Processing of DNA Topoisomerase II–DNA–Protein Crosslinks Associated With Anticancer Drugs

ABSTRACT

During cell division and gene expression, the DNA double‐helical structure unwinds, thereby generating torsional stress. DNA topoisomerases are enzymes that relieve this stress. During this process, topoisomerases form temporary covalent bonds with the phosphate backbone of DNA, generating DNA strand breaks and relieving torsional stress. Topoisomerases then dissociate from DNA after rejoining the DNA breaks. Torsional stress associated with replication or transcription is primarily relieved by topoisomerase I (TOP1) and II (TOP2). Some anticancer drugs targeting topoisomerases, known as topoisomerase poisons, trap the topoisomerase reaction intermediates and cause DNA strand breaks bearing topoisomerase–DNA–protein crosslinks (TOP–DPCs). TOP1 poisons, such as camptothecin, cause DNA single‐strand breaks bearing TOP1–DPCs, which are converted to DNA double‐strand breaks (DSBs) when they collide with DNA replication forks. In contrast, TOP2 poisons, such as etoposide, directly induce DSBs in TOP2–DPCs. However, to elicit a DSB response, TOP2–DPC must first be removed from the DSB ends. Cells possess various pathways to remove TOP2–DPC, and these pathways are thought to function in coordination depending on the situation. This review summarizes these sophisticated TOP2–DPC removal pathways and discusses the clinical applications of TOP2 poison as an anticancer drug, as well as the related challenges.

Topoisomerase II (TOP2) relieving DNA torsional stress is trapped by TOP2 poisons, such as etoposide, inducing DNA double‐strand breaks (DSBs) bearing TOP2–DNA–protein crosslinks (TOP2–DPCs), which must be removed before DSB repair. This review summarizes the TOP2–DPC resolution processes mediated by multiple pathways, including proteolysis and nucleolysis.

1. Introduction

The cells in our bodies contain DNA, which carries genetic information and is stably maintained inside their nuclei. Depending on the state of the cell, DNA undergoes significant changes in its topology. When cells divide to proliferate, they must replicate the DNA to double its quantity. Additionally, the transcription of many genes is spatially and temporally regulated to maintain cellular functions. DNA replication and transcription require the unwinding of double‐stranded DNA. After replication or transcription, the DNA must return to a double‐stranded state. Changes in the topology of the DNA double helix may influence chromatin structure and potentially affect gene expression and genomic stability. Alterations in the topology of the DNA double helix occur naturally during the unwinding and renaturation processes, and DNA topoisomerases relieve such torsional stresses. Vertebrates, including humans, have six DNA topoisomerase genes: TOP1, TOP2A, TOP2B, TOP3A, TOP3B, and TOP1MT (seven if the meiosis‐specific SPO11 is included). TOP1MT functions in the mitochondria, whereas the others function in the nucleus. TOP1, TOP3α, and TOP3β are classified as type I topoisomerases, whereas TOP2α and TOP2β are classified as type II topoisomerases. All topoisomerases transiently cleave DNA by forming covalent bonds between a tyrosine residue of the topoisomerase and the phosphate backbone of the DNA (Pommier et al. 2022). After the topological stress is relieved, the DNA strand breaks are rejoined and the topoisomerase dissociates from the DNA.

There are inhibitors that target TOP1 and TOP2, known as TOP1 and TOP2 poisons, respectively. These inhibitors trap reaction intermediates and induce DNA strand breaks bearing topoisomerase–DNA–protein crosslinks (TOP–DPCs). TOP–DPCs have been widely referred to as TOP–DNA cleavage complexes (TOPccs). In this review, TOPccs trapped by TOP poisons are denoted as TOP–DPCs to distinguish them from spontaneous TOPccs that occur during the native reaction process. TOP2 poisoning induces DSBs bearing TOP2–DPCs. Although the DSB repair and TOP2–DPC removal are closely related, they are distinct processes, each mediated by particular factors, and must be considered separately. Recent studies have revealed that efficient DSB repair requires TOP2–DPC resolution, which is regulated in a complex manner by multiple pathways. Therefore, in this review, we particularly focus on TOP2 and summarize how cells respond to TOP2–DPC induced by TOP2 poisons such as etoposide.

