BACKGROUND: The clonal evolution of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast, tumor epigenetic states are reversible and sensitive to the tumor microenvironment, presumably precluding the use of epigenetics to discover the clonal and sub-clonal evolution of tumors. We recently used mutation patterns to learn how low grade gliomas evolve over time, and also discovered that temozolomide treatment of LGG is associated with TMZ-induced hypermutation and malignant progression to GBM (BE Johnson et al, Science, 2014). METHODS: We used exome and RNA sequencing, DNA methylation arrays, along with computational analysis to construct phylogenetic and phyloepigenetic relationships among co-evolving tumor cell populations. RESULTS: Here we examined the spatial and temporal dynamics of DNA methylation in our clinically and genetically characterized cohort of IDH1-mutant low-grade gliomas and their patient-matched recurrences. While all tumors exhibited the hypermethylation signature associated with IDH1 mutation, low-grade gliomas that underwent spontaneous or temozolomide-associated malignant progression to high-grade glioblastoma multiforme (GBM) had a unique signature of DNA hypomethylation enriched for both active enhancers, and sites of age-related hypermethylation in the brain. Genes with promoter hypomethylation and concordant transcriptional upregulation during evolution to GBM were enriched in cell cycle function, evolving in concert with genetic alterations that deregulate the G1/S cell cycle checkpoint. Despite the plasticity of tumor epigenetic states, phyloepigenetic relationships robustly recapitulated phylogenetic patterns inferred from somatic mutations in the same patients. CONCLUSIONS: These findings highlight widespread co-dependency of genetic and epigenetic events throughout the clonal evolution of initial and recurrent gliomas.
The evolutionary history of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast to stable genetic events, epigenetic states are reversible and sensitive to the microenvironment, prompting the question whether epigenetic information can similarly be used to discover tumor phylogeny. We examined the spatial and temporal dynamics of DNA methylation in a cohort of low-grade gliomas and their patient-matched recurrences. Genes transcriptionally upregulated through promoter hypomethylation during malignant progression to high-grade glioblastoma were enriched in cell cycle function, evolving in parallel with genetic alterations that deregulate the G1/S cell cycle checkpoint. Moreover, phyloepigenetic relationships robustly recapitulated phylogenetic patterns inferred from somatic mutations. These findings highlight widespread co-dependency of genetic and epigenetic events throughout brain tumor evolution.
Hypoxia-Inducible Gene Domain Family Member 1A (HIGD1A) is a survival factor induced by Hypoxia-inducible Factor-1 (HIF1). HIF1 regulates many responses to oxygen deprivation but viable cells within hypoxic perinecrotic solid tumor regions frequently lack HIF1α. HIGD1A is induced in these HIF-deficient extreme environments and interacts with the mitochondrial electron transport chain to repress oxygen consumption, enhance AMPK activity and lower cellular ROS levels. Importantly, HIGD1A decreases tumor growth but promotes tumor cell survival in vivo. The human Higd1a gene is located on chromosome 3p22.1, where many tumor suppressor genes reside. Consistent with this, the Higd1a gene promoter is differentially methylated in human cancers, preventing its hypoxic induction. When hypoxic tumor cells are confronted with glucose deprivation, however, DNA methyltransferase activity is inhibited, enabling HIGD1A expression, metabolic adaptation and possible dormancy induction. Our findings therefore reveal important new roles for this family of mitochondrial proteins in cancer biology.
Analysis of the 3D structure of DNA in tumour cells reveals how mutations
in the IDH1 gene, and associated changes in methyl groups
attached to DNA, elevate the expression of cancer-promoting genes.
