Prognostic and therapeutic relevance of c-FLIP in acute myeloid leukaemia

Chemoresistance is a major contributor to the aggressiveness of AML and is often due to insufficient apoptosis. The CFLAR gene is expressed as long and short splice forms encoding the anti-apoptotic proteins c-FLIP(L) and c-FLIP(S) (CFLAR(L) and CFLAR(S) , respectively) that play important roles in drug resistance. In univariate analyses of CFLAR mRNA expression in adult AML patients, those individuals with higher than median mRNA expression of the long splice form CFLAR(L) (but not the short splice form) had significantly lower 3 year overall survival (P = 0·04) compared to those with low expression. In cell line studies, simultaneous down-regulation of c-FLIP(L) and c-FLIP(S) proteins using siRNA induced apoptosis in U937 and NB-4 AML cells, but not K562 or OCI-AML3 cells. However, dual c-FLIP(L/S) downregulation sensitized all four cell lines to apoptosis induced by recombinant tumour necrosis factor-related apoptosis-inducing ligand (rTRAIL). Moreover, specific downregulation of c-FLIP(L) was found to recapitulate the phenotypic effects of dual c-FLIP(L/S) downregulation. The histone deacetylase (HDAC)1/2/3/6 inhibitor Vorinostat was found to potently down-regulate c-FLIP(L) expression by transcriptional and post-transcriptional mechanisms and to sensitize AML cells to rTRAIL. Further analyses using more selective HDAC inhibitors revealed that HDAC6 inhibition was not required for c-FLIP(L) down-regulation. These results suggest that c-FLIP(L) may have clinical relevance both as a prognostic biomarker and potential therapeutic target for HDAC inhibitors in AML although this requires further study.

RUNX3 downregulation in human lung adenocarcinoma is independent of p53, EGFR or KRAS status

RUNX3 aberrations play a pivotal role in the oncogenesis of breast, gastric, colon, skin and lung tissues. The aim of this study was to characterize further the expression of RUNX3 in lung cancers. To achieve this, a lung cancer tissue microarray (TMA), frozen lung cancer tissues and lung cell lines were examined for RUNX3 expression by immunohistochemistry, while the TMA was also examined for EGFR and p53 expression. RUNX3 promoter methylation status, and EGFR and KRAS mutation status were also investigated. Inactivation of RUNX3 was observed in 70% of the adenocarcinoma samples, and this was associated with promoter hypermethylation but not biased to EGFR/KRAS mutations. Our results suggest a central role of RUNX3 downregulation in pulmonary adenocarcinoma, which may not be dependent of other established cancer-causing pathways and may have important diagnostic and screening implications.

JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

We developed an analytic strategy that correlates gene expression and clinical outcomes as a means to identify novel candidate oncogenes operative in breast cancer. This analysis, followed by functional characterization, resulted in the identification of Jumonji Domain Containing 6 (JMJD6) protein as a novel driver of oncogenic properties in breast cancer.

Small RNA-mediated chromatin silencing directed to the 3' region of the Arabidopsis gene encoding the developmental regulator, FLC.

Small RNA-mediated chromatin silencing is well characterized for repeated sequences and transposons, but its role in regulating single-copy endogenous genes is unclear. We have identified two small RNAs (30 and 24 nucleotides) corresponding to the reverse strand 3' to the canonical poly(A) site of FLOWERING LOCUS C (FLC), an Arabidopsis gene encoding a repressor of flowering. Genome searches suggest that these RNAs originate from the FLC locus in a genomic region lacking repeats. The 24-nt small RNA, which is most abundant in developing fruits, is absent in mutants defective in RNA polymerase IVa, RNA-DEPENDENT RNA POLYMERASE 2, and DICER-LIKE 3, components required for RNAi-mediated chromatin silencing. The corresponding genomic region shows histone 3 lysine 9 dimethylation, which was reduced in a dcl2,3,4 triple mutant. Investigations into the origins of the small RNAs revealed a polymerase IVa-dependent spliced, antisense transcript covering the 3' FLC region. Mutation of this genomic region by T-DNA insertion led to FLC misexpression and delayed flowering, suggesting that RNAi-mediated chromatin modification is an important component of endogenous pathways that function to suppress FLC expression.

