REVIEW

Cell cycle responses to Topoisomerase II inhibition:
Molecular mechanisms and clinical implications
Tanya N. Soliman1, Daniel Keifenheim2, Peter J. Parker3, and Duncan J. Clarke2

Introduction
Chromosome segregation errors result in aneuploidy that causes
infertility, birth defects, and cancer (Klaasen and Kops, 2022).
Aneuploidy has an enormous impact on human health: about
30% of miscarriages are due to aneuploidy; 1/1,000 live-bornbabies are aneuploid, suffering a variety of developmental disorders (see Brosens et al., 2022; McCoy, 2017); in adults, segregation errors drive cancer (Klaasen and Kops, 2022). A
prominent cause of chromosome segregation errors is failure ofcell cycle control mechanisms that ensure the fidelity of genometransmission, including controls that act upon entry into andprogression through mitosis (Abbas et al., 2013; Potapova et al.,2013).
DNA replication produces physically entangled (catenated)sister DNA helices that must be resolved to allow chromosome
segregation (Fig. 1 A; Cook, 1991). Topoisomerase IIA (Topo IIA)
is the only enzyme that can decatenate these topological links
between sister chromatids, rendering the enzyme and its uniquestrand passage reaction (SPR) essential for mitosis (Brown andCozzarelli, 1979; Holm et al., 1985; Liu et al., 1979; Wang, 2002).
When Topo IIA is perturbed, chromosomes can missegregate,
leading to formation of micronuclei. Even a single micronucleus
containing just one chromosome can cause genome instability
via mechanisms such as chromothripsis, promoting cancer(Dahiya et al., 2022; De Marco Zompit and Stucki, 2021; Umbreit
et al., 2020). It is critical for genome integrity that Topo IIA

resolves all catenation before anaphase. Furthermore, because
one class of Topo IIA inhibitors are workhorse cancer drugs(Nitiss, 2009), it has been important to understand the biochemical and structural basis of the SPR, which is targeted incancer treatments (Dong and Berger, 2007; Ling et al., 2022;
Vanden Broeck et al., 2021; Wendorff et al., 2012).
The SPR is performed by a symmetrical homodimeric Topo
IIA holoenzyme requiring hydrolysis of two ATP molecules(Fig. 1 B; Abbas et al., 2013; Wang, 2002). Topo IIA makes atransient double-strand break (DSB) in one DNA helix, referred
to as the gated-segment (G-segment), passes a second helix
through the break (the transported-segment or T-segment), andthen re-ligates the G-segment (Brown and Cozzarelli, 1979; Liuet al., 1979; Wang, 2002). The central region of the enzyme forms
the catalytic core, which binds and cuts the G-segment allowing
passage of the T-segment through the enzyme. While this intricate structural view of Topo IIA explains how DNA molecules
are decatenated, to prevent erroneous chromosome segregation,
control systems are needed to ensure decatenation is completed
before anaphase (Andrews et al., 2006; Clarke et al., 2006;
Downes et al., 1994; Furniss et al., 2013; Pandey et al., 2020).
The Topo IIA G2-phase (decatenation) checkpoint
The DNA damage response (DDR) pathway is a G2 checkpoint
responsible for arresting cells in G2 in response to DNA damage(Fig. 2). The DDR involves the activation of ataxia-telangiectasia

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DNA Topoisomerase IIA (Topo IIA) is an enzyme that alters the topological state of DNA and is essential for the separation of
replicated sister chromatids and the integrity of cell division. Topo IIA dysfunction activates cell cycle checkpoints, resulting inarrest in either the G2-phase or metaphase of mitosis, ultimately triggering the abscission checkpoint if non-disjunction
persists. These events, which directly or indirectly monitor the activity of Topo IIA, have become of major interest as many
cancers have deficiencies in Topoisomerase checkpoints, leading to genome instability. Recent studies into how cells sense
Topo IIA dysfunction and respond by regulating cell cycle progression demonstrate that the Topo IIA G2 checkpoint is distinct
from the G2-DNA damage checkpoint. Likewise, in mitosis, the metaphase Topo IIA checkpoint is separate from the spindle
assembly checkpoint. Here, we integrate mechanistic knowledge of Topo IIA checkpoints with the current understanding of
how cells regulate progression through the cell cycle to accomplish faithful genome transmission and discuss theopportunities this offers for therapy.

.............................................................................................................................................................................
1Barts Cancer Institute, Queen Mary University London, London, UK;USA;

2Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN,

3

The Francis Crick Institute, London, UK.

Correspondence to Peter J. Parker: peter.parker@crick.ac.uk; Duncan J. Clarke: clark140@umn.edu.
© 2023 Soliman et al. This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).
Rockefeller University Press
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https://doi.org/10.1083/jcb.202209125

1 of 14mutated kinase (ATM)/ATM-related (ATR) and both Chk1 andp53/Chk2 pathways acting redundantly in G2 (see Lockwood
et al., 2022) to trigger cell cycle arrest, allowing for DNA repair and subsequent cell cycle re-entry (Ciccia and Elledge,2010). Topo IIA poisons, such as Etoposide, trap the covalent
Topo IIA–DNA intermediate following strand passage, but prior
to the DSB re-ligation step (see above, Fig. 1 B iii), triggering a
DDR-related G2 arrest (Nam et al., 2010). This DNA damage–associated behavior has influenced perception of the Topo IIA–
dependent checkpoint in G2 (Downes et al., 1994; Kaufmann andKies, 1998), which likely reflects the physiological process of
sensing ongoing post–S-phase decatenation of sister chromatids
to avoid non-disjunction. Topo IIA responsive cell cycle controls
Soliman et al.
Topoisomerase II checkpoint controls