Mutations in isocitrate dehydrogenase 1 (IDH1) are characteristic of low-grade gliomas. We recently showed that mutant IDH1 cells reprogram cellular metabolism by down-regulating pyruvate dehydrogenase (PDH) activity. Reduced pyruvate metabolism via PDH could lead to increased pyruvate conversion to lactate. The goal of this study was therefore to investigate the impact of the IDH1 mutation on the pyruvate-to-lactate flux. We used 13C magnetic resonance spectroscopy and compared the conversion of hyperpolarized [1-13C]-pyruvate to [1-13C]-lactate in immortalized normal human astrocytes expressing mutant or wild-type IDH1 (NHAIDHmut and NHAIDHwt). Our results indicate that hyperpolarized lactate production is reduced in NHAIDHmut cells compared to NHAIDHwt. This reduction was associated with lower expression of the monocarboxylate transporters MCT1 and MCT4 in NHAIDHmut cells. Furthermore, hyperpolarized lactate production was comparable in lysates of NHAIDHmut and NHAIDHwt cells, wherein MCTs do not impact hyperpolarized pyruvate delivery and lactate production. Collectively, our findings indicated that lower MCT expression was a key contributor to lower hyperpolarized lactate production in NHAIDHmut cells. The SLC16A3 (MCT4) promoter but not SLC16A1 (MCT1) promoter was hypermethylated in NHAIDHmut cells, pointing to possibly different mechanisms mediating reduced MCT expression. Finally analysis of low-grade glioma patient biopsy data from The Cancer Genome Atlas revealed that MCT1 and MCT4 expression was significantly reduced in mutant IDH1 tumors compared to wild-type. Taken together, our study shows that reduced MCT expression is part of the metabolic reprogramming of mutant IDH1 gliomas. This finding could impact treatment and has important implications for metabolic imaging of mutant IDH1 gliomas.
MCT1; MCT4; mutant IDH1; metabolic reprogramming; magnetic resonance spectroscopy
Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm (MPN) of childhood with a poor prognosis. Mutations in NF1, NRAS, KRAS, PTPN11 and CBL occur in 85% of patients, yet there are currently no risk stratification algorithms capable of predicting which patients will be refractory to conventional treatment and therefore be candidates for experimental therapies. In addition, there have been few other molecular pathways identified aside from the Ras/MAPK pathway to serve as the basis for such novel therapeutic strategies. We therefore sought to genomically characterize serial samples from patients at diagnosis through relapse and transformation to acute myeloid leukemia in order to expand our knowledge of the mutational spectrum in JMML. We identified recurrent mutations in genes involved in signal transduction, gene splicing, the polycomb repressive complex 2 (PRC2) and transcription. Importantly, the number of somatic alterations present at diagnosis appears to be the major determinant of outcome.
Temozolomide (TMZ) increases the overall survival of patients with glioblastoma (GBM), but its role in the clinical management of diffuse low-grade gliomas (LGG) is still being defined. DNA hypermethylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter is associated with an improved response to TMZ treatment, while inactivation of the DNA mismatch repair (MMR) pathway is associated with therapeutic resistance and TMZ-induced mutagenesis. We previously demonstrated that TMZ treatment of LGG induces driver mutations in the RB and AKT-mTOR pathways, which may drive malignant progression to secondary GBM. To better understand the mechanisms underlying TMZ-induced mutagenesis and malignant progression, we explored the evolution of MGMT methylation and genetic alterations affecting MMR genes in a cohort of 34 treatment naïve LGGs and their recurrences. Recurrences with TMZ-associated hypermutation had increased MGMT methylation compared to their untreated initial tumors and higher overall MGMT methylation compared to TMZ-treated non-hypermutated recurrences. A TMZ-associated mutation in one or more MMR genes was observed in five out of six TMZ treated, hypermutated recurrences. In two cases, pre-existing heterozygous deletions encompassing MGMT, or an MMR gene, were followed by TMZ-associated mutations in one of the genes of interest. These results suggest that tumor cells with methylated MGMT may undergo positive selection during TMZ treatment in the context of MMR deficiency.
