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BackgroundRecent studies have suggested that fatty acid oxidation (FAO) is a key metabolic pathway for the growth of triple negative breast cancers (TNBCs), particularly those that have high expression of MYC. However, the underlying mechanism by which MYC promotes FAO remains poorly understood.MethodsWe used a combination of metabolomics, transcriptomics, bioinformatics, and microscopy to elucidate a potential mechanism by which MYC regulates FAO in TNBC.ResultsWe propose that MYC induces a multigenic program that involves changes in intracellular calcium signalling and fatty acid metabolism. We determined key roles for fatty acid transporters (CD36), lipases (LPL), and kinases (PDGFRB, CAMKK2, and AMPK) that each contribute to promoting FAO in human mammary epithelial cells that express oncogenic levels of MYC. Bioinformatic analysis further showed that this multigenic program is highly expressed and predicts poor survival in the claudin-low molecular subtype of TNBC, but not other subtypes of TNBCs, suggesting that efforts to target FAO in the clinic may best serve claudin-low TNBC patients.ConclusionWe identified critical pieces of the FAO machinery that have the potential to be targeted for improved treatment of patients with TNBC, especially the claudin-low molecular subtype.

Mutations in both RAS and the PTEN/PIK3CA/AKT signaling module are found in the same human tumors. PIK3CA and AKT are downstream effectors of RAS, and the selective advantage conferred by mutation of two genes in the same pathway is unclear. Based on a comparative molecular analysis, we show that activated PIK3CA/AKT is a weaker inducer of senescence than is activated RAS. Moreover, concurrent activation of RAS and PIK3CA/AKT impairs RAS-induced senescence. In vivo, bypass of RAS-induced senescence by activated PIK3CA/AKT correlates with accelerated tumorigenesis. Thus, not all oncogenes are equally potent inducers of senescence, and, paradoxically, a weak inducer of senescence (PIK3CA/AKT) can be dominant over a strong inducer of senescence (RAS). For tumor growth, one selective advantage of concurrent mutation of RAS and PTEN/PIK3CA/AKT is suppression of RAS-induced senescence. Evidence is presented that this new understanding can be exploited in rational development and targeted application of prosenescence cancer therapies. © 2011 Elsevier Inc.

Summary Approximately 10% of human colorectal cancer (CRC) are associated with activated BRAFV600E mutation, typically in absence of APC mutation and often associated with a CpG island methylator (CIMP) phenotype. To protect from cancer, normal intestinal epithelial cells respond to oncogenic BRAFV600E by activation of intrinsic p53 and p16-dependent tumor suppressor mechanisms, such as cellular senescence. Conversely, CIMP is thought to contribute to bypass of these tumor suppressor mechanisms, e.g. via epigenetic silencing of tumor suppressor genes, such as p16. It has been repeatedly proposed that DNMT3B is responsible for BRAFV600E-induced CIMP in human CRC. Here we set out to test this by in silico, in vitro, and in vivo approaches. We conclude that although both BRAFV600E and DNMT3B harbor oncogenic potential in vitro and in vivo and show some evidence of cooperation in tumor promotion, they do not frequently cooperate to promote CIMP and human intestinal cancer., Graphical Abstract, Highlights • DNMT3B antagonizes BRAFV600E-induced senescence-like state • DNMT3B accelerates BRAFV600E-induced intestinal cancer • Other studies do not support a role for DNMT3B in CIMP and cooperation with BRAFV600E • On balance, BRAFV600E and DNMT3B are unlikely to cooperate in human intestinal cancer, Biological Sciences; Molecular Biology; Cancer

Cellular senescence is a stable proliferation arrest that suppresses tumorigenesis. Cellular senescence and associated tumor suppression depend on control of chromatin. Histone chaperone HIRA deposits variant histone H3.3 and histone H4 into chromatin in a DNA replication-independent manner. Appropriately for a DNA replication-independent chaperone, HIRA is involved in control of chromatin in nonproliferating senescent cells, although its role is poorly defined. Here, we show that nonproliferating senescent cells express and incorporate histone H3.3 and other canonical core histones into a dynamic chromatin landscape. Expression of canonical histones is linked to alternative mRNA splicing to eliminate signals that confer mRNA instability in nonproliferating cells. Deposition of newly synthesized histones H3.3 and H4 into chromatin of senescent cells depends on HIRA. HIRA and newly deposited H3.3 colocalize at promoters of expressed genes, partially redistributing between proliferating and senescent cells to parallel changes in expression. In senescent cells, but not proliferating cells, promoters of active genes are exceptionally enriched in H4K16ac, and HIRA is required for retention of H4K16ac. HIRA is also required for retention of H4K16ac in vivo and suppression of oncogene-induced neoplasia. These results show that HIRA controls a specialized, dynamic H4K16ac-decorated chromatin landscape in senescent cells and enforces tumor suppression.

