Jialin Feng, Oliver J. Read, and Albena T. Dinkova-Kostova. Mol. Cells -0001;0:. https://doi.org/10.14348/molcells.2023.2183

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.

CD5 antigen-like (CD5L), also known as apoptosis inhibitor expressed by macrophages (AIM), is a secreted protein produced by macrophages, which has both cell-autonomous and non-autonomous functions: it has been implicated in multiple biological processes, including inflammation, infection, and fibrosis. The transcriptional regulation of CD5L is complex, and a comparison of the proteomes of bone marrow-derived macrophages from mice with graded expression of transcription factor NF-E2 p45-related factor 2 (Nrf2) indicated that Nrf2 controls expression of the CD5L gene. Using genetic and pharmacological means to increase or decrease Nrf2 activity, we found a correlation between the abundance of Nrf2 and CD5L expression in both murine macrophages and primary human monocyte-derived macrophages. Furthermore, the potency of small-molecule Nrf2 activators with distinct mechanisms of action and different potencies, correlated with the extent of CD5L expression, both at the mRNA and the secreted protein levels. Conversely, depletion of Nrf2 resulted in a significant decrease in CD5L mRNA levels. Chip-seq analysis showed Nrf2 binding 20,000 bp upstream of the Cd5l promoter, a region containing sequences resembling the antioxidant response element (ARE, 5'-TGACNNNGC-3') Nrf2-binding motif. Deletion of two of these sequences by CRISPR/Cas9 gene editing led to a profound decrease in CD5L mRNA levels, confirming the critical role of these ARE-like sequences in controlling CD5L expression. Recombinant CD5L (rCD5L) suppressed transforming growth factor β (TGFβ)-directed fibrogenic responses in human lung fibroblasts, suggesting that Nrf2 activators, such as the clinically used omaveloxolone (RTA-408), could protect against pulmonary fibrosis by boosting the levels of CD5L.

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide with 5-FU still the primary chemotherapeutic of choice. With the increasing use of immunotherapies, much research is focused on the ability to make tumours more immunogenic, thereby rationalising combination with immunotherapy. Here we investigate whether NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine (FUDR), enhances immunogenicity in CRC cell lines and facilitates lymphocyte mediated cell death in vitro. At sub IC₅₀ doses NUC-3373 upregulates damage associate molecular patterns (DAMPs) in both HCT116 and SW480 cells and increases surface expression of MHCII and PD-L1. Pre-treatment with NUC-3373 and subsequent coculture with NK-92 MI natural killer cells caused an increase in LAMP1 expression (degranulation), production of IFN-γ, and NK-mediated cytotoxicity compared to vehicle controls. Cocultures with patient-derived PBMCs with heterologous CRC cells pre-treated with NUC-3373 demonstrated increased cell death compared to both vehicle controls and monocultures of CRC cells exposed to NUC-3373. Lastly, the PD-1 immune checkpoint inhibitor nivolumab showed synergistic activity when HCT116 cells were pre-treated with NUC-3373. To conclude, we show that NUC-3373 can modulate immune signaling and may therefore facilitate immune mediated tumour cell death in vitro.

Introduction: Two clinical case reports of humans with mutations in Itch reported distinct morphological defects such as stunted growth, macrocephaly, and dysmorphic features indicating a role for Itch in bone remodelling. Studies in mice have found that the encoded E3 ubiquitin ligase acts as a negative regulator of osteoclastogenesis, however no studies have investigated whether this is translatable to a human model. Experimental procedures: Human peripheral blood monocytes were separated from whole blood and grown in M-CSF containing media. Media was later supplemented with RANKL to promote osteoclast differentiation. Transient siRNA-mediated Itch knockdown (si-Itch) in monocytes was verified by qPCR and western blot to confirm reduction in both Itch mRNA and protein respectively. Monocytes were aliquoted onto 96-well plates where confluence and osteoclast formation were analysed using automated cytometry analysis before and after staining for tartrate resistant acid phosphatase activity (TRAP). Cells were also stained with Hoechst33342 to look for multinucleate cells. Results: Cells treated with si-Itch showed an 80% knockdown in Itch mRNA and > 75% reduction in protein. Following the 7-day differentiation period, si-Itch caused a 47% increase in multinucleate cells and a 17% increase in numbers of large cellular bodies and, indicating an overall increase in mature osteoclast formation. Conclusions: Our preliminary data shows silencing Itch expression increases the potential of primary human monocytes to differentiate into osteoclast-like cells in vitro.

Background: NUC-3373 is a phosphoramidate modification of fluorodeoxyuridine-monophosphate (FUDR-MP), the active anti-cancer metabolite of fluorouracil (5-FU), which binds and inhibits thymidylate synthase (TS), disrupting DNA synthesis and repair. NUC-3373 is designed to bypass 5-FU resistance mechanisms associated with transport, activation and breakdown, reduce generation of toxic metabolites, and deliver higher levels of active metabolite FUDR-MP to tumors. Our previous work demonstrated that NUC-3373 causes colorectal cancer (CRC) cell lines to release damage associated molecular patterns (DAMPs)- molecular signals that activate circulating immune cells leading to immunogenic cell death (ICD). NUC-3373 promotes activation of co-cultured NK-92 MI cells, a natural killer cell line, by upregulating degranulation and IFN-γ production and downregulating the immune checkpoint molecule TIGIT. This study aims to test the hypothesis that NUC-3373 induced DAMPs promote ICD in co-cultures of CRC cells with peripheral blood mononuclear cells (PBMCs). We also explore the dynamics of PD-L1 and cytokine expression and combination with a PD-1 checkpoint inhibitor. Methods: HCT116 cells were exposed to increasing concentrations of NUC-3373 or vehicle control for 24 hours prior to co-culture with healthy patient-derived PBMCs. At the time of co-culture, cells were treated with 10 υg/ml nivolumab to assess the effect of an anti-PD-1 antibody being combined with NUC-3373. CRC and PBMC co-cultures were continued for up to 72 hours. Gene expression of PD-L1 and cytokines (TNF-α, IFN-γ, and IL-2) was assessed by RT-PCR. PD-L1 surface expression was determined by flow cytometry. Confluence was determined by automated cytometry analysis (Celigo) and cell viability of CRC cells was determined by sulforhodamine-B assay (SRB). Results: NUC-3373 increased the gene expression of cytokines and PD-L1 over 48 hours of co-culture. Surface expression of PD-L1 was increased on both PBMCs and CRC cells in co-culture, as well as on CRC cells cultured without PBMCs, indicating that global increase in PD-L1 is NUC-3373 mediated. CRC cells pre-treated with NUC-3373 and co-cultured with PBMCs displayed a reduction in cell viability compared to monocultured CRC cells. Furthermore, CRC and PBMC co-cultures treated with anti-PD-1 antibody showed increased cytotoxicity when CRC cells were pre-treated with NUC-3373. Conclusions: NUC-3373 induces DAMPs and PD-L1 expression in CRC cells and promotes pro-immune cytokine production from PBMCs resulting in ICD in vitro. Furthermore, the addition of anti-PD-1 antibody to co-cultures of NUC-3373-treated CRC cells and PMBCs enhanced this ICD. With an improved clinical safety profile (NCT03428958), including less haematologic toxicity, and a more convenient dosing regimen than 5-FU, NUC-3373 is an attractive combination partner for immune checkpoint inhibitors. Citation Format: Oliver James Read, Jennifer Bré, David J. Harrison. NUC-3373 potentiates immune-mediated cytotoxicity of CRC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1113.