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Despite decades of research, acute myeloid leukemia (AML) remains a remarkably lethal malignancy. While pediatric AML (pAML) carries a more favorable prognosis than adult AML, the past 25 years of large clinical trials have produced few improvements in pAML survival. Nowhere is this more evident than in patients carrying a t(16;21)(p11;q22) translocation, which yields the FUS::ERG fusion transcript. Patients with FUS::ERG -positive AML are often primary refractory, and most responders quickly relapse. In COG clinical trials, allogeneic stem cell transplantation was of no benefit to FUS::ERG pAML patients; 100% of transplanted patients succumbed to their disease. Expression of major histocompatibility complex (MHC) class I & II and costimulatory molecules is absent at diagnosis in FUS::ERG AML, mirroring the epigenetic mechanism of post-transplant relapse seen in adult AML and its associated dismal outcomes. Here we show that this class-defining immune-repressive phenotype is driven by overexpression of the EZH2 histone lysine methyltransferase in vitro and in multiple clinical cohorts. We show that treatment with the FDA-approved EZH2 inhibitor tazemetostat along with IFN-γ reverses this phenotype, re-establishes MHC presentation, and severely impairs the viability of FUS::ERG AML cells. EZH2 inhibitors may thus provide the first targeted therapeutic option for patients with this high-risk subtype of pAML, with particular benefit as a bridge to successful allogeneic stem cell transplantation.

Deconvolution methods infer quantitative cell type estimates from bulk measurement of mixed samples including blood and tissue. DNA methylation sequencing measures multiple CpGs per read, but few existing deconvolution methods leverage this within-read information. We develop CelFiE-ISH, which extends an existing method (CelFiE) to use within-read haplotype information. CelFiE-ISH outperforms CelFiE and other existing methods, achieving 30% better accuracy and more sensitive detection of rare cell types. We also demonstrate the importance of marker selection and of tailoring markers for haplotype-aware methods. While here we use gold-standard short-read sequencing data, haplotype-aware methods will be well-suited for long-read sequencing., (© 2024. The Author(s).)

Cell-to-cell communication through secreted Wnt ligands that bind to members of the Frizzled (Fzd) family of transmembrane receptors is critical for development and homeostasis. Wnt9a signals through Fzd9b, the co-receptor LRP5 or LRP6 (LRP5/6), and the epidermal growth factor receptor (EGFR) to promote early proliferation of zebrafish and human hematopoietic stem cells during development. Here, we developed fluorescently labeled, biologically active Wnt9a and Fzd9b fusion proteins to demonstrate that EGFR-dependent endocytosis of the ligand-receptor complex was required for signaling. In human cells, the Wnt9a-Fzd9b complex was rapidly endocytosed and trafficked through early and late endosomes, lysosomes, and the endoplasmic reticulum. Using small-molecule inhibitors and genetic and knockdown approaches, we found that Wnt9a-Fzd9b endocytosis required EGFR-mediated phosphorylation of the Fzd9b tail, caveolin, and the scaffolding protein EGFR protein substrate 15 (EPS15). LRP5/6 and the downstream signaling component AXIN were required for Wnt9a-Fzd9b signaling but not for endocytosis. Knockdown or loss of EPS15 impaired hematopoietic stem cell development in zebrafish. Other Wnt ligands do not require endocytosis for signaling activity, implying that specific modes of endocytosis and trafficking may represent a method by which Wnt-Fzd specificity is established.

