Abstract 493: NRF2 modulates sensitivity to thymidylate synthase inhibitors in colon cancer cells

Thymidylate synthase (TYMS) catalyzes the reductive methylation of dUMP, and is the sole de novo source of thymidine for DNA replication and repair. As such, it is an important target of chemotherapeutic drugs, particularly the fluoropyrimidines 5-fluorouracil (FUra) and 5-fluoro-2’-deoxyuridine (FdUrd), as well as the folate analog raltitrexed (RTX). In cells, these drugs are metabolized to derivatives that bind to and inhibit TYMS, leading to depletion of thymidine levels, dysregulation of redox metabolism, generation of oxidative stress, and, eventually, apoptotic cell death. Gene expression profiles of FUra-treated HCT116 cells (a human colon cancer cell line) showed that a number of genes involved in redox metabolism are up-regulated following drug exposure. Several of these were recognized as known targets of nuclear factor-erythroid-2-related factor 2 (NRF2), a member of the cap ‘n’ collar family of bZIP transcription factors that confers protection against oxidative and electrophilic stress. Quantitative PCR assays verified that FUra, FdUrd, and RTX do indeed induce several NRF2-regulated genes, including AKR1B10, ALDH3A1, NQO1, and SERPINE1, among others. Overproduction of NRF2 via DNA transfection increased expression of some, but not all, of these genes, a phenomenon that was reversed by Kelch-like ECH-associated protein-1 (KEAP1), a known negative regulator of NRF2. The impact of TYMS inhibitors on expression of NRF2 target genes was maintained in the presence of exogenous thymidine and the antioxidant N-acetylcysteine, even though these agents completely ameliorated drug-induced apoptosis. This suggests that the response of NRF2 target genes to TYMS-directed drugs occurs independently of TYMS inhibition and subsequent oxidative stress. Utilizing siRNA technology, we examined the impact of NRF2 knockdown on drug-induced apoptosis, and found that increases in the apoptotic index following exposure to TYMS inhibitors were greater in NRF2-knockdown cells as compared to control cells. This was also observed in TYMS-overproducing cells that are profoundly drug resistant, and indicates that reduced NRF2 expression sensitizes cells to these TYMS inhibitors. Overall, we conclude that the induction of NRF2 target genes is a constraining factor in the cytotoxic response to TYMS inhibitors, and that inhibition of the transcription factor may be an effective strategy to enhance sensitivity to these agents. Citation Format: Sarah A. Clinton, Karen W. Barbou