Nuclear factor erythroid 2-related factor 1 (NFE2L1, NRF1) and nuclear factor erythroid 2-related factor 2 (NFE2L2, NRF2) are distinct oxidative stress response transcription factors, both of which have been shown to perform cytoprotective functions, modulating cell stress response and homeostasis. NAD(P)H:quinone oxidoreductase (NQO1) is a mutual downstream antioxidant gene target that catalyzes the two-electron reduction of an array of substrates, protecting against reactive oxygen species (ROS) generation. NQO1 is upregulated in non-small cell lung cancer (NSCLC) and is proposed as a predictive biomarker and therapeutic target. Antioxidant protein expression of immune cells within the NSCLC tumor microenvironment (TME) remains undetermined and may affect immune cell effector functions and survival outcomes. Multiplex immunofluorescence was performed to examine the co-localization of NQO1, NRF1 and NRF2 within the tumor and TME of 162 chemotherapy-naïve, early-stage NSCLC patients treated by primary surgical resection. This study demonstrates that NQO1 protein expression is high in normal, tumor-adjacent tissue and that NQO1 expression varies depending on the cell type. Inter and intra-patient heterogenous NQO1 expression was observed in lung cancer. Co-expression analysis showed NQO1 is independent of NRF1 and NRF2 in tumors. Density-based co-expression analysis demonstrated NRF1 and NRF2 double-positive expression in cancer cells is associated with improved overall survival.
Background: Immune checkpoint inhibitors (ICIs), including PD-(L)1 pathway inhibitors, are now standard of care for a variety of cancers. Characterization of PD-L1 protein has revealed multiple secreted forms; soluble (sPD-L1) and exosomal (ExoPD-L1) variants. In the tumor microenvironment these interfere with T-cell activation, facilitating tumor immune evasion. High circulating levels of sPD-L1 are correlated with advanced disease, a worse prognosis and/or poor response to ICIs. High levels of ExoPD-L1 are reported to contribute to T-cell dysfunction. NUC-7738, a ProTide transformation of 3'-deoxyadenosine (3’-dA), currently in Phase 1/2 in patients with solid tumors (NuTide:701 NCT03829254), has shown encouraging efficacy signals in several tumor types, including melanoma. This study investigates the dynamic between NUC-7738 and secreted forms of PD-L1 in a melanoma cell line and patients. Material and Methods: A375 malignant melanoma cells were treated with NUC-7738 (10 μM) or DMSO for 6 - 72 hours. Cell supernatant was collected for sPD-L1 and ExoPD-L1 analysis and cells were measured for mRNA and metabolites. sPD-L1 mRNA and protein expression were measured in supernatant using RT-qPCR and sandwich ELISA, respectively. NUC-7738 and 3’-dATP were measured by LC-MS. ExoPD-L1 protein levels were assessed by Jess Western analysis (normalized to CD81) and PD-L1 cell surface expression by flow cytometry. Findings were validated in patient plasma samples taken before and 24 hours after C1D1 treatment with NUC-7738 and prior to C2D1of a 14-day cycle. Results: NUC-7738 was converted into anti-cancer metabolite 3’-dATP within 6 hours of treatment with an average concentration of 80 pmoles/106 cells, which was maintained for at least 24 hours and decreased by approximately 50% by 72 hours. The levels are comparable to those measured in PBMCs from patients treated with NUC-7738. NUC-7738 treatment reduced sPD-L1 mRNA expression by up to 40% and caused a time dependent decrease in sPD-L1 protein in the media supernatant by up to 3-fold. NUC-7738 also reduced ExoPD-L1 protein by 50%. NUC-7738 did not alter cell surface PD-L1 expression. Preliminary studies in serum from 4 study patients treated with NUC-7738 showed reductions in Exo-PD-L1 of ≤50% compared to pre-treatment levels. Conclusion: NUC-7738 reduces secreted forms of PD-L1 whilst having no effect on cell surface protein levels. These in vitro results (melanoma cell line) were validated in the clinical setting, whereby ExoPD-L1 was reduced in plasma samples from patients treated with NUC-7738. These findings indicate that, by reducing sPD-L1 levels, NUC-7738 may have the potential to act as an immune sensitizer. We propose that NUC-7738 given in combination with PD-(L)1 pathway inhibitors may offer a promising treatment option, and are currently exploring this in patients who have experienced therapeutic resistance to ICI treatment. Citation Format: Mustafa Elshani, Ying Zhang, Boback Kaghazchi, Alison L. Dickson, Sarah P. Blagden, Stefan Symeonides, Ruth Plummer, David J. Harrison. NUC-7738 causes reduction of soluble and exosome-associated PD-L1 in melanoma cell lines and patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5962.
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