Data from both bulk and single-cell whole-genome DNA methylation experiments are under-utilized in many ways. This is attributable to inefficient mapping of methylation sequencing reads, routinely discarded genetic information, and neglected read-level epigenetic and genetic linkage information. We introduce the BISulfite-seq Command line User Interface Toolkit (BISCUIT) and its companion R/Bioconductor package, biscuiteer, for simultaneous extraction of genetic and epigenetic information from bulk and single-cell DNA methylation sequencing. BISCUIT's performance, flexibility and standards-compliant output allow large, complex experimental designs to be characterized on clinical timescales. BISCUIT is particularly suited for processing data from single-cell DNA methylation assays, with its excellent scalability, efficiency, and ability to greatly enhance mappability, a key challenge for single-cell studies. We also introduce the epiBED format for single-molecule analysis of coupled epigenetic and genetic information, facilitating the study of cellular and tissue heterogeneity from DNA methylation sequencing., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Purpose: Optimized strategies for risk classification are essential to tailor therapy for patients with biologically distinctive disease. Risk classification in pediatric acute myeloid leukemia (pAML) relies on detection of translocations and gene mutations. Long noncoding RNA (lncRNA) transcripts have been shown to associate with and mediate malignant phenotypes in acute myeloid leukemia (AML) but have not been comprehensively evaluated in pAML., Methods: To identify lncRNA transcripts associated with outcomes, we evaluated the annotated lncRNA landscape by transcript sequencing of 1,298 pediatric and 96 adult AML specimens. Upregulated lncRNAs identified in the pAML training set were used to establish a regularized Cox regression model of event-free survival (EFS), yielding a 37 lncRNA signature (lncScore). Discretized lncScores were correlated with initial and postinduction treatment outcomes using Cox proportional hazards models in validation sets. Predictive model performance was compared with standard stratification methods by concordance analysis., Results: Training set cases with positive lncScores had 5-year EFS and overall survival rates of 26.7% and 42.7%, respectively, compared with 56.9% and 76.3% with negative lncScores (hazard ratio, 2.48 and 3.16; P < .001). Pediatric validation cohorts and an adult AML group yielded comparable results in magnitude and significance. lncScore remained independently prognostic in multivariable models, including key factors used in preinduction and postinduction risk stratification. Subgroup analysis suggested that lncScores provide additional outcome information in heterogeneous subgroups currently classified as indeterminate risk. Concordance analysis showed that lncScore adds to overall classification accuracy with at least comparable predictive performance to current stratification methods that rely on multiple assays., Conclusion: Inclusion of the lncScore enhances predictive power of traditional cytogenetic and mutation-defined stratification in pAML with potential, as a single assay, to replace these complex stratification schemes with comparable predictive accuracy.

The SOX transcription factor family is pivotal in controlling aspects of development. To identify genotype-phenotype relationships of SOX proteins, we performed a non-biased study of SOX using 1890 open-reading frame and 6667 amino acid sequences in combination with structural dynamics to interpret 3999 gnomAD, 485 ClinVar, 1174 Geno2MP, and 4313 COSMIC human variants. We identified, within the HMG (High Mobility Group)- box, twenty-seven amino acids with changes in multiple SOX proteins annotated to clinical pathologies. These sites were screened through Geno2MP medical phenotypes, revealing novel SOX15 R104G associated with musculature abnormality and SOX8 R159G with intellectual disability. Within gnomAD, SOX18 E137K (rs201931544), found within the HMG box of ~0.8% of Latinx individuals, is associated with seizures and neurological complications, potentially through blood-brain barrier alterations. A total of 56 highly conserved variants were found at sites outside the HMG-box, including several within the SOX2 HMG-box-flanking region with neurological associations, several in the SOX9 dimerization region associated with Campomelic Dysplasia, SOX14 K88R (rs199932938) flanking the HMG box associated with cardiovascular complications within European populations, and SOX7 A379V (rs143587868) within an SOXF conserved far C-terminal domain heterozygous in 0.716% of African individuals with associated eye phenotypes. This SOX data compilation builds a robust genotype-to-phenotype association for a gene family through more robust ortholog data integration.