SAHA overcomes FLIP-mediated inhibition of SMAC mimetic-induced apoptosis in mesothelioma

Malignant pleural mesothelioma (MPM) is a highly pro-inflammatory malignancy that is rapidly fatal and increasing in incidence. Cytokine signaling within the pro-inflammatory tumor microenvironment makes a critical contribution to the development of MPM and its resistance to conventional chemotherapy approaches. SMAC mimetic compounds (SMCs) are a promising class of anticancer drug that are dependent on tumor necrosis factor alpha (TNFa) signaling for their activity. As circulating TNFa expression is significantly elevated in MPM patients, we examined the sensitivity of MPM cell line models to SMCs. Surprisingly, all MPM cell lines assessed were highly resistant to SMCs either alone or when incubated in the presence of clinically relevant levels of TNFa. Further analyses revealed that MPM cells were sensitized to SMC-induced apoptosis by siRNA-mediated downregulation of the caspase 8 inhibitor FLIP, an antiapoptotic protein overexpressed in several cancer types including MPM. We have previously reported that FLIP expression is potently downregulated in MPM cells in response to the histone deacetylase inhibitor (HDACi) Vorinostat (SAHA). In this study, we demonstrate that SAHA sensitizes MPM cells to SMCs in a manner dependent on its ability to downregulate FLIP. Although treatment with SMC in the presence of TNFa promoted interaction between caspase 8 and the necrosis-promoting RIPK1, the cell death induced by combined treatment with SAHA and SMC was apoptotic and mediated by caspase 8. These results indicate that FLIP is a major inhibitor of SMC-mediated apoptosis in MPM, but that this inhibition can be overcome by the HDACi SAHA. © 2013 Macmillan Publishers Limited All rights reserved.

Variants in the catechol-o-methyltransferase (COMT) gene are associated with schizophrenia in Irish high-density families

The enzyme catechol-o-methyltransferase (COMT) transfers a methyl group from adenosylmethionine to catecholamines including the neurotransmitters dopamine, epinephrine and norepinephrine. This methylation results in the degradation of catecholamines. The involvement of the COMT gene in the metabolic pathway of these neurotransmitters has made it an attractive candidate gene for many psychiatric disorders. In this article, we reported our study of association of COMT with schizophrenia in Irish families with a high density of schizophrenia. Three single nucleotide polymorphisms (SNPs) were genotyped for the 274 such families and within-family transmission disequilibrium tests were performed. SNP rs4680, which is the functional Val/Met polymorphism, showed modest association with the disease by the TRANSMIT, FBAT and PDT programs, while the other two SNPs were negative. These SNPs showed lower level of LDs with each other in the Irish subjects than in Ashkenazi Jews. Haplotype analysis indicated that a haplotype, haplotype A-G-A for SNPs rs737865-rs4680-rs165599, was preferentially transmitted to the affected subjects. This was different from the reported G-G-G haplotype found in Ashkenazi Jews, but both haplotypes shared the Val allele. We concluded that COMT gene is associated with schizophrenia and carries a small but significant risk to the susceptibility in the Irish subjects.

DNA hypermethylation and DNA hypomethylation is present at different loci in chronic kidney disease

Genetic risk factors for chronic kidney disease (CKD) are being identified through international collaborations. By comparison, epigenetic risk factors for CKD have only recently been considered using population-based approaches. DNA methylation is a major epigenetic modification that is associated with complex diseases, so we investigated methylome-wide loci for association with CKD. A total of 485,577 unique features were evaluated in 255 individuals with CKD (cases) and 152 individuals without evidence of renal disease (controls). Following stringent quality control, raw data were quantile normalized and β values calculated to reflect the methylation status at each site. The difference in methylation status was evaluated between cases and controls with resultant P values adjusted for multiple testing. Genes with significantly increased and decreased levels of DNA methylation were considered for biological relevance by functional enrichment analysis using KEGG pathways in Partek Genomics Suite. Twenty-three genes, where more than one CpG per loci was identified with Padjusted < 10−8, demonstrated significant methylation changes associated with CKD and additional support for these associated loci was sought from published literature. Strong biological candidates for CKD that showed statistically significant differential methylation include CUX1, ELMO1, FKBP5, INHBA-AS1, PTPRN2, and PRKAG2 genes; several genes are differentially methylated in kidney tissue and RNA-seq supports a functional role for differential methylation in ELMO1 and PRKAG2 genes. This study reports the largest, most comprehensive, genome-wide quantitative evaluation of DNA methylation for association with CKD. Evidence confirming methylation sites influence development of CKD would stimulate research to identify epigenetic therapies that might be clinically useful for CKD.

