Global Loss Of DNA Methylation Research Articles

Increased mtDNA mutation frequency in oocytes causes epigenetic alterations and embryonic defects.

Mitochondria are essential for female reproductive processes, yet the function of mitochondrial DNA (mtDNA) mutation in oocytes remains elusive. By employing an mtDNA mutator (Polgm) mouse model, we found the fetal growth retardation and placental dysfunction in post-implantation embryos derived from Polgm oocytes. Remarkably, Polgm oocytes displayed the global loss of DNA methylation; following fertilization, zygotic genome experienced insufficient demethylation, along with dysregulation of gene expression. Spindle-chromosome exchange experiment revealed that cytoplasmic factors in Polgm oocytes are responsible for such a deficient epigenetic remodeling. Moreover, metabolomic profiling identified a significant reduction in the α-ketoglutarate (αKG) level in oocytes from Polgm mice. Importantly, αKG supplement restored both DNA methylation state and transcriptional activity in Polgm embryos, consequently preventing the developmental defects. Our findings uncover the important role of oocyte mtDNA mutation in controlling epigenetic reprogramming and gene expression during embryogenesis. αKG deserves further evaluation as a potential drug for treating mitochondrial dysfunction-related fertility decline.

Epigenomic profiling indicates a role for DNA methylation in the postnatal liver and pancreas development of giant pandas

Giant pandas are unique within Carnivora with a strict bamboo diet. Here, the epigenomic profiles of giant panda liver and pancreas tissues collected from three important feeding stages were investigated using BS-seq. Few differences in DNA methylation profiles were exhibited between no feeding and suckling groups in both tissues. However, we observed a tendency toward a global loss of DNA methylation in the gene-body and promoter region of metabolism-related genes from newborn to adult. Correlation analysis revealed a significant negative correlation between the changes in methylation levels within gene promoters and gene expression. The majority of genes related to nutrition metabolism had lost DNA methylation with increased mRNA expression in adult giant pandas. The few galactose metabolism and unsaturated fatty acid metabolism related genes that were hypomethylated and highly-expressed at early stages of giant panda development may meet the nutritional requirement of this species' highly altricial neonates.

Abstract PD3-04: Multi-omics characterization of triple-negative breast cancer identifies therapeutic vulnerabilities and epigenetic immune suppression in the mesenchymal subtype

Abstract Triple-negative breast cancer (TNBC) is a collection of biologically diverse cancers characterized by distinct transcriptional patterns, biology and immune composition. TNBCs display transcriptional diversity with at least four tumor-intrinsic subtypes that include two basal-like (BL1, BL2), a mesenchymal (M) and a luminal androgen receptor (LAR) subtype. Using integrative analyses of transcriptomic, epigenetic, proteomic and phospho-proteomic patterns we have identified subtype-specific vulnerabilities, which advanced our understanding of the cellular origins of TNBC subtypes. In mesenchymal subtype tumors we observed high mutation load and genomic instability, absence of immune cells, low PD-L1 expression, loss of global DNA methylation and transcriptional repression of antigen presentation genes through the polycomb repressor complex 2 (PRC2). Using cell line models, we demonstrate that MHC-I expression is epigenetically silenced by the PRC2 in mesenchymal TNBC cell lines and inhibition of EZH2 restores expression. Pharmacological inhibition of EZH2 enhances chemotherapy efficacy in syngeneic murine tumor models providing a rationale for using PRC2 inhibitors in PD-L1 negative mesenchymal tumors. Citation Format: Brian D Lehmann, Antonio Colaprico, Tiago C Silva, Jianjiao Chen, Hanbing An, Yuguang Ban, Lily Wang, Jamaal L James, Justin Balko, Paula I Gonzalez-Ericsson, Melinda E Sanders, Bing Zhang, Jennifer A Pietenpol, Xi S Chen. Multi-omics characterization of triple-negative breast cancer identifies therapeutic vulnerabilities and epigenetic immune suppression in the mesenchymal subtype [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-04.