Summary: CTCF (CCCTC-binding factor) is an 11-zinc-finger DNA binding protein which regulates much of the eukaryotic genome's 3D structure and function. The diversity of CTCF binding motifs has led to a fragmented landscape of CTCF binding data. We collected position weight matrices of CTCF binding motifs and defined strand-oriented CTCF binding sites in the human and mouse genomes, including the recent Telomere to Telomere and mm39 assemblies. We included selected experimentally determined and predicted CTCF binding sites, such as CTCF-bound cis-regulatory elements from SCREEN ENCODE. We recommend filtering strategies for CTCF binding motifs and demonstrate that liftOver is a viable alternative to convert CTCF coordinates between assemblies. Our comprehensive data resource and usage recommendations can serve to harmonize and strengthen the reproducibility of genomic studies utilizing CTCF binding data., Availability and Implementation: https://bioconductor.org/packages/CTCF. Companion website: https://dozmorovlab.github.io/CTCF/; Code to reproduce the analyses: https://github.com/dozmorovlab/CTCF.dev., Supplementary Information: Supplementary data are available at Bioinformatics Advances online., (© The Author(s) 2022. Published by Oxford University Press.)

Competing Interests: I have read the journal’s policy and have the following conflicts: AG filed a patent application with the Wisconsin Alumni Research Foundation related to classifying activated T cells. KHY is the inventor of a quantitative pathology analytical system (U.S. Patent 10,832,406). This invention is not related to this work. EJF is a Scientific Advisory Board member at Viosera Therapeutics. AAK is a co-inventor on 4 patent applications related to machine learning applications in biology.

Infant Acute Myeloid Leukemia (AML) is a poorly-addressed, heterogeneous malignancy distinguished by surprisingly few mutations per patient but accompanied by myriad age-specific translocations. These characteristics make treatment of infant AML challenging. While infant AML is a relatively rare disease, it has enormous impact on families, and in terms of life-years-lost and life limiting morbidities. To better understand the mechanisms that drive infant AML, we performed integrative analyses of genome-wide mRNA, miRNA, and DNA-methylation data in diagnosis-stage patient samples. Here, we report the activation of an onco-fetal B-cell developmental gene regulatory network in infant AML. AML in infants is genomically distinct from AML in older children/adults in that it has more structural genomic aberrations and fewer mutations. Differential expression analysis of ~1500 pediatric AML samples revealed a large number of infant-specific genes, many of which are associated with B cell development and function. 18 of these genes form a well-studied B-cell gene regulatory network that includes the epigenetic regulators BRD4 and POU2AF1, and their onco-fetal targets LIN28B and IGF2BP3. All four genes are hypo-methylated in infant AML. Moreover, micro-RNA Let7a-2 is expressed in a mutually exclusive manner with its target and regulator LIN28B. These findings suggest infant AML may respond to bromodomain inhibitors and immune therapies targeting CD19, CD20, CD22, and CD79A., Competing Interests: ML is an employee of and has equity ownership in Hematologics Inc. and LP is an employee of Hematologics Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The remaining authors declare no competing financial interests.

Rhabdoid tumor (RT) is a pediatric cancer characterized by the inactivation of SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex. Although this deletion is the known oncogenic driver, there are limited effective therapeutic options for these patients. Here we use unbiased screening of cell line panels to identify a heightened sensitivity of rhabdoid tumor to mithramycin and the second-generation analogue EC8042. The sensitivity of MMA and EC8042 was superior to traditional DNA damaging agents and linked to the causative mutation of the tumor, SMARCB1 deletion. Mithramycin blocks SMARCB1-deficient SWI/SNF activity and displaces the complex from chromatin to cause an increase in H3K27me3. This triggers chromatin remodeling and enrichment of H3K27ac at chromHMM-defined promoters to restore cellular differentiation. These effects occurred at concentrations not associated with DNA damage and were not due to global chromatin remodeling or widespread gene expression changes. Importantly, a single 3-day infusion of EC8042 caused dramatic regressions of RT xenografts, recapitulated the increase in H3K27me3, and cellular differentiation described in vitro to completely cure three out of eight mice., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Benign peripheral nerve sheath tumors are the clinical hallmark of Neurofibromatosis Type 1. They account for substantial morbidity and mortality in NF1. Cutaneous (CNF) and plexiform neurofibromas (PNF) share nearly identical histology, but maintain different growth rates and risk of malignant conversion. The reasons for this disparate clinical behavior are not well explained by recent genome or transcriptome profiling studies. We hypothesized that CNFs and PNFs are epigenetically distinct tumor types that exhibit differential signaling due to genome-wide and site-specific methylation events. We interrogated the methylation profiles of 45 CNFs and 17 PNFs from NF1 subjects with the Illumina EPIC 850K methylation array. Based on these profiles, we confirm that CNFs and PNFs are epigenetically distinct tumors with broad differences in higher-order chromatin states and specific methylation events altering genes involved in key biological and cellular processes, such as inflammation, RAS/MAPK signaling, actin cytoskeleton rearrangement, and oxytocin signaling. Based on our identification of two separate DMRs associated with alternative leading exons in MAP2K3, we demonstrate differential RAS/MKK3/p38 signaling between CNFs and PNFs. Epigenetic reinforcement of RAS/MKK/p38 was a defining characteristic of CNFs leading to pro-inflammatory signaling and chromatin conformational changes, whereas PNFs signaled predominantly through RAS/MEK. Tumor size also correlated with specific CpG methylation events. Taken together, these findings confirm that NF1 deficiency influences the epigenetic regulation of RAS signaling fates, accounting for observed differences in CNF and PNF clinical behavior. The extension of these findings is that CNFs may respond differently than PNFs to RAS-targeted therapeutics raising the possibility of targeting p38-mediated inflammation for CNF treatment.

