Persistent interferon signaling and clonal expansion mark early events in DNA methylation damage-induced liver cancer

<i>N</i>-Nitrosodimethylamine (NDMA), a probable human carcinogen, induces toxic and mutagenic DNA <i>O</i> ⁶-methylguanine (<i>O</i> ⁶MeG) adducts that are repaired by <i>O</i> ⁶-methylguanine-DNA methyltransferase (MGMT). To elucidate how early-life environmental mutagenic exposures promote latent liver tumorigenesis, we performed longitudinal studies in wild-type and MGMT-deficient (<i>Mgmt</i> ^-/-) mice. Neonatal mice received NDMA intraperitoneally on postnatal days 8 and 15 and were followed for up to 10 months post-exposure. Phenomics, transcriptomics, phosphoproteomics, spatial transcriptomics, and histopathology revealed that NDMA exposure in <i>Mgmt</i> ^-/- mice produced a nine-fold increase in liver tumors, including hepatocellular carcinomas and adenomas. Early molecular profiling revealed elevated <i>O</i> ⁶MeG adducts, sustained γH2AX activation, and increased micronucleus formation in <i>Mgmt</i> ^-/- livers. Transcriptomics showed persistent interferon response and immune cell infiltration, particularly in <i>Mgmt</i> ^-/- mice, up to 10 months post-exposure. Clonal expansion, quantified by RaDR-GFP expression, was significantly higher in NDMA-exposed <i>Mgmt</i> ^-/- mice. Spatial transcriptomics of clonally expanded cells demonstrated activation of oncogenic and inflammatory pathways. These findings demonstrate that acute early-life NDMA exposure drives persistent DNA damage signaling, chronic interferon activation, and clonal expansion that together promote MGMT-dependent hepatocarcinogenesis. Collectively, these processes link early environmental mutagen exposure to long-term premalignant evolution and tumor emergence, and define mechanistic biomarkers and potential interception targets for NDMA-induced liver cancer.