Abstract B020: Dual checkpoint inhibition using anti-PD-1 and anti-CTLA-4 in combination with entinostat increases treatment efficacy and decreases suppressive signaling between myeloid cells and T cells in breast-to-lung metastasis
Authors: Gonzalez Edgar , Kreger Jesse , Liu Yingtong , Baugh Aaron , Jang Julie , Al-Zubeidy Batul , Danilova Ludmila , Shin Sarah M , Barbetta Arianna , Bangerth Sara-Elisa B , Jacobo Matthew , Castanon Sofi , Stearns Vered , Connolly Roisin M , Ho Won , Emaumaullee Juliet , MacLean Adam , Roussos-Torres Evanthia T 
Published: February 2025

Abstract
Most patients with breast cancer eventually succumb to disease due to health complications caused by metastasis, with the lungs being one of the most common sites of metastasis. Immune checkpoint inhibitors (ICIs) can reactivate T cells to eliminate cancer cells, but they have proved ineffective against metastatic breast cancer on their own. Immunosuppressive myeloid cells in the tumor microenvironment (TME), such as tumor associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), are abundant in breast tumors and metastases, which inhibit anti-tumoral responses by T cells and are implicated in resistance to treatment with ICIs. In our Phase Ib trial (NCI-9844), the combination of the histone deacetylase inhibitor, entinostat, with dual ICIs anti-PD-1 and anti-CTLA-4 increased survival in patients with metastatic HER2-negative breast cancer, resulting in an overall 25% objective response rate (ORR) and a 40% ORR in patients with TNBC. Our group and others have shown that this treatment combination inhibits breast-to-lung metastasis in the 4T1 model of triple-negative breast cancer (TNBC) and increases survival both in 4T1 and the NT2.5 model of HER2 + breast cancer, with an associated decrease in either MDSC infiltration or suppression of T cell proliferation. In this study, we examined spontaneous breast-to-lung metastases in two syngeneic metastatic mouse models, 4T1 and NT2.5LM, to gain a comprehensive understanding of changes to the metastatic TME induced by entinostat and maintained with the addition of anti-PD-1 and anti-CTLA-4. Survival was increased in the NT2.5LM model, with no resultant changes in infiltration of immune cells either by single cell RNA-sequencing (scRNA-seq) or flow cytometry, as opposed to the 4T1 model, where there was an increase in effector CD8+ T cells within metastases. CellChat analysis revealed decreased signaling between myeloid cells and T cells for multiple ligand-receptor pairs associated with immunosuppression, including Tgfb1_Tgfbr1_Tgfbr2, Entpd1_Adora2a, and Icam1_Itgam_Itgb2. Myeloid immunosuppression was assessed by co-culturing TAMs or granulocytic MDSCs (G-MDSCs) from lung metastases with CD8+ T cells ex-vivo and assessing T cell proliferation. G-MDSCs and MHC-IIhi expressing TAMs suppressed T cell proliferation and did not change with treatment. In addition, tissue samples from our phase 1b trial were analyzed via imaging mass cytometry (IMC), and analysis is underway to validate preclinical findings in patients. Preliminary results demonstrate increased distances between CD8+ T cells and both CD68+ TAMs and CD15+ G-MDSCs in a responder, with no changes observed in a non-responder. Our results suggest that entinostat rewires CD8+ T cell interactions with TAMs and G-MDSCs, potentially increasing the efficacy of ICIs by decreasing myeloid suppression and highlighting specific targets, such as ICAM-1, which we will continue to investigate for mechanistic relevance and ability to serve as a therapeutic targets or biomarkers of response to treatment.
Citation Format: Edgar Gonzalez, Jesse Kreger, Yingtong Liu, Aaron Baugh, Julie Jang, Batul Al-Zubeidy, Ludmila Danilova, Sarah M Shin, Arianna Barbetta, Sara-Elisa B Bangerth, Matthew Jacobo, Sofi Castanon, Vered Stearns, Roisin M Connolly, Won Ho, Juliet Emaumaullee, Adam MacLean, Evanthia T Roussos-Torres. Dual checkpoint inhibition using anti-PD-1 and anti-CTLA-4 in combination with entinostat increases treatment efficacy and decreases suppressive signaling between myeloid cells and T cells in breast-to-lung metastasis [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B020.