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Gmeiner. Cancer Drug Resist 2019;2:994-1001 I http://dx.doi.org/10.20517/cdr.2019.95 Page 995

INTRODUCTION
Targeting DNA is one of the all-time most successful strategies for cancer treatment. Radiation and DNA
damaging drugs such as cyclophosphamide have remained among the most widely used therapeutic
modalities for cancer treatment for decades. A major advance for targeting DNA for cancer treatment was
[1]
the discovery that camptothecin (CPT), a natural product identified by Wall and Wani in a screen for
[2]
natural products with anti-cancer activity, targeted DNA topoisomerase 1 (Top1) . In human cells, Top1,
[3]
[5]
a TypeIB topoisomerase , relieves superhelical density generated during replication and transcription
[4]
[6]
by nicking supercoiled DNA to allow for spontaneous relaxation of supercoils (i.e., “controlled rotation” ),
and then re-ligating the relaxed DNA. CPT analogs stabilize the nicked DNA with Top1 covalently bound
[7]
in a ternary cleavage complex (Top1cc) that consists of Top1, DNA, and CPT . The resulting Top1cc is
converted into a DNA double strand break (DSB) upon collision with either an advancing replication
[8,9]
fork or the transcriptional machinery [10,11] . Accumulation of unrepaired DNA DSBs stimulates activation
of apoptosis.
With the realization that CPT analogs such as topotecan and Irinotecan (CPTs) displayed anti-cancer
[12]
activity during clinical trials in the early 1990s , there was growing appreciation that, in addition to its role
as the sole target for CPTs, Top1 also mediated the cytotoxic effects of diverse treatments. These included
[15]
generic DNA damage due to reactive oxygen species [13,14] , and nucleoside analogs used for cancer
treatment. Studies from the Pommier lab showed that perturbations to DNA structure could result in either
increased susceptibility to Top1-mediated DNA damage, or cause Top1 to be refractory from accessing sites
with certain types of DNA damage. For example, abasic sites that may occur spontaneously in genomic
DNA or as intermediates during base excision repair were shown to cause position-specific changes in
[16]
Top1 cleavage activity . Abasic sites within the first four bases 5’ to the Top1 cleavage site suppressed Top1
cleavage at the preferred site, but stimulated Top1 cleavage at alternative, nearby sites. However, an abasic
site immediately 3’ to the Top1 cleavage site resulted in formation of a stable Top1cc even in the absence
of CPT. Further studies showed that DNA with nicks or gaps with 5’-phosphate termini that could be
generated by ionizing radiation or by the processing of abasic sites also modulated Top1 activity . Nicks
[17]
upstream of a preferred Top1 site suppressed Top1 activity, but irreversible Top1cc formed if nicks were
positioned opposite to the Top1 cleavage site in the non-scissile strand. Top1 trapping was also detected at
base mismatches [16,18] . These findings implicated Top1 in modulating repair of damaged DNA and indicated
Top1cc could be intermediates leading to DNA DSBs induced by diverse treatments and conditions not
[13]
previously realized to target Top1. Further studies investigated 8OxoG , benzo[a]pyrene diol epoxide
adducts , and other types of damage , and demonstrated these either trapped Top1 or inhibited Top1
[19]
[20]
cleavage, in a position-dependent manner. The paradoxical position-dependent enhancement/inhibition
effects of damaged bases on Top1 activity were investigated using X-ray structural analysis. These studies
revealed Top1 binds DNA in an inactive conformation and rearrangement of the active site is required for
catalysis. It was found that 8-OxoG at the +1 position of the scissile strand stabilized the inactive, DNA-
[21]
bound state .

POISONING OF TOP1 WITH NUCLEOSIDE ANALOGS
Nucleoside analogs such as cytarabine (AraC) are among the most active anti-cancer drugs , and are
[22]
used to treat diverse malignancies including front-line treatment for acute myeloid leukemia. AraC is
phosphorylated [by deoxycytidine kinase (dCK)], and AraCTP (AraC 5’-O-triphosphate) is incorporated
[23]
[24]
into nascent DNA during replication, which alters DNA conformation and stability and inhibits
strand elongation. AraC thus serves as a chain-terminating nucleoside analog . Studies with the same
[25]
model system derived from Tetrahymena hexadecameric rDNA sequence described by Westergaard and
[26]
co-workers were used to study the effects of DNA damage on Top1 activity [Figure 1]. These studies
revealed that AraC at the +1 position relative to the Top1 cleavage site enhanced Top1cc formation 4-6-