With epigenetic therapy emerging as a potential therapy for solid tumors and recent reports that indicate an influence of hypomethylating agents on the PD-1/PD-L1 expression in tumor tissue, epigenetic therapy in combination with immune-modulatory compounds might open-up new avenues for cancer treatment. To investigate the influence of hypomethylating agents on the PD-1/PD-L1 signaling cascade in breast cancer and its implication on possible treatment options, we determined the effect of 5-Azacytidine (5-AZA), decitabine (DAC) and anti-PD1 in mono- and combined therapy on the mouse breast cancer model 4T1 in vitro and in vivo. In a life cell imaging based 2D system we evaluated the influence of the different treatment arms on tumor growth, methylation status and PD-L1 expression. Furthermore, we determined the effect of 5-AZA and DAC on autologous T cells in the presence and absence of 4T1 cancer cells. The combination of 5-AZA and anti-PD1 treatment was further evaluated in an in vivo setting. 24 4T1 bearing balb/c mice were treated with either an isotype control, 5-AZA, anti-PD1 or a combination of the latter. Tumor growth was monitored by caliper measurement. At the end of the experiment tumor and hematopoietic organs were analyzed by flow cytometry. A panel of 36 murine cytokines was determined in the murine serum at different time points. Low dose 5-AZA (IC10) and DAC (IC2.5) induced a down regulation of PD-L1 on 4T1 cells in vitro. Interestingly, this effect was most prominent at the dose level and time point when the hypomethylation effect of the respective compound was most pronounced (day 7). 5-AZA and DAC induced an upregulation of PD-1 on autologous CD4+ as well as CD8+ T cells. Of note, 4T1 cells reduced the expression of PD-1 on autologous CD4+ and CD8+ T cells. Antitumoral activity in vitro was determined in all treatment arms. An additive effect of the combination was specifically determined when combining 5-AZA and anti PD-1. Hence, 5-AZA and anti PD-1 were investigated in vivo more in detail. 4T1 is characterized by an immune suppressive tumor microenvironment as indicated by the in vitro results: we determined a reduced CD4/CD8 ratio and a high percentage of MDSC. These features were reverted by the applied treatments. Along the same line, 5-AZA in mono- and in combination therapy with anti-PDL1 specifically increased the G-CSF levels in murine serum. In addition, the PDL-1 downregulation on the tumor cells was reproduced in vivo. Finally, the combination arm displayed the most prominent antitumoral activity. In summary, we have strong evidence that low dose hypomethylating agents influence the immune landscape of breast cancer via the modulation of PD-1/PD-L1 signaling. The combination with checkpoint inhibitors was superior to the monotherapy and might lead to new treatment options for breast cancer patients.

Citation Format: Julia Schüler, Agon Kolica, Hung Ngyuen, Anna Edinger, Kanstantsin Lashuk, Eva Oswald. Hypomethylating agents modulate the PD-1/PD-L1 signaling in a murine breast cancer model and show additive effects in the combination with PD-1 blockage in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1349.