Neighbors, Jeffrey D.

Abstract Background Glioblastoma (GBM) is the most common malignant primary brain tumor and is associated with a poor prognosis. Repurposed posaconazole (PCZ) has demonstrated efficacy in vitro by targeting key metabolic pathways. The aim of this study was to determine the neuropharmacological profile of PCZ in patients with GBM through a Phase 0 first-in-human trial for this indication (NCT04825275). Methods We conducted an open-label, non-randomized, parallel-arm trial in participants with primary or recurrent GBM. Patients in the treatment arm received oral PCZ for 7-10 days presurgically to achieve steady-state concentrations. Microdialysis catheters were implanted to measure interstitial drug levels. Postoperative tissue and fluid samples were analyzed to evaluate PCZ pharmacokinetics (mass spectrometry) and pharmacodynamics, including vascular density, apoptosis, and metabolite concentrations (lactate and pyruvate) compared to controls. Results The trial was closed early due to slow accrual, enrolling 2 participants in the PCZ arm and three in the control arm. No drug-related safety issues were observed. Although PCZ was undetectable in microdialysate fluid, it accumulated in tumor tissue, with concentrations showing an association with CD31-measured endothelial content. PCZ-treated tumors exhibited trends toward lower BCL2 expression indicative of increased apoptosis, and significantly lower pyruvate levels in the tumor periphery compared to controls. Plasma lactate levels in treated patients normalized to control levels over 24 h. Conclusions Despite limited accrual, this trial provides the first clinical evidence that oral PCZ accumulates in human GBM tissue and shows preliminary signals of metabolic modulation. These findings provide biological rationale warranting further clinical investigation, potentially utilizing direct tissue sampling rather than microdialysis.

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.

AbstractThe schweinfurthins have potent antiproliferative activity in multiple glioblastoma multiforme (GBM) cell lines; however, the mechanism by which growth is impeded is not fully understood. Previously, we demonstrated that the schweinfurthins reduce the level of key isoprenoid intermediates in the cholesterol biosynthetic pathway. Herein, we describe the effects of the schweinfurthins on cholesterol homeostasis. Intracellular cholesterol levels are greatly reduced in cells incubated with 3‐deoxyschweinfurthin B (3dSB), an analog of the natural product schweinfurthin B. Decreased cholesterol levels are due to decreased cholesterol synthesis and increased cholesterol efflux; both of these cellular actions can be influenced by liver X‐receptor (LXR) activation. The effects of 3dSB on ATP‐binding cassette transporter 1 levels and other LXR targets are similar to that of 25‐hydroxycholesterol, an LXR agonist. Unlike 25‐hydroxycholesterol, 3dSB does not act as a direct agonist for LXR α or β. These data suggest that cholesterol homeostasis plays a significant role in the growth inhibitory activity of the schweinfurthins and may elucidate a mechanism that can be targeted in human cancers such as GBM.