The Transcriptomic Landscape of Mismatch Repair-Deficient Intestinal Stem Cells

Lynch Syndrome (LS) is the most common cause of hereditary colorectal cancer (CRC) and is secondary to germline alterations in one of four DNA mismatch repair (MMR) genes. Here we aimed to provide novel insights into the initiation of MMR deficient (MMRd) colorectal carcinogenesis by characterizing the expression profile of MMRd intestinal stem cells (ISC). A tissue-specific MMRd mouse model (Villin-Cre;Msh2LoxP/LoxP) was crossed with a reporter mouse (Lgr5-EGFP-IRES-creERT2) to trace and isolate ISCs (Lgr5+) using flow cytometry. Three different ISC genotypes (Msh2-KO, Msh2-HET, and Msh2-WT) were isolated and processed for mRNAseq and mass spectrometry followed by bioinformatic analyses to identify expression signatures of complete MMRd and haplo-insufficiency. These findings were validated using qRT-PCR, immunohistochemistry, and whole transcriptomic sequencing in mouse tissues, organoids, and a cohort of human samples, including normal colorectal mucosa, pre-malignant lesions, and early-stage CRC from LS patients and Familial Adenomatous Polyposis (FAP) patients as controls. Msh2-KO ISCs clustered together with differentiated intestinal epithelial cells from all genotypes. Gene set enrichment analysis indicated inhibition of replication, cell cycle progression, and the Wnt pathway and activation of epithelial signaling and immune reaction. An expression signature derived from MMRd ISCs successfully distinguished MMRd neoplastic lesions of LS patients from FAP controls. SPP1 was specifically upregulated in MMRd ISCs and colocalized with LGR5 in LS colorectal pre-malignant lesions and tumors. These results show that expression signatures of MMRd ISC recapitulate the initial steps of LS carcinogenesis and have the potential to unveil novel biomarkers of early cancer initiation.