 healthy subjects to identify prognostic lincRNAs predictive of patient survival. Computational analysis entailing integration of expression and copy number alteration data revealed five prognostic lincRNAs: RBPMS-AS1, TDRKH-AS1, LINC00578, RP11-470 M17.2 and LINC00941. The copy number alterations in the LINC00578 and RP11-470 M17.2 genes were positively associated with the longer overall survival of LUAD patients. The CNA in LINC00941 was negatively associated with the longer overall survival. Copy number amplification significantly correlated with increased expression of TDRKH-AS1, which regulates telomere organization and EZH2-mediated epigenetic silencing of CDKN1A, CDKN1B and IL24. Decreased survival of LUAD patients was associated with high LINC00941 expression. The LINC00941 regulates the PI3K-AKT signaling pathway, focal adhesion by influencing potential targets, such as KRAS proto-oncogene GTPase and VEGFC. These lincRNA-based prognostic biomarkers may destroy important cancer-related biological processes contributing to LUAD prognosis. In summary, we demonstrate the prognostic potential of four differentially expressed lincRNAs with copy number alterations (RBPMS-AS1, TDRKH-AS1, LINC00578 and RP11-470 M17.2) that are positively associated with longer overall survival of LUAD patients. One differentially expressed lincRNA LINC00941 with copy number alterations was negatively associated with longer overall survival of LUAD patients.