Peter W. Laird - US grants

DESCRIPTION (provided by applicant): We have developed a mouse model system, using hypomorphic alleles of the major DNA methyltransferase Dnmt1, in which we can assess the role of DNA methylation in oncogenesis and investigate various mechanisms by which DNA methylation and/or Dnmt1 may affect the cancer process. We have recently shown that combinations of Dnmt1 hypomorphic alleles can achieve complete genetic suppression of ApcMin/+ induced polyp formation, suggesting that sufficient levels of Dnmt1 expression are required for intestinal polyp development. This is further supported by our observation that intestinal tumorigenesis in mismatch-repair deficient Mih1-/- mice is also suppressed by low levels of Dnmt1. However, lymphomagenesis is increased in these same mice, suggesting that modulation of Dnmt1 levels can have opposing effects on oncogenesis in vivo. The molecular basis for this strong effect of Dnmt1 levels on various models of oncogenesis is not understood. We have recently shown in a tissue-culture model system that Dnmt1 deficiency causes a reduction in methylation-dependent genetic events, such as methylcytosine deamination, and a reduction in methylation-dependent epigenetic events, such as transcriptional silencing by promoter CpG island hypermethylation. In this competing continuation, we propose to 1) expand our analysis of the effects of in vivo modulation of DNA methylation on cancer model systems and 2) analyze the influence of DNA methylation and/or methyltransferases on mutation frequencies in vivo, and 3) analyze sequence features affecting de novo methylation in ES cells. In summary, we propose to continue to investigate the causal effects of DNA methylation in cancer and to analyze both genetic and epigenetic mechanisms by which DNA methylation and/or DNA methyltransferases could affect oncogenesis.

[unreadable] DESCRIPTION (provided by applicant): In recent years, it has become clear that molecular events leading to oncogenesis include not only genetic mutations, but also epigenetic alterations, such as the DNA methylation of promoter CpG islands. We have shown that DNA methylation changes are both causal contributors to neoplasia, and potential biomarkers for cancer, including adenocarcinoma of the esophagus. We have developed a sensitive, quantitatively accurate, automated DNA methylation analysis technique, called MethyLight, with which we have shown that DNA methylation profiles can discriminate between normal squamous mucosa of the esophagus, intestinal metaplasia, and dysplasia. These preliminary results suggest that DNA methylation markers could eventually be exploited as clinical tools in the early detection of esophageal adenocarcinoma and/or in risk assessment in surveillance programs. [unreadable] [unreadable] Since the 1970s, incidence rates for esophageal and gastric cardia adenocarcinomas have risen substantially, particularly among white males in the United States. Reasons for the increase of these tumor types are not well understood. We have conducted a case-control study to determine the role of smoking, alcohol use, body size characteristics, and other risk factors in the etiology of adenocarcinoma of the esophagus and gastric cardia. [unreadable] [unreadable] Here we propose to use automated MethyLight technology to generate extensive methylation profiles for tissue samples retrieved from this population-based case-control study. This will allow us to identify superior methylation markers for further development and testing as clinical tools in the early detection of esophageal adenocarcinoma, and for the differentiation of Barrett's cases that are at high risk for further progression. We will also investigate whether factors known to increase the risk of adenocarcinoma of the esophagus, increase the risk of DNA methylation in normal tissue, or in the resulting adenocarcinoma. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Epithelial ovarian carcinoma is the leading cause of death from gynecologic cancer in the US. Approximately 75% of patients present with advanced stage disease, for which the five-year survival rate remains below 30%, whereas the five-year survival rate for stage I disease is 93%. Therefore, it is anticipated that effective methods of early detection of ovarian cancer would substantially reduce overall mortality rates for this disease. Despite much effort, there are currently no reliable procedures for the early detection of ovarian cancer available. [unreadable] [unreadable] In the past decade, a substantial amount of evidence for the occurrence of extensive alterations of DNA methylation patterns in cancer cells has accumulated. We and others have recently shown that these abnormal DNA methylation patterns can be detected in tumor-derived DNA in the serum and plasma of cancer patients. [unreadable] [unreadable] We propose to use a sophisticated automated methylation analysis technology that we have developed, called MethyLight, to screen a large panel of genes to identify markers specific for ovarian cancer, compared to non-neoplastic ovarian tissue. This will allow the correlation of methylated markers in ovarian tumors with clinicopathological features, and with response to chemotherapy and overall survival. Subsequently, we can use the information obtained in this screen of tissue samples to develop markers for the detection of ovarian tumor-derived DNA in the serum of patients with existing or recurring disease. Finally, we propose to use the most promising biomarkers to evaluate the capacity to detect preclinical relapse of disease, as a function of time before clinical diagnosis of relapse.

