Activation of STING-Dependent Innate Immune Signaling By S-Phase-Specific DNA Damage in Breast Cancer

Abstract

Background: Previously we identified a DNA damage response–deficient (DDRD) molecular subtype within breast cancer. A 44-gene assay identifying this subtype was validated as predicting benefit from DNA-damaging chemotherapy. This subtype was defined by interferon signaling. In this study, we address the mechanism of this immune response and its possible clinical significance. Methods: We used immunohistochemistry (IHC) to characterize immune infiltration in 184 breast cancer samples, of which 65 were within the DDRD subtype. Isogenic cell lines, which represent DDRD-positive and -negative, were used to study the effects of chemokine release on peripheral blood mononuclear cell (PBMC) migration and the mechanism of immune signaling activation. Finally, we studied the association between the DDRD subtype and expression of the immune-checkpoint protein PD-L1 as detected by IHC. All statistical tests were two-sided. Results: We found that DDRD breast tumors were associated with CD4+ and CD8+ lymphocytic infiltration (Fisher’s exact test P < .001) and that DDRD cells expressed the chemokines CXCL10 and CCL5 3.5- to 11.9-fold more than DNA damage response–proficient cells (P < .01). Conditioned medium from DDRD cells statistically significantly attracted PBMCs when compared with medium from DNA damage response–proficient cells (P < .05), and this was dependent on CXCL10 and CCL5. DDRD cells demonstrated increased cytosolic DNA and constitutive activation of the viral response cGAS/STING/TBK1/IRF3 pathway. Importantly, this pathway was activated in a cell cycle–specific manner. Finally, we demonstrated that S-phase DNA damage activated expression of PD-L1 in a STING-dependent manner. Conclusions: We propose a novel mechanism of immune infiltration in DDRD tumors, independent of neoantigen production. Activation of this pathway and associated PD-L1 expression may explain the paradoxical lack of T-cell-mediated cytotoxicity observed in DDRD tumors. We provide a rationale for exploration of DDRD in the stratification of patients for immune checkpoint–based therapies.

DNA Damage and Repair Biomarkers of Immunotherapy Response

“…DDRD tumors were defined by a 44-gene expression signature associated with loss of the S-phase-specific DNA damage response and improved response to DNA-damaging chemotherapy. [102] In the study, DDRD tumors had increased IFN-related gene expression compared to non-DDRD tumors as well as increased levels of CD4 + and CD8 + T cells in the tumor and stroma[103]. Subsequent cell-based assays demonstrated that expression of the chemokines CXCL10 and CCL5, which play a key role in CD4 + and CD8 + T cell chemotaxis, increased following siRNA-mediated depletion of individual DNA repair genes (such as BRCA1 , BRCA2 , and FANCD2 ).…”

“…[103, 104] DNA-damaging agents such as cisplatin and etoposide can also increase cytosolic DNA levels in the absence of known DNA repair defects. [108] However, the mechanism by which this free DNA arises is currently not understood.…”

Targeted therapies in gynaecological cancers

“…Firstly, tumours with a BRCA1/2 mutation or an HRR defect have a higher mutational burden and therefore an elevated neo‐antigen load, which is thought to elicit an increased anti‐tumour immune response . Secondly, PARP inhibitor treatment up regulates PD‐L1 expression in vivo and in vitro , and in the absence of a functional BRCA pathway there is activation of the innate immune response via the STING/TKB1/IRF3 response, which might influence the antitumor effect of a PARP inhibitor immunotherapy combination. Several different combinations of immunotherapy and PARP inhibitors are currently under evaluation in both the first‐line and relapsed settings in a variety of gynaecological cancers.…”

Danger signals: Chemotherapy enhancers?

“…Enhanced expression of PD ‐L1 was also observed upon cisplatin and hydroxyurea treatments and such expression is dependent of STING activation. This observation suggests that when T‐cell infiltration and activation is therapeutically enhanced, the ensuing adaptive resistance can be overcome by a combination with PD ‐L1/ PD ‐1 blockade . (e) The anticancer efficacy of anti‐ CD 47 antibodies relies on the STING ‐dependent activation of adaptive immune responses: Chemotherapy treatment with cyclophosphamide or paclitaxel given before anti‐ CD 47 has synergistic anticancer effects, possibly due to increased DNA release from tumor cells…”

“…Genomically unstable breast cancers such as BRCA1‐mutant are characterized by interferon signaling and lymphocytic infiltration . Accordingly, Parkes et al . showed that in DDRD cancer cells, the presence of cytosolic DNA activates the STING/TBK1/IRF3 pathway.…”