Low-grade glioma; Temozolomide; Hypermutator; Mismatch Repair; MGMT
DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Many studies have mapped DNA methylation changes associated with embryogenesis, cell differentiation, and cancer at a genome-wide scale. Our understanding of genome-wide DNA methylation changes in a developmental or disease-related context has been steadily growing. However, the investigation of which CpGs are variably methylated in different normal cell or tissue types is still limited. Here, we present an in-depth analysis of 54 single-CpG-resolution DNA methylomes of normal human cell types by integrating high-throughput sequencing-based methylation data. We found that the ratio of methylated to unmethylated CpGs is relatively constant regardless of cell type. However, which CpGs made up the unmethylated complement was cell-type specific. We categorized the 26,000,000 human autosomal CpGs based on their methylation levels across multiple cell types to identify variably methylated CpGs and found that 22.6% exhibited variable DNA methylation. These variably methylated CpGs formed 660,000 variably methylated regions (VMRs), encompassing 11% of the genome. By integrating a multitude of genomic data, we found that VMRs enrich for histone modifications indicative of enhancers, suggesting their role as regulatory elements marking cell type specificity. VMRs enriched for transcription factor binding sites in a tissue-dependent manner. Importantly, they enriched for GWAS variants, suggesting that VMRs could potentially be implicated in disease and complex traits. Taken together, our results highlight the link between CpG methylation variation, genetic variation, and disease risk for many human cell types.
DNA methylomes; methylCRF; methylation dynamics; regulatory elements
Glioblastoma multiforme (GBM) contains a population of cells that exhibit stem cell phenotypes. These cancer stem cells (CSCs) may be a source of therapeutic resistance, although support for this important concept is limited.
We determined whether early-passage GBM CSCs respond differently than patient-matched, genotypically similar non-CSCs to clinically relevant single or serial doses of temozolomide (TMZ), radiation therapy (XRT), or alternating TMZ treatment and XRT, which is the standard of care for GBM patients.
Despite the phenotypic differences, including the presence of stem cell markers and formation of intracranial tumors, the CSCs and matched non-CSCs were equally resistant to TMZ in a majority of patients, using 2 independent assays. TMZ response was consistent with methylated O6-DNA methylguanine-methyltransferase (MGMT) and MGMT protein levels in both culture types. In contrast, CSCs were unexpectedly more responsive to XRT compared with matched non-CSCs from 2 patients despite having relatively equal resistance to TMZ. However, for the majority of culture pairs from individual patients, responses in CSCs were indistinguishable from non-CSC cultures.
In our patient-matched primary cultures, response to TMZ was tightly linked to the individual tumor's MGMT status and independent of their phenotypic differences. TMZ and XRT together revealed no additive benefit compared with monotherapy for either culture type, in contrast to the notion that the CSC population is more resistant to XRT. If the tumor cell response in vitro mirrors therapeutic response in larger patient cohorts, these rapid assays in primary cultures could allow empirical selection of efficacious therapeutic agents on a patient-specific basis.
cancer stem cell; glioblastoma multiforme; radiation response
The clonal evolution of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast, tumor epigenetic states, including DNA methylation, are reversible and sensitive to the tumor microenvironment, presumably precluding the use of epigenetics to discover tumor phylogeny. Here we examined the spatial and temporal dynamics of DNA methylation in a clinically and genetically characterized cohort of IDH1-mutant low-grade gliomas and their patient-matched recurrences. WHO grade II gliomas are diffuse, infiltrative tumors that frequently recur and may undergo malignant progression to a higher grade with a worse prognosis. The extent to which epigenetic alterations contribute to the evolution of low-grade gliomas, including malignant progression, is unknown. While all gliomas in the cohort exhibited the hypermethylation signature associated with IDH1 mutation, low-grade gliomas that underwent malignant progression to high-grade glioblastoma (GBM) had a unique signature of DNA hypomethylation enriched for active enhancers, as well as sites of age-related hypermethylation in the brain. Genes with promoter hypomethylation and concordant transcriptional upregulation during evolution to GBM were enriched in cell cycle function, evolving in concert with genetic alterations that deregulate the G1/S cell cycle checkpoint. Despite the plasticity of tumor epigenetic states, phyloepigenetic trees robustly recapitulated phylogenetic trees derived from somatic mutations in the same patients. These findings highlight widespread co-dependency of genetic and epigenetic events throughout the clonal evolution of initial and recurrent glioma.