DESCRIPTION (provided by applicant): Recent investigations of the joint effects of genetics and environmental exposures on human health have focused on the role of germline polymorphisms in DNA sequences, including single nucleotide polymorphisms (SNPs) and differences in the number of nucleotide repeats. Although such mutations and their interactions with environmental exposures clearly play a role in human diseases, a growing body of evidence indicates that epigenetic changes, especially changes in patterns of DNA methylation, are important contributors to the pathogenesis of human disease. DNA methylation adds a new dimension to the understanding of gene-environment interactions. DNA methylation is uniquely positioned as both an additional source of genetic modification of the response to environmental influences, and is also a potential biomarker of environmental exposure. Despite the evolving evidence for the importance of DNA methylation in human disease pathogenesis, little is known about the environmental determinants of methylation or how gene polymorphisms influence the patterns of epigenetic changes following exposure. The need for a better understanding of the interactions between environmental exposures, polymorphisms and DNA methylation is clear. In this application, the investigators propose to take the next steps beyond studies of the effects of environmental exposures and genetic variation on human diseases, by integrating information on genomic methylation patterns. The broad objectives of this planning application are to develop a framework that will foster a collaborative interdisciplinary research program to study environmental and genetic determinants of DNA methylation patterns and to understand the influence of these epigenetic changes on human disease occurrence in the context of germline DNA variation. The collaborations to be built during the 3-year planning period bring together experienced investigators to develop and utilize evolving biostatistical, molecular, genetic, and epidemiologic methods. At the end of the planning period, they intend to submit an application to establish a Center for Environmental Epigenomics, to be affiliated with the Environmental Genome Project.

[unreadable] DESCRIPTION (provided by applicant): Human colorectal cancer arises as a consequence of both genetic and epigenetic alterations, including promoter CpG island hypermethylation. A subset of colorectal tumors has been described to have an unusually high number of hypermethylated CpG islands, leading to the definition of a distinct phenotype, referred to as "CpG Island Methylator Phenotype", or "CIMP". The long-term objective of this proposal is to study the association between CIMP status and molecular, demographic, and histopathologic features, and environmental risk factors, using colorectal cancer samples collected through the Cooperative Family Registry for Colorectal Cancer Studies (Colon CFR), an NCI-supported consortium intended as a resource to promote collaborative and interdisciplinary studies in the genetic epidemiology of colorectal cancer. We have recently published an improved DMA methylation marker set and analysis technology with which CIMP can be efficiently defined with high accuracy in archival colorectal cancer specimens. We propose to 1) estimate the association between CIMP status and age, sex, family history, race and country of origin, using 4,943 population-based colorectal cancer samples collected through the Colon CFR, 2) estimate the association between CIMP status and tumor location, grade, invasive margin, lymphocytic infiltration, direct spread, lymph node spread, venous spread and type of residual adjacent polyp, if present, and 3) estimate the association between CIMP status and selected risk factors, both genetic and environmental/lifestyle factors, including somatic mutations in BRAF, germline mutations in the MMR genes, smoking history, red meat and alcohol intakes, dietary folate intake, folate metabolic enzyme polymorphisms and history of hormone use. This study will contribute to our understanding of the etiology of CIMP, and its relationship to other molecular and histopathologic features of colorectal cancer. Colorectal cancer involves changes to genes that control cell growth and division. These changes can be structural, as in the case of genetic mutations, or they can reflect an alteration in how actively the gene is being used, referred to as an epigenetic change. This study will investigate how some colorectal tumors acquire an unusually high number of epigenetic changes, with the long-term goal of using this knowledge to block or reverse these types of deleterious changes. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): In the past two decades, it has become clear that cancer arises not only as a consequence of genetic events, such as mutations, copy number alterations, and sequence rearrangements, but also as a result of extensive changes to the epigenome. Epigenetic alterations contribute causally to the origin and malignant progression of cancer, and affect clinical outcome. The USC-JHU Cancer Epigenome Characterization Center specializes in the production and analysis of genome-scale cancer epigenetic data. During the pilot phase of The Cancer Genome Atlas, our center has been responsible for the production and deposition of all high-throughput epigenomic data within TCGA. A key outcome of the TCGA pilot was our finding of a link in glioblastoma multiforme between MGMT promoter methylation, treatment with alkylating agents, and a hypermutator phenotype associated with mismatch repair deficiency, an observation with potential clinical implications. For the next phase of TCGA, we propose the following three objectives. Objective 1 is to characterize the DNA methylation status of 27,578 CpG dinucleotides located in 14,495 gene promoters in at least 10,000 human cancer samples and 1,000 control samples using the lllumina Infinium DNA Methylation analysis platform. Objective 2 is to transition epigenomic data production in TCGA to whole genome shotgun bisulfite sequence analysis to provide single-base-pair resolution DNA methylation data for TCGA cancer samples. Objective 3 is to implement quality control and quality assurance measures to ensure that epigenomic data deposited for public dissemination meets rigorous standards. Our group has considerable expertise in cancer epigenomics, with two founders of the field of cancer epigenetics and with members who collectively have pioneered the majority of widely used DNA methylation analysis techniques, and who have provided many of the biological insights. Epigenomic data are a necessary component for a full understanding of the relationship between alterations in the cancer genome and the origin and clinical diversity of individual tumors.

Most men assume they will be able to conceive a child with no difficulty. However, 10-20% of all couples attempting pregnancy are infertile, with male factor infertility accounting for 40-50% of the cases. For most male infertility cases, the etiology is unknown and specific therapy is not available. Diagnosis of infertility can be a distressing life crisis, and the absence of a clear understanding of the pathophysiology of the disease and the corresponding lack of efficacious treatment for most male-factor infertility underscores the need for novel approaches to better understand this condition. This proposal seeks to improve the understanding of the pathophysiology of male factor infertility and the ability to diagnose male infertility by investigating the epigenetic state of human spermatozoa and defining its physiologic role in male infertility. Epigenetic programming (altering heritable biological information without changing DNA nucleotide sequence) is a normal process that leads to modifications of gene activity that can be transmitted to daughter cells. Extensive epigenetic reprogramming occurs during normal maturation of germ cells and spermatogenesis. However, improper epigenetic programming can have adverse health effects. Our preliminary data indicate that levels of one epigenetic indicator, DNA methylation, are elevated in spermatozoa of men who have abnormal sperm numbers, motility and morphology. This pattern may be expected if disrupted epigenetic programming predisposes to male infertility. This is particularly intriguing in light of preliminary animal studies suggesting that toxic exposures that negatively affect semen parameters also alter sperm DNA methylation. We propose that assessment of DNA methylation profiles in the male germ line provides the opportunity to better understand the physiologic mechanisms leading to idiopathic male infertility and to develop sensitive biomarkers of disrupted epigenetic programming. We anticipate that these markers will provide a mechanistic explanation for a category of idiopathic infertility and improve the diagnosis of and ultimately the treatment of male infertility by allowing us to study the epigenetic state of the germ cell and its relationship to fertility outcomes. This project should improve our ability to diagnose male factor infertility by identifying an epigenetic role in the pathogenesis of male factor infertility and lead to the development of a biomarker of abnormal sperm function that can be used as 1) a screening test for male factor infertility;2) a predictor of fertility outcomes in an ART setting, and 3) a marker/predictor of adverse fertility outcomes following toxic exposures.

