Combinatorial clinical trials of PARP inhibitors with immunotherapies are ongoing, yet the immunomodulatory effects of PARP inhibition have been incompletely studied. Here, we sought to dissect the mechanisms underlying PARP inhibitor-induced changes in the tumor microenvironment of BRCA1-defi cient triple-negative breast cancer (TNBC). We demonstrate that the PARP inhibitor olaparib induces CD8 + T-cell infi ltration and activation in vivo , and that CD8 + T-cell depletion severely compromises antitumor effi cacy. Olaparib-induced T-cell recruitment is mediated through activation of the cGAS/STING pathway in tumor cells with paracrine activation of dendritic cells and is more pronounced in HR-defi cient compared with HR-profi cient TNBC cells and in vivo models. CRISPR-mediated knockout of STING in cancer cells prevents proinfl ammatory signaling and is suffi cient to abolish olaparib-induced T-cell infi ltration in vivo. These fi ndings elucidate an additional mechanism of action of PARP inhibitors and provide a rationale for combining PARP inhibition with immunotherapies for the treatment of TNBC. SIGNIFICANCE: This work demonstrates cross-talk between PARP inhibition and the tumor microenvironment related to STING/TBK1/IRF3 pathway activation in cancer cells that governs CD8 + T-cell recruitment and antitumor effi cacy. The data provide insight into the mechanism of action of PARP inhibitors in BRCA-associated breast cancer.

DNA polymerase theta (POLθ) is synthetic lethal with Homologous Recombination (HR) deficiency and thus a candidate target for HR-deficient cancers. Through high-throughput small molecule screens we identified the antibiotic Novobiocin (NVB) as a specific POLθ inhibitor that selectively kills HR-deficient tumor cells in vitro and in vivo . NVB directly binds to the POLθ ATPase domain, inhibits its ATPase activity, and phenocopies POLθ depletion. NVB kills HR-deficient breast and ovarian tumors in GEMM, xenograft and PDX models. Increased POLθ levels predict NVB sensitivity, and BRCA-deficient tumor cells with acquired resistance to PARP inhibitors (PARPi) are sensitive to NVB in vitro and in vivo. Mechanistically, NVB-mediated cell death in PARPi-resistant cells arises from increased double-strand break end resection, leading to accumulation of single-strand DNA intermediates and non-functional RAD51 foci. Our results demonstrate that NVB may be useful alone or in combination with PARPi in treating HR-deficient tumors, including those with acquired PARPi resistance. (151/150)

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