Regulation of mRNA splicing, a critical and tightly regulated cellular function, underlies the majority of proteomic diversity, and is frequently disrupted in disease. Using an integrative genomics approach, we combined both genome and exon level transcriptome data in two somatic tissues (cerebella and peripheral ganglia) from a transgenic mouse model of neuroblastoma, a tumor that arises from peripheral neural crest. Here we describe splicing quantitative trait loci (sQTL) associated with differential splicing across the genome that we use to identify genes with previously unknown functions within the splicing pathway and to define de novo intronic splicing motifs that influence splicing from hundreds of bases away. Our results show that these splicing motifs represent sites for functional recurrent mutations and highlight novel candidate genes in human cancers, including childhood neuroblastoma.
Alternative splicing; neuroblastoma; sQTL; MYC; FUBP1
While the methylation of DNA in 5′ promoters suppresses gene expression, the role of DNA methylation in gene bodies is unclear1–5. In mammals, tissue- and cell type-specific methylation is present in a small percentage of 5′ CpG island (CGI) promoters, while a far greater proportion occurs across gene bodies, coinciding with highly conserved sequences5–10. Tissue-specific intragenic methylation might reduce,3 or, paradoxically, enhance transcription elongation efficiency1,2,4,5. Capped analysis of gene expression (CAGE) experiments also indicate that transcription commonly initiates within and between genes11–15. To investigate the role of intragenic methylation, we generated a map of DNA methylation from human brain encompassing 24.7 million of the 28 million CpG sites. From the dense, high-resolution coverage of CpG islands, the majority of methylated CpG islands were revealed to be in intragenic and intergenic regions, while less than 3% of CpG islands in 5′ promoters were methylated. The CpG islands in all three locations overlapped with RNA markers of transcription initiation, and unmethylated CpG islands also overlapped significantly with trimethylation of H3K4, a histone modification enriched at promoters16. The general and CpG-island-specific patterns of methylation are conserved in mouse tissues. An in-depth investigation of the human SHANK3 locus17,18 and its mouse homologue demonstrated that this tissue-specific DNA methylation regulates intragenic promoter activity in vitro and in vivo. These methylation-regulated, alternative transcripts are expressed in a tissue and cell type-specific manner, and are expressed differentially within a single cell type from distinct brain regions. These results support a major role for intragenic methylation in regulating cell context-specific alternative promoters in gene bodies.
Intragenic DNA methylation; alternate promoters; comparative epigenomics; SHANK3
The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but a similar reference has lacked for epigenomic studies. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection to-date of human epigenomes for primary cells and tissues. Here, we describe the integrative analysis of 111 reference human epigenomes generated as part of the program, profiled for histone modification patterns, DNA accessibility, DNA methylation, and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically-relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation, and human disease.
Summary: We present methylC track, an efficient mechanism for visualizing single-base resolution DNA methylation data on a genome browser. The methylC track dynamically integrates the level of methylation, the position and context of the methylated cytosine (i.e. CG, CHG and CHH), strand and confidence level (e.g. read coverage depth in the case of whole-genome bisulfite sequencing data). Investigators can access and integrate these information visually at specific locus or at the genome-wide level on the WashU EpiGenome Browser in the context of other rich epigenomic datasets.
Availability and implementation: The methylC track is part of the WashU EpiGenome Browser, which is open source and freely available at http://epigenomegateway.wustl.edu/browser/. The most up-to-date instructions and tools for preparing methylC track are available at http://epigenomegateway.wustl.edu/+/cmtk.
Supplementary data are available at Bioinformatics online.
Both the epidermal growth factor receptor and vascular endothelial growth factor pathways are frequently overexpressed in glioblastoma multiforme. This study combined bevacizumab, a vascular endothelial growth factor inhibitor, and erlotinib, an epidermal growth factor receptor inhibitor, with standard radiation and temozolomide (TMZ), with the goal of improving overall survival (OS).