seem to detect a stalled SPR intermediate known as the Topo IIA
closed clamp (Fig. 1 B iv; Furniss et al., 2013; Pandey et al., 2020).
The closed clamp is a natural structural conformation that exists
transiently at the end of the normal enzyme cycle (Roca et al.,
1994; Wang, 2002). Following re-ligation of the cut DNA helix(i.e., in the absence of any DNA damage), a Topo IIA closed
clamp is trapped on DNA until both ATP molecules have beenhydrolyzed, which allows opening of the N-terminal andC-terminal gates of the enzyme. This step must occur for Topo
IIA to release from DNA and begin a new SPR cycle (Roca et al.,
1994; Wang, 2002). The prolongation of this sensing checkpoint
can be triggered pharmacologically by catalytic inhibitors of the
ICRF-193 class, which block ATPase activity in this final step ofJournal of Cell Biology
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Figure 1. Decatenation by Topo IIA is disrupted by small molecules. (A) DNA replication produces catenated (entangled) sister chromatid DNA molecules.
Topo IIA is highly conserved and is the only eukaryotic enzyme that can resolve catenations to permit the separation and segregation of sister chromatids
during anaphase of mitosis. Decatenation is achieved progressively, being initiated during DNA replication, continuing in G2-phase, and being completed inmitosis. The activity of Topo IIA can be perturbed by small molecules categorized into two distinct classes dependent on their mode of action: poisons andcatalytic inhibitors. (B) The unique SPR of DNA Topo II decatenates DNA helices. (i–iv) Top: SPR cycle: (i) G-segment double-helical DNA binds at the catalytic
core (orange/yellow); (ii) T-segment DNA captured by the enzyme N-gate (purple), G-segment cleavage; (iii) T-segment transport; (iv) G-segment religation—transient closed clamp enzyme conformation until ATP hydrolysis opens N-gate and C-gate (green). The SPR can be interrupted by small
molecules at various points in the cycle including, but not limited to, those shown for Etoposide and ICRF-193 (red). The Topo II poison Etoposide blocks the religation step, while the catalytic inhibitor ICRF-193 inhibits ATP hydrolysis. Bottom: Domain structure (colors match domains above). The CTD has not been
crystalized. Table: Two categories of small molecules that target Topo IIA and their cell cycle consequences. One category, poisons, prevents G-segment
ligation (step iii to iv in panel B), which generates double-stranded DNA breaks and triggers the G2-phase DNA damage checkpoint. Major chemical classes are
anthracyclines (e.g., Doxorubicin, Epirubicin, and Daunorubicin) and Epipodophyllotoxins (e.g., Etoposide, Teniposide). The other category, catalytic inhibitors,
traps Topo II on DNA, forming a closed clamp complex (iv in panel B) triggering the G2-phase decatenation and metaphase Topo IIA checkpoints. Bisdioxopiperazines are the most widely studied (e.g., ICRF-193, ICRF-187/Dexrazoxane, Sobuzoxane/MST-16). The focus of this review is cellular responses tocatalytic inhibition, where cycle checkpoints are triggered by DNA–Topo IIA closed clamps. These structures arise naturally but are stabilized by treatment withcatalytic inhibitors.

2 of 14the catalytic cycle of Topo IIA, preventing Topo IIA release fromchromatin (Fig. 1 B iv). The distinction between Topo IIA poisonsand catalytic inhibitors and the cell cycle responses they elicit issummarized in Box 1.
In untransformed cells that display a sustained G2 arrest in
response to ICRF-193, there is no sustained DNA damage as determined by γH2AX immunostaining; the limited extent ofγH2AX staining on exit from S-phase is resolved shortly after
S-phase in a Topo IIA–independent manner, and this resolution
does not influence the ICRF-193–induced G2 arrest (Lockwood
et al., 2022; it is noted that transformed cells that progress
through the cell cycle despite ICRF-193 exposure typically undergo aberrant cell divisions triggering DNA damage). The ICRF193–induced G2 arrest is dependent upon the expression of Topo
IIA (Bower et al., 2010a; Furniss et al., 2013); knock-down ofTopo IIA bypasses the arrest while leaving chromosome

Box 1. Definitions and scope of the review
Topo II is the only enzyme in eukaryotes that can disentangle intertwined
DNA duplexes, i.e., catenated DNA. Many inhibitors have been identified directed at Topo II (reviewed in Larsen et al., 2003), including those thatsuppress association to DNA, prevent ATP binding, block the re-ligation step,and inhibit the ATPase activity required for enzyme release from DNA (seescheme in Fig. 1). Pertinent to this review are the ligation inhibitors typicallyreferred to as Topo II poisons that trap the enzyme–DNA intermediate,
prevent re-ligation of the Topo IIA–implemented DSB, and hence trigger a
DNA damage response (see text and Fig. 1 for further details). These agents,
which include Etoposide, are used as cytotoxic agents in the treatment ofsome cancers. The focus of attention here is, however, on the ATPase inhibitors that trap Topo II on DNA at the end of its catalytic cycle; it is thisclass of inhibitors that trigger specific Topo II–dependent checkpoint delaysin the cell cycle. These checkpoints act in G2, M, or at cytokinesis, reflecting
the natural cellular monitoring of catenation by Topo II occupancy on chromatin, a process amplified by the inhibition of its catalytic activity. Ourcurrent understanding of the underlying controls operating in these checkpoints is reviewed here. The idiosyncrasies of these Topo II–dependent
checkpoint controls may lend themselves to distinctive anticancer approaches as discussed.

Soliman et al.
Topoisomerase II checkpoint controls

catenations (Bower et al., 2010a; Brownlow et al., 2014). A similar pattern of behavior occurs in budding yeast (Baxter andDiffley, 2008; Furniss et al., 2013). This provides evidence thatthe arrest is not simply triggered by a physical constraint onintertwined chromatin nor by some independent sensing ofcatenated chromatin, but is in fact relayed to the cell cycle machinery in a manner dependent upon Topo IIA and by inference
its specific conformation and juxta-positioning on chromatin at
G2 (i.e., post-replication). Topo IIA is in effect used both as thesensor of retained catenation and as the executor of its resolution. The existence of a Topo IIA–linked signaling pathway responsible for implementing this arrest (see below) formalizes
this as a Topo IIA–dependent G2 checkpoint. As noted above, thepurpose of this checkpoint is to ensure chromatin catenationassociated disjunction prior to M-phase entry (replication stressand associated non-disjunction do not trigger the same signaling
relay) (recently reviewed in Saxena and Zou, 2022).
In early studies, specific regulators were shown to be involved in the implementation of the Topo IIA–dependent
checkpoint, and these included p53 (Doherty et al., 2003;
Kaufmann et al., 2002). Recent genome-wide screening has
elucidated a more detailed signal relay responsible for implementing the Topo IIA–dependent G2 checkpoint. This has
elaborated a pathway involving the DNA-interacting SMC5/6
complex subunits, the protein kinases ATM/ATR (acting redundantly), alongside the tumor suppressor p53 and the cdk
inhibitor p21 (Deiss et al., 2019). Contrasting with the DDR(Stracker et al., 2009), this relay does not trigger activation of, or
require, Chk1/2 (Deiss et al., 2019; Deming et al., 2001), although
the downstream p53-p21 module in this checkpoint pathway
overlaps with one of the redundant DDR pathways acting in G2(see Lockwood et al., 2022). The upstream triggers define thedistinctiveness of these G2 checkpoints with, for example,
the SMC5/6 complex and Topo IIA proteins being essential for
the ICRF-193–induced arrest while not impacting the DDR; theJournal of Cell Biology
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Figure 2. Checkpoints regulating mitotic progression. Topo IIA checkpoints operate in parallel with the G2 DDR and the SAC to regulate the G2-to-M andmetaphase-to-anaphase cell cycle transitions. Key components required for each checkpoint response are shown. When non-disjunction occurs leading to anunresolved chromatin bridge, the abscission checkpoint delays cytokinesis until bridge resolution, after which PKCε phosphorylates Aurora B on S227 to permit
abscission.