Background Age-associated epigenetic changes are implicated in aging. Notably, age-associated DNA methylation changes comprise a so-called aging “clock”, a robust biomarker of aging. However, while genetic, dietary and drug interventions can extend lifespan, their impact on the epigenome is uncharacterised. To fill this knowledge gap, we defined age-associated DNA methylation changes at the whole-genome, single-nucleotide level in mouse liver and tested the impact of longevity-promoting interventions, specifically the Ames dwarf Prop1 df/df mutation, calorie restriction and rapamycin. Results In wild-type mice fed an unsupplemented ad libitum diet, age-associated hypomethylation was enriched at super-enhancers in highly expressed genes critical for liver function. Genes harbouring hypomethylated enhancers were enriched for genes that change expression with age. Hypermethylation was enriched at CpG islands marked with bivalent activating and repressing histone modifications and resembled hypermethylation in liver cancer. Age-associated methylation changes are suppressed in Ames dwarf and calorie restricted mice and more selectively and less specifically in rapamycin treated mice. Conclusions Age-associated hypo- and hypermethylation events occur at distinct regulatory features of the genome. Distinct longevity-promoting interventions, specifically genetic, dietary and drug interventions, suppress some age-associated methylation changes, consistent with the idea that these interventions exert their beneficial effects, in part, by modulation of the epigenome. This study is a foundation to understand the epigenetic contribution to healthy aging and longevity and the molecular basis of the DNA methylation clock. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1185-3) contains supplementary material, which is available to authorized users.

Altered DNA methylation and associated destabilization of genome integrity and function is a hallmark of cancer. Replicative senescence is a tumour suppressor process that imposes a limit on the proliferative potential of normal cells that all cancer cells must bypass. Here we show by whole-genome single-nucleotide bisulfite sequencing that replicative senescent human cells exhibit widespread DNA hypomethylation and focal hypermethylation. Hypomethylation occurs preferentially at gene-poor, late-replicating, lamin-associated domains and is linked to mislocalization of the maintenance DNA methyltransferase (DNMT1) in cells approaching senescence. Low-level gains of methylation are enriched in CpG islands, including at genes whose methylation and silencing is thought to promote cancer. Gains and losses of methylation in replicative senescence are thus qualitatively similar to those in cancer, and this ‘reprogrammed’ methylation landscape is largely retained when cells bypass senescence. Consequently, the DNA methylome of senescent cells might promote malignancy, if these cells escape the proliferative barrier. Cancer is associated with altered DNA methylation. Using whole-genome single-nucleotide sequencing, Adams and colleagues reveal that senescent cells, as well as cells that have bypassed senescence through p53 and pRB inactivation, exhibit methylation changes similar to those seen in cancer.

Cellular senescence is a stable proliferation arrest associated with an altered secretory pathway (senescence-associated secretory phenotype). Cellular senescence is also a tumor suppressor mechanism, to which both proliferation arrest and senescence-associated secretory phenotype are thought to contribute. The melanocytes within benign human nevi are a paradigm for tumor-suppressive senescent cells in a premalignant neoplasm. Here a comparison of proliferating and senescent melanocytes and melanoma cell lines by RNA sequencing emphasizes the importance of senescence-associated proliferation arrest in suppression of transformation. Previous studies showed that activation of the Wnt signaling pathway can delay or bypass senescence. Consistent with this, we present evidence that repression of Wnt signaling contributes to melanocyte senescence in vitro. Surprisingly, Wnt signaling is active in many senescent human melanocytes in nevi, and this is linked to histological indicators of higher proliferative and malignant potential. In a mouse, activated Wnt signaling delays senescence-associated proliferation arrest to expand the population of senescent oncogene-expressing melanocytes. These results suggest that Wnt signaling can potentiate nevogenesis in vivo by delaying senescence. Further, we suggest that activated Wnt signaling in human nevi undermines senescence-mediated tumor suppression and enhances the probability of malignancy.