Treatment consisted of fractionated radiotherapy to 60 Gy, with daily TMZ at 75 mg/m2/d and erlotinib 150–200 mg/d (or 500–600 mg/d for patients on enzyme-inducing antiepileptic drugs). Bevacizumab was given at 10 mg/kg every 2 weeks, starting ≥4 weeks after surgery. After radiotherapy, adjuvant TMZ was given at 200 mg/m2/d × 5d per 28-day cycle, with unchanged erlotinib and bevacizumab doses. Treatment continued until progression or for 12 months. Efficacy was compared against an institutional historical control. A sample of 55 patients was calculated to provide 85% power to detect a hazard ratio of 0.67 for OS.
Fifty-nine patients were enrolled for efficacy analysis after a 15-patient safety lead-in. For the efficacy group, median age was 54 years; median KPS was 90. Gross total and subtotal resections were achieved in 33% and 53%, respectively. The most frequent related grade 3/4 adverse effects were lymphopenia, thrombocytopenia, neutropenia, diarrhea, weight loss, and fatigue. One patient died of disseminated aspergillosis. Median OS was 19.8 months (vs 18 mo for HC, P = .33) and median progression-free survival was 13.5 months (vs 8.6 mo for HC, P = .03).
The combination of bevacizumab, erlotinib, TMZ, and radiotherapy appears to be well tolerated and improved progression-free survival but did not reach the primary endpoint of improved OS.
bevacizumab; erlotinib; glioblastoma; radiation; temozolomide
Reactivation of telomerase reverse transcriptase (TERT) expression enables cells to overcome replicative senescence and escape apoptosis, fundamental steps in the initiation of human cancer. Multiple cancer types, including up to 83% of glioblastomas (GBM), harbor highly recurrent TERT promoter mutations of unknown function but specific to two nucleotide positions. We identify the functional consequence of these mutations in GBM to be recruitment of the multimeric GABP transcription factor specifically to the mutant promoter. Allelic recruitment of GABP is consistently observed across four cancer types, highlighting a shared mechanism underlying TERT reactivation. Tandem flanking native ETS motifs critically cooperate with these mutations to activate TERT, likely by facilitating GABP heterotetramer binding. GABP thus directly links TERT promoter mutations to aberrant expression in multiple cancers.
Developmental history shapes the epigenome and biological function of differentiated cells. Epigenomic patterns have been broadly attributed to the three embryonic germ layers. Here we investigate how developmental origin influences epigenomes. We compare key epigenomes of cell types derived from surface ectoderm (SE), including keratinocytes and breast luminal and myoepithelial cells, against neural crest-derived melanocytes and mesoderm-derived dermal fibroblasts to identify SE differentially methylated regions (SE-DMRs). DNA methylomes of neonatal keratinocytes share many more DMRs with adult breast luminal and myoepithelial cells than with melanocytes and fibroblasts from the same neonatal skin. This suggests that SE origin contributes to DNA methylation patterning, while shared skin tissue environment has limited effect on epidermal keratinocytes. Hypomethylated SE-DMRs are in proximity to genes with SE relevant functions. They are also enriched for enhancer- and promoter-associated histone modifications in SE-derived cells, and for binding motifs of transcription factors important in keratinocyte and mammary gland biology. Thus, epigenomic analysis of cell types with common developmental origin reveals an epigenetic signature that underlies a shared gene regulatory network.
The role of intermediate methylation states in DNA is unclear. Here, to comprehensively identify regions of intermediate methylation and their quantitative relationship with gene activity, we apply integrative and comparative epigenomics to 25 human primary cell and tissue samples. We report 18,452 intermediate methylation regions located near 36% of genes and enriched at enhancers, exons, and DNase I hypersensitivity sites. Intermediate methylation regions average 57% methylation, are predominantly allele-independent, and are conserved across individuals and between mouse and human, suggesting a conserved function. At enhancers, these regions have an intermediate level of active chromatin marks and their associated genes have intermediate transcriptional activity. Exonic intermediate methylation correlates with exon inclusion at the level between that of fully methylated and unmethylated exons, highlighting gene context-dependent functions. We conclude that intermediate DNA methylation is a conserved signature of gene regulation and exon usage.
