@article {ChristensonMOLPHARM-AR-2020-000191, author = {Eric S. Christenson and Anthony Gizzi and Junru Cui and Matthew Egleston and Kyle J. Seamon and Michael DePasquale and Benjamin Orris and Ben H. Park and James T. Stivers}, title = {Inhibition of hUNG2 Sensitizes a Large Fraction of Colorectal Cancer Cells to 5-fluorodeoxyuridine (FdU) and Raltitrexed (RTX) but not Fluorouracil (FU)}, elocation-id = {MOLPHARM-AR-2020-000191}, year = {2021}, doi = {10.1124/molpharm.120.000191}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {Previous shRNA knockdown studies have established that depletion of human uracil DNA glycosylase 2 (hUNG2) sensitizes some cell lines to 5-fluorodeoxyuridine (FdU). Here we selectively inhibit the catalytic activity of hUNG2 by lentiviral transduction of UNG inhibitor protein (UGI) into a large panel of cancer cell lines under control of a doxycycline-inducible promoter. This induced inhibition strategy better assesses the therapeutic potential of small molecule targeting of hUNG2. In total, six of eleven colorectal lines showed large increases in FdU potency upon hUNG2 inhibition ("responsive"). This hUNG2 dependent response was not observed with fluorouracil (FU), indicating that FU does not operate through the same DNA repair mechanism as FdU. Potency of the thymidylate synthase inhibitor raltitrexed (RTX), which elevates dUTP levels, was only incrementally enhanced upon hUNG2 inhibition, suggesting that responsiveness is associated with incorporation and persistence of FdU in DNA rather than dU. The importance of FdU/G lesions in toxicity is supported by the observation that dT supplementation completely rescued the toxic effects of dU/A lesions resulting from RTX, but dT only increased the IC50 for FdU, which forms abundant FdU/A and FdU/G mismatches. Contrary to previous reports, cellular responsiveness to hUNG2 inhibition did not correlate with p53 status or thymine DNA glycosylase expression. A model is suggested where the persistence of FU/G base pairs in the absence of hUNG2 activity elicits a unique DNA damage response in a significant fraction of colorectal lines. Significance Statement The pyrimidine base 5-fluorouracil is a mainstay chemotherapeutic for treatment of advanced colorectal cancer. Here we show that its deoxynucleoside form, 5-fluorodeoxyuridine (FdU), operates by a distinct DNA incorporation mechanism that is strongly potentiated by inhibition of the DNA repair enzyme uracil DNA glycosylase (hUNG). The UNG-dependent mechanism was present in over 50\% of colorectal cell lines tested, suggesting that a significant fraction of human cancers may be sensitized to FdU in the presence of a small molecule UNG inhibitor.}, issn = {0026-895X}, URL = {https://molpharm.aspetjournals.org/content/early/2021/03/31/molpharm.120.000191}, eprint = {https://molpharm.aspetjournals.org/content/early/2021/03/31/molpharm.120.000191.full.pdf}, journal = {Molecular Pharmacology} }