Project Summary/Abstract Histone modification and cytosine-5 DNA methylation represent two important epigenetic marks. These two very distinct modifications work in close cooperation to control transcriptional potential, thereby influencing a wide diversity of biological processes. The past decade has seen an explosion of interest in the role of these epigenetic modifications in human heath and disease. The goal of the proposed application is to advance our understanding of these two modifications, by developing and applying two novel in vivo imaging technologies capable of visualizing the epigenetic and even genetic consequences of these modifications. The first specific aim is to develop an in vivo imaging system to assess the causal influence of individual epigenetic modifications. The technology is designed to both achieve selective introduction of a desired modification at a predetermined genomic locus, and to produce a quantitative optical readout of the effect of the epigenetic modification on transcription. This is achieved through the sequence-specific recruitment of epigenetic modifier proteins to a promoter driving a fluorescence-luminescence fusion reporter. The power of this approach comes from the combination of the quantitative optical analysis and the localized recruitment. This will allow high-throughput evaluation of the primary causal effects of individual epigenetic modifications. The second specific aim is to develop an imaging assay for the in vivo visualization of CpG transition mutations. Transition mutations at the epigenetic DNA methylation mark are responsible for approximately one-third of all human hereditary disease mutations and for nearly 50% of all p53 point mutations found in human colorectal cancer. However, the lack of imaging tools for this type of epigenetically induced mutagenesis has held back our understanding of the timing and cell-type specificity of this event in vivo. The proposed system is designed to provide direct visualization of the result of the mutation event. This is achieved through the use of a mutant green fluorescent protein that produces a fluorescent signal upon CpG transition mutation in the chromophore region. The conversion of a specific genetic mutation into an optical signal provides an attractive opportunity to analyze the mutation without employing direct sequencing. The in situ analysis of mutagenesis would allow us to not only determine tissue-specific and cell-type specific in vivo CpG mutation frequencies, but also to analyze mutation kinetics.

DESCRIPTION (provided by applicant): In this application, we propose to address PQ10: As we improve methods to identify epigenetic changes that occur during tumor development, can we develop approaches to discriminate between driver and passenger epigenetic events?. Epigenetic mechanisms exert a strong effect on gene expression potential, and have been shown to undergo widespread change in most human cancers. In contrast to most mutational events, epigenetic events often display a high degree of correlation, with a large number of defined alterations that appear to be passenger events without functional contribution to the cancer process. We propose to develop an integrated computational and experimental validation pipeline to identify epigenetic driver events in cancer. In Aim 1 we will develop a probabilistic framework for predicting and prioritizing candidate epigenetic driver loci. This approach is unique in that it fully integrates the wealth of available data, using complementary data types derived from primary genomic data, experimental data, and supporting curated information, resulting in a composite Epigenetic Driver Score (EDS), reflecting the posterior probability that each gene is an epigenetic driver. Aim 2 will provide experimental data on epigenetic addiction, using cell lines depleted of DNA methyltransferases, and thus selected to retain only the most essential silencing events, in addition to data obtained with embryonic and adult stem-cell and progenitors. These experimental data sets will be used to complement primary epigenomic data we have generated in the context of TCGA, to provide Epigenetic Driver Scores for each locus in each tumor type, using the methodology developed in Aim 1. In Aim 3a we will functionally test the top-ranked candidate epigenetic drivers of colon, breast, and lung cancer in vitro, by reintroducing expression of candidate genes into appropriate human cancer cells lines containing the relevant silencing events. These experiments will be complemented by shRNA approaches in cell lines to modulate the functional expression of the candidate epigenetic drivers. In vitro proliferation and apoptosis assays will be used to assess phenotypic effects. In Aim 3b we will assess the functional contributions of the candidate epigenetic drivers in vivo, using the stable cell lines created in Aim 3a in xenograft mouse models. The results of these validation experiments will be used to iteratively train the EDS model. Given the sensitivity of learning algorithms to their training data, we anticipate an improvement in performance as the number of training examples increases. By performing data-driven modeling in a probabilistic framework and computationally- directed experimentation the available data will be utilized to the fullest extent, while allowing for the addition of new data types and expert curation. The role of epigenetic events in cancer is increasingly appreciated, but the challenge of distinguishing drivers from passengers has not yet been adequately addressed. The systematic validation pipeline proposed here will address a large unmet need, and yield insights into the complementary roles of epigenetic and genetic events in key signaling pathways that drive tumorigenesis. PUBLIC HEALTH RELEVANCE: Cancer arises from loss of the growth control of cells. Although most attention has focused on genetic mutations as the origin of these defects, in recent years, it has become increasingly appreciated that changes in the epigenetic control of gene activity also contribute to this loss of growth control. This application addresses an unmet need to develop methods of finding out which epigenetic changes contribute directly to cancer formation.

