Brca2- deficiency promotes increased cGAS-STING pathway activation and an immunosuppressive tumor immune microenvironment in prostate cancer

Background : Combined germline and somatic tumor profiling of advanced metastatic prostate cancer has revealed that ~20% of metastatic tumors have mutations in DNA repair genes such as BRCA2 . Germline variants in BRCA2 predispose to aggressive high-risk prostate cancer and more advanced disease at diagnosis. BRCA2 is vital to the repair of DNA double-strand breaks (DSB) by homologous recombination (HR) and deficiency leads to genomic instability which has been shown to drive inflammation via the cGAS-STING pathway. The implications of loss of BRCA2 function in prostate cancer on immune activation and the tumor microenvironment (TME) are unknown. This represents a significant knowledge gap towards devising therapies that effectively target the immunosuppressive TME of HR-deficient prostate cancer. Methods : We have engineered an immunocompetent Brca2 -deficient prostate cancer model with loss of function frame shift (fs) mutations ( Brca2 fs ) using CRISPR/Cas9 to address the critical gap in current preclinical models. We validated BRCA2 loss of function and characterized innate immune signaling through the cGAS-STING pathway. We performed spectral flow cytometry and single-cell RNA sequencing (scRNA-seq) on Brca2 WT and Brca2 fs tumors from immunocompetent mice to assess the TME. Mice bearing Brca2 fs tumors were treated with anti-CCR8 monoclonal antibodies with and without anti-PD-1 to test the effectiveness of targeting the CCR8-axis in Brca2 -deficient prostate tumors. Results : Brca2 fs cells show hallmarks of increased DNA damage and cGAS-STING pathway activation compared to Brca2 WT cells. Analysis of implanted Brca2 fs tumors demonstrated enrichment of CD4+ T cells and immunosuppressive CCR8+ regulatory T cells (Treg) compared to Brca2 WT tumors. scRNA-seq of Brca2 fs tumors implanted in immunocompetent mice showed a significant increase in the interferon expression signature that is likely the result of increased cGAS-STING pathway activation in tumor cells. Additionally, tumor associated macrophages (TAMs) from Brca2 fs tumors showed a greater immunosuppressive phenotype with an increase in M2 polarization (pro-tumor) and high expression of Ccl8 , the CCR8 receptor ligand. Combination anti-CCR8 and anti-PD-1 therapy significantly reduced tumor growth in mice bearing Brca2 fs tumors. Conclusions : Our analysis has revealed enrichment of immunosuppressive CCR8+ Tregs and Ccl8 -high expressing TAMs in Brca2 -deficient but not Brca2 -proficient prostate cancer tumors. Anti-CCR8 therapy, to deplete CCR8+ Tregs, in combination with anti-PD-1 therapy significantly reduced Brca2 fs tumor growth in our immunocompetent mouse model. These findings present the exciting possibility of targeting the CCR8-axis in BRCA2 -deficient prostate tumors to overcome these immunosuppressive mechanisms and enhance activity of tumor-reactive T cells.