Telomere Maintenance Pathways Research Articles

Mutations inhibiting KDM4B drive ALT activation in ATRX-mutated glioblastomas

Alternative Lengthening of Telomeres (ALT) is a telomere maintenance pathway utilised in 15% of cancers. ALT cancers are strongly associated with inactivating mutations in ATRX; yet loss of ATRX alone is insufficient to trigger ALT, suggesting that additional cooperating factors are involved. We identify H3.3G34R and IDH1/2 mutations as two such factors in ATRX-mutated glioblastomas. Both mutations are capable of inactivating histone demethylases, and we identify KDM4B as the key demethylase inactivated in ALT. Mouse embryonic stem cells inactivated for ATRX, TP53, TERT and KDM4B (KDM4B knockout or H3.3G34R) show characteristic features of ALT. Conversely, KDM4B over-expression in ALT cancer cells abrogates ALT-associated features. In this work, we demonstrate that inactivation of KDM4B, through H3.3G34R or IDH1/2 mutations, acts in tandem with ATRX mutations to promote ALT in glioblastomas.

Meta-Analysis and Systematic Review of the Genomics of Mucosal Melanoma.

Mucosal melanoma is a rare subtype of melanoma. To date, there has been no comprehensive systematic collation and statistical analysis of the aberrations and aggregated frequency of driver events across multiple studies. Published studies using whole genome, whole exome, targeted gene panel, or individual gene sequencing were identified. Datasets from these studies were collated to summarize mutations, structural variants, and regions of copy-number alteration. Studies using next-generation sequencing were divided into the "main" cohort (n = 173; fresh-frozen samples), "validation" cohort (n = 48; formalin-fixed, paraffin-embedded samples) and a second "validation" cohort comprised 104 tumors sequenced using a targeted panel. Studies assessing mutations in BRAF, KIT, and NRAS were summarized to assess hotspot mutations. Statistical analysis of the main cohort variant data revealed KIT, NF1, BRAF, NRAS, SF3B1, and SPRED1 as significantly mutated genes. ATRX and SF3B1 mutations occurred more commonly in lower anatomy melanomas and CTNNB1 in the upper anatomy. NF1, PTEN, CDKN2A, SPRED1, ATM, CHEK2, and ARID1B were commonly affected by chromosomal copy loss, while TERT, KIT, BRAF, YAP1, CDK4, CCND1, GAB2, MDM2, SKP2, and MITF were commonly amplified. Further notable genomic alterations occurring at lower frequencies indicated commonality of signaling networks in tumorigenesis, including MAPK, PI3K, Notch, Wnt/β-catenin, cell cycle, DNA repair, and telomere maintenance pathways. This analysis identified genomic aberrations that provide some insight to the way in which specific pathways may be disrupted. IMPLICATIONS: Our analysis has shown that mucosal melanomas have a diverse range of genomic alterations in several biological pathways. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/6/991/F1.large.jpg.

Implication of therapeutic outcomes associated with molecular characterization of paediatric aplastic anaemia

ObjectivesSevere aplastic anemia is characterized by a hypocellular bone marrow and peripheral cytopenia. Mesenchymal stem cells (MSCs) play a crucial role in haematopoietic stem cells (HSCs) development and the development of microenvironment suitable for hematopoiesis. Molecular characterization of telomere maintenance pathway and gene expression profiling of MSCs can be important for the therapeutic interventions among paediatric aplastic anaemia patients. MethodsThe study involved paediatric aplastic anaemia patients (n = 10) and age matched paediatric healthy donors (n = 8). Peripheral blood samples were collected from the individuals. Average leucocyte telomere length and gene expression of the telomere maintenance genes were determined by quantitative real time PCR. Microarray based gene expression profiles (GSE33812) of MSCs for five paediatric aplastic anaemia patients were analyzed compared to five healthy controls and the data was downloaded from the GEO database. ResultsThe telomere length was significantly shorter among paediatric AA patients compared to age matched healthy donors. Interestingly, one subgroup (n = 2) of paediatric AA patients has moderate telomere length comparable to age matched healthy donors. Based on the gene expression analysis of telomere maintenance pathway, TERF2 was significantly downregulated among paediatric patients with shorter telomere length but not among paediatric patients with moderate telomere length. Gene expression profiling of MSCs revealed three differentially expressed genes (GAS2L3, MK167 and TMSB15A) among the patients and was associated with therapeutic outcome. ConclusionTelomere length estimation and gene expression patterns of the MSCs and telomere length maintenance pathway may serve as a potential biomarker and could be associated with therapeutic choice of paediatric aplastic anaemia patients.