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Soliman et al.
Topoisomerase II checkpoint controls

short-term, caffeine-sensitive (requiring ATM or ATR) delay in
G2 progression even in the absence of p53 (Kaufmann et al.,2002). It has been established that this transient delay is distinguished from the DDR and requires Topo IIA (Kaufmannet al., 2002). A distinctive element relaying this response is
the phosphorylation of Topo IIA on S1524 and the recruitment of
MDC1 through its BRCA1 C-terminal (BRCT) domain (Luo et al.,2009). MDC1 is itself required for the DNA damage response;
however, the Topo IIA–MDC1 complex is not (Luo et al., 2009).
Whether these responses involve the SMC5/6 complex remains
to be determined, although it would be attractive to posit thebifurcation of signals from SMC5/6-Topo IIA leading to a G2
delay on the one hand (p53 independent) and a program of longterm arrest and senescence on the other (p53 dependent). The
differential localization of these proteins (e.g., in or out of PML
bodies) and the potential distinctive coassociation of other regulators perhaps determine these outputs much as ALT cells
display distinctive ICRF-193–induced downstream events (discussed above).
Irrespective of the details that have yet to be resolved, theevidence indicates that there is a checkpoint in G2 that monitors
the Topo IIA closed clamp occupation of chromatin. The specificcircumstances evidently impact the particular pathways engaged but the implication remains the same; if Topo IIA in thisconformation is sensed on chromatin associated with the SMC5/6
complex, then there is a delay to G2 progression. The existence of
these regulatory processes suggests that in the absence of any
experimental manipulation, the steady-state association of Topo
IIA with chromatin is exploited by cells to sense ongoing decatenation, the associated potential for non-disjunction, and hence
the need for cell cycle delay and catenane resolution.
Engagement of the metaphase Topo IIA checkpoint is distinct
from the spindle assembly checkpoint (SAC) and is associatedwith promotion of catenation resolution
The mitotic apparatus, the spindle and kinetochores, is mechanically complex, but without cell cycle controls, chromosome
segregation is error prone. In early mitosis, the SAC senses
chromosome biorientation, deciding when anaphase can proceed accurately (Foley and Kapoor, 2013; Lara-Gonzalez et al.,2021; Musacchio, 2015). These controls sense microtubule attachments to kinetochores and tension forces to ascertain ifbiorientation has been achieved (reviewed in Chacón et al.,2014). In parallel, cells monitor Topo IIA to ensure that the sister chromatids can be efficiently separated before chromosome
segregation is initiated in anaphase (Fig. 2). Like the G2-phase
Topo IIA checkpoint, this metaphase Topo IIA checkpoint seems
to be activated by Topo IIA–DNA complexes, recruiting cell cycle
regulators to chromosomes to stop cells initiating anaphase.
These Topo IIA complexes can be generated experimentally,
either by blocking ATP hydrolysis with catalytic Topo IIA inhibitors (e.g., ICRF-193) or genetically by mutating residues ofTopo IIA leading to perturbed ATPase hydrolysis (Brownlow
et al., 2014; Clarke et al., 2006; Iwai et al., 1997; Kelly et al.,2020; Skoufias et al., 2004; Toyoda and Yanagida, 2006).
The mechanism of the metaphase Topo IIA checkpoint is atleast partially conserved from yeast to human cells (Andrews
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converse being true for DNA-dependent protein kinase (DNAPK)–dependent sensing of chemically induced DSBs (reviewed inJackson and Bartek, 2009). The requirement for the SMC5/6
complex in the Topo IIA–dependent checkpoint has been demonstrated by manipulation of subunit expression (all six mammalian subunits; SMC5–6 and NSMCE1–4) in cells and also
verified in cells derived from a patient with a germline mutationin the NSMCE2 subunit of the complex (Deiss et al., 2019).
NSMCE2 is an E3 ligase responsible for the ICRF-193–induced
SUMOylation of a non-canonical site, K1520, at the C-terminusof Topo IIA, and defects in this activity lead to a failure to sustain
a G2 arrest in response to ICRF-193 (Deiss et al., 2019).
The implementation of the Topo IIA checkpoint in G2 is associated with the accumulation of Topo IIA and the SMC5/6
complex in promyelocytic leukemia protein (PML) bodies. The
colocalization of these proteins is likely important in promoting
NSMCE2-directed SUMOylation of Topo IIA, although no formal
evidence of higher-order complex formation/physical contact
has yet been seen within these nuclear structures. Nevertheless,given the pattern of S-phase chromatin loading and M-phase
unloading of the SMC5/6 complex (Gallego-Paez et al., 2014;
Tsuyama et al., 2006), a role in supporting the sensing andcompletion of post-replicative sister chromatid disjunction is an
attractive proposition. While there are numerous roles assigned
to the SMC5/6 complex (Palecek, 2018), it is of interest thatbased on separation of function alleles, the essential role for the
related SMC5/6 complex in yeast is manifest specifically in G2/M
(Menolfi et al., 2015).
Interestingly, in transformed cells engaging alternative
lengthening of telomeres (ALT cells), ICRF-193 induces accumulation of Topo IIA and the SMC5/6 complex in a subset of
PML bodies, termed APBs (ALT-associated promyelocytic leukemia nuclear bodies), and this is associated with a G2 arrest(Potts and Yu, 2007). However, in this context, the arrest isdriven in a Chk1-dependent manner and is independent of p53(Lockwood et al., 2022), consistent with the association of p53
inaction in ALT tumors (Barthel et al., 2017). The arrest requires
the RecQ-like helicase Bloom’s syndrome protein (BLM)(Lockwood et al., 2022), and perhaps, related to this, it has beenshown that Topo IIA can be found associated with BLM andtelomere-binding protein TRF2 in ALT cells (Bhattacharyya
et al., 2009; Lockwood et al., 2022). It is likely that Topo IIA
contributes to the resolution of the recombination intermediates
associated with this specific telomere elongation process (see,for example, Hou et al., 2022), and hence that this is sensed
when it becomes trapped on the telomeric structures by ICRF193. The implication is that the signaling relays triggeringarrest downstream of SMC5/6-Topo IIA are exquisitely afunction of the context, i.e., the corecruited regulators. This
context-dependent behavior provides a rationalization for theidentification of other players that have been identified in
ICRF-193–induced G2 arrest(s): BRCA1, WRN, MDC1, Plk1,
MCPH1, and Chk1 (Arroyo et al., 2019, 2020; Deming et al.,2001, 2002; Lou et al., 2005; Luo et al., 2009; Robinson et al.,2007; Spoerri et al., 2016).
While untransformed cells can engage a long-term G2 arrestin response to ICRF-193, cells have also been reported to trigger a