SummaryThe HIRA chaperone complex, comprised of HIRA, UBN1, and CABIN1, collaborates with histone-binding protein ASF1a to incorporate histone variant H3.3 into chromatin in a DNA replication-independent manner. To better understand HIRA’s function and mechanism, we integrated HIRA, UBN1, ASF1a, and histone H3.3 chromatin immunoprecipitation sequencing and gene expression analyses. Most HIRA-binding sites colocalize with UBN1, ASF1a, and H3.3 at active promoters and active and weak/poised enhancers. At promoters, binding of HIRA/UBN1/ASF1a correlates with the level of gene expression. HIRA is required for deposition of histone H3.3 at its binding sites. There are marked differences in nucleosome and coregulator composition at different classes of HIRA-bound regulatory sites. Underscoring this, we report physical interactions between the HIRA complex and transcription factors, a chromatin insulator and an ATP-dependent chromatin-remodeling complex. Our results map the distribution of the HIRA chaperone across the chromatin landscape and point to different interacting partners at functionally distinct regulatory sites.

On acquisition of an oncogenic mutation, primary human and mouse cells can enter oncogene-induced senescence (OIS). OIS is characterized by a stable proliferation arrest and secretion of proinflammatory cytokines and chemokines, the senescence-associated secretory phenotype. Proliferation arrest and the senescence-associated secretory phenotype collaborate to enact tumor suppression, the former by blocking cell proliferation and the latter by recruiting immune cells to clear damaged cells. However, the interactions of OIS cells with the immune system are still poorly defined. Here, we show that engagement of OIS in primary human melanocytes, specifically by melanoma driver mutations NRASQ61K and BRAFV600E, causes expression of the major histocompatibility class II antigen presentation apparatus, via secreted IL-1ß signaling and expression of CIITA, a master regulator of major histocompatibility class II gene transcription. In vitro, OIS melanocytes activate T-cell proliferation. In vivo, nonproliferating oncogene-expressing melanocytes localize to skin-draining lymph nodes, where they induce T-cell proliferation and an antigen presentation gene expression signature. In patients, expression of major histocompatibility class II in melanoma is linked to favorable disease outcome. We propose that OIS in melanocytes is accompanied by an antigen presentation phenotype, likely to promote tumor suppression via activation of the adaptive immune system.

Senescence is thought to play an important role in the progressive age-related decline in tissue integrity and concomitant diseases, but not much is known about the complex interplay between upstream regulators and downstream effectors. We profiled whole genome gene expression of non-stressed and rotenone-stressed human fibroblast strains from young and oldest old subjects, and measured senescence associated β-gal activity. Microarray results identified gene sets involved in carbohydrate metabolism, Wnt/β-catenin signaling, the cell cycle, glutamate signaling, RNA-processing and mitochondrial function as being differentially regulated with chronological age. The most significantly differentially regulated mRNA corresponded to the p16 gene. p16 was then investigated using qPCR, Western blotting and immunocytochemistry. In conclusion, we have identified cellular pathways that are differentially expressed between fibroblast strains from young and old subjects.

MYC is one of the most commonly mutated and highly amplified oncogenes in human breast cancer. MYC amplifications occur most frequently in triple-negative breast cancers (TNBCs). TNBCs can be divided into two molecular subtypes: basal-like and claudin-low breast cancers. These cancers tend to be extremely aggressive and are strongly associated with disease recurrence, poor prognosis and high mortality. In particular, claudin-low tumors are classified by a loss of tight junctions and cell-to-cell contacts and an enrichment for genes associated with an epithelial-to-mesenchymal transition (EMT) and mammary stem cells (also known as tumor-initiating cells). Despite the high level of disease severity, there are no targeted therapies for claudin-low TNBCs. To address this unmet need, we utilized human mammary epithelial cells (HuMECs) that express oncogenic levels of MYC and a mutant MYC (T58A) to characterize the behavioral and metabolic changes that occur during the formation of MYC-driven breast cancers. We found that MYC regulates the expression of genes associated with cell stemness, EMT, lipid metabolism, and calcium (Ca2+) signaling and that the expression of this gene signature promotes cell growth, survival, migration, and metabolic plasticity. The gene signature of MYC-expressing HuMECs highly correlates with the gene signature of claudin-low breast cancers, therefore highlighting the relevance of our HuMEC model to human claudin-low breast cancer. We found the major drivers underlying the MYC-dependent changes in cell behavior to be stimulation of Ca2+ signaling and strong activation of lipid metabolism. Ca2+ signaling is stimulated through the MYC-dependent repression of Ca2+ efflux mechanisms; elevated cytosolic Ca2+ then consequently stimulates a Ca2+/calmodulin kinase kinase 2 (CAMKK2)/AMPK signaling axis that activates fatty acid scavenging and transport, as well as β-oxidation. Enhanced lipid metabolism thereby provides the necessary biomass (fatty acids) for phospholipid biosynthesis and energy (ATP) to support the metabolically demanding processes of cell growth, proliferation, and migration. In all, our findings provide a strong rationale for targeting lipid metabolism and the Ca2+/CAMKK2/AMPK signaling axis in MYC-driven, and potentially claudin-low, breast cancers. Citation Format: Jessica C. Casciano, Adam Cohen-Nowak, Johan Vande Voorde, Qifeng Zhang, Susan Chalmers, Mairi Sandison, Ann Hedley, Tony McBryan, Thomas Beer, Hsin-Yao Tang, David W. Speicher, Peter Adams, Xiufeng Liu, Richard Schlegel, John McCarron, Michael J. Wakelam, Eyal Gottlieb, Zachary T. Schug. MYC expression promotes lipid metabolism and metabolic plasticity in human mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1441.

