Histone H4 lysine 20 tri-methylation safeguards breast cancer lineage fidelity and unfolded protein response.

Triple-negative breast cancer (TNBC) has poor outcomes and few effective treatments, partly because of epigenetic heterogeneity. We show that the heterochromatin mark H4K20me3 is more variable than H3K9me3 across normal and malignant breast tissues, and that H4K20me3-enriched regions are linked to more open chromatin and active transcription. Pharmacologic inhibition of the H4K20me3 methyltransferases KMT5B and KMT5C with A-196 decreases H4K20me3 and increases H4K20me1 at loci involved in lineage identity, stress responses, and the cell cycle. A-196 suppresses tumor growth in vivo while promoting mesenchymal features and the unfolded protein response (UPR). Multi-omic profiling shows that mesenchymal TNBCs have the lowest H4K20me3 levels and the greatest sensitivity to UPR stress. In basal TNBC, KMT5B&5 C inhibition drives mesenchymal transition and elevated endoplasmic reticulum stress-associated growth arrest via E2F4. Combining A-196 with the HSP90, IRAK4 inhibitors, or STING agonist synergistically reduces TNBC tumor growth. These findings nominate KMT5B&5C as promising therapeutic targets in TNBC.