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et al., 2006; Clarke et al., 2006; Furniss et al., 2013; Pandey et al.,2020), revealing that it is a fundamental aspect of mitotic regulation. Studies in human cells initially used catalytic inhibitors
such as ICRF-193 to inhibit Topo IIA ATPase activity in metaphase, which resulted in delayed anaphase onset (Brownlow
et al., 2014; Clarke et al., 2006; Iwai et al., 1997; Kelly et al.,2020; Pandey et al., 2020; Skoufias et al., 2004; Toyoda andYanagida, 2006). In yeast genetic screens, top2 mutants that
activate the metaphase Topo IIA checkpoint were isolated andfound to be defective in ATPase activity (Andrews et al., 2006;
Furniss et al., 2013). Thus, the yeast top2 mutants genetically
mimic ICRF-193 treatment in human cells. Subsequent studies
confirmed that the metaphase Topo IIA checkpoint mechanism
is distinct from the canonical SAC (Andrews et al., 2006;
Brownlow et al., 2014; Clarke et al., 2006; Furniss et al., 2013;
Kelly et al., 2020; Pandey et al., 2020; Skoufias et al., 2004). Core
components required for metaphase Topo IIA checkpoint activation are Haspin kinase, Aurora B, and protein kinase Cε(PKCε; Brownlow et al., 2014; Pandey et al., 2020). Both SAC andmetaphase Topo IIA checkpoints ultimately activate Mad2 toprevent anaphase onset but by using distinct mechanisms(Andrews et al., 2006; Skoufias et al., 2004; Toyoda andYanagida, 2006). The SAC recruits Mad2 to kinetochores
where it is activated and then released. The SAC therefore requires intact kinetochores for activation (see Rieder et al., 1995;
Tavormina and Burke, 1998). In contrast, Mad2 does not localize
to kinetochores when the metaphase Topo IIA checkpoint is
active (Brownlow et al., 2014; Skoufias et al., 2004; Toyoda andYanagida, 2006; Fig. 3), and (at least in yeast) kinetochores are
not required for metaphase Topo IIA checkpoint activation(Andrews et al., 2006; Skoufias et al., 2004; Toyoda andYanagida, 2006). In summary, while the SAC monitors chromosome spindle attachment, the metaphase Topo IIA checkpoint
monitors ongoing Topo IIA (closed clamp) occupation of chromatin to ensure sister chromatids can separate in anaphase(Furniss et al., 2013).
Factors required for the metaphase Topo IIA checkpoint in
human cells were initially identified based on analysis of the
Soliman et al.
Topoisomerase II checkpoint controls

conserved mechanism in yeast (Andrews et al., 2006; Furniss
et al., 2013), which among other factors, identified Ipl1 (yeast
ortholog of Aurora B kinase) as an essential component. Anaphase onset is delayed when the Topo IIA catalytic inhibitor
ICRF-193 is added to metaphase human cells, but this was bypassed by Aurora B inhibitors (Pandey et al., 2020). At the same
concentrations, these inhibitors could not bypass the SAC(induced with nocodazole that depolymerizes microtubules),demonstrating the SAC and metaphase Topo IIA checkpoint
have distinct mechanisms (Pandey et al., 2020). In these experiments, because ICRF-193 was added to metaphase cells,
chromosome biorientation had already occurred, satisfying the
SAC (Pandey et al., 2020). This might mean there is a window oftime in metaphase after SAC inactivation when the Topo IIA
checkpoint can still be engaged. Nevertheless, a fraction of cells
proceeds quickly into anaphase when ICRF-193 is added inmetaphase, suggesting those cells had already passed a commitment point to allow the metaphase–anaphase transition(Pandey et al., 2020).
Given that ICRF-193 does not activate the canonical SAC, an
important question is how Aurora B is activated to induce themetaphase Topo IIA checkpoint. Clues came from the fact that
ICRF-193 induces SUMOylation of the Topo IIA C-terminal domain (CTD) specifically on mitotic chromosomes and that inboth interphase and mitotic cells, SUMOylation initiates repair
pathways that have evolved to remove trapped Topo IIA complexes from DNA (Agostinho et al., 2008; Hassebroek et al.,2020; Isik et al., 2003; Schellenberg et al., 2017; Tian et al.,2021; Xiao et al., 2003; Yoshida et al., 2016). This led to theprediction that SUMOylation of Topo IIA would also activate themetaphase Topo IIA checkpoint to provide extra time for removal of the trapped complexes and for decatenation before
anaphase onset. Consistent with this hypothesis, engineered
human cells where endogenous Topo IIA is replaced with amutant lacking three conserved SUMOylated lysines (Topo II3KR) were partially defective in metaphase Topo IIA checkpoint
activation (Pandey et al., 2020), demonstrating that SUMOylation in response to ICRF-193 is likely involved in Aurora B
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Figure 3. Chromosomal localization of mitotic checkpoint regulators. (A) Cartoon
showing relative location in mitosis of sister kinetochores (K; ovals), sister chromatid cores(chromosome cores), and the inner centromere(i-CEN; gray). The entire chromosome arm
width is indicated by light blue–shaded regions.
(B) Localization patterns of Mad2, Bub1, BubR1,
Aurora B, Topo IIA, and SUMO2/3 in mitosis after
nocodazole treatment (SAC activation) versus
ICRF-193 treatment (Topo IIA checkpoint activation). Mad2 localizes to kinetochores and Aurora B remains at the inner centromere after
nocodazole treatment, while SUMO2/3 is distributed diffusely. After a brief (30 min) ICRF-193
treatment in metaphase, Aurora B is recruitedaway from centromeres to the chromosome
cores along with SUMO2/3. Consistent withchromosome biorientation being maintained,
Mad2 is not recruited to kinetochores, but Bub1
and BubR1 are retained, dependent on PKCε.