Background. \ud Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are neoplastic disorders of hematopoietic stem cells. DNA methyltransferase inhibitors (DNMTi), 5-azacytidine (AzaC) and 5-aza-2’-deoxycytidine (Decitabine), benefit some MDS/AML patients. However, the role of DNMTi-induced DNA hypomethylation in regulation of gene expression in AML is unclear.\ud Results. \ud We compared the effects of AzaC on DNA methylation and gene expression using whole-genome single-nucleotide bisulfite-sequencing (WGBS) and RNA-sequencing in OCI-AML3 (AML3) cells. For data analysis, we used an approach recently developed for discovery of differential patterns of DNA methylation associated with changes in gene expression, that is tailored to single-nucleotide bisulfite-sequencing data (Washington University Interpolated Methylation Signatures (WIMSi)). By this approach, a subset of genes upregulated by AzaC was found to be characterized by AzaC-induced signature methylation loss flanking the transcription start site. These genes are enriched for genes increased in methylation and decreased in expression in AML3 cells compared to normal hematopoietic stem and progenitor cells. Moreover, these genes are preferentially upregulated by Decitabine in human primary AML blasts, and control cell proliferation, death and development. \ud Conclusions.\ud Our WGBS and WIMSi data analysis approach has identified a set of genes whose is methylation and silencing in AML is reversed by DNMTi. These genes are good candidates for direct regulation by DNMTi, and their reactivation by DNMTi may contribute to therapeutic activity. This study also demonstrates the ability of WIMSi to reveal relationships between DNA methylation and gene expression, based on single-nucleotide bisulfite-sequencing and RNA-seq data.

Cellular senescence is a stable proliferation arrest associated with an altered secretory pathway, the senescence-associated secretory phenotype. However, cellular senescence is initiated by diverse molecular triggers, such as activated oncogenes and shortened telomeres, and is associated with varied and complex physiological endpoints, such as tumor suppression and tissue aging. The extent to which distinct triggers activate divergent modes of senescence that might be associated with different physiological endpoints is largely unknown. To begin to address this, we performed gene expression profiling to compare the senescence programs associated with two different modes of senescence, oncogene-induced senescence (OIS) and replicative senescence (RS [in part caused by shortened telomeres]). While both OIS and RS are associated with many common changes in gene expression compared to control proliferating cells, they also exhibit substantial differences. These results are discussed in light of potential physiological consequences, tumor suppression and aging.

Epigenetics is the study of heritable genome functions that do not depend directly on DNA sequence. Commonly studied epigenetic mechanisms include DNA methylation, histone variants and histone modifications. This chapter will describe the basic mechanisms underlying DNA methylation, histone structure and variants and post translational modifications. A summary of the known effects and disease relationships will be provided and methods of measuring and manipulating these epigenetic marks will be discussed. Finally, a number of epigenetic disorders and emerging therapies will be presented.

