Abstract B10: HDAC inhibition modulates immune checkpoint pathway in triple-negative breast cancer
Authors: Barberio Manuela Terranova , Thomas Scott , Ali Niwa , Pawlowska Nela , Park Jeenah , Rosenblum Michael D. , Budillon Alfredo , Munster Pamela N. 
Published: February 2019

Abstract
Background: Breast cancer remains one of the most serious and common diseases, second only to lung cancer as a leading cause of cancer death in women, accounting for 30% of new diagnoses. In particular, hormone-sensitive metastatic breast cancer and triple-negative breast cancer (TNBC) remains a therapeutic challenge. Despite aggressive therapeutic options, resistance develops in almost all patients and survival remains poor, prompting the need for novel approaches.
This project focuses on the characterization of the molecular mechanisms underlying epigenetic modifiers and in particular Histone deacetylase inhibitors (HDACi) as priming modulators of immunotherapy, mainly focusing on the diversity between TNBC and hormone responsive breast cancer.
HDACi represent a new class of anticancer agents that can affect multiple cancer pathways, including reverse hormone therapy resistance, resulting in prolonged antitumor responses in patients. In addition to their effects on tumor signaling, HDACi have immune modulatory functions, including modulation of regulatory T-cells, Foxp3 expression, changes in tumor-infiltrating lymphocyte composition and induction of PD-L1 expression. The PD-1/PD-L1 pathway is an immune checkpoint inhibitor pathway that under normal conditions down-regulates cytotoxic T-cell activity to maintain immune homeostasis. Cancer cells take advantage of this pathway to suppress immune control and inhibit the anti-tumor immune response by deactivating cytotoxic T-cells in the tumor microenvironment.
Breast cancer is considered a less immunogenic cancer compared to melanoma and lung cancer. PD-L1 expression is mainly found in TNBC, HER2+, ER- and PR- tumors. Increased PD-L1 expression correlates with higher tumor infiltrating lymphocytes and those data together correlate with better response in breast cancer patients.
PD-1/PD-L1 pathway represents one of the primary immunosuppressive drivers in multiple types of cancer. Thus, inhibiting PD-1/PD-L1 interactions may prevent T-cell suppression and reactivate immune-surveillance.
Methods and Results: Evaluating PD-L1 basal expression in a different set of human and mouse breast cancer cell lines by Western blotting and real-time PCR, we found TNBC and HER2+ cells express the highest PD-L1 levels. Testing different epigenetic modifiers, we found that HDACi were able to up-regulate PD-L1 mRNA and protein expression in a time-dependent manner up to 72 hours in the TNBC MDA-MB231 cells. Co-culturing tumor cells with human peripheral blood mononuclear cell (PBMCs), we performed comprehensive flow cytometric immunophenotyping to define the role of epigenetic priming in promoting immune cell activation. We found that HDACi upregulate PD-L1 and HLA-DR expression on MDA-MB231 tumor cells independently from PBMCs presence. Opposite effects were observed with hormone responsive ER+ MCF-7 cells, where PD-L1 was not significantly increased and HLA-DR was down-regulated upon HDACi treatment. These effects were associated with reduced Foxp3 expression on CD4+ Tregs in vitro after HDACi treatment. Importantly, HDACi significantly enhanced the in vivo response to PD-1 and CTLA-4 blockade in the triple-negative 4T1 breast cancer mouse model. This was associated with increased tumor infiltrating T cells, increased survival, and tumor growth inhibition in vivo.
Conclusion: Overall, this study suggests that the combination of HDACi with immune checkpoint inhibitors identify a novel therapeutic strategy and warrants further clinical evaluation for the treatment of TNBC.
Citation Format: Manuela Terranova Barberio, Scott Thomas, Niwa Ali, Nela Pawlowska, Jeenah Park, Michael D. Rosenblum, Alfredo Budillon, Pamela N. Munster. HDAC inhibition modulates immune checkpoint pathway in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B10.