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Table 1. Topo IIA SUMOylation site mutants and phenotypes
Species

Lysine(s) Phenotypes/Functions

References

Yeast (S. cerevisiae) 1220
SUMOylation of these lysine residues is implicated in: (1) regulating sister centromere(1) Bachant et al. (2002)
1246 1247 cohesion, (2) recruitment of Ipl1 (yeast ortholog of Aurora B) to centromeres in mitosis, and (3) (2) Edgerton et al. (2016)
1277
inducing ubiquitination of Top2-DNA cleavage complexes.
(3) Sun et al. (2020)
1278

Human

Required for stable maintenance of minichromosomes. Triple K-to-R mutant stabilizes
dicentric chromosomes, indicating reduced kinetochore function.

Takahashi et al. (2006)

660

Inhibits Topo IIA decatenation activity. Regulates resolution of centromeric catenations inmitosis.

Ryu et al. (2010)

1235
Recruitment of (1) Claspin, (2) Haspin, and Aurora B to mitotic chromosomes.
1276 1298(1) Ryu et al. (2015)(2) Yoshida et al. (2016)

662

Topo IIA-K662R has reduced Topo IIA centromere localization and increased chromosome
segregation defects.

Antoniou-Kourounioti et al.
(2019)

1228
1240

SUMOylation by ZATT/ZNF541 ligase induced by replication fork stalling (hydroxyurea
treatment in S-phase) promotes fork reversal and recruitment of PICH DNA translocase.

Tian et al. (2021)

1240

Topo IIA-K1240R mutant has reduced Topo IIA centromere localization.

Antoniou-Kourounioti et al.
(2019)

1240
1267
1286

Triple (K-to-R) mutant reduces Aurora B recruitment to mitotic chromosomes induced by
ICRF-193 and partially bypasses the metaphase Topo IIA checkpoint.

Pandey et al. (2020)

1520

SUMOylated by NSE2 (Smc5/6 complex) E3 ligase induced by ICRF-193. Topo IIA-K1520R
Deiss et al. (2019)
mutant increases chromosome segregation defects but does not affect the G2-phase Topo IIA
checkpoint, unlike depletion of Smc5/6 complex components, which does lead to a failure tosustain G2 arrest in response to ICRF-193.

activation. The partial metaphase Topo IIA checkpoint bypass
may indicate that additional lysines in the CTD are SUMOylated
in the presence of ICRF-193, consistent with other studies (seeTable 1; Antoniou-Kourounioti et al., 2019; Ryu et al., 2010;
Schellenberg et al., 2017; Tian et al., 2021).
The evidence implicating Topo IIA SUMOylation in themetaphase checkpoint activation is important because it provides a direct link to Aurora B activation. In yeast and Xenopus
egg extracts, Topo IIA SUMOylation recruits Haspin kinase tochromosomes in mitosis (Edgerton et al., 2016; Yoshida et al.,2016), and Haspin generates a binding site for Aurora B byphosphorylating histone H3 at Threonine 3 (H3T3-phos; Fig. 4).
Haspin has SUMO interacting motifs (SIMs) that mediate a direct interaction with SUMOylated Topo IIA, explaining how
trapped Topo IIA complexes recruit Haspin to chromosomes(Yoshida et al., 2016). H3T3 is the only known substrate ofHaspin, and when phosphorylated, it interacts directly with
Survivin, a subunit of the chromosome passenger complex(CPC) of which Aurora B is also a component (Dai et al., 2005; De
Antoni et al., 2012; Kelly et al., 2010; Wang et al., 2010, 2011,2012). Crucially, Haspin inhibitors were found to bypass metaphase Topo IIA checkpoint activation, like Aurora B inhibitors(O’Connor et al., 2015; Pandey et al., 2020). Consistent with thiscascade being responsible for metaphase Topo IIA checkpoint
activation, both Haspin kinase activity and Topo IIA SUMOylation are required for efficient recruitment of Aurora B to chromosomes in mitotic cells treated with ICRF-193. Strikingly,
Aurora B was recruited to chromosome cores, where trapped
Topo IIA complexes are most abundant (Fig. 3). This contrasts
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with the localization of Aurora B at inner centromeres and kinetochores when the SAC is active (Broad et al., 2020; Campbell
and Desai, 2013; Carmena et al., 2012; Dai et al., 2006; Petsalaki
and Zachos, 2016; Yamagishi et al., 2010; Fig. 3). Finally, ICRF193 treatment in mitosis also induced a dramatic increase of
SUMO2/3 at chromosome cores. Chromosome cores are thecentral region of chromatid arms where Topo IIA is concentrated, and indeed SUMO2/3, Aurora B, and Topo IIA localizedwith the same pattern at the cores after ICRF-193 treatment(Fig. 3; Pandey et al., 2020). Altogether, the evidence supports amodel where the metaphase Topo IIA checkpoint is activated by
SUMOylated Topo IIA, which recruits Haspin to then recruit
Aurora B (Fig. 4).
Additional evidence has indicated that nucleosome binding of
Topo IIA may facilitate activation of the metaphase Topo IIA
checkpoint. Aurora B recruitment seems to rely on H3T3
phosphorylation by Haspin, leading to the prediction that Haspin must not only be recruited to chromosomes but must be inclose proximity to nucleosomes. The discovery of a nucleosome
binding region of Topo IIA, the chromatin tether (ChT) domain(Lane et al., 2013), could provide this missing link. Deletion of
the ChT results in chromosome segregation errors, the expectedphenotype if the checkpoint requires the ChT to efficiently enact
Haspin-mediated H3T3 phosphorylation (Sundararajan et al.,2023). It will be important to determine if the ChT is required
for the activation of the checkpoint.
Another major gap in understanding is how Aurora B activates the metaphase Topo IIA checkpoint via Mad2 activation.
Because there is no direct connection between Aurora B and
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Frog (X. Laevis egg
extracts)