Identifying regulators of parental imprinting by CRISPR/Cas9 screening in haploid human embryonic stem cells

In mammals, imprinted genes are regulated by differentially methylated regions (DMRs) that are inherited from germ cells, leading to monoallelic expression in accordance with parent-of-origin. Yet, it is largely unknown how imprinted DMRs are maintained in human embryos despite global DNA demethylation following fertilization. Here, we explored the mechanisms involved in imprinting regulation by employing human parthenogenetic embryonic stem cells (hpESCs), which lack paternal alleles. We show that although global loss of DNA methylation in hpESCs affects most imprinted DMRs, many paternally-expressed genes (PEGs) remain repressed. To search for factors regulating PEGs, we performed a genome-wide CRISPR/Cas9 screen in haploid hpESCs. This revealed ATF7IP as an essential repressor of a set of PEGs, which we further show is also required for silencing sperm-specific genes. Our study reinforces an important role for histone modifications in regulating imprinted genes and suggests a link between parental imprinting and germ cell identity.

Multiomics Analysis of Neuroblastoma Cells Reveals a Diversity of Malignant Transformations

Neuroblastoma (NB) is a pediatric cancer of the developing sympathetic nervous system that exhibits significant variation in the stage of differentiation and cell composition of tumors. Global loss of DNA methylation and genomic 5-hydroxymethylcytosine (5hmC) is a hallmark of human cancers. Here, we used our recently developed single-base resolution approaches, hmTOP-seq and uTOP-seq, for construction of 5hmC maps and identification of large partially methylated domains (PMDs) in different NB cell subpopulations. The 5hmC profiles revealed distinct signatures characteristic to different cell lineages and stages of malignant transformation of NB cells in a conventional and oxygen-depleted environment, which often occurs in tumors. The analysis of the cell-type-specific PMD distribution highlighted differences in global genome organization among NB cells that were ascribed to the same lineage identity by transcriptomic networks. Collectively, we demonstrated a high informativeness of the integrative epigenomic and transcriptomic research and large-scale genome structure in investigating the mechanisms that regulate cell identities and developmental stages of NB cells. Such multiomics analysis, as compared with mutational studies, open new ways for identification of novel disease-associated features which bring prognostic and therapeutic value in treating this aggressive pediatric disease.

Acute Deletion of Tet2 or Tet3 Transiently Dysregulate Murine Erythropoiesis

The majority of palindromic CpG dinucleotides in mammalian DNA are symmetrically methylated on cytosine (5mC). Erythroid differentiation is associated with replication-dependent genome-wide loss of ~30-50% of all 5mC (1). One potential mechanism of demethylation involves Tet dioxygneases, which oxidize 5 methylcytosine (5mC) to 5-hydroxy-methylcytosine (5hmC), thereby interfering with maintenance methylation by DNMT1 (2). Tet2 and Tet3 are the two major Tet proteins expressed in hematopoietic cells. Tet2 is one of the most commonly mutated genes in hematological malignancies. Germline deletion of Tet2 results in increased hematopoietic stem cell self-renewal, erythroid and myeloid hyperplasia and myeloid and lymphoid malignancies (3, 4). Germline deletion of Tet3 is embryonic lethal (5). Here we investigated the potential role of Tet2 and Tet3 in the global loss of DNA methylation during erythroid differentiation, using mouse genetic models.We bred Tet2fl/fl and Tet3fl/fl mice onto the Rosa26-CreERT2 background, enabling acute deletion in vivo of either Tet2, Tet3 or both genes in adult and fetal mice. Within 4 to 10 days of deletion, we noted a rapid decline in total bone marrow cellularity in all genotypes; bone-marrow erythroblasts decreased to 15% of control value in Tet2-deleted (Tet2del) mice (p<0.0001), and to 43% (p=0.05) and 20% (p<0.0001) of controls in the Tet3 del and Tet2/Tet3 double deleted mice (DKO), respectively. These findings were associated with decreased reticulocyte counts in peripheral blood, and a decline in hematocrit, in Tet2del (36% vs. 46% in controls, p<0.004) and the DKO (31%, p=0.03). Bone-marrow CFU-e progenitors, but not BFU-e progenitors, also decreased in number. These losses partially improved over the next 2 to 4 weeks in mice deleted for either Tet2 or Tet3, but not in the DKO mice, in which anemia persisted. Megakaryocyte CFU-Mk progenitors increase in the DKO but are unaffected in the single deletions. Lymphoid lineage cells (CD4+, CD8+ and CD19+) showed similar transient losses, but the numbers of granylocytic and monocytic progenitors in the bone marrow was not affected. Similarly, fetal liver cell number decreased in all genotypes within 4 days of deletion, compared with controls, in all genotypes, and most severely in the DKO.We used three approaches to determine the potential effect of Tet proteins on erythroid DNA demethylation: Mass spectrometry, to measure global levels of 5mC and 5hmC in fetal liver erythroblasts; 5hMe-DIP-seq (5hyrdoxymethyl-DNA immune precipitation- sequencing), to determine the genomic distribution of hydroxymethylation in erythroblasts; and pyrosequencing for DNA methylation/hydroxymethylation at specific genomic loci including repetitive LINE_1 elements. We found that 5hmC is highly enriched in previously identified erythroid enhancers. Tet2 accelerates, but is not essential, for DNA demethylation of specific erythroid enhancers. Neither Tet2 nor Tet3 are required for global DNA demethylation.Taken together, we conclude that Tet2 and Tet3 are required during erythropoiesis, where they play partially redundant roles, possibly contributing to the changing chromatin environment during activation of erythroid terminal differentiation. Their acute deletion leads to transient loss of erythroblast viability and to anemia, but this is followed by a process of adaptation and chronically compensated erythropoiesis. These results raise the possibility that the adaptation required for the rapid partial recovery in the erythroid and lymphoid lineages following acute Tet2 loss may contribute, over the long term, to the development of myeloid and lymphoid malignancies.1. J. R. Shearstone et al., Global DNA demethylation during mouse erythropoiesis in vivo. Science334, 799-802 (2011).2. M. Ko et al., Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature468, 839-843 (2010).3. M. Ko et al., TET proteins and 5-methylcytosine oxidation in hematological cancers. Immunol Rev263, 6-21 (2015).4. J. An et al., Acute loss of TET function results in aggressive myeloid cancer in mice. Nature communications6, 10071 (2015).5. T. P. Gu et al., The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature477, 606-610 (2011). DisclosuresNo relevant conflicts of interest to declare.