A phase II study of vorinostat (MK-0683) in patients with polycythaemia vera and essential thrombocythaemia

Inhibition of histone deacetylases may be an important target in patients with myeloproliferative neoplasms. This investigator-initiated, non-randomized, open-label phase II multi-centre study included 63 patients (19 essential thrombocythaemia, 44 polycythaemia vera) from 15 centres. The primary objective was to evaluate if vorinostat was followed by a decline in clonal myeloproliferation as defined by European Leukaemia Net. Thirty patients (48%) completed the intervention period (24 weeks of therapy). An intention-to-treat response rate of 35% was identified. Pruritus was resolved [19% to 0% (P = 0·06)] and the prevalence of splenomegaly was lowered from 50% to 27% (P = 0·03). Sixty-five per cent of the patients experienced a decrease in JAK2 V617F allele burden (P = 0·006). Thirty-three patients (52% of patients) discontinued study drug before end of intervention due to adverse events (28 patients) or lack of response (5 patients). In conclusion, vorinostat showed effectiveness by normalizing elevated leucocyte and platelet counts, resolving pruritus and significantly reducing splenomegaly. However, vorinostat was associated with significant side effects resulting in a high discontinuation rate. A lower dose of vorinostat in combination with conventional and/or novel targeted therapies may be warranted in future studies.

Genetic examination of SETD7 and SUV39H1/H2 methyltransferases and the risk of diabetes complications in patients with type 1 diabetes

Hyperglycemia plays a pivotal role in the development and progression of vascular complications, which are the major sources of morbidity and mortality in diabetes. Furthermore, these vascular complications often persist and progress despite improved glucose control, possibly as a result of prior episodes of hyperglycemia. Epigenetic modifications mediated by histone methyltransferases are associated with gene-activating events that promote enhanced expression of key proinflammatory molecules implicated in vascular injury. In this study, we investigated genetic polymorphisms of the SETD7, SUV39H1, and SUV39H2 methyltransferases as predictors of risk for micro- and macrovascular complications in type 1 diabetes.

A randomised, phase II trial of the DNA-hypomethylating agent 5-aza-2'-deoxycytidine (decitabine) in combination with carboplatin vs carboplatin alone in patients with recurrent, partially platinum-sensitive ovarian cancer.

Background: Our previous laboratory and clinical data suggested that one mechanism underlying the development of platinum resistance in ovarian cancer is the acquisition of DNA methylation. We therefore tested the hypothesis that the DNA hypomethylating agent 5-aza-2'-deoxycytodine (decitabine) can reverse resistance to carboplatin in women with relapsed ovarian cancer.

Methods: Patients progressing 6-12 months after previous platinum therapy were randomised to decitabine on day 1 and carboplatin (AUC 6) on day 8, every 28 days or carboplatin alone. The primary objective was response rate in patients with methylated hMLH1 tumour DNA in plasma.

Results: After a pre-defined interim analysis, the study closed due to lack of efficacy and poor treatment deliverability in 15 patients treated with the combination. Responses by GCIG criteria were 9 out of 14 vs 3 out of 15 and by RECIST were 6 out of 13 vs 1 out of 12 for carboplatin and carboplatin/decitabine, respectively. Grade 3/4 neutropenia was more common with the combination (60% vs 15.4%) as was G2/3 carboplatin hypersensitivity (47% vs 21%).

Conclusions: With this schedule, the addition of decitabine appears to reduce rather than increase the efficacy of carboplatin in partially platinum-sensitive ovarian cancer and is difficult to deliver. Patient-selection strategies, different schedules and other demethylating agents should be considered in future combination studies.

Use of the γ-H2AX Assay to Investigate DNA Repair Dynamics Following Multiple Radiation Exposures

Radiation therapy is one of the most common and effective strategies used to treat cancer. The irradiation is usually performed with a fractionated scheme, where the dose required to kill tumour cells is given in several sessions, spaced by specific time intervals, to allow healthy tissue recovery. In this work, we examined the DNA repair dynamics of cells exposed to radiation delivered in fractions, by assessing the response of histone-2AX (H2AX) phosphorylation (γ-H2AX), a marker of DNA double strand breaks. γ-H2AX foci induction and disappearance were monitored following split dose irradiation experiments in which time interval between exposure and dose were varied. Experimental data have been coupled to an analytical theoretical model, in order to quantify key parameters involved in the foci induction process. Induction of γ-H2AX foci was found to be affected by the initial radiation exposure with a smaller number of foci induced by subsequent exposures. This was compared to chromatin relaxation and cell survival. The time needed for full recovery of γ-H2AX foci induction was quantified (12 hours) and the 1:1 relationship between radiation induced DNA double strand breaks and foci numbers was critically assessed in the multiple irradiation scenarios.

Functional Consequences of Splicing of the Antisense Transcript COOLAIR on FLC Transcription

Antisense transcription is widespread in many genomes; however, how much is functional is hotly debated. We are investigating functionality of a set of long noncoding antisense transcripts, collectively called COOLAIR, produced at Arabidopsis FLOWERING LOCUS C (FLC). COOLAIR initiates just downstream of the major sense transcript poly(A) site and terminates either early or extends into the FLC promoter region. We now show that splicing of COOLAIR is functionally important. This was revealed through analysis of a hypomorphic mutation in the core spliceosome component PRP8. The prp8 mutation perturbs a cotranscriptional feedback mechanism linking COOLAIR processing to FLC gene body histone demethylation and reduced FLC transcription. The importance of COOLAIR splicing in this repression mechanism was confirmed by disrupting COOLAIR production and mutating the COOLAIR proximal splice acceptor site. Our findings suggest that altered splicing of a long noncoding transcript can quantitatively modulate gene expression through cotranscriptional coupling mechanisms.

