Multi-omics elucidation of KDM5C, KDM6A, and KMT2B roles in cancer epigenetic dysregulation and transcriptional reprogramming.

Histone-modifying enzymes (HMEs) are critical regulators of tumorigenesis through epigenetic reprogramming. While mutations in HMEs are recognized drivers of cancer epigenome dysregulation, systematic comparative analyses of their mutational impacts and functional divergence across malignancies remain underexplored. Here, we investigated three HMEs frequently mutated in diverse cancers: KMT2B (H3K4me3 methyltransferase), KDM5C (H3K4me3 demethylase), and KDM6A (H3K27me3 demethylase). Using CRISPR/Cas9-engineered HEK293T knockout cell lines, we performed integrated multi-omics profiling that combined genome-wide chromatin accessibility, transcriptomics, and chromatin-bound proteomics. Contrary to expectations that KMT2B loss (H3K4me3 depletion) and KDM5C loss (H3K4me3 accumulation) would induce opposing transcriptional programs, or that KDM6A deficiency (H3K27me3 accumulation) would exhibit distinct regulatory effects, our analyses revealed distinct effect of all three HME modulations in terms of both transcriptional output and chromatin-associated proteomic state. Functionally, KDM5C loss upregulated FOXF2 and downregulated KLF5, implicating the dysregulation of G protein-coupled receptor pathways; KDM6A loss upregulated JUNB and downregulated TP73, affecting extracellular matrix regulation; and KMT2B loss upregulated JUN and downregulated HOXA10, impacting on cytokine signaling. Notably, transcription factors such as PATZ1 and GATA2 were commonly altered across knockouts. In PANC-1 pancreatic cancer cells, we further confirmed that KDM6A regulates CDH family genes controlling cell adhesion, thereby promoting migration and invasion. Finally, integrative analyzes demonstrated strong correlations between promoter accessibility, transcription factor occupancy, and gene expression, and uncovered cooperation between epigenetic and genetic drivers. Together, these findings reveal context-dependent functional hierarchies among HMEs and underscore the necessity of multi-layered analyses to resolve the complexity of epigenetic regulation in cancer.