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Gmeiner. Cancer Drug Resist 2019;2:994-1001 I http://dx.doi.org/10.20517/cdr.2019.95 Page 997

Table 1. Summary of nucleoside analogs and other DNA damage effects on Top1cc
Substitution Effects on Top1cc Ref.
Abasic site Position-dependent, stable Top1cc in absence of CPT or suppression of Top1 cleavage [16,17]
8-Oxo-dG Position-dependent effects +1 scissile strand stabilized inactive Top1 [13,21]
BaP Position-dependent effects [19]
AraC AraC in either scissile or non-scissile strand enhanced Top1cc; CPT-resistant cells are cross- [27]
resistant to AraC
GEM Position-dependent effects including inducing new Top1cc sites; CPT-resistant cells are [39]
cross-resistant to GEM
FdU FdU substitution +1 or +2 in non-scissile strand inhibited re-ligation step of Top1 catalysis; [46,48,52,53,55,57]
Top1cc important for F10 biological activity; opposite effect of Tdp1 knockout relative to CPT

CPT: camptothecin; Top1: topoisomerase 1; AraC: cytarabine; GEM: gemcitabine; FdU: 5-fluoro-2’-deoxyuridine; Tdp1: tyrosyl DNA
phosphodiesterase 1; Top1cc: Top1 cleavage complex

[39]
formation . GEM substitution at the +1 site increased Top1cc formation 5-7-fold while GEM at the -5
position of the non-scissile strand induced a new Top1 cleavage site adjacent to GEM substitution. GEM
effects were site-specific and GEM at -3 or +2 of the scissile strand had no effect on Top1cc formation.
Similar to AraC, GEM inhibited the re-ligation step of Top1 catalysis and Top1-deficient P388 CPT-
resistant murine leukemia cells were cross-resistant to GEM, consistent with the in vivo significance of
Top1cc for anti-tumor activity.

DUAL TARGETING OF THYMIDYLATE SYNTHASE/TOP1 WITH F10
[40]
Fluoropyrimidine drugs (FPs) are used to treat > 2 million cancer patients worldwide each year .
Although the deoxynucleotide metabolites FdUMP and FdUTP are primarily responsible for anti-cancer
activity, FPs are administered either as the nucleobase (5-FU) or as 5-FU pro-drugs (e.g., capecitabine),
because the deoxynucleoside FdU is very rapidly converted to 5-FU in the liver . A principal target for FP
[41]
[42]
chemotherapy is thymidylate synthase (TS) , which is required for de novo thymidine (Thy) biosynthesis
to support rapid proliferation of malignant cells. 5-FU is inefficiently converted to the TS inhibitory
[43]
metabolite FdUMP with ~85% of the dose administered to humans degraded or excreted intact . Among
anabolic metabolites, ribonucleotides that contribute primarily to systemic toxicities are produced at
[44]
higher levels than deoxynucleotides . The Gmeiner lab developed FP polymers (e.g., F10) to efficiently
generate FdUMP . F10 displayed improved cytotoxicity relative to 5-FU in the NCI60 cell line panel [46,47] ,
[45]
[48]
is preferentially taken up by malignant cells , and thus does not require extracellular degradation to
monomers for biological activity.

Analysis of F10’s mechanism based on the response profile across the NCI60 cell line screen using the
[49]
COMPARE algorithm revealed similarities to Top1 poisons [46,47] . In contrast, 5-FU was distantly related to
both F10 and Top1 poisons. In collaboration with Pommier, we showed F10 induced Top1cc in malignant
cells and that CPT-resistant cells were cross-resistant to F10 as well as to FdUMP, FdU, and raltitrexed (an
[46]
anti-folate TS inhibitor) . FdU mismatched base pairs in the non-scissile strand at positions +1 or +2
relative to a model Top1 cleavage site trapped Top1cc, and inhibited the re-ligation step of Top1 catalysis.
These studies used the same model duplex used to study Top1cc by AraC and GEM [Figure 1]. We went
on to show that FdU-dG mismatched base pair destabilized duplex DNA, which may explain why FdU
substitution in the non-scissile strand perturbed Top1-mediated re-ligation [50,51] . The cytotoxic effects of F10
require TS inhibition and are reversible with exogenous Thy; however, Thy reversibility is limited to < 16 h of
treatment , a time that corresponds to Top1cc formation, after which F10’s cytotoxic effects are no longer
[52]
reversible with Thy. Thus, Top1cc formation appears to be an irreversible step in F10’s mechanism leading
to cell death.

Top1cc induced by F10 differ fundamentally from CPT analogs because repair occurs under thymineless
conditions, which renders repair ineffective since FdU or dU are re-incorporated at the lesion site [Figure 2A