DESCRIPTION (provided by applicant): The past decade has seen an explosion of interest in the role of epigenetics in cancer. Tumor suppressor gene silencing by promoter methylation is one of the key epigenetic mechanisms that contributes to tumorigenesis. The goal of the proposed application is to expand our understanding of the role of DNA methylation in tumor maintenance, by reducing DNA methylation in established colorectal tumors, using an innovative mouse model system in which the DNA methylation process can be reversibly and tightly inhibited through transcriptional repression of the endogenous Dnmt1 DNA methyltransferase gene. The first specific aim is to determine the role of DNA methylation in the maintenance of colorectal tumors. Our transcriptional repression technology enables us to circumvent cell lethality imposed by both conventional and conditional knock-out of Dnmt1, and allows us to produce the first mouse model with the capacity to conditionally and reversibly transcriptionally repress endogenous Dnmt1. The ability of our technology to suppress the DNA methylation process after the tumor has developed will allow tumors to develop under normal epigenetic influence, permitting an assessment of the role of DNA methylation in tumor maintenance without affecting tumor initiation. The inhibition of DNA methylation through transcriptional repression rather than the use of toxic compounds will allow a more specific assessment of the role of DNA methylation. The use of mouse cancer models for which the contribution of DNA methylation has been demonstrated will increase the likelihood of observing promising anti-neoplastic responses. The ability to temporally manipulate DNA methylation levels in tightly tissue-controlled context will provide unique abilities to investigate the role o epigenetics at defined time windows of tumor initiation, progression, invasion, and metastasis. The second specific aim is to comprehensively map aberrant DNA methylation changes involved in the colorectal tumor maintenance and development, and to identify candidate epigenetic driver events by comparison to human primary colorectal tumors. A combined genomics approach of whole genome shotgun bisulfite sequencing (WGSBS) and gene expression microarray analysis will allow us to efficiently map tumor- specific DNA methylation changes with an accompanying gene expression change. Comprehensive genome- wide DNA methylation data of 500 human primary colorectal cancer samples being produced in the lab in the context of TCGA will allow us to perform extensive validation of results obtained in our proposed mouse study. This proposed application will likely yield valuable insights into epigenetic contributions to the maintenance of established tumors, and will produce a genome-wide view of epigenetic alterations involved in the development and maintenance of colorectal tumors.

PROJECT SUMMARY / ABSTRACT The past three decades have witnessed an accumulating body of evidence that epigenetic mechanisms play an instrumental role in human cancer. Epigenetic alterations can serve as driver events in cancer by inactivating tumor-suppressor genes. The finding that these silencing events are mutually exclusive with structural or mutational inactivation of the same gene reinforces the functional significance of epigenetic silencing. The majority of cases of microsatellite instability in sporadic human tumors can be attributed to epigenetic silencing of the MLH1 mismatch repair gene. One of the most striking discoveries to emerge from cancer genome projects has been the previously unappreciated preponderance of somatic mutations in epigenetic regulators in most types of human cancer. Clearly, epigenetic mechanisms play a key role in human cancer, and a comprehensive molecular characterization of cancer should include epigenomic profiling. We propose to create an Integrative Cancer Epigenomic Data Analysis Center (ICE-DAC) to provide specialized analysis pipelines and expertise as part of the Genome Data Analysis Network (GDAN). We anticipate that epigenomic data will be provided as bisulfite-based sequence data or as DNA methylation BeadArray data, and we provide an analysis workflow that can accommodate either. We propose to apply specialized epigenetic analyses we have developed for both data types in our extensive experience in cancer genome consortia. In Specific Aim 1, we will develop, improve and implement analytic bioinformatic tools for epigenomic data analysis, including improvements to analysis tools for processing bisulfite sequence data. We will continue the development of analysis tools that use DNA methylation data to analyze tumor heterogeneity and subclonal structure, including the deconstruction of non-malignant cellular composition of the tumor. In Specific Aim 2, we will provide advanced specialized analysis of cancer epigenomic data generated by the Genome Characterization Center and/or provided through the Data Processing GDAC. Our automated workflow will provide timely primary data analysis for AWGs, and can accommodate both sequence-based or array-based DNA methylation data. This workflow will call differentially methylated regions (DMRs), identify cancer subtypes through stratified cluster analysis, analyze CpH methylation, and analyze tumor purity and subclonal heterogeneity. Performing variant analysis from bisulfite sequence data allows us to determine the impact of non-coding mutations on epigenetic state. In Specific Aim 3, we will integrate epigenomic data with other genomic, transcriptomic, proteomic, and clinical data to derive biologically and clinically relevant novel insights. Integration of DNA methylation and RNA-Seq data will be used for epigenetic silencing calls and for our custom enhancer identification ELMER pipeline, both of which will feed into pathway and network analyses.