Inhibition of the alternative lengthening of telomeres pathway by subtelomeric sequences in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, telomerase is constitutively active and is essential for chromosome end protection and illimited proliferation of cell populations. However, upon inactivation of telomerase, alternative mechanims of telomere maintenance allow proliferation of only extremely rare survivors. S. cerevisiae type I and type II survivors differ by the nature of the donor sequences used for repair by homologous recombination of the uncapped terminal TG1-3 telomeric sequences. Type I amplifies the subtelomeric Y' sequences and is more efficient than type II, which amplifies the terminal TG1-3 repeats. However, type II survivors grow faster than type I survivors and can easily outgrow them in liquid cultures. The mechanistic interest of studying S. cerevisiae telomeric recombination is reinforced by the fact that type II recombination is the equivalent of the alternative lengthening of telomeres (ALT) pathway that is used by 5–15 % of cancer types as an alternative to telomerase reactivation. In budding yeast, only around half of the 32 telomeres harbor Y' subtelomeric elements. We report here that in strains harboring Y' elements on all telomeres, type II survivors are not observed, most likely due to an increase in the efficiency of type I recombination. However, in a temperature-sensitive cdc13-1 mutant grown at semi-permissive temperature, the increased amount of telomeric TG1-3 repeats could overcome type II inhibition by the subtelomeric Y’ sequences. Strikingly, in the 100 % Y’ strain the replicative senescence crisis normally provoked by inactivation of telomerase completely disappeared and the severity of the crisis was proportional to the percentage of chromosome-ends lacking Y’ subtelomeric sequences. The present study highlights the fact that the nature of subtelomeric elements can influence the selection of the pathway of telomere maintenance by recombination, as well as the response of the cell to telomeric damage caused by telomerase inactivation.

Genomic Analysis of Localized High-Risk Prostate Cancer Circulating Tumor Cells at the Single-Cell Level.

Accurate risk classification of men with localized high-risk prostate cancer directly affects treatment management decisions and patient outcomes. A wide range of risk assessments and classifications are available. However, each one has significant limitations to distinguish between indolent and aggressive prostate cancers. Circulating tumor cells (CTCs) may provide an alternate additional source, beyond tissue biopsies, to enable individual patient-specific clinical assessment, simply because CTCs can reveal both tumor-derived and germline-specific genetic information more precisely than that gained from a single diagnostic biopsy. In this study, we combined a filtration-based CTC isolation technology with prostate cancer CTC immunophenotyping to identify prostate cancer CTCs. Next, we performed 3-D telomere profiling prior to laser microdissection and single-cell whole-exome sequencing (WES) of 21 CTCs and 4 lymphocytes derived from 10 localized high-risk prostate cancer patient samples. Localized high-risk prostate cancer patient CTCs present a high number of telomere signals with lower signal intensities (short telomeres). To capture the genetic diversity/heterogeneity of high-risk prostate cancer CTCs, we carried out whole-exome sequencing. We identified 202,241 single nucleotide variants (SNVs) and 137,407 insertion-deletions (indels), where less than 10% of these genetic variations were within coding regions. The genetic variation (SNVs + indels) and copy number alteration (CNAs) profiles were highly heterogeneous and intra-patient CTC variation was observed. The pathway enrichment analysis showed the presence of genetic variation in nine telomere maintenance pathways (patients 3, 5, 6, and 7), including an important gene for telomere maintenance called telomeric repeat-binding factor 2 (TRF2). Using the PharmGKB database, we identified nine genetic variations associated with response to docetaxel. A total of 48 SNVs can affect drug response for 24 known cancer drugs. Gene Set Enrichment Analysis (GSEA) (patients 1, 3, 6, and 8) identified the presence of CNAs in 11 different pathways, including the DNA damage repair (DDR) pathway. In conclusion, single-cell approaches (WES and 3-D telomere profiling) showed to be useful in unmasking CTC heterogeneity. DDR pathway mutations have been well-established as a target pathway for cancer therapy. However, the frequent CNA amplifications found in localized high-risk patients may play critical roles in the therapeutic resistance in prostate cancer.

Telomere Length Measurement by Molecular Combing.