Sex-specific effects of in vitro fertilization on adult metabolic outcomes and hepatic transcriptome and proteome in mouse.

Although in vitro fertilization (IVF) is associated with adverse perinatal outcomes, there is increasing concern about the long-term and sex-specific health implications. Augmenting our IVF mouse model to longitudinally investigate metabolic outcomes in offspring from optimal neonatal litter sizes, we found sex-specific metabolic outcomes in IVF offspring. IVF-conceived females had higher body weight and cholesterol levels compared to naturally conceived females, whereas IVF-conceived males had higher levels of triglycerides and insulin, and increased body fat composition. Through adult liver transcriptomics and proteomics, we identified sexually dimorphic dysregulation of the sterol regulatory element-binding protein (SREBP) pathways that are associated with the sex-specific phenotypes. We also found that global loss of DNA methylation in placenta was linked to higher cholesterol levels in IVF-conceived females. Our findings indicate that IVF procedures have long-lasting sex-specific effects on metabolic health of offspring and lay the foundation to utilize the placenta as a predictor of long-term outcomes.

Roles of DNA Methylation in Cold Priming in Tartary Buckwheat.

Plants experience a wide array of environmental stimuli, some of which are frequent occurrences of cold weather, which have priming effects on agricultural production and agronomic traits. DNA methylation may act as an epigenetic regulator for the cold response of Tartary buckwheat (Fagopyrum tataricum). Combined with long-term field observation and laboratory experiments, comparative phenome, methylome, and transcriptome analyses were performed to investigate the potential epigenetic contributions for the cold priming of Tartary buckwheat variety Dingku1. Tartary buckwheat cv. Dingku1 exhibited low-temperature resistance. Single-base resolution maps of the DNA methylome were generated, and a global loss of DNA methylation was observed during cold responding in Dingku1. These sites with differential methylation levels were predominant in the intergenic regions. Several hundred genes had different DNA methylation patterns and expressions in different cold treatments (cold memory and cold shock), such as CuAO, RPB1, and DHE1. The application of a DNA methylation inhibitor caused a change of the free lysine content, suggesting that DNA methylation can affect metabolite accumulation for Tartary buckwheat cold responses. The results of the present study suggest important roles of DNA methylation in regulating cold response and forming agronomic traits in Tartary buckwheat.