2. DNA Topoisomerase II

The unwinding of double‐helical DNA during replication and transcription elicits alterations in DNA topology. When the double helix unwinds, the genomic DNA cannot rotate freely, resulting in a situation referred to as positive supercoils (Sc⁺), where the pitch of the twisting becomes denser ahead of the unwinding machinery. Contrastingly, behind the unwinding machinery, the DNA twists loosen, leading to a state referred to as negative supercoils (Sc⁻) (Figure 1). TOP2 can resolve some types of DNA topological stresses, including Sc⁺ and Sc⁻, and as well as tangles involving two strands of DNA, such as DNA catenanes and knots (Nitiss 2009a; Pommier et al. 2022). TOP2 uses adenosine triphosphate (ATP) hydrolysis as an energy source to catalyze the cleavage and rejoining of double‐stranded DNA. TOP2 temporarily forms a phospho‐tyrosyl bond with the 5′ end of the DNA when cleavage occurs. TOP2 functions as a homodimer; therefore, a DNA DSB is induced by the two monomers, each cleaving one strand of DNA. After a DSB is generated, the torsional stress is relieved by the passage of another DNA strand through the DSB, and the DSB is quickly rejoined.

FIGURE 1.

TOP2 and DNA supercoils associated with DNA transactions. During DNA replication and transcription, DNA unwinding inevitably alters DNA topology, generating DNA supercoils. The DNA double helix becomes overwound ahead of the unwinding machinery, generating positive supercoils (Sc⁺), whereas it becomes underwound behind the unwinding machinery, generating negative supercoils (Sc⁻). These DNA topological alterations are resolved by TOP2.

In vertebrates, including humans, two paralogs of the TOP2 gene exist: TOP2A and TOP2B, which encode the TOP2α and TOP2β proteins, respectively. Although TOP2α and TOP2β share similar catalytic properties, they have different functions (Nitiss 2009a). TOP2β is expressed throughout the cell cycle, whereas TOP2α levels peak during the S and G2/M phases of the cell cycle and are associated with cell proliferation (Heck et al. 1988; Kimura et al. 1994). In addition to its role in resolving torsional stress associated with replication fork progression, TOP2α plays a significant role in chromosomal condensation and separation during mitosis (McClendon et al. 2005; Li et al. 2008; Farr et al. 2014; Nielsen et al. 2020). TOP2β activity is primarily associated with transcription. It participates in the regulation of chromatin structure around promoter regions of specific genes and transcription elongation (Ju et al. 2006; Lyu et al. 2006; King et al. 2013; Madabhushi et al. 2015; Uuskula‐Reimand and Wilson 2022; Yao et al. 2025). In addition, it has also been reported that TOP2α is involved in transcriptional regulation by interacting with RNA polymerase II (Mondal and Parvin 2001; Herrero‐Ruiz et al. 2021). Furthermore, TOP2α has been reported to be necessary for the expression of specific genes in embryonic stem cells and during their differentiation (Thakurela et al. 2013). These findings indicate that TOP2α also contributes to the regulation of transcriptional activity in concert with TOP2β. In addition to TOP2, TOP1 also contributes to the resolution of the supercoils associated with replication or transcription. The coordinated actions of TOP2α, TOP2β, and TOP1 efficiently resolve the torsional stress generated by DNA transactions.

3. Targeting TOP2 as Anticancer Drugs

Drugs that target TOP2 are widely used in chemotherapy to treat blood cancers, such as leukemia and lymphoma, as well as solid tumors, such as breast and lung cancers. These drugs induce DNA damage by compromising TOP2 function and cause cell death in cancer cells. These include TOP2 poisons and catalytic inhibitors that interfere with TOP2 activity via mechanisms different from those of the poisons (Nitiss 2009b; Pommier et al. 2010; Vann et al. 2021; Jang et al. 2025).

