Effect of programmed cell death protein ligand 1 nuclear translocation mediated by Enolase on urothelial carcinoma immune evasion.

Abstract

788 Background: The efficacy of programmed death-ligand 1 (PD-L1)-based immunotherapy is limited in urothelial carcinoma (UC), one of the most aggressive solid tumors characterized by a lactic acid (LA)-enriched tumor microenvironment (TME) in favor of tumor immune evasion. In addition to protecting tumor cells from immune surveillance, PD-L1 can translocate to the nucleus and promote tumor development through diverse pathways. However, whether nuclear PD-L1 can impose an effect on LA accumulation in the TME to replenish the immune checkpoint function of membrane PD-L1 remains elusive. Methods: α-enolase (ENO1) expression in UC was analyzed via western blotting and immunohistochemistry using high throughput tissue microarray. The in vitro mechanism that ENO1 regulates PD-L1 nuclear translocation and subsequent transcriptional activation of monocarboxylate transporter 4 (MCT4) expression to facilitate LA secretion was investigated using immunofluorescence, nuclear cytoplasm separation, western blotting, Immunoprecipitation, mass spectrometry, GST pull-down assay, T cell cytotoxicity assay, and Seahorse experiment. In vivo efficacy and mechanistic studies, using C57BL/6 mice and nude mice with subcutaneous and orthotopic tumors, employed neutralizing antibody against CD8 and PD-1. Survival curves were constructed for the orthotopic model. Immunohistochemistry and flow cytometry analyzed local and systemic alterations in the tumor microenvironment (TME). Results: ENO1 physically interacts with PD-L1 and transports PD-L1 to the nucleus by recruiting Importin β (KPNB1) independently of its metabolic activity. Mutation of Leu432 and Lys434 in the C-terminal domain of ENO1 abrogates its binding to KPNB1 and subsequent PD-L1 nuclear import. Nuclear PD-L1 further transcriptionally upregulates MCT4 expression to facilitate LA secretion to the TME. Finally, targeting ENO1-mediated PD-L1 nuclear translocation alleviates LA accumulation in the TME, reinvigorates CD8+ T cells-mediated antitumor immune response, and potentiates anti-PD-L1 immunotherapy in UC. Conclusions: ENO1 is a feasible target for blocking PD-L1 nuclear translocation, thereby enhancing the efficacy of immunotherapy in UC.