Immunochemical Detection of Modified Cytosine Species in Mammalian Preimplantation Embryos.

DNA methylation undergoes dynamic changes at the genome-wide scale during the early steps of mammalian embryo development. Immunochemical detection of 5-methylcytosine (5mC) in the zygote has led to the discovery that a global loss of DNA methylation takes place soon after fertilization, occurring rapidly in the paternal pronucleus. Using the same method employed above, which detects modified bases in the denatured single stranded DNA, we showed that this active DNA "demethylation" in the paternal pronucleus involves oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) by the TET3 enzyme. By immunostaining of genetically altered zygotes we revealed that the maternal pronucleus is protected from TET3-mediated oxidation by histone H3K9 methyltransferase enzymes, EHMT2 and SETDB1. The same assays are also applicable for visualizing the temporal and spatial distribution of the modified cytosine residues in preimplantation embryos. Here, we provide a detailed protocol for detecting 5mC, 5hmC, 5fC, and 5caC in mouse zygotes and preimplantation-stage embryos using antibodies raised against modified cytosine species.

The loss of global DNA methylation due to decreased DNMT expression in the postnatal mouse ovaries may associate with infertility emerging during ovarian aging.

Ovarian aging is one of the main causes of female infertility, and its molecular background is still largely unknown. As DNA methylation regulates many oogenesis/folliculogenesis-related genes, the expression levels and cellular localizations of DNA methyltransferases (DNMTs) playing key roles in this process is important in the ovaries from early to aged terms. In the present study, we aimed to evaluate the spatial and temporal expression of the Dnmt1, Dnmt3a, Dnmt3b, and Dnmt3l genes as well as global DNA methylation levels in the mouse ovaries during aging. For this purpose, the following groups were created: young (1- and 2-week old; n = 3 from each week), prepubertal (3- and 4-week-old; n = 3 from each week), pubertal (5- and 6-week-old; n = 3 from each week), postpubertal (16- and 18-week-old; n = 3 from each week), and aged (52-, 60- and 72-week-old; n = 3 from each week). We found here that Dnmt1, Dnmt3a, and Dnmt3l genes' expression at mRNA and protein levels as well as global DNA methylation profiles were gradually and significantly decreased in the postnatal ovaries from young to aged groups (P < 0.05). In contrast, there was a remarkable increase of Dnmt3b expression in the pubertal, postpubertal and aged groups (P < 0.05). Our findings suggest that the significantly altered DNMT expression and global DNA methylation levels during ovarian aging may contribute to female infertility development at the later terms of lifespan. Also, new researches are required to determine the molecular biological mechanism(s) that how altered DNMT expression and decreased DNA methylation lead to ovarian aging.

Transposable element expression in tumors is associated with immune infiltration and increased antigenicity

Profound global loss of DNA methylation is a hallmark of many cancers. One potential consequence of this is the reactivation of transposable elements (TEs) which could stimulate the immune system via cell-intrinsic antiviral responses. Here, we develop REdiscoverTE, a computational method for quantifying genome-wide TE expression in RNA sequencing data. Using The Cancer Genome Atlas database, we observe increased expression of over 400 TE subfamilies, of which 262 appear to result from a proximal loss of DNA methylation. The most recurrent TEs are among the evolutionarily youngest in the genome, predominantly expressed from intergenic loci, and associated with antiviral or DNA damage responses. Treatment of glioblastoma cells with a demethylation agent results in both increased TE expression and de novo presentation of TE-derived peptides on MHC class I molecules. Therapeutic reactivation of tumor-specific TEs may synergize with immunotherapy by inducing inflammation and the display of potentially immunogenic neoantigens.

Abstract 839: Whole genome DNA methylation analysis of multiple myeloma identifies pervasive hypomethylation and biomarkers of survival

