P08.03 Drug screening reveals epigenetic regulators of PD-L1 expression in triple-negative breast cancer

Background Immunotherapy has emerged as an effective cancer treatment by reactivating the immune system against tumor cells. 1 However, tumor-intrinsic upregulation of PD-L1 constitutes a major immune-evasion mechanism, suppressing T cell–mediated antitumor immunity.2 3 Although immune checkpoint blockade with anti–PD-1 or anti–PD-L1 antibodies can restore T cell function, durable clinical responses are achieved in only a subset of patients.4 These limitations underscore the need to identify upstream regulatory mechanisms that control PD-L1 expression in cancer cells. Emerging evidence suggests that epigenetic regulation plays a crucial role in modulating PD-L1 expression and influencing responses to immunotherapy.5 Here, we conducted a systematic pharmacological screen using 145 drugs targeting various epigenetic enzymes to identify key chromatin-based regulators of PD-L1 expression in triple-negative breast cancer (TNBC) cells.Methods A tumor cell line model with high basal PD-L1 expression was selected for this study. MDA-MB-231 cells were seeded in 12-well plates at a density of 50,000 cells/well and treated with 5 µM of the Epigenetic Drug Library (#11076) for 48 hours. Following treatment, cells were fixed with 4% formaldehyde and stained with a cell surface–specific anti–PD-L1 Alexa Fluor 488–conjugated antibody (#25048) diluted 1:200 in 0.5% BSA/PBS buffer. PD-L1 surface expression was quantified by flow cytometry using the FITC channel and compared to vehicle-treated cells. Compounds that significantly modulated PD-L1 surface expression were subsequently identified and reported.Results Based on the screening results, PFI-4 (BRPF1 inhibitor), CPTH2 (GCN5 inhibitor), SGC-CBP30 (CBP/p300 inhibitor), and Bromosporine (BRD4 inhibitor) significantly reduced PD-L1 surface expression in MDA-MB-231 cells.Conclusions Our findings highlight tumor-specific epigenetic vulnerabilities that govern immune checkpoint regulation. Importantly, this study identifies multiple epigenetic targets whose pharmacological inhibition may reduce PD-L1 expression in PD-L1–positive tumors and thereby provide a strong rationale for tumor context–specific combination treatment strategies. Future studies will assess the direct binding of the identified epigenetic regulators to the PD-L1 promoter using ChIP, determine their causal role via CRISPR-mediated gene editing, and evaluate the therapeutic potential of epigenetic–immunotherapy combinations in functional immune assays.References Farkona S, Diamandis EP, Blasutig IM. Cancer immunotherapy: the beginning of the end of cancer? BMC Med. 2016;14(1):73.Cha JH, Chan LC, Li CW, Hsu JL, Hung MC. Mechanisms controlling PD-L1 expression in cancer. Mol Cell. 2019;76(3):359–370.Chen J, Jiang CC, Jin L, Zhang XD. Regulation of PD-L1: a novel role of pro-survival signalling in cancer. Ann Oncol. 2016;27(3):409–416.Ribas A. Tumor immunotherapy directed at PD-1. N Engl J Med. 2012;366(26):2517–2519.Chen X, Pan X, Zhang W, Guo H, Cheng S, He Q, et al. Epigenetic strategies synergize with PD-L1/PD-1 targeted cancer immunotherapies to enhance antitumor responses. Acta Pharm Sin B. 2020;10(5):723–733.