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.