Abstract Multiple myeloma is a malignancy of terminally differentiated, antibody secreting B cells known as plasma cells. Normal plasma cell differentiation and cell fate are coupled to epigenetic and transcriptional reprogramming, including a proliferation-dependent global loss of DNA methylation. However, relatively little is known about the epigenetic changes that underlie myelomagenesis and how these contribute to disease etiology. To this end, we have analyzed the DNA methylome of 119 myeloma specimens from the CoMMpass study (NCT01454297) by whole genome bisulfite sequencing (WGBS) and more than 90% of these same specimens were also characterized for structural variants (long-insert whole genome sequencing) and gene expression (RNA-seq). Unsupervised hierarchical clustering grouped together specimens with t(4;14) translocations that upregulate the H3K36 dimethyltransferase NSD2 (also known as MMSET and WHSC1), which likely impacts the DNA methylation state through epigenetic cross-talk. These data also revealed a dramatic genome-wide hypomethylation where myeloma samples had a median global CpG methylation level of 41% as compared to 71% and 89% in normal plasma cells and B cells, respectively. Demethylation of the myeloma methylome occurred in large megabase domains encompassing genes that were devoid of gene expression. Conversely, DNA methylation remained mostly unchanged in the bodies of genes that were highly expressed. Although the majority of these hypomethylated domains were common across myelomas, many regions of variable methylation exist and these differences corresponded with proximal gene expression differences. These variably methylated regions were compared to PFS and OS and this identified 6,314 CpG loci where the level of DNA methylation was prognostic of outcome (P≤0.00001). These loci were clustered into discrete regions and in the majority of cases (79%), reduced DNA methylation at these loci corresponded with poor outcome. For example, several loci in the gene bodies of PRKCE, MGMT, FHIT, WWOX were prognostic of poor survival. Interestingly, myeloma t(14;16) translocations disrupt the tumor suppressor WWOX and induce the oncogene MAF. These data suggest that WWOX expression may also be lost by epigenetic mechanisms. Finally, we analyzed the DNA methylome of primary and relapsed samples for 22 patients, including multiple relapsed samples for 2 patients. These data identified genome-wide DNA methylation remodeling with a median of 1.9 million differential methylated CpGs between the newly diagnosed and relapsed specimens. These relapse differentially methylated loci coincided at the same regions in several patients and significantly overlapped loci where the DNA methylation level was prognostic of outcome. Integrative genetic, epigenetic, and transcriptional analyses for 120 myeloma samples will be presented. Citation Format: Benjamin G. Barwick, Doris R. Powell, Daniel Penaherrera, Sheri Skerget, Jonathan J. Keats, Daniel Auclair, Sagar Lonial, Lawrence H. Boise, Paula M. Vertino. Whole genome DNA methylation analysis of multiple myeloma identifies pervasive hypomethylation and biomarkers of survival [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 839.

Partially methylated domains are hypervariable in breast cancer and fuel widespread CpG island hypermethylation

Global loss of DNA methylation and CpG island (CGI) hypermethylation are key epigenomic aberrations in cancer. Global loss manifests itself in partially methylated domains (PMDs) which extend up to megabases. However, the distribution of PMDs within and between tumor types, and their effects on key functional genomic elements including CGIs are poorly defined. We comprehensively show that loss of methylation in PMDs occurs in a large fraction of the genome and represents the prime source of DNA methylation variation. PMDs are hypervariable in methylation level, size and distribution, and display elevated mutation rates. They impose intermediate DNA methylation levels incognizant of functional genomic elements including CGIs, underpinning a CGI methylator phenotype (CIMP). Repression effects on tumor suppressor genes are negligible as they are generally excluded from PMDs. The genomic distribution of PMDs reports tissue-of-origin and may represent tissue-specific silent regions which tolerate instability at the epigenetic, transcriptomic and genetic level.

Abstract P5-04-03: Molecular characterisation of ER+ breast cancer dormancy and acquired resistance using a clinical model: Potential involvement of epigenetic regulation

