Characterization of the immune tumor microenvironment of HER2-positive breast cancer following treatment with entinostat and immune checkpoint inhibition.

e15061 Background: HER2+ breast cancers are known to be less-immunogenic and associated with low response rates to immune checkpoint inhibitors (ICIs). A combination of immunosuppressive signals that prevent cytotoxic T cells from infiltrating the tumor microenvironment (TME) and, low tumor antigen expression, contribute to immunotherapy resistance in this population. Epigenetic modulators can both reexpress tumor antigens and rewire the immunosuppressive environment. We previously used a histone deacetylase inhibitor, entinostat (ENT), in combination with ICIs to reverse the immunosuppressive TME and increase tumor antigen expression in a NeuN HER2+ mouse model of breast cancer. Our results showed that ENT in combination with anti-PD-1, anti-CTLA-4, provided a significant survival benefit compared to either treatment alone. Methods: This current study employs single cell RNA-sequencing on whole tumor samples from mice treated with ICIs and entinostat to investigate the role of epigenetic inhibitors in rewiring the expression of tumor antigens and the cellular landscape of the TME. We generate single cell data over 54,000 cells from 20 tumors treated with entinostat alone or in combination with anti-PD1 and anti-CTLA4 and their combination. Results: Analysis of cells in the TME identifies consistent proportion of monocytes, macrophages, T-cells, Myeloid Derived Suppressor Cells (MDSCs) and Cancer Associated Fibroblasts (CAFs) before and after treatment. Differential expression analysis within the cell types identifies distinct subpopulations and we explore those that are either proportionally higher or lower in each treatment group. Notably, pathway analysis on differentially expressed genes of each cell type identified that combination entinostat and checkpoint treatment increased T cell activation, leukocyte proliferation, myeloid leukocyte and neutrophil migration, and decreased Wnt signaling and histone modifications in tumor cells. These results are being corroborated in patient samples from a parallel clinical trial to provide translational relevance. Conclusions: Our current work provides insights into the transcriptional network within a breast tumor after treatment with ENT+ICIs. We predict our findings will bring us closer to identifying additional therapeutic targets and ultimately improve survival rates of patients with less-immunogenic tumors.