Over the past two decades, progress in tumor immunology and targeted therapy has reshaped oncology, and in many cases, reshaped the course of once-intractable diseases. Yet the cost of its clinical manifestation has created a disease of its own: "financial toxicity," the burden of drugs such as imatinib, where a year's supply can easily cost as much as a house. Equally rapid progress in mathematical oncology over this time period has often come in the form of fundamental, rather than applied, advances. However, in new work by Hähnel and colleagues, we can see the outlines of a viable treatment for financial toxicity: precise, dynamic, clinically validated, and immune-aware models, able to accurately identify patients who remain disease-free in the months and years after discontinuing effective, but pricey, targeted therapies. See related article by Hähnel et al., p. 2394 ., (©2020 American Association for Cancer Research.)

Purpose: A cryptic inv(16)(p13.3q24.3) encoding the CBFA2T3-GLIS2 fusion is associated with poor outcome in infants with acute megakaryocytic leukemia. We aimed to broaden our understanding of the pathogenesis of this fusion through transcriptome profiling., Experimental Design: Available RNA from children and young adults with de novo acute myeloid leukemia (AML; N = 1,049) underwent transcriptome sequencing (mRNA and miRNA). Transcriptome profiles for those with the CBFA2T3-GLIS2 fusion ( N = 24) and without ( N = 1,025) were contrasted to define fusion-specific miRNAs, genes, and pathways. Clinical annotations defined distinct fusion-associated disease characteristics and outcomes., Results: The CBFA2T3-GLIS2 fusion was restricted to infants <3 years old ( P < 0.001), and the presence of this fusion was highly associated with adverse outcome ( P < 0.001) across all morphologic classifications. Further, there was a striking paucity of recurrent cooperating mutations, and transduction of cord blood stem cells with this fusion was sufficient for malignant transformation. CBFA2T3-GLIS2 positive cases displayed marked upregulation of genes with cell membrane/extracellular matrix localization potential, including NCAM1 and GABRE . Additionally, miRNA profiling revealed significant overexpression of mature miR-224 and miR-452 , which are intronic miRNAs transcribed from the GABRE locus. Gene-set enrichment identified dysregulated Hippo, TGFβ, and hedgehog signaling, as well as NCAM1 (CD56) interaction pathways. Therapeutic targeting of fusion-positive leukemic cells with CD56-directed antibody-drug conjugate caused significant cytotoxicity in leukemic blasts., Conclusions: The CBFA2T3-GLIS2 fusion defines a highly refractory entity limited to infants that appears to be sufficient for malignant transformation. Transcriptome profiling elucidated several highly targetable genes and pathways, including the identification of CD56, providing a highly plausible target for therapeutic intervention., (©2019 American Association for Cancer Research.)

