Abstract 4209: Targeting RNase H2: a dual mechanism strategy to elevate replication stress, DNA damage, and antitumor immunity in triple-negative breast cancer

Abstract The inhibition of Ribonuclease H2 (RNase H2) activity offers a promising therapeutic approach for suppressing tumor growth in triple-negative breast cancers (TNBCs) by elevating DNA replication stress, inducing DNA damage, inducing cell death, and enhancing sensitivity to immunotherapy. RNase H2, a key enzyme responsible for removing misincorporated ribonucleotides from the genome, plays a pivotal role in maintaining genomic integrity. Overexpression of the RNASEH2A gene, which encodes the catalytic subunit of the RNase H2 complex, has been observed in breast tumors, with the highest levels detected in TNBC tumors and cell lines, making it a potential target for therapeutic intervention. This study explores the impact of RNase H2 inhibition on tumor proliferation, antitumor immunity, and its potential synergy with other DNA replication stress inhibitors. Our findings demonstrate that silencing RNASEH2A expression or pharmacologically inhibiting RNase H2 activity with the R14 inhibitor significantly increased levels of pRPA32 and γH2AX, indicating elevated DNA replication stress and DNA double-strand breaks. This was associated with substantial suppression of tumor growth and prolonged survival in TNBC models, while non-tumorigenic mammary epithelial cells remained unaffected. RNA sequencing analysis revealed that knockout of RNASEH2A in TNBC cell lines activated immune system signaling pathways, including increased production of T-cell-attracting chemokines such as CCL5, CXCL10, and CXCL11. This increase in immunostimulatory cytokine expression was attributed to the accumulation of cytosolic single-stranded DNA, which enhances innate immune responses. Moreover, pharmacological suppression of RNase H2 by the R14 inhibitor improved the efficacy of immune checkpoint blockade by promoting the infiltration of antitumor immune cells into the tumor microenvironment. This indicates that RNase H2 inhibition not only directly suppresses tumor growth but also modulates the tumor immune landscape, rendering TNBCs more responsive to immunotherapy. Additionally, the R14 inhibitor exhibited synergistic effects when combined with DNA replication stress inhibitors, including ATR inhibitors, PARP inhibitors, and their combination, consistent with the known role of RNase H2 deficiency in sensitizing cells to these therapeutic strategies. In conclusion, targeting RNase H2 activity represents a novel and dual-faceted therapeutic strategy in TNBCs, combining direct tumor growth suppression through increased DNA damage with enhanced antitumor immunity. This approach offers a promising avenue for combination therapy with immune checkpoint inhibitors and DNA replication stress modulators. These findings provide a rationale for the clinical development of RNase H2 inhibitors as part of an integrated treatment strategy for TNBC. Citation Format: Thai Quynh Anh Nguyen, Daniel J. McGrail, Hui Dai, Aysegul Cifci, Yong Du, Funda Meric-Bernstam, Shiaw-Yih Lin. Targeting RNase H2: a dual mechanism strategy to elevate replication stress, DNA damage, and antitumor immunity in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4209.