Abstract 1555: Single cell level treatment-specific characterization of HER2+ breast cancers treated with immune checkpoint inhibitors and entinostat
Authors: Sidiropoulos Dimitrios N. , Davis-Marcisak Emily , Rafie Christine , Kagohara Luciane T. , Sharma Gaurav , Connolly Roisin M. , Stearns Vered , Yegnasubramanian Srinivasan , Jaffee Elizabeth M. , Fertig Elana J. , Torres Evanthia T. Roussos 
Published: September 2020

Abstract
HER2+ breast cancers are known to be less-immunogenic and associated with low response rates to ICIs such as anti-PD-1, anti-PDL-1 or anti-CTLA-4. 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 benzamide histone deacetylase inhibitor, entinostat, 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 entinostat in combination with anti-PD-1, anti-CTLA-4, or both provided a significant survival benefit compared to either treatment alone. This current study employs single cell RNA-seq 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 of over 54,000 cells from 20 tumors treated with entinostat alone or in combination with anti-PD-1 and anti-CTLA-4 and their combination. 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. Further analysis of the tumor cells from these data and additional ATAC-seq data will enable us to further test the role of antigen reexpression in this TME of activated tumors. We also used the CoGAPS matrix factorization algorithm and RNA velocity analysis to identify transcriptional patterns that are enriched in response to combination treatment. Our current work provides insights into the transcriptional network within a breast tumor after treatment with entinostat. Our follow-up studies in patient samples from a corresponding clinical trial will allow us to map the role of epigenetic modulation in breast tumors. We predict our findings will bring us closer to identifying additional therapeutic targets and ultimately improve survival rates of patients with less-immunogenic tumors. NCT02453620.
Citation Format: Dimitrios N. Sidiropoulos, Emily Davis-Marcisak, Christine Rafie, Luciane T. Kagohara, Gaurav Sharma, Roisin M. Connolly, Vered Stearns, Srinivasan Yegnasubramanian, Elizabeth M. Jaffee, Elana J. Fertig, Evanthia T. Roussos Torres. Single cell level treatment-specific characterization of HER2+ breast cancers treated with immune checkpoint inhibitors and entinostat [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1555.