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Lotz et al. Cancer Drug Resist 2020;3:149-60 I http://dx.doi.org/10.20517/cdr.2019.114 Page 155

of Top2 also contributes to the level of drug resistance in solid tumors since proteasome inhibition leads
[57]
to etoposide resistance . In addition, the E3 ubiquitin ligase Bmi1/Ring1A controls the proteasomal
[58]
degradation of Top2cc in HeLa cells upon teniposide treatment .
Evidence for a functional and physical interplay between ubiquitination and SUMOylation have been
reported at a larger scale, suggesting a coordination for proteasome degradation and the regulation of
[59]
ubiquitin modifiers . The Small Ubiquitin-like Modifier or SUMO proteins are considered as members
[60]
of the ubiquitin-like protein family, although they are not directly related to protein degradation . It was
shown that the resolution of TOP2cc by tyrosyl-DNA phosphoesterase 2 is controlled by the SUMO ligase
[61]
ZATT . SUMOylation play a critical role in DNA condensation and chromosome segregation. Top2a is
directed to the inner centromere via the E3 ligase RanBP2-mediated SUMOylation for the resolution of
[62]
sister centromeres . The E3 ligase PIASγ was also shown to regulate the catalytic activity of Top2a at the
[63]
centromere for the proper segregation of chromosome . Evidence of a crosstalk between phosphorylation
and SUMOylation was shown in cancer cells, which targets the CTD [64,65] .

Strikingly, most of the SUMO sites in the catalytic domains were identified along the dimeric interface of the
Top2a structure [Figure 3]. Structure determination of Top2 has shown that the subunits form an intertwined
dimer structure and that the buried surface could be accessible during the catalytic cycle [Figure 1B] [66-69] .

SUMOylation, similar to other post-translational modifications, is impacted by chemical adjuvants but
little information is available thus far. Conjugation to the SUMO2/3 by the SUMO ligase PIASg in response
[70]
to Top2 inhibitors alters the Top2 decatenation activity, essential for chromatid arm separation at mitosis .
Interestingly, SUMOylation in Top2a is increased upon ICRF-193 or teniposide exposure, as well as
[71]
following oxidative stress or heat shock .

Altogether, ubiquitination and SUMOylation are important modifications that can have a direct impact on
Top2a levels, interplay with other PTM, and consequently affect drug response.

TOP2a MUTATIONS IN RESISTANT CELLS
Point mutations in the Top2a gene that lead to podophyllotoxin resistance mostly affect the drug binding
site or are located on the TOPRIM domain [Figure 4A]. A recent study in yeast identified mutations of
[72]
Top2 conferring resistance against vosaroxin, a quinolone derivative in a phase II clinical trial . Some
mutations also appeared in the unstructured C-terminal region of human Top2a, indicating that residues
that are external to the binding pockets of the drugs can contribute to resistance mechanisms, due to the
allosteric properties of the protein.

Point mutations conferring resistance to the bispiperazine compounds were found in the ATPase domain
of Top2a in small cell lung cancer and Chinese hamster ovary cells . These mutations impact the dimeric
[73]
[74]
interface and the formation of the ICRF binding pocket [Figure 4]. Another study in yeast showed that
drug resistance mutations are not restricted to the N-terminal domain but can also be found in the DNA-
[75]
gate [Figure 4B]. Single point mutations located in the N-terminal domain display a more resistant
phenotype compared with those in the DNA-gate, with the exception of Gly551, a conserved residue in
eukaryotic Top2. Interestingly, a Gly551Ser mutation confers resistance to both Top2 poison etoposide and
the catalytic inhibitor ICRF [72,75] . The dual resistance could be explained by the proximity of the etoposide-
binding site in the DNA groove and the allosteric properties of the enzyme, since movements of the DNA-
gate are coupled to ATP hydrolysis [Figure 1B].

Although no drug resistant mutations targeting known PTM sites could be found in the literature except for
[72]
vosaroxin resistant yeast cells , it cannot be excluded that such events occur in the Top2a gene of resistant