Histone H4 lysine 20 tri-methylation safeguards breast cancer lineage fidelity and unfolded protein response [ChIP-seq/ATAC-seq]

Triple-negative breast cancer (TNBC) is a breast cancer subtype with worse prognosis and limited therapeutic options, partly due to significant epigenetic heterogeneity both within and between tumors. We previously reported that histone H4 lysine 20 trimethylation (H4K20me3) is the most variable histone modification in TNBC. H4K20me3 showed an opposing genomic distribution to other H4 acetylations and was essential for histone integration with a subset of distal intergenic genomic regions. Unlike H3K9me3, H4K20me3 exhibited greater heterogeneity across tumors and normal mammary glands. Regions uniquely enriched for H4K20me3 have higher epigenetic and transcriptional activity. Inhibition of the H4K20me3 methyltransferase, KMT5B/5C using A-196 led to substantial H4K20me3 loss and H4K20me1 gain at regions associated with lineage-specificity, stress response, and cell cycle regulation. A-196 treatment moderately suppressed tumor growth in vivo, inducing mesenchymal transition and unfolded protein response (UPR) in cancer cells, and increased proinflammatory macrophage infiltration into the tumor microenvironment. Integrated multi-omic analysis revealed that mesenchymal TNBCs, with the lowest H4K20me3 levels, have more active chromatin at UPR genes and stronger responses to ER stress inducers. Consistently, KMT5B/5C inhibition in basal TNBC induced mesenchymal-like features, heightened ER stress response and the associated cell proliferative suppression through increased G1 arrest, driven by the de novo recruitment of the transcriptional repressor E2F4. Combining A-196 with the HSP90 inhibitor 17AAG synergistically reduced TNBC tumor growth. Overall, our study identifies H4K20me3 as a facultative heterochromatin mark regulating lineage identity and UPR in TNBC, offering novel epigenetic therapeutic strategies targeting KMT5B/5C.