Little is known about the spectrum of mitochondrial DNA (mtDNA) mutations across pediatric malignancies. In this study, we analyzed matched tumor and normal whole genome sequencing data from 616 pediatric patients with hematopoietic malignancies, solid tumors, and brain tumors. We identified 391 mtDNA mutations in 284 tumors including 45 loss-of-function mutations, which clustered at four statistically significant hotspots in MT-COX3 , MT-ND4 , and MT-ND5 , and at a mutation hotspot in MT-tRNA-MET . A skewed ratio (4.83) of nonsynonymous versus synonymous (dN/dS) mtDNA mutations with high statistical significance was identified on the basis of Monte Carlo simulations in the tumors. In comparison, opposite ratios of 0.44 and 0.93 were observed in 616 matched normal tissues and in 249 blood samples from children without cancer, respectively. mtDNA mutations varied by cancer type and mtDNA haplogroup. Collectively, these results suggest that deleterious mtDNA mutations play a role in the development and progression of pediatric cancers. SIGNIFICANCE: This pan-cancer mtDNA study establishes the landscape of germline and tumor mtDNA mutations and identifies hotspots of tumor mtDNA mutations to pinpoint key mitochondrial functions in pediatric malignancies., (©2019 American Association for Cancer Research.)

Senescence, a state of permanent cell cycle arrest, can be induced by DNA damage. This process, which was initially described in fibroblasts, is now recognized to occur in stem cells. It has been well characterized in cell lines, but there is currently very limited data available on human senescence in vivo. We recently reported that the expression of transposable elements (TE), including endogenous retroviruses, was up-regulated along with inflammatory genes in human senescent hematopoietic stem and progenitor cells (HSPCs) in vivo. The mechanism of regulation of TE expression is not completely understood, but changes in DNA methylation and chromatin modifications are known to alter their expression. In order to elucidate the molecular mechanisms for TE up-regulation after senescence of HSPCs, we employed whole-genome bisulfite sequencing in paired senescent and active human HSPCs in vivo from healthy subjects. We found that the senescent HSPCs exhibited hypomethylated regions in the genome, which were enriched for TEs. This is the first report characterizing the methylome of senescent human HSPCs.

We report a new class of artifacts in DNA methylation measurements from Illumina HumanMethylation450 and MethylationEPIC arrays. These artifacts reflect failed hybridization to target DNA, often due to germline or somatic deletions and manifest as incorrectly reported intermediate methylation. The artifacts often survive existing preprocessing pipelines, masquerade as epigenetic alterations and can confound discoveries in epigenome-wide association studies and studies of methylation-quantitative trait loci. We implement a solution, P-value with out-of-band (OOB) array hybridization (pOOBAH), in the R package SeSAMe. Our method effectively masks deleted and hyperpolymorphic regions, reducing or eliminating spurious reports of epigenetic silencing at oft-deleted tumor suppressor genes such as CDKN2A and RB1 in cases with somatic deletions. Furthermore, our method substantially decreases technical variation whilst retaining biological variation, both within and across HM450 and EPIC platform measurements. SeSAMe provides a light-weight, modular DNA methylation data analysis suite, with a performant implementation suitable for efficient analysis of thousands of samples.

