Targeting DNA Polymerase Epsilon Leads to Tumor Clearance and Activation of an NF-?B-mediated inflammatory response in Triple Negative Breast Cancer

Breast cancer remains the second leading cause of cancer-related mortality among women, with triple-negative breast cancer (TNBC) exhibiting a particularly poor five-year prognosis1. Here, we demonstrate that among genetic and pharmacological perturbations targeting DNA replication, suppression of DNA polymerase epsilon (POLE) in TNBC, induces a potent, TNBC-specific gene expression signature enriched in inflammatory cytokines that are transcriptional targets of NF-?B. TNBC cells exhibit markedly higher levels of DNA damage and canonical NF-?B activation compared to luminal breast cancer cells. Notably, NF-?B activation in this context depends on the canonical component RELA, but not the non-canonical component RELB. Mechanistically, ATM, STING, and RIG-I each contribute to NF-?B activation following POLE suppression. In vivo, POLE suppression in a murine TNBC model leads to cancer cell-intrinsic elimination of tumor burden and increased immune cell infiltration. Together, these findings support a model in which replication stress from POLE inhibition triggers robust NF-?B–mediated inflammation and immune microenvironment remodeling in TNBC and can independently trigger tumor eradication. These results suggest a potential therapeutic avenue for targeting POLE in TNBC. Overall design: Cancer cell lines MDA-MB-231, Hs578t, or MCF7, each expressing either a control vector or a POLE cDNA, and an shRNA targeting POLE, 6 days after infection. Cancer cell line MDA-MB-231 treated with aphidicolin (APH; 2 µg/mL), which inhibits DNA polymerases a, d, and e; hydroxyurea (HU; 2 mM), which depletes nucleotide pools by inhibiting ribonucleotide reductase; paclitaxel (PTX; 4 nM), which induces mitotic stress that indirectly perturbs DNA replication by arresting cells in metaphase; cycloheximide (CHX; 2 µM), which indirectly impairs replication by blocking protein synthesis and depleting essential replication factors; and cisplatin (CP; 12 µM), for 24 hours.