Abstract 1848: NUC-3373 induces ER stress and the release of damage associated molecular patterns in colorectal cancer cells

Abstract

Abstract Background: NUC-3373 is a ProTide transformation of fluorodeoxyuridine monophosphate (FUDR-MP), the active anti-cancer metabolite of 5-fluorouracil (5-FU). NUC-3373 can overcome key cancer resistance mechanisms and the rapid degradation known to impact 5-FU's clinical utility. Clinical studies are currently investigating NUC-3373 as monotherapy (solid tumors) and in combination with agents commonly used to treat patients with colorectal cancer (CRC). Previous studies revealed ultrastructural adaptations in CRC cell lines exposed to NUC-3373 that were indicative of endoplasmic reticulum (ER) stress. NUC-3373 was also shown to induce the formation of long-lasting ternary thymidylate synthase (TS) complexes. The induction of ER stress may promote the release of damage-associated molecular patterns (DAMPs) which have the potential to evoke immunogenic cell death (ICD). This study examines the ability of NUC-3373 to induce both ER stress and the release of DAMPs in CRC. Methods: Immunoglobulin-binding protein (BiP) was used as a marker of ER stress induction. BiP and TS (free and ternary complex) expression were measured by Western blot and quantified using Licor Odyssey. TS was knocked down using TYMS-targeting siRNA and lipofectamine-based transfection. DAMPs, active release of nuclear high mobility group box protein 1 (HMGB1) and cell surface exposure of calreticulin (CRT), were assessed by fluorescence microscopy and flow cytometry respectively. Results: NUC-3373 resulted in the rapid formation of TS ternary complexes, which was associated with ER stress as assessed by BiP protein expression. siRNA knockdown studies confirmed that TS ternary complex formation is necessary for the induction of ER stress. A strong correlation between BiP and TS ternary complex formation (R2=0.97) was observed. Furthermore, NUC-3373 induced the release of DAMPs, with increased expression of CRT at the cell surface and concomitant loss of nuclear HMGB1. Conclusions: NUC-3373, a potent inhibitor of TS activity, also induces ER stress, upregulates cell surface CRT, and decreases nuclear HMGB1 in CRC cells. The aforementioned DAMPs have been shown to cause ICD. In addition to being an effective cytotoxic agent, these findings suggest that NUC-3373 has the potential to evoke a host immune response and enhance the clinical utility of immunotherapy. Citation Format: Fiona G. McKissock, Oliver J. Read, David J. Harrison. NUC-3373 induces ER stress and the release of damage associated molecular patterns in colorectal cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1848.