Abstract Background: The risk of recurrence for oestrogen receptor positive (ER+) breast cancer patients treated with 5 years of adjuvant endocrine therapy persists for many years or even decades following surgery and apparently successful adjuvant therapy. This period of dormancy and acquired resistance is inherently difficult to investigate. Therefore, previous efforts have been limited to in vitro or in vivo approaches. In this study sequential tumour samples from patients receiving extended neoadjuvant endocrine treatment were characterised as a novel clinical model of ER+ breast cancer dormancy and acquired resistance. Methods: Consecutive tumour samples from 62 patients undergoing extended (4-45 months) neoadjuvant letrozole therapy were subjected to transcriptomic and proteomic analysis, representing pre- (before treatment), early-on (13-120 days) and long-term (&amp;gt;120 days) neoadjuvant letrozole treatment. Patients with at least a 40% initial reduction in tumour size by 4 months of treatment were included. Of these, 42 patients with no subsequent progression were classified as “dormant”, and the remaining 20 patients as “acquired resistant”. Expression analysis was performed by using Illumina BeadChips. R and BioConductor packages were used for analysis. Differentially expressed genes were determined by using paired Rank Products (FDR, 5%). Results: Multidimensional scaling using most variant 500 genes demonstrated that long-term treated dormant samples clustered separately from their matched pre- and early-on samples whereas long-term treated resistant samples were indistinguishable from their pre-treatment counterparts. Therapy-induced changes in resistant tumours were common features of treatment, rather than being specific to resistant phenotype. Comparative analysis of long-term treated dormant and resistant tumours highlighted changes in epigenetics pathways including DNA methylation and histone acetylation. DNA methylation marks 5-methylcytosine and 5-hydroxymethylcytosine were significantly reduced in resistant tumours compared to dormant tissues after extended letrozole treatment. Decrease in 5-hydroxymethylcytosine were significant early-on. Conclusions: This is the first patient-matched gene expression study investigating long-term aromatase inhibitor-induced dormancy and acquired resistance in breast cancer. Dormant tumors exhibit distinct molecular changes under extended treatment whereas acquired resistant tumors are more similar to matched diagnostic samples supporting the molecular concordance between primary tumors and metastases. Global loss of DNA methylation was observed in resistant tumours under extended treatment which can be predicted within first 4 months of therapy. Epigenetic alterations may lead to escape from dormancy and drive acquired resistance in a subset of patients supporting a potential role for therapy targeted at these epigenetic alterations in the management of endocrine resistant breast cancer. Funding: This work was supported by Marie Skłodowska-Curie Individual Fellowship [H2020-MSCA-IF, 658170] and Welcome Trust Institutional Fund (ISSF3) to CS and AHS, Breast Cancer Now to AHS. Citation Format: Selli C, Turnbull AK, Pearce D, Fernando A, Renshaw L, Thomas JS, Dixon MJ, Sims AH. Molecular characterisation of ER+ breast cancer dormancy and acquired resistance using a clinical model: Potential involvement of epigenetic regulation [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-03.

Global increase in DNA methylation during orange fruit development and ripening

DNA methylation is an important epigenetic mark involved in many biological processes. The genome of the climacteric tomato fruit undergoes a global loss of DNA methylation due to active DNA demethylation during the ripening process. It is unclear whether the ripening of other fruits is also associated with global DNA demethylation. We characterized the single-base resolution DNA methylomes of sweet orange fruits. Compared with immature orange fruits, ripe orange fruits gained DNA methylation at over 30,000 genomic regions and lost DNA methylation at about 1,000 genomic regions, suggesting a global increase in DNA methylation during orange fruit ripening. This increase in DNA methylation was correlated with decreased expression of DNA demethylase genes. The application of a DNA methylation inhibitor interfered with ripening, indicating that the DNA hypermethylation is critical for the proper ripening of orange fruits. We found that ripening-associated DNA hypermethylation was associated with the repression of several hundred genes, such as photosynthesis genes, and with the activation of hundreds of genes, including genes involved in abscisic acid responses. Our results suggest important roles of DNA methylation in orange fruit ripening.

Molecular changes during extended neoadjuvant letrozole treatment of breast cancer: distinguishing acquired resistance from dormant tumours

