Targeting RNase H2: A Dual Mechanism Strategy to Elevate Replication Stress, DNA Damage, and Antitumor Immunity in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) lacks effective targeted therapies and carries a poor prognosis. TNBC cells escape oncogene-induced senescence and adapt to elevated replication stress. Here, we show that cells escaping senescence depend on overexpression of RNase H2, which removes misincorporated ribonucleotides from genomic DNA. RNASEH2A, the catalytic subunit of RNase H2, is overexpressed in TNBC tumors and correlates with poor survival. Genetic silencing or pharmacological inhibition of RNase H2 selectively impairs TNBC viability while sparing non-tumorigenic mammary epithelial cells and suppresses tumor growth in vivo. Mechanistically, RNase H2 inhibition increases replication stress, DNA damage, and cytosolic single-stranded DNA accumulation, triggering innate immune activation and upregulation of T cell–recruiting chemokines. RNase H2 inhibition synergizes with ATR and PARP inhibitors and enhances immune checkpoint blockade efficacy. Together, these findings identify RNase H2 as a therapeutic vulnerability in TNBC and support combined strategies integrating DNA damage modulation and immunotherapy.