Genomic transposable elements (TEs) constitute the majority of the genome. Expression of TEs is known to activate the double-stranded RNA recognition pathway ("viral mimicry"), leading to the activation of interferon-stimulated genes, inflammation, and immune-mediated cell death. Recently, we showed that the expression of TEs is suppressed along with immune pathways in leukemic stem cells (LSCs) in acute myeloid leukemia, suggesting a potential mechanism for immune escape of LSCs. This indicated that, during oncogenesis, where there is escape from senescence, expression of TEs is suppressed. Senescence is known to activate the interferon response and inflammatory cytokines, known as the senescence-associated secretory phenotype (SASP). We characterized the transcriptome of senescent and active human hematopoietic stem and progenitor cells (HSPCs) in vivo and showed co-occurrence of overexpression of TEs, SASP genes, and gene pathways of inflammation in senescence. The percentage of circulating senescent HSPCs (s-HSPCs) did not increase with age, indicating active clearance. Induction of senescence in human HSPCs in vitro showed increased expression of TE and SASP genes. SASP is known to mediate clearance of senescent cells and active clearance of senescent cells has been shown to increase organismal fitness. We speculate that the expression of TEs in s-HSPCs could contribute to orderly clearance of the cells via activation of immune pathways, warranting further mechanistic studies. This is the first study to characterize the transcriptome of human s-HSPCs in vivo, revealing activated expression of TEs and inflammatory genes., (Copyright © 2018 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Background: Decitabine is a deoxycytidine nucleoside derivative inhibitor of DNA-methyltransferases, which has been studied extensively and is approved for myelodysplastic syndrome in adults but with less focus in children. Accordingly, we conducted a phase 1 multicenter, randomized, open-label study to evaluate decitabine pre-treatment before standard induction therapy in children with newly diagnosed AML to assess safety and tolerability and explore a number of biologic endpoints., Results: Twenty-four patients were fully assessable for all study objectives per protocol (10 in Arm A = epigenetic priming induction, 14 in Arm B = standard induction). All patients experienced neutropenia and thrombocytopenia. The most common grade 3 and 4 non-hematologic adverse events observed were gastrointestinal toxicities and hypophosphatemia. Plasma decitabine PK were similar to previously reported adult data. Overall CR/CRi was similar for the two arms. MRD negativity at end-induction was 85% in Arm A versus 67% in Arm B patients. DNA methylation measured in peripheral blood over the course of treatment tracked with blast clearance and matched marrow aspirates at day 0 and day 21. Unlike end-induction marrow analyses, promoter methylation in blood identified an apparent reversal of response in the lone treatment failure, 1 week prior to the patient's marrow aspirate confirming non-response. Decitabine-induced effects on end-induction (day 35-43 following initiation of treatment) marrows in Arm A were reflected by changes in DNA methylation in matched paired marrow diagnostic aspirates., Conclusions: This first-in-pediatrics trial demonstrates that decitabine prior to standard combination chemotherapy is feasible and well tolerated in children with newly diagnosed AML. Pre-treatment with decitabine may represent a newer therapeutic option for pediatric AML, especially as it appears to induce important epigenetic alterations. The novel biological correlates studied in this trial offer a clinically relevant window into disease progression and remission. Additional studies are needed to definitively assess whether decitabine can enhance durability responses in children with AML., Trial Registration: NCT01177540.

EPHB4, an ephrin type B receptor, is implicated in the growth of several epithelial tumors and is a promising target in cancer therapy; however, little is known about its role in hematologic malignancies. In this article, we show that EPHB4 is highly expressed in ∼30% of acute myeloid leukemia (AML) samples. In an unbiased RNA interference screen of primary leukemia samples, we found that EPHB4 drives survival in a subset of AML cases. Knockdown of EPHB4 inhibits phosphatidylinositol 3-kinase/AKT signaling, and this is accompanied by a reduction in cell viability, which can be rescued by a constitutively active form of AKT. Finally, targeting EPHB4 with a highly specific monoclonal antibody (MAb131) is effective against AML in vitro and in vivo . EPHB4 is therefore a potential target in AML with high EPHB4 expression., Competing Interests: Conflict-of-interest disclosure: A.A.M. received research funding and served as a consultant for Pfizer, Inc. J.W.T. received research support from Aptose, Array, AstraZeneca, Constellation, Genentech, Gilead, Incyte, Janssen, Seattle Genetics, Syros, and Takeda and is on the Scientific Advisory Board for Leap Oncology. The remaining authors declare no competing financial interests.

