Abstract Human epidermal growth factor receptor 2 (HER2), which gene is often amplified in breast cancer (BC), promotes metastatic phenotype and confers aggressive tumor behavior. Progress made in recent years by therapeutic strategies using monoclonal antibodies has increased survival rates of HER2-positive BC patients. However, current clinical trials in BC combining immunotherapy (inhibition of PD-1/PD-L1) with anti-HER2 antibodies, resulted only in a relatively modest overall response rate. This underlines that rise of resistance, recurrence, and metastatic processes remain the main hindrance for successful therapy and complete cancer remissions. Thus, there is an urgent need to bridge a therapeutic gap for the development of targeted therapies, especially for HER2-positive metastatic BC. Direct targeting of amplified cancer genes to invoke apoptosis through the activation of DNA damage response offers an alternative mechanism to induce tumor-specific cell death. The technology recently developed in our laboratory utilizes triplex-forming oligonucleotides (TFOs) to form triplex structures at amplified oncogenic loci. Excessive distortion to the helical DNA structure caused by the TFOs leads to immoderate DNA damage and apoptosis, but only in response to the formation of multiple triplex structures. As a result, normal cells having only two copies of the gene can efficiently handle a low level of triplex-induced DNA damage by employing nucleotide excision repair (NER). This phenomenon provides an opportunity to specifically target and induce apoptosis in BC cells with amplifications of the HER2 gene. Intriguingly, recent studies provide strong evidence that genomic DNA damage leads to activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway and facilitates antitumor immune responses. HER2 also directly participates in a disruption of STING signaling, suppressing antitumor immunity and provides a survival advantage, conferring resistance to trastuzumab. Therefore, the crosstalk between stimulation of innate immune signaling and DNA damage response opens an interesting perspective of combination therapy strategies focusing on both DNA-damaging and immune system-activating therapies in BC. In this work, we discovered that TFO-induced DNA damage leads to activation of innate immune signaling in BC cell line models. First, we characterized HER2 amplification and expression of cGAS-STING pathway in numerous BC cell lines and assessed levels of triplex-induced DNA damage and apoptosis. Next, we evaluated activation of the cGAS-STING pathway in BC cell lines by analyzing STING downstream targets following TFO treatment. We discovered that TFO-induced DNA damage response in BC cells had limited effect on canonical STING signaling as measured by stimulation of TBK1-IRF3 downstream targets. However, TFO treatment induced phosphorylation of NF-κB p65 transcription factor. We validated STING-dependency in the stimulation of innate immune system following TFO treatment and confirmed that this activation can also be achieved with TFOs targeting various genomic regions including exons and introns within the HER2 gene. To reveal mechanistic landscape of the TFO-induced activation of innate immune signaling we performed analysis of gene expression using bulk RNA sequencing. In summary, we have validated that HER2-targeted TFOs can induce activation of the innate immune system signaling in BC cell lines. Our work contributes to understanding of the crosstalk between DNA repair, apoptosis, and innate immune signaling. This will aid in the design of novel combinations of therapeutic approaches to increase responsiveness of BCs to immune-targeted treatments through activation of DNA damage response, ultimately overcoming therapy resistance mechanisms. Citation Format: A. Krysztofiak, A. Brown, A. Minnah, F. Rogers. Direct targeting of amplified HER2 gene activates immune signaling through DNA damage response abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-04-18.
Krysztofiak et al. (Tue,) studied this question.