Introduction Vorinostat is a small molecule inhibitor of class I and II histone deacetylases with preclinical activity in melanoma. Methods We evaluated 32 patients with advanced primary cutaneous or ocular melanoma in a multi-institutional setting (PMH Phase II Consortium) with continuous daily oral vorinostat 400 mg. The primary endpoint was response rate by RECIST, with time to progression as a secondary endpoint. The study was designed to distinguish a response rate of 20 % from a RR of 5 % and to distinguish a 2 month median progression-free survival (PFS), from one of 3.1 months. The study proceeded to stage 2 following 2 of 16 responses.. We also assessed VEGF, FGF levels, P52 polymorphisms and chromatin-associated proteins as potential biomarkers. Results Therapy was associated with significant side effects, including fatigue, nausea, lymphopenia, and hyperglycemia. Eleven patients experienced at least one grade 3 or higher adverse event. There were two confirmed PRs in patients with cutaneous melanoma. Sixteen patients had stable disease and 14 patients had progressive disease for best response. In addition, two patients with cutaneous melanoma scored as stable disease had early unconfirmed partial responses with subsequent progression. Patients with stable disease or partial response (n = 18) had a median progression free survival of 5 months. (range 2–12 months). Conclusions Vorinostat demonstrated some early responses and a high proportion of patients with stable disease, but did not meet its primary endpoint of response. Different schedules of this agent with BRAF mutation status and markers of histone acetylation could be explored in melanoma.

The Wnt signaling pathway is involved in the regulation of tissue patterning and organ development during embryogenesis and continues to contribute to the maintenance of tissue homeostasis in adulthood. Recently, Wnt signaling has also been implicated in the establishment and progression of replicative cellular senescence. Given the known roles of tissue homeostasis and cellular senescence in aging, we sought to determine whether Wnt signaling changes with age. We examined the expression of 84 Wnt pathway-related genes in the liver, lung, skeletal muscle, and brain tissue from young and old mice. Expression changes were compared with those seen in cellular senescence, and transcription factors that might mediate these changes were predicted bioinformatically. In aggregate, our data are indicative of a general decrease in Wnt signaling with age, especially in the lung and brain. Furthermore, the set of genes that are differentially expressed with age is distinct from the genes differentially expressed in cellular senescence. The transcription factors predicted to regulate these changes, Nf-κB, Myb, Nkx2-1, Nr5a2, and Ep300, are known to regulate inflammation, differentiation, lipid metabolism, and chromatin remodeling, all of which have previously been implicated in aging. Although our study does not address whether altered Wnt signaling is a cause or an effect of aging, the presence of a relationship between the two provides a starting point for further investigation.

Introduction: Differentiation of vascular endothelial cells (ECs) in clinically relevant numbers for injection into ischaemic areas could offer therapeutic potential in the treatment of cardiovascular conditions, including myocardial infarction, peripheral vascular disease and stroke. While we and others have demonstrated successful generation of functional endothelial-like cells from human embryonic stem cells (hESCs), little is understood regarding the complex transcriptional and epigenetic changes that occur during differentiation, in particular during early commitment to a mesodermal lineage.\ud Methods: We performed the first gene expression microarray study of hESCs undergoing directed differentiation to ECs using a monolayer-based, feeder-free and serum-free protocol. Microarray results were confirmed by quantitative RT-PCR and immunocytochemistry, and chromatin immunoprecipitation (ChIP)-PCR analysis was utilised to determine the bivalent status of differentially expressed genes.\ud \ud Results: We identified 22 transcription factors specific to early mesoderm commitment. Among these factors, FOXA2 was observed to be the most significantly differentially expressed at the hESC–EC day 2 timepoint. ChIP-PCR analysis revealed that the FOXA2 transcription start site is bivalently marked with histone modifications for both gene activation (H3K4me3) and repression (H3K27me3) in hESCs, suggesting the transcription factor may be a key regulator of hESC differentiation.\ud Conclusion: This enhanced knowledge of the lineage commitment process will help improve the design of directed differentiation protocols, increasing the yield of endothelial-like cells for regenerative medicine therapies in cardiovascular disease.

