Chemotherapeutic induction of cytosolic single-stranded DNA accumulation sensitizes triple-negative breast cancer to immunotherapy.

BACKGROUND

Despite the widespread adoption of chemoimmunotherapy in triple-negative breast cancer (TNBC), the mechanisms by which cytotoxic chemotherapy engages antitumor immunity remain poorly defined. Identifying tumor-intrinsic immunogenic programs that predict and enhance responsiveness to immune checkpoint blockade (ICB) is therefore of critical clinical importance.

METHODS

Transcriptomic signatures of TREX1 deficiency were generated from CRISPR-engineered TNBC models and applied to multiple independent TNBC cohorts treated with chemoimmunotherapy. Cytosolic single-stranded DNA (ssDNA) accumulation was quantified using a flow cytometry-based assay to functionally screen chemotherapeutic agents. Immune activation and therapeutic efficacy were evaluated using in vitro assays, syngeneic mouse tumor models, flow cytometry, and single-cell RNA sequencing. Statistical analyses included two-sided t tests or Wilcoxon tests, analysis of variance where appropriate, and receiver operating characteristic analyses.

RESULTS

An in vivo TREX1-deficiency transcriptional signature, reflecting ssDNA-driven immune programs rather than TREX1 expression alone, robustly predicted clinical response to chemoimmunotherapy across independent TNBC cohorts. Functional screening identified LP-184, an acylfulvene-derived alkylating agent in clinical development, as a potent inducer of cytosolic ssDNA and type I interferon signaling. LP-184 enhanced antigen presentation, reduced immunosuppressive M2-like macrophages, promoted CD8+ T-cell priming, and synergized with anti-programmed cell death protein 1 therapy in vivo. Exogenous ssDNA recapitulated key immunostimulatory effects of LP-184, supporting ssDNA accumulation as a central mechanistic mediator.

CONCLUSIONS

These findings establish cytosolic ssDNA-driven immune programs as a mechanistic link between DNA damage and antitumor immunity, providing both a predictive biomarker and a therapeutic axis for chemoimmunotherapy. Pharmacologic induction of ssDNA represents a rational strategy to enhance ICB efficacy and expand immunotherapy responsiveness in TNBC.