While significant effort has been dedicated to the characterization of epigenetic changes associated with pre-natal differentiation relatively little is known about the epigenetic changes that accompany post-natal differentiation where fully functional differentiated cell types with limited lifespans arise. Here we sought to address this gap by generating epigenomic and transcriptional profiles from primary human breast cell types isolated from disease-free human subjects. From these data we define a comprehensive human breast transcriptional network, including a set of myoepithelial- and luminal epithelial- specific intronic retention events. Intersection of epigenetic states with RNA expression from distinct breast epithelium lineages demonstrates that mCpG provides a stable record of exonic and intronic usage whereas H3K36me3 is dynamic. We find a striking asymmetry in epigenomic reprogramming between luminal and myoepithelial cell types, with the genomes of luminal cells harboring more than twice the number of hypomethylated enhancer elements compared to myoepithelial cells.
The role of intermediate methylation states in DNA is unclear. Here, to comprehensively identify regions of intermediate methylation and their quantitative relationship with gene activity, we apply integrative and comparative epigenomics to 25 human primary cell and tissue samples. We report 18,452 intermediate methylation regions located near 36% of genes and enriched at enhancers, exons and DNase I hypersensitivity sites. Intermediate methylation regions average 57% methylation, are predominantly allele-independent and are conserved across individuals and between mouse and human, suggesting a conserved function. These regions have an intermediate level of active chromatin marks and their associated genes have intermediate transcriptional activity. Exonic intermediate methylation correlates with exon inclusion at a level between that of fully methylated and unmethylated exons, highlighting gene context-dependent functions. We conclude that intermediate DNA methylation is a conserved signature of gene regulation and exon usage.
Many loci in the mammalian genome are intermediately methylated. Here, by comprehensively identifying these loci and quantifying their relationship with gene activity, the authors show that intermediate methylation is an evolutionarily conserved epigenomic signature of gene regulation.
While significant effort has been dedicated to the characterization of epigenetic changes associated with prenatal differentiation, relatively little is known about the epigenetic changes that accompany post-natal differentiation where fully functional differentiated cell types with limited lifespans arise. Here we sought to address this gap by generating epigenomic and transcriptional profiles from primary human breast cell types isolated from disease-free human subjects. From these data we define a comprehensive human breast transcriptional network, including a set of myoepithelial- and luminal epithelial-specific intronic retention events. Intersection of epigenetic states with RNA expression from distinct breast epithelium lineages demonstrates that mCpG provides a stable record of exonic and intronic usage, whereas H3K36me3 is dynamic. We find a striking asymmetry in epigenomic reprogramming between luminal and myoepithelial cell types, with the genomes of luminal cells harbouring more than twice the number of hypomethylated enhancer elements compared with myoepithelial cells.
Epigenetic changes associated with post-natal differentiation have been characterized. Here the authors generate epigenomic and transcriptional profiles from primary human breast cells, providing insights into the transcriptional and epigenetic events that define post-natal cell differentiation in vivo.
Diffuse gliomas consist of both low- and high-grade varieties, each with distinct morphological and biological features. The often extended periods of relative indolence exhibited by low-grade gliomas (LGG; WHO grade II) differ sharply from the aggressive, rapidly fatal clinical course of primary glioblastoma (GBM; WHO grade IV). Nevertheless, until recently, the molecular foundations underlying this stark biological contrast between glioma variants remained largely unknown. The discoveries of distinctive and highly recurrent genomic and epigenomic abnormalities in LGG have both informed a more accurate classification scheme and pointed to viable avenues for therapeutic development. As such, the field of neuro-oncology now seems poised to capitalize on these gains to achieve significant benefit for LGG patients. This report will briefly recount the proceedings of a workshop held in January 2013 and hosted by Accelerate Brain Cancer Cure (ABC2) on the subject of LGG. While much of the meeting covered recent insights into LGG biology, its focus remained on how best to advance the clinical management, whether by improved preclinical modeling, more effective targeted therapeutics and clinical trial design, or innovative imaging technology.
clinical trials; genomics; low-grade glioma; personalized medicine