Telomeres are repetitive regions of DNA bound by specialized proteins at the termini of linear chromosomes that prevent the natural chromosome ends from being recognized as DNA double strand breaks. Telomeric DNA is gradually eroded with each round of cell division, resulting in the accumulation of critically short or dysfunctional telomeres that eventually trigger cellular senescence. Consequently, telomere length is indicative of the proliferative capacity of a cell. Multiple methods exist to measure telomere length and telomere content, but a simple and reliable technique to accurately measure individual telomere lengths is currently lacking. We have developed the Telomere length Combing Assay (TCA) to measure telomere length on stretched DNA fibers. We used TCA to measure telomere erosion in primary human fibroblasts, and to detect telomere lengthening in response to activation of telomere maintenance pathways. TCA was also used to accurately measure telomere length in healthy individuals, and to identify critically short telomeres in patients with telomere biology disorders. TCA is performed on isolated DNA, negating the need for cycling cells. TCA is amenable to semi-automated image analysis, and can be fully automated using the Genomic Vision molecular combing platform. This not only precludes sampling bias, but also provides the potential for high-throughput applications and clinical development. TCA is a simple and versatile technique to measure the distribution of individual telomere lengths in a cell population, offering improved accuracy, and more detailed biological insight for telomere length measurement applications.

ORCA/LRWD1 Regulates Homologous Recombination at ALT-Telomeres by Modulating Heterochromatin Organization.

SummaryTelomeres are maintained by telomerase or in a subset of cancer cells by a homologous recombination (HR)-based mechanism, Alternative Lengthening of Telomeres (ALT). The mechanisms regulating telomere-homeostasis in ALT cells remain unclear. We report that a replication initiator protein, Origin Recognition Complex-Associated (ORCA/LRWD1), by localizing at the ALT-telomeres, modulates HR activity. ORCA's localization to the ALT-telomeres is facilitated by its interaction to SUMOylated shelterin components. The loss of ORCA in ALT-positive cells elevates the levels of two mediators of HR, RPA and RAD51, and consistent with this, we observe increased ALT-associated promyelocytic leukemia body formation and telomere sister chromatid exchange. ORCA binds to RPA and modulates the association of RPA to telomeres. Finally, the loss of ORCA causes global chromatin decondensation, including at the telomeres. Our results demonstrate that ORCA acts as an inhibitor of HR by modulating RPA binding to ssDNA and inducing chromatin compaction.

Telomere Maintenance Genes are associated with Type 2 Diabetes Susceptibility in Northwest Indian Population Group

Telomere length attrition has been implicated in various complex disorders including Type 2 Diabetes (T2D). However, very few candidate gene association studies have been carried out worldwide targeting telomere maintenance genes. In the present study, variants in various critical telomere maintenance pathway genes for T2D susceptibility in Northwest Indian population were explored. With case-control candidate gene association study design, twelve variants from seven telomere maintenance genes were evaluated. Amongst these five variants, rs9419958 (OBFC1), rs4783704 (TERF2), rs16847897 (TERC/LRRC31), rs10936599 (TERC/MYNN), and rs74019828 (CSNK2A2) showed significant association with T2D (at p-value ≤ 0.003, threshold set after Bonferroni correction) in the studied population. In silico analyses of these variants indicated interesting functional roles that warrant experimental validations. Findings showed that variants in telomere maintenance genes are associated with pathogenesis of T2D in Northwest Indian population. We anticipate further, such candidate gene association studies in other Indian populations and worldwide would contribute in understanding the missing heritability of T2D.

Structural basis for stabilization of human telomeric G-quadruplex [d-(TTAGGGT)]4 by anticancer drug adriamycin

