Background
Immune checkpoint blockade (ICB) has shown unprecedented success in improving clinical outcomes for cancer patients, but many patients still fail to respond to treatment.1 Studies by our lab have demonstrated that DNA Damage Repair (DDR) pathways play a key role in determining response to ICB. More granular assessment shows that certain DDR alterations are more immunogenic, such as BRCA2 compared to BRCA1, despite being part of the same pathway.2 To better characterize the effect of these alternations on the tumor immune microenvironment (TME) and define key determinants of ICB response, we created isogenic knockouts of BRCA2 and BRCA1 in a murine 4T1 metastatic TNBC background to assess how these tumor-intrinsic programs influence the TME and poise tumors for immunotherapy response.
Methods
We analyzed whole tumors by bulk RNAseq and enriched for CD45+ cells by scRNAseq and flow cytometry. Single-cell analysis was performed using Seurat and Cell Chat. We established a genetic interferon reporter assay to measure interferon-driven inflammation in cis and in trans with tumor-associated macrophages by flow cytometry and used this to assess isogenic cell lines, tumor-intrinsic gene knockouts, and a panel of inhibitors to define determinants of response. Cell fractionation experiments were used to quantify cytosolic DNA and R-loops. In vivo murine experiments with aCSF1R depletion were performed to evaluate monocyte necessity in ICB response.
Results
Differential immune landscapes identified via scRNAseq in Brca2-mutant tumors at baseline showed key differences in the myeloid compartment and interferon-stimulated gene (ISG) expression in tumor-infiltrating myeloid cells from Brca2 mutant tumors. Bulk RNAseq analysis showed increased production of T cell trafficking chemokines that could poise the tumor for response to ICB. Interferon reporter assay results suggested tumor-intrinsic cGAS drives trans-activation of myeloid STING and IFNb1. Further assessment of tumor cell lines via cellular fractionation experiments identified the presence of both DNA and R-loops in the cytoplasm of BRCA2-mutant cell lines that serve to active cGAS/STING. In vivo experiments with tumor intrinsic knockouts showed that tumor STING signaling and cytokine production were dispensable for ICB response while depletion of monocytes via CSF1R blockade showed a complete reversal of ICB response, underlining the essential role of monocytes in poising the tumor for ICB response.
Conclusions
Taken together, our results have important implications for understanding the key drivers of ICB response and how specific patient mutations differentially the microenvironment for immunotherapy response.
References
Hirsch L, Zitvogel L, Eggermont A, et al. PD-Loma: a cancer entity with a shared sensitivity to the PD-1/PD-L1 pathway blockade. Br J Cancer 2019;120:3–5.
Samstein RM, Krishna C, Ma X, et al. Mutations in BRCA1 and BRCA2 differentially affect the tumor microenvironment and response to checkpoint blockade immunotherapy. Nat Cancer 2020;1:1188–1203.