BackgroundThe risk of recurrence for endocrine-treated breast cancer patients persists for many years or even decades following surgery and apparently successful adjuvant therapy. This period of dormancy and acquired resistance is inherently difficult to investigate; previous efforts have been limited to in-vitro or in-vivo approaches. In this study, sequential tumour samples from patients receiving extended neoadjuvant aromatase inhibitor therapy were characterised as a novel clinical model.MethodsConsecutive tumour samples from 62 patients undergoing extended (4–45 months) neoadjuvant aromatase inhibitor therapy with letrozole were subjected to transcriptomic and proteomic analysis, representing before (≤ 0), early (13–120 days), and long-term (> 120 days) neoadjuvant aromatase inhibitor therapy with letrozole. Patients with at least a 40% initial reduction in tumour size by 4 months of treatment were included. Of these, 42 patients with no subsequent progression were classified as “dormant”, and the remaining 20 patients as “acquired resistant”.ResultsChanges in gene expression in dormant tumours begin early and become more pronounced at later time points. Therapy-induced changes in resistant tumours were common features of treatment, rather than being specific to the resistant phenotype. Comparative analysis of long-term treated dormant and resistant tumours highlighted changes in epigenetics pathways including DNA methylation and histone acetylation. The DNA methylation marks 5-methylcytosine and 5-hydroxymethylcytosine were significantly reduced in resistant tumours compared with dormant tissues after extended letrozole treatment.ConclusionsThis is the first patient-matched gene expression study investigating long-term aromatase inhibitor-induced dormancy and acquired resistance in breast cancer. Dormant tumours continue to change during treatment whereas acquired resistant tumours more closely resemble their diagnostic samples. Global loss of DNA methylation was observed in resistant tumours under extended treatment. Epigenetic alterations may lead to escape from dormancy and drive acquired resistance in a subset of patients, supporting a potential role for therapy targeted at these epigenetic alterations in the management of resistance to oestrogen deprivation therapy.

Whole Genome DNA Methylation Analysis of Commpass Identifies Biomarkers of Multiple Myeloma Survival

Multiple myeloma is a malignancy of terminally differentiated, antibody secreting B cells known as plasma cells. Normal B cell differentiation and cell fate are coupled to epigenetic and transcriptional reprogramming, including a proliferation-dependent global loss of DNA methylation (Barwick et al., 2016, 2018). However, relatively little is known about the epigenetic changes that underlie myelomagenesis and how these may contribute to pathogenesis. To this end, we are analyzing the DNA methylome of myeloma specimens from the MMRF CoMMpass trial (NCT01454297), which has already characterized the mutational, structural, and transcriptional landscape of nearly 1,000 myelomas from newly diagnosed patients.CoMMpass specimens were obtained from a centralized biobank with approval from the CoMMpass Tissue Use Committee and Emory IRB. DNA isolated from CD138+ myeloma specimens was subjected to reduced representation bisulfite sequencing (RRBS) or whole genome bisulfite sequencing (WGBS). In total, DNA methylation was derived for over 24 million CpGs with an average of 18x coverage. WGBS data from normal B cells and plasma cells was obtained with permission from the BluePrint project (Agirre et al., 2015) via the European Genome Archive. DNA methylation levels were associated with PFS and OS using a cox proportional regression.We have determined the DNA methylome for 36 primary myeloma specimens and an additional 84 specimens are currently being sequenced. Relative to normal B cells that had an average DNA methylation level of 89.1%, plasma cells and myelomas exhibited a progressive demethylation with mean levels of 71.3% and 43.7%, respectively. While this is consistent with previous observations (Agirre et al., 2015; Salhia et al., 2010), WGBS revealed that myeloma in particular was characterized by large hypomethylated domains. These large hypomethylated domains encompassed genes that were devoid of gene expression whereas DNA methylation remained unchanged in the bodies of genes that were highly expressed. Although the majority of these hypomethylated domains were common across myelomas, there existed many regions where methylation levels varied between myelomas and these differences commonly corresponded with local gene expression differences. To understand if these specific patterns of DNA methylation were indicative of disease pathogenesis, DNA methylation levels were compared to PFS and OS. This identified 2,594 regions where the level of DNA methylation was prognostic of outcome (P≤0.001). Reduced DNA methylation corresponded with poor outcome at 88.5% (N=2,298) of these regions, which included loci proximal to cell cycle genes such as MYC, E2F3, CCND1, and CCNE1. Only 11.5% (N=296) of regions associated with outcome had higher levels of DNA methylation associated with poor prognosis. These regions tended to be proximal to genes involved in B cell receptor signaling, such as PLCG2 and VAV2. Although the expression of several of these genes was also prognostic of survival, the majority were not, indicating that the epigenetic state contains a unique prognostic value.These data indicate that myeloma undergoes profound epigenetic remodeling that is co-ordinate with changes in gene expression. Perhaps the most striking feature were megabase domains of hypomethylation. That DNA methylation was preferentially retained in the bodies of expressed genes suggests that a molecular mechanism and/or cellular selection occurs to maintain methylation at genes whose expression is required for myeloma cell survival. Despite the small number (N=36) of myeloma specimens analyzed thus far, the large number of regions associated with survival indicates the potential prognostic value of DNA methylation in myeloma. Furthermore, DNA methylation indicative of outcome only partially overlapped with the prognostic value of gene expression, indicating DNA methylation has independent value as a biomarker of outcome in myeloma. This may be due, in part, to the fact that DNA methylation is a very stable modification that not only reflects the current gene expression program, but is also indicative of the cell history and potential. Integrative genetic, epigenetic, and transcriptional analysis from WGBS of 120 CoMMpass myeloma specimens will be presented, including matched baseline and relapsed specimens from 25 patients. DisclosuresLonial:Amgen: Research Funding. Boise:Abbvie: Consultancy; AstraZeneca: Honoraria.