The mammalian HIRA/UBN1/ASF1a complex is a histone chaperone complex that is conserved from yeast (Saccharomyces cerevisiae) to humans. This complex preferentially deposits the histone variant H3.3 into chromatin in a DNA replication-independent manner and is implicated in diverse chromatin regulatory events from gene activation to heterochromatinization. In yeast, the orthologous complex consists of three Hir proteins (Hir1p, Hir2p, and Hir3p), Hpc2p, and Asf1p. Yeast Hir3p has weak homology to CABIN1, a fourth member of the human complex, suggesting that Hir3p and CABIN1 may be orthologs. Here we show that HIRA and CABIN1 interact at ectopic and endogenous levels of expression in cells, and we isolate the quaternary HIRA/UBN1/CABIN1/ASF1a (HUCA) complex, assembled from recombinant proteins. Mutational analyses support the view that HIRA acts as a scaffold to bring together UBN1, ASF1a, and CABIN1 into a quaternary complex. We show that, like HIRA, UBN1, and ASF1a, CABIN1 is involved in heterochromatinization of the genome of senescent human cells. Moreover, in proliferating cells, HIRA and CABIN1 regulate overlapping sets of genes, and these genes are enriched in the histone variant H3.3. In sum, these data demonstrate that CABIN1 is a functional member of the human HUCA complex and so is the likely ortholog of yeast Hir3p.

This paper describes an approach to user configuration of activity awareness. This approach offers users increased flexibility by allowing the use of multiple methods of configuration to be used within a unified framework; these configuration methods can include context, policy rules and automatic techniques of configuring behaviour. In this paper we describe the benefits of this flexibility and discuss a model of configuration that can be used to support these features.

There are many sources of change within the domain of home care. People have changing needs, beliefs, and preferences regarding their care plan and how they might want to interact with existing and emerging home care technologies. The devices and services available to the user are likely to change over time depending on a person's capabilities or location within the home and the current devices and services available. The resulting interaction methods can therefore also change in accordance with the room location, available devices or displays, or preferred modalities. Home care systems therefore need to offer configuration possibilities that support this change. Computer systems offer methods and tools to support configuration in the short term, but do not provide mechanisms for supporting configuration over both short and long term. This paper presents an approach that addresses this issue in the home care domain by integrating methods for interaction requirements engineering with system support for turning those requirements into a working configuration. Both the methods and system support are designed to address a gradual process of change -- 'interaction evolution' in home care. We present the key features of our approach using a home care scenario and consider our progress to date in implementing and validating the approach.

Background Acute myeloid leukaemia (AML) is one of the commonest haemopoietic cancers; despite intensive clinical trial developments there has been little change in survival over the past 20 years. A major focus of research is the identification of less toxic regimens that offer the possibility of response above that of supportive care. Epigenetic therapies, such as the DNA methyltransferase inhibitor azacitidine, offer a novel approach to the treatment of this disease. Despite increasing use in the clinic, the mechansim by which azacitidine regulates gene transcription remains unclear—in particular, whether induction of gene expression change is directly methylation-dependent or secondary to indirect downstream effects. We aimed to investigate the genome-wide effects of azacitidine on DNA methylation and gene transcription in vitro. Methods Using an acute leukaemia cell line (OCI-AML3), we compared methylation and gene expression in OCI-AML3 cells treated with and without azacitidine. For the best possible comparison, we used whole genome bisulfite sequencing and RNA-Seq. Using these data, we aimed to identify the molecular variables that determine a gene's responsiveness to azacitidine. Findings We found that a low dose of azacitidine, which resulted in DNA hypomethylation but was not toxic enough to acutely arrest DNA synthesis and cell proliferation, induced widespread methylation loss, of about 50%, across the genome. This demethylation was uniform, rather than selectively targeted, across all genomic features. Hence, the absolute loss of methylation was proportional to the starting density of methylation in a given region of the genome. Azacitidine induced significant gene expression change in 3·4% of all genes with both losses and gains of gene expression (false discovery rate r 2 =−0·252, p=1·46 × 10 −14 ). In line with this finding, a high level of gene expression in untreated cells also correlated with azacitidine-induced alteration in gene expression. Neither of these variables was specifically associated with either upregulation or downregulation of gene expression. Interpretation We found an inconsistency between the widespread extent of DNA methylation loss and the limited gene expression changes in this model. Therefore, the simple view that DNA methyltransferase inhibitor-induced demethylation at CpG island promoters is primarily responsible for reactivation of previously silenced genes could be an oversimplification. We conclude that azacitidine interacts with other, as yet uncharacterised, factors that contribute to transcriptional changes in hypomethylated CpG island containing genes. This observation provides a clinical rationale for the combination of DNA methyltransferase inhibitors with other epigenetic agents whose function could be synergistically exploited to modify the transcriptional profile of the leukaemia genome. Funding None.