Besides inhibiting DNA duplication, DNA dependent RNA synthesis and topoisomerase-II enzyme action, anticancer drug adriamycin is found to cause telomere dysfunction and shows multiple strategies of action on gene functioning. We present evidence of binding of adriamycin to parallel stranded intermolecular [d-(TTAGGGT)]4 G-quadruplex DNA comprising human telomeric DNA by proton and phosphorus-31 nuclear magnetic resonance spectroscopy. Diffusion ordered spectroscopy shows formation of complex between the two molecules. Changes in chemical shift and line broadening of DNA and adriamycin protons suggest participation of specific chemical groups/moieties in interaction. Presence of sequential nuclear Overhauser enhancements at all base quartet steps and absence of large downfield shifts in 31P resonances give clear proof of absence of intercalation of adriamycin chromophore between base quartets. Restrained molecular dynamics simulations using observed 15 short intermolecular inter proton distance contacts depict stacking of ring D of adriamycin with terminal G6 quartet by displacing T7 base and external groove binding close to T1–T2–A3 bases. The disappearance of imino protons monitored as a function of temperature and differential scanning calorimetry experiments yield thermal stabilization of 24 °C, which is likely to come in the way of telomerase association with telomeres. The findings pave the way for design of alternate anthracycline based drugs with specific modifications at ring D to enhance induced thermal stabilization and use alternate mechanism of binding to G-quadruplex DNA for interference in functional pathway of telomere maintenance by telomerase enzyme besides their well known action on duplex DNA. Communicated by Ramaswamy H. Sarma

Binding of anticancer drug adriamycin to parallel G-quadruplex DNA [d-(TTAGGGT)]4 comprising human telomeric DNA leads to thermal stabilization: A multiple spectroscopy study.

Adriamycin is known to exert its anti cancer action by inhibiting DNA duplication, RNA transcription and topoisomerase-II enzyme action. Recent findings of its binding to G-quadruplex DNA resulting in telomere dysfunction indicated multiple strategies of its action. The interaction of anticancer drug adriamycin with parallel stranded inter molecular G-quadruplex DNA [d-(TTAGGGT)]4 comprising human telomeric DNA sequence TTAGGG was investigated by absorption, fluorescence, circular dichroism and nuclear magnetic resonance spectroscopy to understand mode of their interaction. The adriamycin binds as monomer to G-quadruplex DNA with affinity (Kb1 = 9.8x105 M-1 and Kb2 = 6.7x105 M-1 ) higher than that reported for daunomycin, at two independent sites, mainly in terminal stacking and groove binding modes. The bound complex formed as a result of specific interactions induces thermal stabilization of DNA by 12.5-28.1°C, which is likely to hinder telomere association with telomerase enzyme and contribute significantly to adriamycin-induced apoptosis in cancer cell lines. The findings have therapeutic potential towards drug designing by way of altering substituent groups on anthracyclines to enhance efficacy using additional mechanism of targeting pathway of telomere maintenance by disrupting telomerase association with telomeres.

The Landscape of Genome Wide Somatic Alterations Identifies a Good-Risk Group in Newly Diagnosed Multiple Myeloma

Multiple Myeloma (MM) is a plasma cells malignancy with number of recent therapeutic options that has improved outcomes with median survival now stretching beyond 8 years. There has been an intense search to identify genomic and laboratory correlates of outcome for high risk patients. However, a subgroup of patients have a long survival but genomic segmentation of this important group which can be potentially cured has not been identified. We here described first such attempt at identifying a subset of patients with long survival. We have analyzed data from 205 newly-diagnosed uniformly-treated MM patients using both deep whole genome (WGS) (80x) and whole transcriptome sequencing (RNAseq). Median number of SNVs, small insertion and deletion per megabase were 2.18 [0.49 - 14.52], 0.077 [0.024 - 0.17] and 0.12 [0.03 - 0.26] respectively. MM subgroups defined by FISH or copy number alterations (CNAs) had significantly different mutational load (Kruskal-Wallis p-value = 0.003). In general, Hyperdiploid MM (HMM) patients had lower SNV load compared to other subgroups and t(14;16) had the highest mutational load per megabase (Dunn test FDR < 0.25). These subgroups also showed significant differences for mutational process utilization. Mutational processes associated spontaneous deamination of 5-methylcytosine to thymine was significantly high in HMM, APOBEC activity was very strong in t(14;16) MM and DNA repair was significantly different in t(6;14), del17p and t(11;14). Importantly, the mutations associated with clonal cell population and thus in early phases of MM cell progression were driven by spontaneous or enzymatic deamination (Signature 1), somatic hypermutation in lymphoid cells (Signature 9) and Signature 17 (ANOVA p value < 1e-16) and surprisingly APOBEC activity was constant among the clones (ANOVA p value > 0.05). However, mutations associated with homologous recombination (HR) and nucleotide excision repair (NER) activity were significantly enriched in the subclonal mutations (ANOVA p value < 1e-16) suggesting their role in later stages of MM progression. Based on this information, we next calculated the homologous recombination repair defects using copy number alterations from WGS data. We identified that HR score generated using WGS copy number information predicted outcome in newly diagnosed myeloma. Further analysis of this data identified that newly diagnosed MM patients with hyperdiploid MM (HMM) and no HR- repair deficiency have superior outcome with 6 year overall survival (OS) probability for this of 100% in IFM/DFCI 2009 cohort (Logrank test p value = 0.0012). We next validated this finding using MMRF CoMMpass dataset and we confirmed 6-yr OS probability of 95% (Logrank test p value = 0.0016) in this independent dataset (Figure 1). We further investigated RNAseq data between these genomically defined groups and identified that chromosome and telomere maintenance pathways (FDR < 0.01) and low bone disease associated genes described by Zhan et al. (FDR = 0.004) were up regulated in low-risk group. Finally, patients who were able to maintain HR, had lower mutation rate for TP53 (0% vs 9%, FDR < 0.05), CSMD1 (0% vs 6%, FDR < 0.05), FAT3/FAT4 mutations (1.7% vs 15%, FDR < 0.05), however surprisingly higher mutation rate for NRAS (42% vs 14%, p value < 0.001). In addition, DNA damage associated processes predicted by SNVs trinucleotides were significantly lower in the low-risk group (12% vs. 20%, p value < 1e-05) providing further support to this data. In conclusion, we report a detailed genomic profile using deep DNA and RNA sequencing and identify a genomically-defined sub group that is predicted to have a long survival. This study also identifies the role of HR in myeloma with potential for its translational application in both prognosis as well as therapy. Figure 1 Disclosures Richardson: Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. Moreau:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Thakurta:Celgene: Employment, Equity Ownership. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Avet-Loiseau:celgene: Consultancy, Other: travel fees, lecture fees, Research Funding; takeda: Consultancy, Other: travel fees, lecture fees, Research Funding. Munshi:Takeda: Consultancy; Oncopep: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Janssen: Consultancy.

