Acute degron-mediated RUNX1 loss reprograms enhancer activity associated with epithelial destabilization and initiation of cancer hallmarks.

RUNX1 coordinates phenotypic gene expression, and its suppression or perturbations are associated with breast tumor initiation and progression. Mechanisms governing the dual roles of RUNX1 in sustaining the mammary epithelial phenotype while suppressing initiation of cancer-compromised gene expression are unclear. Degron-mediated acute, selective, and complete RUNX1 ablation in human mammary epithelial cells reveals promoter and distal enhancer-driven expression of a gene cohort. RUNX1 ablation rapidly and selectively decreases chromatin accessibility and H3K27ac at RUNX1-bound enhancers but not promoters. While differentially initiated and expressed genes contacted by RUNX1-bound enhancers are enriched in epithelial maintenance and stemness pathways, genes with RUNX1-promoter occupancy support DNA damage responsiveness. Modified cell morphology, metabolic control, breast cancer stemness, plasticity, anchorage-independent survival, chemoresistance, and perturbed DNA damage reactivity are observed upon RUNX1 ablation. These findings define RUNX1 as a tumor suppressor maintaining epithelial cell state, preserving enhancer activity to prevent gene expression associated with cancer hallmarks.