1220
1246
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Mad2, the relevant substrates of Aurora B during metaphase
Topo IIA checkpoint activation are unknown. There may be
novel Aurora B substrates involved since Aurora B is recruited toa distinctive location at chromosome cores where establishedkinetochore substrates are not known to be present. Further,
metaphase Topo IIA checkpoint activation requires PKCε, which
triggers a switch in Aurora B substrate selection (Brownlow
et al., 2014; Kelly et al., 2020). PKCε phosphorylates a secondsite in the activation loop of Aurora B (S227), and this specificallyoccurs in the chromosome-associated fraction of Aurora B inmitosis upon metaphase Topo IIA checkpoint activation (Fig. 4;
Kelly et al., 2020). S227 phosphorylation induces a significant
conformational change in the activation loop, and as a result,
there is a dramatic change in Aurora B substrate specificity(Kelly et al., 2020; Pike et al., 2016). Importantly, knock-down of
the WT protein and replacement with an Aurora B S227A mutant
led to increased chromosome segregation errors and ultrafine
DNA bridges (UFBs) in anaphase and was also deficient in metaphase Topo IIA checkpoint activation. This evidence demonstrates that the Aurora B substrate specificity switch is essential
for proper engagement of the metaphase Topo IIA checkpoint,
but the relevant Aurora B substrate(s) are not known. The Aurora B specificity switch has an additional crucial consequence:increased decatenation. The specificity switch promotes Aurora
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B phosphorylation of Topo IIA at Serine 29, which substantially
increases Topo IIA activity facilitating resolution of this topological block to cell division (Fig. 4; Kelly et al., 2020). It appears
that the sensing, arrest, and resolution are integrated into a coherent set of events that are by their very nature self-limiting.
Given that the effectors of Aurora B remain to be identified,further studies are needed to determine how Mad2 is activated.
The identity of the Topo IIA SUMO E3 ligase that activates thecheckpoint is also unknown. Several E3 ligases have been shown
to SUMOylate Topo IIA, including PIASy and RanBP2 (Azuma
et al., 2005; Dawlaty et al., 2008; Dı́az-Martı́nez et al., 2006;
Navarro and Bachant, 2008), ZNF451/ZATT (Schellenberg et al.,2017) and, as described above, NSMCE2 (Deiss et al., 2019). ZATT
is known to SUMOylate Topo IIA in interphase and functions inremoval of trapped Topo IIA complexes from DNA (Riccio et al.,2020; Schellenberg et al., 2017; Tian et al., 2021). NSMCE2 is acomponent of SMC5/6 complexes that can SUMOylate Topo IIA,
and as discussed above is known to be required for the G2-phase
Topo IIA checkpoint activated by ICRF-193 (Deiss et al., 2019). It
will be important to determine which of these E3 ligases act inmitosis and which of the known Topo IIA SUMOylation sites canrecruit Haspin to chromosomes. It is already evident that SUMOylation of different lysine residues has different outcomes(see Table 1).
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Figure 4. Metaphase Topo II checkpoint model. Closed clamp Topo II complexes are SUMOylated, recruiting Haspin via its SIMs to Topo IIA–bound nucleosomes. Haspin generates H3T3p, which recruits CPC–Aurora B. PKCε phosphorylates Aurora B–S227, inducing a substrate-specificity switch required for
metaphase checkpoint activation and increased Topo IIA activity. In parallel, PKCε phosphorylates SERBP1, which is sequestered in M-bodies, repressing
translation, needed for successful cell division.

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events remain to be elucidated. Nevertheless, it is establishedthat upon sensing DNA trapped in the cytokinetic furrow, Aurora
B signaling is sustained, maintaining a stabilized intercellular
canal (Steigemann et al., 2009) and preventing localization of the
ESCRTIII complex, in particular CHMP4C, to the midbody ringand site of abscission (Carlton et al., 2012; Pike et al., 2016; the
ESCRT machinery is involved in multiple aspects of membrane
remodeling; see Olmos, 2022). This is reported to be antagonizedby the UFB binding protein, RIF1, recruiting PP1 to unresolved
DNA bridges during cytokinesis (Bhowmick et al., 2019). Thiscounters the action of Aurora B to delay abscission and allow forbridge resolution. Exit from this arrest is associated with phosphorylation of Aurora B on S227 by a PKCε-14.3.3 complex at the
midbody (Watson et al., 2021), switching specificity toward S165
on the CPC subunit, Borealin (Pike et al., 2016). The extent towhich PP1 and the S227 kinase antagonism acts alongside
CHMP4C (Carlton et al., 2012) and clk1/2/4 (Petsalaki and Zachos,
2016) to determine timing is unclear. It will be of interest to establish whether PP1 inactivation/mislocalization triggers Aurora
B S227 and/or Borealin S165 phosphorylation, and where PKCε is
not required (e.g., in ALT cells, see above), what acts to trigger
S227 phosphorylation or is there an alternative route for abscission checkpoint exit? Ultimately, if bridging cannot be resolved,
cells will fail cytokinesis through collapse of the midbody resulting in a binucleated cell or cells will divide despite the bridge
leading to DNA breakage, accumulation of DNA damage, and ultimately aneuploidy (see, for example, Thoresen et al., 2014).
Defective decatenation checkpoints have been observed in a
number of tumor types including lung (Lockwood et al., 2022;
Nakagawa et al., 2004), melanoma (Brooks et al., 2014a, 2014b),
colon (Jain et al., 2015), bladder (Doherty et al., 2003), and breast(Bower et al., 2017). Loss of the Topo IIA (decatenation) checkpoint in G2 correlates with inactivation or loss-of-function of p53(Doherty et al., 2003; Kaufmann et al., 2002; Lockwood et al.,2022), known to be a common feature of not only these tumor
types but more broadly; TP53 is mutated in >50% of all humantumors (Bykov et al., 2018). A variety of effectors had been implicated in the G2-phase Topo IIA checkpoint response prior towider genome screening, including BRCA1 (Lou et al., 2005),
Chk1 (Robinson et al., 2007), WRN (Franchitto et al., 2003), andATM (Bower et al., 2010b). ATM appears to act redundantly with
ATR in normal epithelial cells (Deiss et al., 2019); however, itremains likely that particular transformed cell models exploit
distinct arrest pathways as discussed above. Indeed, the identification of Chk1 as a requirement for ICRF-193–induced G2 arrest
in DT40 cells (Robinson et al., 2007) would suggest operation of
the ALT pathway in these cells; despite expressing telomerase
there is evidence that this is the case (O’Hare and Delany, 2011).
The implication is that for catalytic inhibition of Topo IIA,
there may be a number of different conditional dependencies
in transformed cells that are particular to the transformation.
This poses a challenge in exploiting these pathways but is equallyan opportunity for highly selective interventions.