PROJECT SUMMARY / ABSTRACT Cancer is thought to start with the loss of function of key gatekeeper genes, such as APC in intestinal tumorigenesis. Sporadic cases of colorectal cancer in humans are thought to acquire somatic genetic hits for both alleles, while cases of Familial Adenomatous Polyposis are thought to acquire the first hit through germline inheritance of a defective allele, followed by somatic loss of heterozygosity of the remaining wildtype allele. Although loss of Apc function is a powerful early oncogenic event in intestinal tumorigenesis, it does not by itself appear to be sufficient to drive progression in all intestinal stem cells. The past three decades have witnessed an accumulating body of evidence that epigenetic mechanisms play an instrumental role in human cancer. Epigenetic alterations can serve as driver events in cancer by inactivating tumor-suppressor genes. The finding that these silencing events are mutually exclusive with structural or mutational inactivation of the same gene reinforces the functional significance of epigenetic silencing. Most epigenetic silencing events appear to be clonal, suggesting that these alterations occur very early in tumorigenesis, possibly preceding clonal expansion. DNA methylation silencing events in human and mouse tumors are enriched for gene targets of Polycomb repression in stem cells that are normally required for differentiation. We hypothesize that stem cells with appropriate pre- existing DNA methylation alterations, possibly resulting in impaired differentiation capability are primed for rapid progression upon loss of Apc function. We propose to use an innovative visualization system in the ApcMin mouse model of intestinal tumorigenesis to study the earliest events in cancer initiation, characterize the rates of progression, and identify cooperating genetic and epigenetic events in early tumorigenesis. Specific Aim 1 will be to determine the rate of Loss of Heterozygosity of Apc in intestinal epithelium. Our strategy to visualize loss of the wildtype Apc allele is to incorporate a transcriptional repressor into the endogenous Apc locus through gene targeting, without disrupting Apc function (Aim 1a). We will use this system to determine the rate of Apc loss of heterozygosity in the intestinal epithelium and in early tumors (Aim 1b). Specific Aim 2 will be to define genetic and epigenetic events in early progressing lesions. We will use fluorescence-based cell sorting and Whole Genome Bisulfite Sequencing (WGBS) to identify DNA methylation alterations in early lesions, as well as somatic mutation discovery and copy number alterations. Larger numbers of more advanced lesions will be analyzed for recurrent DNA methylation abnormalities using a custom mouse DNA methylation Infinium array that we have designed in collaboration with Illumina. Specific Aim 3 will be to identify early candidate driver events that cooperate with Loss of Apc using human DNA methylation, mutation, copy number and expression data from TCGA, as well as our mouse DNA methylation and gene expression data to prioritize candidates (Aim 3a). We will use a novel CRISPR/Cas9-based DNA methylation editing system in mouse adenoma and human cancer cell line to validate candidate drivers.