Deleterious Germline Variants, Especially in the DNA Repair Pathway, Are Common in Patients with Non-Related Multiple Cancers, One of Them Being Hematological Malignancy

Approximately 8% of un-selected pediatric (Zhang et al, NEJM 2015) and adult (Huang et al, Cell 2018) cancer patients have a deleterious germline (GL) variant. However, the frequency in patients with >2 non-related cancers, a combination of different hematological malignancies (HM) or a HM and a non-hematological malignancy, is not known. We hypothesize that genetic predisposition is higher in patients with multiple cancers, compared to historical literature for patients with a single cancer. Method: Clinical and laboratory information on 213 cancer patients, enrolled in the South Australian MDS (SA-MDS) registry (n=90) and University of Chicago clinic (n=123) were analyzed. Germline variants were identified by sequencing paired bone marrow and germline samples for 217-300 cancer related genes and annotated using American College of Medical Genetics (ACMG) 2015 guidelines as pathogenic or likely pathogenic (i.e. deleterious). Additionally, somatic mutations in 240 genes were identified in the SA-MDS cohort (n=90). Results: The median age at diagnosis was 64 years (interquartile range, 61-76 years). One-hundred and five patients had therapy-related myeloid neoplasm (T-MN), following treatment with chemo- and/or radiotherapy for an unrelated prior malignancy. While 100 patients had non-related multiple cancers (MC) one of them being HM without prior exposure to chemotherapy or radiotherapy, and, 8 patients had only myelodysplastic syndrome or acute myeloid leukemia. Overall, 20.5% (42/205) patients harbored 46 deleterious GL variants, annotated according to ACMG guidelines (Fig 1A). Most GL variants were in DNA repair pathway (26/46, 57%) followed by TP53, telomere maintenance and drug transport pathways (4/46, 9% each; Fig 1B). Three DDX41 variants were also seen. The frequency of patients with deleterious GL variants was similar in T-MN (21/105; 20%) and MC (21/100, 21%; Fig 1C). Similarly, the frequency of patients with deleterious GL variants in DNA repair pathways was also similar in T-MN (13/105, 12%) and MC (11/100, 11%). Of the patients with available cytogenetic data, complex cytogenetics were more frequent in patients with a deleterious GL variant compared to those without (11/28, 39% vs 28/125, 22%; p=0.06). At the time of analysis, somatic and germline mutation data on 240 genes was available in the SA-MDS cohort only (n=90). Somatic TP53 mutations were frequent in patients with deleterious GL variant (8/15, 53%) compared to patients without deleterious GL variant (11/75, 15%; p=0.003) (Fig 1D). Interestingly, 80% (4/5) patients with deleterious GL variant in a DNA repair pathway had a somatic TP53 mutation compared to 40% (4/10) with deleterious GL variant in non-DNA repair pathways. Conclusions: Our results from a large cohort show that patients with >2 non-related cancers have a higher frequency of deleterious germline variants compared to previous reports from patients with single cancer (20.5% vs 8%). Variants in the DNA repair pathway are the most common. The presence of deleterious germline variants is associated with genetic instability as evidenced by high frequency of complex karyotypes and somatic TP53 mutations. Disclosures Scott: Celgene: Honoraria. Godley:UpToDate, Inc.: Patents & Royalties: receives royalties from a coauthored article on inherited hematopoietic malignancies ; Invitae, Inc.: Membership on an entity's Board of Directors or advisory committees.

