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BACKGROUND & AIMS: Aberrant epigenetic programs that suppress differentiation and enhance plasticity drive colorectal cancer (CRC), yet the molecular determinants underlying these processes remain elusive. We aimed to identify and characterize epigenetic regulators of CRC differentiation, uncovering mechanisms that reprogram cancer cell states.METHODS: A small-molecule library targeting epigenetic regulators was screened using an endogenous dual-reporter system. We evaluated lead compounds in mouse and human CRC models via histopathology, cellular assays, epigenetic studies, mass spectrometry-based histone modification profiling, and single-cell RNA sequencing. Integrative analyses of drug-induced chromatin dynamics, gene expression, target engagement, and histone marks elucidated molecular mechanisms. Focused genetic screens were conducted to identify regulators of histone deacetylase (HDAC)1/2-mediated differentiation.RESULTS: We found that inhibition of the HDAC1/2 catalytic domain promotes CRC differentiation and suppresses tumor growth. Unbiased profiling of histone modifications identified acetylation of lysine 27 on histone H3 protein subunit (H3K27ac) and acetylation of lysine 9 on histone H3 protein subunit (H3K9ac) as critical regulatory marks, with genome-wide analyses demonstrating their enrichment at HDAC1/2-bound regions associated with open chromatin and up-regulated differentiation genes. Disrupting H3K27ac by targeted degradation of acetyltransferase E1A binding protein P300 reversed the differentiation phenotype induced by HDAC1/2 inhibition in a patient-derived CRC organoid. Genetic screens revealed that death-associated protein kinase 3 contributes to H3K27ac-mediated CRC differentiation induced by HDAC1/2 inhibition.CONCLUSIONS: Our findings establish histone acetylation as a chemically targetable mechanism governing CRC cell fate and demonstrate that epigenetic reprogramming can be leveraged as a therapeutic strategy. By identifying HDAC1/2 inhibition as a driver of differentiation and revealing H3K27ac as a key regulatory mark, this study provides a framework for targeting chromatin-modifying enzymes to counteract CRC plasticity and improve treatment outcomes.