PROJECT SUMMARY / ABSTRACT Clonal expansion is a pivotal characteristic of cancer, and is thought to be initiated by a genetic alteration in a key gatekeeper driver gene. However, not all normal cells appear to be susceptible to malignant transformation following such an event. Multiple lines of evidence suggest that epigenetic heterogeneity among normal cells may affect their cancer-initiating potential. This is a particularly challenging concept to investigate, since there is no straightforward way a priori to identify which cells may be susceptible or resistant to transformation prior to clonal expansion. We propose an innovative series of experiments to test the hypothesis that pre-existing epigenetic heterogeneity among normal cells affects the likelihood of malignant transformation and further progression upon genetic alteration of a cancer driver gene. In Specific Aim 1, we will use an improved protocol for single-cell whole genome bisulfite sequencing to document the degree of DNA methylation heterogeneity among flow-sorted mouse colon stem cells. For the first time, this will provide insight into whether low-level DNA methylation abnormalities observed in normal tissues are stochastically distributed, or whether subsets of stem cells bear multiple concerted epigenetic abnormalities. In Specific Aim 2, we will first generate large numbers of individual clonally expanded mouse colon organoids harboring conditional alleles for Apc, Trp53, Kras and Braf. Each organoid will be cryopreserved and concurrently subjected to whole genome bisulfite sequencing to delineate each methylome. Organoids representing diverse methylomes will be thawed, the cancer drivers activated, and then tested for tumorigenicity by colon enema engraftment. This innovative experiment aspires to provide the first direct evidence for the contribution of pre-existing epigenetic heterogeneity to cancer predisposition. In Specific Aim 3, we will provide empirical evidence for this concept by using a novel in vivo transcriptional control system to transiently up- or down-regulate DNA methyltransferase activity in mice, prior to activation of conditional key cancer drivers. This aim should provide a proof of concept for a causal role of pre-existing epigenetic variation affecting cancer propensity. This proposal addresses a concept for which there is considerable indirect evidence, but which remains a poorly studied area because of the technical and conceptual challenges presented by the premise. Our proposed experiments are intricate and complex, but we have considerable expertise in all areas of the proposal, and have developed cutting-edge solutions to many of the technical hurdles. We may not be able to conclusively delineate in detail all epigenetic variations that predispose to malignancy, but this exploratory project should provide a proof of principle for the importance of pre-existing epigenetic heterogeneity in cancer susceptibility. The outcome of this study should have a substantial impact on our understanding of lifetime accumulation of cancer risk, with implications for cancer screening and prevention.

PROJECT SUMMARY / ABSTRACT Cellular and molecular alterations that accumulate during cell division are considered to be contributors to aging phenotypes. Changes in epigenetic marks, including DNA methylation, have been widely documented in aging. This has spurred the development of epigenetic molecular clocks, which rely primarily on replication- independent gain of methylation at CpG islands. As molecular clocks tuned to chronological time across different tissue types with varying mitotic rates and histories, these clocks are not directly linked to mitotic cell division. In contrast, we have found that loss of DNA methylation at lamina-attached, late-replicating regions of the genome is closely tied to apparent mitotic history. We propose to use primary human cell culture to experimentally validate that mitosis is a driver of hypomethylation, and to explore the underlying mechanisms and consequences of this hypomethylation, to define genomic and chromatin features driving hypomethylation, and use this data to construct a mitotic molecular clock. In Specific Aim 1 we propose to use primary human cell culture to provide experimental evidence for the contribution of mitosis to PMD hypomethylation, and to disentangle the time-dependency and mitosis- dependency of the phenomenon using cell cycle inhibition. We will also investigate whether enhancing maintenance methylation machinery is able to counteract the progressive loss of DNA methylation. In Specific Aim 2 we propose to investigate whether DNA hypomethylation contributes to replicative senescence or associated phenotypes in primary human cell culture. We will investigate whether inhibition of maintenance methylation accelerates senescence, and whether progressive hypomethylation is extended in telomerase- immortalized primary human fibroblasts, contributing to premalignant phenotypes associated with such immortalization. We will also investigate whether accelerated senescence by progerin expression or supra- physiologic O2 leads to accelerated DNA hypomethylation. In Specific Aim 3 we will characterize the genomic and chromatin features that influence individual CpG hypomethylation rates. We will measure the rates of DNA hypomethylation of individual CpGs in six different primary human cell types and use this information to identify cell-type-specific, as well as universal genomic and chromatin drivers of hypomethylation. Finally, we will use elastic net regression on the assembled data to construct cell-type-specific and universal epigenetic mitotic clocks in Specific Aim 4. The preliminary studies strongly support the concept and feasibility of a DNA hypomethylation-based mitotic clock. The outcome of this proposed research could have important impacts on our understanding of the contribution of widespread hypomethylation to aging phenotypes, with potential implications for aging interventions. The availability of accurate molecular clocks specifically designed to measure mitotic history would provide a valuable molecular tool to characterize cells and tissues in human health and disease.