The level of activity of the alternative lengthening of telomeres correlates with patient age in IDH-mutant ATRX-loss-of-expression anaplastic astrocytomas

All cancer cells need to maintain functional telomeres to sustain continuous cell division and proliferation. In human diffuse gliomas, functional telomeres are maintained due either to reactivation of telomerase expression, the main pathway in most cancer types, or to activation of a mechanism called the alternative lengthening of telomeres (ALT). The presence of IDH1/2 mutations (IDH-mutant) together with loss of ATRX expression (ATRX-lost) are frequently associated with ALT in diffuse gliomas. However, detection of ALT, and a fortiori its quantification, are rarely, if ever, measured in neuropathology laboratories. We measured the level of ALT activity using the previously described quantitative “C-circle” assay and analyzed it in a well characterized cohort of 104 IDH-mutant and ATRX-lost adult diffuse gliomas. We report that in IDH-mutant ATRX-lost anaplastic astrocytomas, the intensity of ALT was inversely correlated with age (p < 0.001), the younger the patient, the higher the intensity of ALT. Strikingly, glioblastomas having progressed from anaplastic astrocytomas did not exhibit this correlation. ALT activity level in the tumor did not depend on telomere length in healthy tissue cells from the same patient. In summary, we have uncovered the existence, in anaplastic astrocytomas but not in glioblastomas with the same IDH and ATRX mutations, of a correlation between patient age and the level of activity of ALT, a telomerase-independent pathway of telomere maintenance.

Dual mode of binding of anti cancer drug epirubicin to G-quadruplex [d-(TTAGGGT)]4 containing human telomeric DNA sequence induces thermal stabilization

Epirubicin exerts its anti cancer action by blocking DNA/RNA synthesis and inhibition of topoisomerase-II enzyme. Recent reports on its influence on telomere maintenance, suggest interaction with G-quadruplex DNA leading to multiple strategies of action. The binding of epirubicin with parallel stranded inter molecular G-quadruplex DNA [d-(TTAGGGT)]4 comprising human telomeric DNA sequence TTAGGG was investigated by absorption, fluorescence, circular dichroism and nuclear magnetic resonance spectroscopy. The epirubicin binds as monomer to G-quadruplex DNA with affinity, Kb1 = 3.8 × 106 M−1 and Kb2 = 2.7 × 106 M−1, at two independent sites externally. The specific interactions induce thermal stabilization of DNA by 13.2–26.3 °C, which is likely to come in the way of telomere association with telomerase enzyme and contribute to epirubicin-induced apoptosis in cancer cell lines. The findings pave the way for drug designing in view of the possibility of altering substituent groups on anthracyclines to enhance efficacy using alternate mechanism of its interaction with G4 DNA, causing interference in telomere maintenance pathway by inducing telomere dysfunction.

Cancers after Chornobyl: From Epidemiology to Molecular Quantification.

