Abstract 1008: Characterizing the post-translational regulation of programmed death-ligand 1 (PD-L1) in triple-negative breast cancer: Role of EphA3/Fes/Fer

Abstract

Abstract Background: The programmed death ligand-1 (PD-L1) is a cell surface protein expressed on a variety of antigen-presenting cells for the purpose of controlling the immune response. As such, the role of PD-L1 expression on the surface of cancer cells has been proven to contribute to cancer immune evasion and tumor progression. Triple negative breast cancer has the most abundant tumor infiltrating lymphocytes (TILs) and PD-L1 is found highly upregulated in TNBC, allowing blockade immune checkpoint therapy a potentially effective treatment. However, the overall response rate is less than 20-30%. Over the past years, the literature that implicated the regulation of PD-L1 was primarily on transcription levels. The objective of our present study is to further characterize the post-translational modification of PD-L1 in efforts to better target it in TNBC immunotherapy. Methods and Results: By proteomic screen utilizing tandem affinity purification of interacting proteins using SFB (S, Flag, and Streptavidin binding peptide)-tagged PD-L1, we identified a number of novel interactions between PD-L1 and 67 candidates. A number of exclusive kinases have also shown high-confidence binding with PD-L1 in the proteomic screen. The primary hits were shortlisted and validated by means of co-immunoprecipitation (co-IP). The co-IP has confirmed a number of hits identified in the proteomic screen, including receptor tyrosine kinases (ephrin receptors, EphA3), and non-receptor tyrosine kinases (Fes and Fer) which have been explored in tumor progression and immune cell development. To test whether our candidate kinases can directly phosphorylate PD-L1 in vitro, His tagged-PD-L1 was mixed with active EphA3/Fes/Fer kinases. Phosphorylated PD-L1-His was detected by WB using PNBM treatment and anti-thiophosphate antibody. Kinase assays showed that EphA3/Fes/Fer can directly phosphorylate PD-L1. We also found that Fes/Fer were able to up-regulate PD-L1 protein expression in a dose-dependent manner. Conclusions: PD-L1 exhibits interactions with tyrosine kinases, which suggests PD-L1 phosphorylation as a novel post-translational modification. Our findings provide a rationale for the development of combination kinase inhibitors and immunotherapy in TNBC. Citation Format: Min Ling, Wenqi Wang, Xiaolong Yang. Characterizing the post-translational regulation of programmed death-ligand 1 (PD-L1) in triple-negative breast cancer: Role of EphA3/Fes/Fer [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 1008.