Considerable attention has been focused at improving the outcomes of older patients with Myelodysplastic syndrome/Acute Myeloid Leukemia (MDS/AML). However there remains a lack of consensus regarding how best to approach their management. For a significant proportion of elderly patients, with poor performance status and co-morbidities, the risk of early induction death precludes them from receiving intensive chemotherapy. A major focus of ongoing research remains the identification less toxic regimens which offer the possibility of response above that of supportive care. Epigenetic therapies, such as the DNA methyltransferase inhibitors (DNMTi) offer a novel, less intensive, approach to the treatment of MDS/AML. Despite increasing utilization in the clinic, it is still unclear how Azacitidine (Aza-C) regulates gene transcription; in particular, whether induction of gene expression change is directly methylation-dependent or secondary to indirect downstream effects. Historically, methylation studies attempting to decipher the effects of Aza-C have typically been limited to examining selected cytosine-guanine dinucleotides (CpGs) through the use of CpG microarrays. Newer methods, enriching for methylated DNA fragments by immunoprecipitation or restriction enzyme targeting, both introduce inherent bias to the analysis. Using OCI-AML3 cells, this is the first reported use of bisulfite sequencing of a whole DNMTi treated AML genome and offers a unique insight into the effects of Aza-C at every CpG genome-wide. This study, in parallel with RNA sequencing, has addressed the effects of demethylation on transcriptional change. At a global level we demonstrate that Aza-C, at doses comparable to those used within the Aza-C treated patient population, induces widespread methylation loss, of approximately 50%, across the genome. This demethylation is uniform, rather than selectively targeted, across all genomic features. Hence, the absolute loss of methylation is proportional to the starting density of methylation in a given region of the genome. Aza-C induces significant gene expression change in 3.4% of all genes with both losses and gains of gene expression evident. We found no evidence that Aza-C-induced hypomethylation at CpG islands was preferentially associated with upregulation of gene expression. However, at genes undergoing significant changes in expression, low CpG island methylation in untreated cells weakly correlated with altered transcription. In line with this, a high gene expression level in untreated cells also correlated with Aza-C-induced alteration in gene expression. Neither of these parameters was specifically associated with either up or down regulation of gene expression. This indicates that the simple view that DNMTi-induced demethylation at CpG island promoters is primarily responsible for reactivation of previously silenced genes is an oversimplification. The incongruence between the widespread extent of DNA methylation loss and the relatively limited gene expression changes in this model, leads us to conclude that Aza-C interacts with other, as yet uncharacterized, factors which contribute to transcriptional changes in hypomethylated CpG island containing genes. This provides a clinical rationale for the combination of DNMTis with other epigenetic agents whose function could be synergistically exploited to modify the transcriptional profile of the leukemia genome. Disclosures: No relevant conflicts of interest to declare.

Background Cellular senescence is a permanent growth arrest that occurs in response to cellular stressors, such as telomere shortening or activation of oncogenes. Although the process of senescence growth arrest is somewhat conserved between mouse and human cells, there are some critical differences in the molecular pathways of senescence between these two species. Recent studies in human fibroblasts have defined a cell signaling pathway that is initiated by repression of a specific Wnt ligand, Wnt2. This, in turn, activates a histone chaperone HIRA, and culminates in formation of specialized punctate domains of facultative heterochromatin, called S enescence-A ssociated H eterochromatin F oci (SAHF), that are enriched in the histone variant, macroH2A. SAHF are thought to repress expression of proliferation-promoting genes, thereby contributing to senescence-associated proliferation arrest. We asked whether this Wnt2-HIRA-SAHF pathway is conserved in mouse fibroblasts. Results We show that mouse embryo fibroblasts (MEFs) and mouse skin fibroblasts, do not form robust punctate SAHF in response to an activated Ras oncogene or shortened telomeres. However, senescent MEFs do exhibit elevated levels of macroH2A staining throughout the nucleus as a whole. Consistent with their failure to fully activate the SAHF assembly pathway, the Wnt2-HIRA signaling axis is not overtly regulated between proliferating and senescent mouse cells. Conclusions In addition to the previously defined differences between mouse and human cells in the mechanisms and phenotypes associated with senescence, we conclude that senescent mouse and human fibroblasts also differ at the level of chromatin and the signaling pathways used to regulate chromatin. These differences between human and mouse senescence may contribute to the increased propensity of mouse fibroblasts (and perhaps other mouse cell types) to become immortalized and transformed, compared to human cells.