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Bypassing Topo IIA checkpoints
Repair mechanisms have evolved to disperse trapped Topo IIA
complexes (Agostinho et al., 2008; Hassebroek et al., 2020; Isiket al., 2003; Schellenberg et al., 2017; Tian et al., 2021; Xiao et al.,
2003; Yoshida et al., 2016); however, the extent to which these
operate physiologically to bypass or exit the Topo IIA checkpoints and hence impact the timing of these cell cycle delays
remains to be determined. By contrast, there is clear evidence
for somatic changes influencing checkpoint function and consequently triggering downstream events. Cells with a defective
Topo IIA–dependent G2 checkpoint can enter mitosis with anexcessive amount of unresolved DNA catenanes, and if unresolved prior to anaphase entry, non-disjunction is manifest in
Plk1-interacting checkpint helicase (PICH)-positive UFBs during
anaphase (Wang et al., 2008). Unresolved, this non-disjunction
leads to chromosome segregation errors, division failure, andaneuploidy. It is noted that the retention of some catenated DNA
on entry into mitosis, particularly at the centromere, appears to
be required to maintain chromosome structure (Bauer et al.,2012; Bower et al., 2010a). Hence, a low degree of catenationin mitosis may be “normal.” The monitoring and resolution ofthis residual centromeric catenation may be linked to the timingof anaphase entry/progression, presumably under the control
of Haspin–Aurora B responsible for the Topo IIA metaphasecheckpoint (see above).
There is evidence that the resolution of excessive catenation
appears to engage additional/distinct mitotic pathways that are
determined by the specific G2 trigger. This is exemplified by theengagement of PKCε during mitosis, where loss of the p53dependent checkpoint pathway in G2 is associated with asubsequent dependence on PKCε for effective cell division(Brownlow et al., 2014; Kelly et al., 2020; Lockwood et al., 2022;
Martini et al., 2018). By contrast, in ALT cells, where p53 is
dysfunctional and Chk1 is the key downstream relay for the G2arrest, bypassing this ICRF-193–induced engagement of the G2checkpoint does not engage PKCε and by inference requires
distinct regulators to facilitate subsequent mitotic delay andresolution (Lockwood et al., 2022). The extent to which Haspin
and Aurora B are common to all mitotic Topo IIA–associateddelays, while PKCε and other regulators are idiosyncrasies of thecontext, remains to be determined. Likewise, it remains to be
determined whether Topo IIA activation (Kelly et al., 2020),
translational repression (Martini et al., 2021), and Aurora B S227
phosphorylation (Pike et al., 2016) are required in all circumstances of mitotic delay, and if so what kinases other than PKCεcan execute these actions (see Fig. 4).
Bypassing the Topo IIA checkpoints engenders DNA bridging,which persists into telophase and cytokinesis, activating the
Aurora B–dependent abscission checkpoint to delay cytokinesis(see Fig. 2; Bhowmick et al., 2019; Steigemann et al., 2009). Interestingly, it has been shown very recently that the sensing ofDNA bridging at the abscission checkpoint can be effected
through the detection of trapped Topo IIA–DNA cleavage complexes in the DNA knots that form juxtaposed to the midbody(Petsalaki et al., 2023). This trapping is distinct from the closed
clamp conformer associated with G2- and M-phase Topo IIA
checkpoints, and the proximal signals affecting downstream

Opportunities for intervention
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Box 2. Context of checkpoint interventions in cancer

degree of selectivity offers some interesting opportunities clinically (Box 2). Since “normal” cells typically do not expose
themselves to the lower fidelity, Topo IIA–dependent controls
operating in M-phase and at abscission, then in some cancers
where the G2 checkpoint is dysfunctional (e.g., TP53 mutant,discussed above), there are druggable targets that are unusuallyengaged in these conditional pathways. These could be exploited
in a synthetic lethal approach alongside catalytic Topo IIA inhibitors. There is already good evidence that targeting tumors
with a defective Topo IIA–dependent G2 response may be a viable therapeutic strategy. Checkpoint deficient cell lines including hepatocellular and pancreatic ductal adenocarcinoma(PDAC) (Lee et al., 2022) and colorectal cancer (Jain et al., 2015)lines have been reported to be sensitive to Topo IIA catalytic
inhibition alone, with cells undergoing a catastrophic mitosis
after being unable to decatenate their DNA. Why these cell types
display this sensitivity is not yet understood, although a complete analysis of Topo IIA–associated checkpoint effector expression has yet to be reported. Melanoma cell lines that have adefective G2 decatenation checkpoint have been reported to be
dependent on PI3K signaling and are sensitive to the PI3Kα,γ,and mTOR inhibitor PF-05212384 (Brooks et al., 2014b). Again,
how, if at all, this dependency relates to Topo IIA checkpoint
effector expression is unknown.
Topo IIA catalytic inhibitors are currently used in clinicalpractice alongside anthracycline chemotherapeutics (Martyet al., 2006). In this setting, they provide a cardioprotectivefunction as ROS scavengers (Lebrecht et al., 2007) or by targeting Topo IIB (Deng et al., 2014; Jirkovský et al., 2021). Use of
Topo II catalytic inhibitors, for example, dexrazoxane (ICRF187), in targeting the Topo IIA–dependent G2 checkpoint has notbeen assessed clinically (either as a single agent or in combination), but it is known to have a tolerable toxicity profile inpatients (Macedo et al., 2019). Combining dexrazoxane withtargeting the metaphase Topo IIA checkpoint may present a new
therapeutic opportunity for TP53 mutant cancers. Topo IIA inhibition would arrest normal, healthy cells in the surrounding
tissues in G2-phase, while the checkpoint-deficient tumor cells
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Perspectives on Topo II checkpoints
There are many outstanding questions associated with the operation/inaction of these Topo IIA–dependent checkpoints anddelays that have implications not only for our understanding of
the mechanistic details but also for the deployment of interventions that can exploit the idiosyncratic dependencies oftransformed cells. Some of these issues we have highlighted
above, among others, is the question of timing—in G2, the engagement of a long-term arrest and commitment to a senescenceprogram is not hard-wired, with some transformed cells delaying transit but then progressing through G2- to M-phase, albeit
in a passage-dependent manner (Kaufmann et al., 2002). Whatdictates this pattern of behavior, how is the transient delay
implemented, and why does this erode on passage—what selective advantage does this confer? There is also the question of
the origins of these pathways. Given the existence of a series of
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There are many cytotoxic agents, including the Topo IIA poisons, deployed incancer treatments. These drugs induce DNA damage, which in cycling cells
causes the malfunction of DNA replication, associated disjunction errors,
failed cell division, and cell death. The narrow therapeutic window for theseagents is a consequence of the rapid division of cancers relative to mosttissues. Nevertheless, rapidly turning over tissues, e.g., gut and bone marrow,are associated with the “side effects” of such treatments. More selective
approaches to the blockade of cell division through the specific targeting ofcell cycle controls and checkpoints have promised greater selectivity and abetter therapeutic index compared with these general DNA-damaging agents.
This is the case for the cdk4/6 inhibitor Palbociclib, which is FDA-approvedfor deployment as a combination therapy in HR+ advanced breast cancer (Duet al., 2020). While there has been evidence of poor tolerance for some otherinterventions of this type, the targeting of the cell cycle regulator Aurora B,
for example, still offers promise (see Borah and Reddy, 2021). Related approaches that draw upon dysfunctional properties of tumor cells may alsooffer opportunities for intervention, and given the frequent loss of the G2Topo IIA checkpoint in tumor cells, the downstream reliance on M-phase andanaphase checkpoints to resolve residual catenation yields the potential for agood therapeutic index in targeting these cell cycle controls.