An overview and new data are presented from cancer studies of the most exposed groups of the population after the Chornobyl accident, performed at the National Research Center for Radiation Medicine (NRCRM). Incidence rates of solid cancers were analyzed for the 1990–2016 period in cleanup workers, evacuees, and the general population from the contaminated areas. In male cleanup workers, the significant increase in rates was demonstrated for cancers in total, leukemia, lymphoma, and thyroid cancer, as well as breast cancer rates were increased in females. Significantly elevated thyroid cancer incidence was identified in the male cleanup workers cohort (150,813) in 1986–2012 with an overall standardized incidence ratio (SIR) of 3.35 (95% CI: 2.91–3.80). A slight decrease in incidence rates was registered starting at 25 years after exposure. In total, 32 of 57 deaths in a group of cleanup workers with confirmed acute radiation syndrome (ARS) or not confirmed ARS (ARS NC) were due to blood malignancies or cancer. Molecular studies in cohort members included gene expression and polymorphism, FISH, relative telomere length, immunophenotype, micronuclei test, histone H2AX, and TORCH infections. Analysis of chronic lymphocytic leukemia (CLL) cases from the cohort showed more frequent mutations in telomere maintenance pathway genes as compared with unexposed CLL patients.

Abstract 1571: Telomere maintenance variants and survival after colorectal cancer: Smoking- and sex-specific associations

Abstract Background: Telomeres play an important role in determining colorectal cancer (CRC) prognosis; telomere length varies by smoking status and sex. The association between variation in telomere maintenance genes and survival after CRC remains uncertain; evidence on the interaction between these genes and prognostic factors, such as smoking and sex, is also lacking. Methods: We selected candidates from a subset of the large genome-wide International Survival Analysis in Colorectal Cancer Consortium (ISACC). Common variants (MAF &amp;gt;5%) in 13 telomere maintenance genes (TERT, TERC, TERF1, TERF2, TINF2, TERF2IP, ACD, POT1, TNKS, TNKS2, TNKS1BP1, TEP1, PINX1) were examined. Cox proportional hazard models adjusted for age, sex, study, and genetic ancestry were used to analyze associations of these variants with overall and CRC-specific survival, with additional stratification analyses by prognostic factors, including smoking status and sex. Likelihood ratio tests evaluated the significance of interaction terms. P-values were adjusted for multiple comparisons by the Bonferroni method within each gene. Results: Eight SNPs within TERT, TERF1, TNKS, TNKS1BP1, TEP1 and TERF2 were associated with overall and CRC-specific survival at the P-value threshold of 0.05. The association between rs73202875 (TNKS) and CRC-specific survival varied significantly by smoking pack-years (adjusted Pinteraction =0.024, never smokers vs. ≥45 smoking pack-years). Significant interactions by sex were observed such that the minor allele at rs2975843 (TERF1) was associated with poorer overall and CRC-specific survival in women, but not in men (adjusted Pinteraction =0.002 and 0.019, respectively). The minor allele at rs74429678 (POT1) was associated with increased CRC-specific mortality in women but not in men [HR (95% CI) =1.33(1.07-1.65) and 0.75(0.52-0.97), respectively; adjusted Pinteraction =0.002]. A significant interaction was also observed for overall survival between sex and rs75676021 (POT1, adjusted Pinteraction =0.023). Conclusion: This study showed statistically significant associations between candidate genes in the telomere maintenance pathway with overall and CRC-specific survival. Additionally, we observed a statistically significant interaction for telomere maintenance genes with smoking pack-years (TNKS) and sex (POT1, TERF1), respectively. Validation of these findings in other large studies is warranted; further functional annotation of these SNPs should be conducted. Citation Format: Sheetal Hardikar, Hang Yin, Sara Lindstroem, Li Hsu, Kristin Anderson, Barbara Banbury, Sonja I. Berndt, Andrew T. Chan, Edward L. Giovanucci, Tabitha A. Harrison, Amit D. Joshi, Nan Hongmei, John D. Potter, Lori C. Sakoda, Martha L. Slattery, Robert E. Schoen, Emily White, Ulrike Peters, Polly A. Newcomb. Telomere maintenance variants and survival after colorectal cancer: Smoking- and sex-specific associations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1571.

Exploring the cross-phenotype network region of disease modules reveals concordant and discordant pathways between chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.

Chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are two pathologically distinct chronic lung diseases that are associated with cigarette smoking. Genetic studies have identified shared loci for COPD and IPF, including several loci with opposite directions of effect. The existence of additional shared genetic loci, as well as potential shared pathobiological mechanisms between the two diseases at the molecular level, remains to be explored. Taking a network-based approach, we built disease modules for COPD and IPF using genome-wide association studies-implicated genes. The two disease modules displayed strong disease signals in an independent gene expression data set of COPD and IPF lung tissue and showed statistically significant overlap and network proximity, sharing 19 genes, including ARHGAP12 and BCHE. To uncover pathways at the intersection of COPD and IPF, we developed a metric, NetPathScore, which prioritizes the pathways of a disease by their network overlap with another disease. Applying NetPathScore to the COPD and IPF disease modules enabled the determination of concordant and discordant pathways between these diseases. Concordant pathways between COPD and IPF included extracellular matrix remodeling, Mitogen-activated protein kinase (MAPK) signaling and ALK pathways, whereas discordant pathways included advanced glycosylation end product receptor signaling and telomere maintenance and extension pathways. Overall, our findings reveal shared molecular interaction regions between COPD and IPF and shed light on the congruent and incongruent biological processes lying at the intersection of these two complex diseases.

Histone Modifications and the Maintenance of Telomere Integrity.

Telomeres, the nucleoprotein structures at the ends of eukaryotic chromosomes, play an integral role in protecting linear DNA from degradation. Dysregulation of telomeres can result in genomic instability and has been implicated in increased rates of cellular senescence and many diseases, including cancer. The integrity of telomeres is maintained by a coordinated network of proteins and RNAs, such as the telomerase holoenzyme and protective proteins that prevent the recognition of the telomere ends as a DNA double-strand breaks. The structure of chromatin at telomeres and within adjacent subtelomeres has been implicated in telomere maintenance pathways in model systems and humans. Specific post-translational modifications of histones, including methylation, acetylation, and ubiquitination, have been shown to be necessary for maintaining a chromatin environment that promotes telomere integrity. Here we review the current knowledge regarding the role of histone modifications in maintaining telomeric and subtelomeric chromatin, discuss the implications of histone modification marks as they relate to human disease, and highlight key areas for future research.

Molecular Recognition of Parallel G-quadruplex [d-(TTGGGGT)]₄ Containing Tetrahymena Telomeric DNA Sequence by Anticancer Drug Daunomycin: NMR-Based Structure and Thermal Stability.

The anticancer drug daunomycin exerts its influence by multiple strategies of action to interfere with gene functioning. Besides inhibiting DNA/RNA synthesis and topoisomerase-II, it affects the functional pathway of telomere maintenance by the telomerase enzyme. We present evidence of the binding of daunomycin to parallel-stranded tetramolecular [d-(TTGGGGT)]4 guanine (G)-quadruplex DNA comprising telomeric DNA from Tetrahymena thermophilia by surface plasmon resonance and Diffusion Ordered SpectroscopY (DOSY). Circular Dichroism (CD) spectra show the disruption of daunomycin dimers, suggesting the end-stacking and groove-binding of the daunomycin monomer. Proton and phosphorus-31 Nuclear Magnetic Resonance (NMR) spectroscopy show a sequence-specific interaction and a clear proof of absence of intercalation of the daunomycin chromophore between base quartets or stacking between G-quadruplexes. Restrained molecular dynamics simulations using observed short interproton distance contacts depict interaction at the molecular level. The interactions involving ring A and daunosamine protons, the stacking of an aromatic ring of daunomycin with a terminal G6 quartet by displacing the T7 base, and external groove-binding close to the T1–T2 bases lead to the thermal stabilization of 15 °C, which is likely to inhibit the association of telomerase with telomeres. The findings have implications in the structure-based designing of anthracycline drugs as potent telomerase inhibitors.

Structural and functional characterization of the RBBP4-ZNF827 interaction and its role in NuRD recruitment to telomeres.

The nucleosome remodeling and histone deacetylase (NuRD) complex is an essential multi-subunit protein complex that regulates higher-order chromatin structure. Cancers that use the alternative lengthening of telomere (ALT) pathway of telomere maintenance recruit NuRD to their telomeres. This interaction is mediated by the N-terminal domain of the zinc-finger protein ZNF827. NuRD-ZNF827 plays a vital role in the ALT pathway by creating a molecular platform for recombination-mediated repair. Disruption of NuRD binding results in loss of ALT cell viability. Here, we present the crystal structure of the NuRD subunit RBBP4 bound to the N-terminal 14 amino acids of ZNF827. RBBP4 forms a negatively charged channel that binds to ZNF827 through a network of electrostatic interactions. We identify the precise amino acids in RBBP4 required for this interaction and demonstrate that disruption of these residues prevents RBBP4 binding to both ZNF827 and telomeres, but is insufficient to decrease ALT activity. These data provide insights into the structural and functional determinants of NuRD activity at ALT telomeres.