Abstract 4265: The role of the mevalonate pathway in post-translational modifications of PD-L1

Abstract Introduction: The binding of Programmed Death-Ligand 1 (PD-L1) on the surface of tumor cells to PD-1 on T cells prevents T-cell-mediated anti-cancer immune responses. Immune checkpoint blockades that target the PD-L1/PD-1 interaction have improved survival rates but have several critical limitations. Post-translational modifications (PTMs) of PD-L1, such as glycosylation, are crucial for its stability and engagement with PD-1. Thus, targeting PD-L1 PTMs may be a novel method to improve immune checkpoint blockade efficacy. The mevalonate pathway (MVP) generates various substances involved in PTMs process. Here, we explore the effect of MVP in altering the PTMs of PD-L1 and its impacts on cell surface PD-L1 function. Methods: The triple-negative human breast cancer (TNBC) cell lines MDA-MB-231 and BT549 were treated with the HMG-CoA reductase inhibitor lovastatin (LOV) with or without the addition of IFN-γ and/or MVP intermediates for 48 hours. PD-L1 transcription was evaluated by qPCR. Alterations in PD-L1 size were detected by Western blotting. PD-L1 surface levels were measured by flow cytometry. HMG-CoA reductase (HMGCR) knockdown was performed to validate the non-off-target effect of LOV. The binding of PD-1 protein to the cell surface PD-L1 was measured by PD-1/PD-L1 binding assay. PD-L1 glycosylation levels were measured by Concanavalin A (Con A) lectin binding assay with immunoprecipitated PD-L1 protein. Results: LOV treatment caused a significant molecular weight shift of PD-L1 from approximately 45 kDa to 40 kDa and reduced the cell surface PD-L1 levels. However, PD-L1 transcription was not affected by LOV. HMGCR knockdown produced similar changes, indicating LOV's impact on PD-L1 was not due to off-target effects. The addition of MVP intermediates mevalonate (MEV) or geranylgeraniol, but not farnesol prevented LOV-induced changes in PD-L1 molecular weight and surface levels. Interestingly, the addition of 25-hydroxycholesterol, but not dolichol, partially prevented LOV-induced decreases in PD-L1 surface levels. Geranylgeranyl transferase inhibitor GGTI-286, which prevents protein geranylgeranylation, also altered PD-L1 molecular weight and decreased surface levels. In addition, LOV impaired the binding of PD-1 protein to tumor cells' surface PD-L1, which was reversed by the addition of MVP substrate MEV. Con A lectin-binding of immunoprecipitated PD-L1 showed that the LOV did not reduce the N-glycosylation level of PD-L1. Conclusions: This study reveals the involvement of the MVP in regulating PD-L1 PTMs and highlights the MVP as a potential therapeutic target to modulate PD-L1’s molecular weight and function and improve the efficacy of immunotherapy interventions, presenting a promising avenue for further investigation. Citation Format: Ruoheng Zhang, Emily J. Koubek, Jessie L. Reed, Jeffrey D. Neighbors, Joel M. Reid, Raymond J. Hohl. The role of the mevalonate pathway in post-translational modifications of PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4265.