Marked Global DNA Hypomethylation Is Associated with Constitutive PD-L1 Expression in Melanoma

SummaryConstitutive expression of the immune checkpoint, PD-L1, inhibits anti-tumor immune responses in cancer, although the factors involved in PD-L1 regulation are poorly understood. Here we show that loss of global DNA methylation, particularly in intergenic regions and repeat elements, is associated with constitutive (PD-L1CON), versus inducible (PD-L1IND), PD-L1 expression in melanoma cell lines. We further show this is accompanied by transcriptomic up-regulation. De novo epigenetic regulators (e.g., DNMT3A) are strongly correlated with PD-L1 expression and methylome status. Accordingly, decitabine-mediated inhibition of global methylation in melanoma cells leads to increased PD-L1 expression. Moreover, viral mimicry and immune response genes are highly expressed in lymphocyte-negative plus PD-L1-positive melanomas, versus PD-L1-negative melanomas in The Cancer Genome Atlas (TCGA). In summary, using integrated genomic analysis we identified that global DNA methylation influences PD-L1 expression in melanoma, and hence melanoma's ability to evade anti-tumor immune responses. These results have implications for combining epigenetic therapy with immunotherapy.

Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation

Here we interrogated, using three separate but complementary experimental approaches, the impact of vitamin B12 availability and methotrexate exposure on Daphnia magna, which we hypothesised should have an opposite effect on One carbon metabolism (OCM). OCM is a vital biological process supporting a variety of physiological processes, including DNA methylation. Contrary to mammalian models, this process remains largely unexplored in invertebrates. The purpose of this study was to elucidate the impact of OCM short-term alteration on the fitness and epigenome of the keystone species, Daphnia. We used maternal age at reproduction, brood size and survival rates in combination with DNA methylation sensitive comet assay to determine the effects of vitamin B12 or MTX on fitness and the epigenome. Vitamin B12 had a positive influence on Daphnia fitness and we provide evidence demonstrating that this may be associated with an increased level of genome-wide DNA methylation. Conversely, exposing D. magna to MTX negatively influenced the fitness of the animals and was associated with loss of global DNA methylation, translating in decreased fitness. These results highlight the potential importance of OCM in invertebrates, providing novel evidence supporting a potential role for epigenetic modifications to the genome in D. magna environmental adaptability.

Derivation of ground-state female ES cells maintaining gamete-derived DNA methylation.

Inhibitors of Mek1/2 and Gsk3β, known as 2i, enhance the derivation of embryonic stem (ES) cells and promote ground-state pluripotency in rodents. Here we show that the derivation of female mouse ES cells in the presence of 2i and leukaemia inhibitory factor (2i/L ES cells) results in a widespread loss of DNA methylation, including a massive erasure of genomic imprints. Despite this global loss of DNA methylation, early-passage 2i/L ES cells efficiently differentiate into somatic cells, and this process requires genome-wide de novo DNA methylation. However, the majority of imprinting control regions (ICRs) remain unmethylated in 2i/L-ES-cell-derived differentiated cells. Consistently, 2i/L ES cells exhibit impaired autonomous embryonic and placental development by tetraploid embryo complementation or nuclear transplantation. We identified the derivation conditions of female ES cells that display 2i/L-ES-cell-like transcriptional signatures while preserving gamete-derived DNA methylation and autonomous developmental potential. Upon prolonged culture, however, female ES cells exhibited ICR demethylation regardless of culture conditions. Our results provide insights into the derivation of female ES cells reminiscent of the inner cell mass of preimplantation embryos.