Summary: The minfi package is widely used for analyzing Illumina DNA methylation array data. Here we describe modifications to the minfi package required to support the HumanMethylationEPIC ('EPIC') array from Illumina. We discuss methods for the joint analysis and normalization of data from the HumanMethylation450 ('450k') and EPIC platforms. We introduce the single-sample Noob ( ssNoob ) method, a normalization procedure suitable for incremental preprocessing of individual methylation arrays and conclude that this method should be used when integrating data from multiple generations of Infinium methylation arrays. We show how to use reference 450k datasets to estimate cell type composition of samples on EPIC arrays. The cumulative effect of these updates is to ensure that minfi provides the tools to best integrate existing and forthcoming Illumina methylation array data., Availability and Implementation: The minfi package version 1.19.12 or higher is available for all platforms from the Bioconductor project., Contact: khansen@jhsph.edu., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2016. Published by Oxford University Press.)

Extracellular cues play critical roles in the establishment of the epigenome during development and may also contribute to epigenetic perturbations found in disease states. The direct role of the local tissue environment on the post-development human epigenome, however, remains unclear due to limitations in studies of human subjects. Here, we use an isogenic human ileal neobladder surgical model and compare global DNA methylation levels of intestinal epithelial cells pre- and post-neobladder construction using the Infinium HumanMethylation450 BeadChip. Our study is the first to quantify the effect of environmental cues on the human epigenome and show that the local tissue environment directly modulates DNA methylation patterns in normal differentiated cells in vivo. In the neobladder, the intestinal epithelial cells lose their tissue-specific epigenetic landscape in a time-dependent manner following the tissue's exposure to a bladder environment. We find that de novo methylation of many intestine-specific enhancers occurs at the rate of 0.41% per month (P < 0.01, Pearson = 0.71), while demethylation of primarily non-intestine-specific transcribed regions occurs at the rate of -0.37% per month (P < 0.01, Pearson = -0.57). The dynamic resetting of the DNA methylome in the neobladder not only implicates local environmental cues in the shaping and maintenance of the epigenome but also illustrates an unexpected cross-talk between the epigenome and the cellular environment.

Background: Many mutations that contribute to the pathogenesis of acute myeloid leukemia (AML) are undefined. The relationships between patterns of mutations and epigenetic phenotypes are not yet clear., Methods: We analyzed the genomes of 200 clinically annotated adult cases of de novo AML, using either whole-genome sequencing (50 cases) or whole-exome sequencing (150 cases), along with RNA and microRNA sequencing and DNA-methylation analysis., Results: AML genomes have fewer mutations than most other adult cancers, with an average of only 13 mutations found in genes. Of these, an average of 5 are in genes that are recurrently mutated in AML. A total of 23 genes were significantly mutated, and another 237 were mutated in two or more samples. Nearly all samples had at least 1 nonsynonymous mutation in one of nine categories of genes that are almost certainly relevant for pathogenesis, including transcription-factor fusions (18% of cases), the gene encoding nucleophosmin (NPM1) (27%), tumor-suppressor genes (16%), DNA-methylation-related genes (44%), signaling genes (59%), chromatin-modifying genes (30%), myeloid transcription-factor genes (22%), cohesin-complex genes (13%), and spliceosome-complex genes (14%). Patterns of cooperation and mutual exclusivity suggested strong biologic relationships among several of the genes and categories., Conclusions: We identified at least one potential driver mutation in nearly all AML samples and found that a complex interplay of genetic events contributes to AML pathogenesis in individual patients. The databases from this study are widely available to serve as a foundation for further investigations of AML pathogenesis, classification, and risk stratification. (Funded by the National Institutes of Health.).

We propose a novel approach to background correction for Infinium HumanMethylation data to account for technical variation in background fluorescence signal. Our approach capitalizes on a new use for the Infinium I design bead types to measure non-specific fluorescence in the colour channel opposite of their design (Cy3/Cy5). This provides tens of thousands of features for measuring background instead of the much smaller number of negative control probes on the platforms (n = 32 for HumanMethylation27 and n = 614 for HumanMethylation450, respectively). We compare the performance of our methods with existing approaches, using technical replicates of both mixture samples and biological samples, and demonstrate that within- and between-platform artefacts can be substantially reduced, with concomitant improvement in sensitivity, by the proposed methods.