Intakes of dietary folate and other B vitamins are associated with risks of esophageal adenocarcinoma, Barrett's esophagus, and reflux esophagitis

Folate is implicated in carcinogenesis via effects on DNA synthesis, repair, and methylation. Efficient folate metabolism requires other B vitamins and is adversely affected by smoking and alcohol. Esophageal adenocarcinoma (EAC) may develop through a process involving inflammation [reflux esophagitis (RE)] leading to metaplasia [Barrett’s esophagus (BE)] and carcinoma. Within a population-based, case-control study, we investigated associations between dietary folate and related factors and risks of EAC, BE, and RE. EAC and BE cases had histologically confirmed disease; RE cases had endoscopically visible inflammation. Controls, age-sex frequency matched to EAC cases, were selected through population and general practice registers. Participants underwent structured interviews and completed food-frequency questionnaires. Multivariate ORs and 95% CIs were computed using logistic regression. A total of 256 controls and 223 EAC, 220 BE, and 219 RE cases participated. EAC risk decreased with increasing folate intake (OR highest vs. lowest = 0.56; 95% CI: 0.31, 1.00; P-trend < 0.01). Similar trends were found for BE (P-trend < 0.01) and RE (P-trend = 0.01). Vitamin B-6 intake was significantly inversely related to risks of all 3 lesions. Riboflavin intake was inversely associated with RE. Vitamin B-12 intake was positively associated with EAC. For EAC, there was a borderline significant interaction between folate intake and smoking (P-interaction = 0.053); compared with nonsmokers with high (≥median) folate intake, current smokers with low intakes (<median) had an 8-fold increased risk (OR: 8.15; 95% CI: 3.61, 18.40). The same group had increased BE risk (OR: 2.93; 95% CI: 1.24, 6.92; P-interaction = 0.12). Folate and other dietary methyl-group factors are implicated in the etiology of EAC and its precursors.

Epigenome-Wide Association Study for Parkinson's Disease

A methylation-based EWAS on carefully phenotyped individuals with Parkinson’s disease (PD) was conducted to reveal prioritised genes and pathways with statistically significant and sizable changes in PD and in the anxiety that often accompanies it. This was followed by subsequent replication of top-ranked CpG sites. Using the Infinium® HumanMethylation 450K beadchip (Illumina Inc., USA), twenty unique genes with a sizable difference in methylation (P adjusted < 0.05, Δβ ≥ 0.2), after correction for multiple testing, were identified between PD and controls, while seventeen were identified between PD with anxiety and PD without anxiety. Twelve top ranked, significantly associated loci in PD were evaluated in an independent replicate population using Sequenom EpiTYPER for 219 individuals with similar phenotypes to the cross-sectional case–control discovery design. FANCC cg14115740 and TNKS2 cg11963436 show significant differential methylation between PD cases and controls using both techniques and their Δβ values, which have the same direction of effect, are reasonable to warrant further investigation

Chromosome synapsis and recombination in simple and complex chromosomal heterozygotes of tuco-tuco (Ctenomys talarum: Rodentia: Ctenomyidae):Rodentia: Ctenomyidae)

The chromosomal speciation hypothesis suggests that irregularities in synapsis, recombination, and segregation in heterozygotes for chromosome rearrangements may restrict gene flow between karyotypically distinct populations and promote speciation. Ctenomys talarum is a South American subterranean rodent inhabiting the coastal regions of Argentina, whose populations polymorphic for Robertsonian and tandem translocations seem to have a very restricted gene flow. To test if chromosomal differences are involved in isolation among its populations, we examined chromosome pairing, recombination, and meiotic silencing of unsynapsed chromatin in male meiosis of simple and complex translocation heterozygotes using immunolocalization of the MLH1 marking mature recombination nodules and phosphorylated histone γH2A.X marking unrepaired double-strand breaks. We observed small asynaptic areas labeled by γH2A.X in pericentromeric regions of the chromosomes involved in the trivalents and quadrivalents. We also observed a decrease of recombination frequency and a distalization of the crossover distribution in the heterozygotes and metacentric homozygotes compared to acrocentric homozygotes. We suggest that the asynapsis of the pericentromeric regions are unlikely to induce germ cell death and decrease fertility of the heterozygotes; however, suppressed recombination in pericentromeric areas of the multivalents may reduce gene flow between chromosomally different populations of the Talas tuco-tuco.