Abstract 1655: NUC-3373 induced DAMPs release in CRC cells promotes natural killer cell activation
Authors: Read Oliver James , Harrison David James , Bré Jennifer 
Published: July 2021

Abstract
Background: NUC-3373 is a novel anti-cancer agent designed to replace 5-FU, one of the most widely used anti-cancer drugs globally. Used across a broad range of tumors, including colorectal cancer (CRC), 5-FU (and oral formulation, capecitabine) exerts its main anti-cancer activity via conversion to the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which binds and inhibits thymidylate synthase (TS), disrupting DNA synthesis and repair. NUC-3373, a phosphoramidate transformation of FUDR-MP, is a targeted inhibitor of TS is designed to bypass 5-FU resistance mechanisms associated with transport, activation and breakdown, and reduce the generation of toxic metabolites. Drug-induced cellular stress leads to the release of damage associated molecular patterns (DAMPs), including calreticulin, HMGB1 and ATP, from cancer cells. These DAMPs can be detected by immune cells in the surrounding tumor microenvironment and promote immunogenic cell death (ICD) of the cancer cells. Previous data showed that NUC-3373 induces the release of DAMPs in the human CRC lines HCT116 and SW480. This study aims to demonstrate the association between DAMPs release caused by NUC-3373 and the activation of natural killer (NK) cells.
Methods: CRC cells HCT116, HT-29 and SW480 were exposed to several sub-IC50 doses of NUC-3373. The negative control was DMSO in medium, and the positive controls were oxaliplatin, a known inducer of ICD, and PMA/ionomycin, which causes NK cell degranulation. After 24 h, the culture medium was changed to wash out NUC-3373 and subsequently, cells were co-cultured with NK-92 MI cells for 4 h. NK cells were harvested from the supernatant and labelled with anti-CD56 for analysis by flow cytometry. NK cell activation was assessed by measuring expression of LAMP1 on the cell surface as an indication of degranulation, and intracellular accumulation of interferon gamma (IFNγ) in cells treated with GolgiStop.
Results: Following treatment of HCT116 cells with sub-IC50 doses of NUC-3373, co-cultured NK-92 MI cells displayed a 125% increase in cell surface LAMP1 and a 96% increase in intracellular IFNγ expression compared to the DMSO-treated control. PMA/ionomycin-treated samples displayed a 463% and 271% upregulation in LAMP1 and IFNγ respectively. Degranulation was also observed in NK cells that had been co-cultured with HT-29 and SW480 cells treated at NUC-3373 doses closer to the respective IC50.
Conclusions: NUC-3373 induces endoplasmic reticulum stress in CRC cells, followed by release of DAMPs. This promotes NK activation as shown by increased degranulation and upregulation of IFNγ. In an in vivo system, activation of NK cells can recruit and activate other immune cell subtypes to create a more immunogenic environment. This study suggests that NUC-3373 can modulate the tumor microenvironment to promote ICD, making it an attractive combination partner for immunotherapies such as PD-1/PD-L1 blockade therapy.
Citation Format: Oliver James Read, David James Harrison, Jennifer Bré. NUC-3373 induced DAMPs release in CRC cells promotes natural killer cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1655.