would be primed for targeting the checkpoint relay engaged inmetaphase. Known targets of the metaphase pathway include
PKCε (Brownlow et al., 2014), Aurora B (Pandey et al., 2020),
and PICH (Nielsen et al., 2015); BLM helicase (Nguyen et al.,2013; Russell et al., 2011) and Haspin (Amoussou et al., 2018;
Huertas et al., 2012) could also be actionable targets in TopoIIA–dependent G2 checkpoint deficient tumors.
To date, no combinations drawing upon these findings havebeen tested in patients, and although it is the case that previously available PKCε inhibitors lack the desired specificity forclinical use (Roffey et al., 2009), new-generation inhibitors may
overcome this limitation (Blasio et al., 2018). PKCε appears to beengaged in multiple pathways; however, at least in unstressedlaboratory mice, its complete absence is tolerated (Castrillo et al.,2001), suggesting that its inhibition would not engender ontarget toxicity. Beyond p53 loss-of-function, which tumor
types might offer themselves to a combined PKCε/Topo IIA inhibitor attack remains to be determined. First-generation Aurora B inhibitors have proven to be too toxic (Kovacs et al.,2023); however, more recent inhibitors show promise (Borah
and Reddy, 2021). Again, beyond p53 status, it is not clear
where deployment of a combination treatment might be mosteffective. Targeting PICH may overcome the potential issues ofundesired on-target/off-pathway consequences. PICH is engaged in resolution of non-disjunction structures in anaphase asis the BLM helicase (Rouzeau et al., 2012). PICH acts in part todisperse SUMOylated Topo IIA foci on chromatin (Hassebroek
et al., 2020). Notably, PICH-deficient cell lines are sensitive toTopo IIA inhibition and become binucleated (Nielsen et al.,2015), while PICH depletion in triple-negative breast cancer
cells leads to non-disjunction, cytokinesis failure, and ultimately
apoptosis after mitotic catastrophe (Huang et al., 2019). It has
also been reported that aneuploid or chromosomally unstable
tumors, as might be driven by aberrations/inhibition of TopoIIA–dependent G2 checkpoints, may be targeted by mitotic
checkpoint inhibitors such as Mps1 inhibitors that elicit an accumulation of mitotic defects resulting in more unstable and lessfit karyotypes (Cohen-Sharir et al., 2021). If not driving catastrophic outcomes, would this combination be a driver of neoantigen production prescribing immuno-oncology approaches?

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Acknowledgments
P.J. Parker has been supported by the Francis Crick Institute,which receives its core funding (CC2068 and CC2140) from
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Topoisomerase II checkpoint controls

Cancer Research UK, UK Medical Research Council, and theWellcome Trust. D.J. Clarke and D. Keifenheim were supportedby the National Institutes of Health grant NIGMS R01GM130858. T.N. Soliman acknowledges support from The Bart’s
Charity project grant (DRG2040).
Disclosures: P.J. Parker reported personal fees from Apollo
Therapeutics and Phoremost outside the submitted work. No
other disclosures were reported.
Submitted: 22 July 2023
Revised: 24 October 2023
Accepted: 25 October 2023

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conditional control processes operating in M-phase and then anaphase, what can we conclude about the evolution of these processes? Assuming these did not arise simply to enable transformed
cells to circumvent the Topo IIA–dependent G2 checkpoint, it
seems likely that there are physiological contexts in which normal
cells are programmed to avoid this p53-dependent G2 checkpoint
while retaining some ability to reduce the undesired consequences
through the ensuing mitotic and abscission checkpoints. It is interesting that in mouse stem and progenitor cells, there is a deficiency in this checkpoint, and this is recovered when embryonicstem cells are differentiated in response to retinoic acid (Damelin
et al., 2005). It is hard to rationalize this loss of checkpoint control
in cell types crucial to tissue homeostasis where there is a requirement for complete integrity in cell division. It would be ofinterest to determine whether replication in these cells relied
exclusively on Topo I acting ahead of the replication fork to obviate the need for Topo IIA action after replication. By contrast,
there are circumstances where a lowered tolerance in the requirement for precision might be more easily rationalized; forexample, in a pathogen response where the demand for the very
rapid adaptive immune cell expansion of pathogen-recognizing
clones overrides the more time-demanding process of highfidelity cell division, sacrificing some degree of genomic integrity in a life-and-death struggle (Bonilla and Oettgen, 2010). A
similar rationalization would apply in the context of hyperplasia
associated with response to (life-threatening) injury.
In bypassing the Topo IIA–dependent G2 checkpoint, cells
engage control processes that are not usually required for cell
cycle regulation. This is well characterized for the engagementof PKCε in the Topo IIA–dependent mitotic delay, where thereliance is not a function of somatic mutation as pharmacological bypass in otherwise G2-arrest competent cells still engages
PKCε (Brownlow et al., 2014). It seems likely that this reflects theacute, stress-specific implementation of distinct cell cycle regulatory programs. Related to this, there is no evidence of PKCεengagement in the abscission checkpoint, except in cells with
Topo IIA involvement; similarly, there is no PKCε engagement in
replication stress-driven behaviors in mitosis, despite there
being non-disjunction challenges in the context of collapsed
replication forks and incomplete DNA replication (Lin andPasero, 2021). So what triggers and maintains the selective
downstream program, and are the ultimate targets of these
programs common across cell cycle–associated stresses? Some
indication of downstream overlap comes from the finding thatthe p53–p21 axis is a target of the G2 Topo IIA checkpoint
pathway in common with one of the redundant DDR pathways
triggered in G2 (Lockwood et al., 2022). Equally, it is unknown
how different Topo IIA–associated stress-induced regulatory
programs delay SAC silencing, activate Topo IIA, modify mitotic
translation, and ultimately regulate exit from the abscission
checkpoint. Resolution of these issues will offer precision approaches to interventions.

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