AbstractIntroductionLynch syndrome (LS) is a hereditary condition that increases the risk of colorectal (CRC) and extracolonic cancers that exhibit microsatellite instability (MSI‐H). MSI‐H is driven by defective mismatch repair (dMMR), and approximately 15% of nonhereditary CRCs also exhibit MSI‐H. Here, we aimed to better define mechanisms underlying tumor initiation in LS and MSI‐H cancers through multi‐omic analyses of LS normal colon organoids and MSI‐H tumors.MethodsRight (n= 35) and left (n= 23) colon organoids generated from normal colon biopsies at routine colonoscopy of LS and healthy individuals were subjected to Illumina EPIC array. Differentially methylated region (DMR) analysis was performed by DMRcate. RNA‐sequencing (n= 16) and bisulfite‐sequencing (n= 15) were performed on a subset of right colon organoids. CRISPR‐cas9‐mediated editing of MMR genes in colon organoids of healthy individuals was followed by quantitative PCR ofMSH4. The relationship betweenMSH4expression and tumor mutational burden was further explored in three independent tumor data sets.ResultsWe identified a hypermethylated region ofMSH4in both the right and left colon organoids of LS versus healthy controls, which we validated using bisulfite‐sequencing. DMR analysis in three gastrointestinal and one endometrial data set revealed that this region was also hypermethylated in MSI‐H versus microsatellite stable (MSS) tumors.MSH4expression was increased in colon organoids of LS versus healthy subjects and in publicly available MSI‐H versus MSS tumors across four RNA‐seq and four microarray data sets. CRISPR‐cas9 editing ofMLH1andMSH2, but notMSH6, in normal colon organoids significantly increasedMSH4expression.MSH4expression was significantly associated with tumor mutational burden in three publicly available data sets.ConclusionsOur findings implicate DNA methylation and gene expression differences ofMSH4as a marker of dMMR and as a potential novel biomarker of LS. Our study of LS colon organoids supports the hypothesis that dMMR exists in the colons of LS subjects prior to CRC.