TOP2 poisons such as etoposide and teniposide trap TOP2 reaction intermediates and accumulate TOP2–DPCs bound to the 5′ end of DSBs. Additionally, the DNA intercalator‐type TOP2 poisons, doxorubicin (an anthracycline) and mitoxantrone (an anthraquinone), also cause the accumulation of TOP2–DPCs (Nitiss 2009b). However, these poisons are associated with severe side effects such as cardiotoxicity and secondary cancers accompanied by chromosomal translocations (Cowell and Austin 2012; Zhang et al. 2012; Pendleton et al. 2014; Linders et al. 2024). Conversely, TOP2 catalytic inhibitors reduce enzyme activity. ICRF‐193 and ICRF‐187 inhibit TOP2's ATP hydrolysis, allowing TOP2 to bind to DNA and inhibit the catalytic cycle without forming TOP2ccs. ICRF‐187 is clinically used in combination with doxorubicin and other anthracycline‐based agents to reduce cardiotoxicity (Ishida et al. 1995; Larsen et al. 2003; Lyu et al. 2007; Szponar et al. 2024).

Cells attempt to repair DSBs induced by TOP2 poisons. The repair of TOP2 poison–induced DSBs mainly relies on nonhomologous end‐joining (NHEJ), one of the major pathways for DSB repair, and defects in NHEJ factors, such as DNA ligase IV and DNA‐dependent protein kinase (DNA‐PK), greatly increase cellular sensitivity to etoposide (Adachi et al. 2003; Willmore et al. 2004; Maede et al. 2014). However, the trapped TOP2 by TOP2 poisons such as etoposide becomes an obstacle to DNA repair mechanisms. An in vitro study has shown that DNA‐PK fails to activate in response to DSBs bound by TOP2 (Martensson et al. 2003). Therefore, quickly removing the trapped TOP2 proteins and exposing the free 5′ ends of DNA is necessary for efficient repair. Meanwhile, ectopic rejoining of DSBs can cause leukemia‐inducing chromosomal translocations, which are significant side effects of TOP2 poisons (Cowell and Austin 2012; Olmedo‐Pelayo et al. 2020). A deeper understanding of how TOP2–DPCs are processed could help reduce these side effects and improve the efficacy of chemotherapy involving TOP2 poisons.

4. Proteolytic Resolution of TOP2–DPC

The resolution of TOP2–DPCs induced by TOP2 poisons can be divided into two stages. The first stage is the degradation of TOP2, an obstacle bound to the 5′ end of the DSB. The second stage involves the processing of the DSB ends for subsequent repair. Each of these stages is controlled by multiple pathways (Figure 2).

FIGURE 2.

Pathways for trapped TOP2 proteolysis. TOP2–DPCs caused by TOP2 poisons undergo ubiquitination by BMI1/RING1A or SCF^β‐TrCP, leading to proteasomal degradation and generation of a naked DSB that can trigger DSB responses (left pathway). Alternatively, ZATT and PIAS4 mediate SUMOylation of the trapped TOP2, promoting RNF4‐dependent ubiquitination and subsequent proteasomal proteolysis (central pathway). Additionally, SPRTN contributes to the removal of TOP2–DPCs via a proteasome‐independent mechanism, which may be enhanced by TOP2 ubiquitination (right pathway).

Exposure to TOP2 poisons leads to the formation of TOP2–DPCs, which triggers TOP2 degradation. Previous studies have shown that proteasome inhibition delays the removal of TOP2–DPCs by TOP2 poisons, leading to their accumulation. This suggests that TOP2 poisons‐induced degradation of TOP2 is proteasome‐dependent (Mao et al. 2001; Xiao et al. 2003; Zhang et al. 2006; Azarova et al. 2007). However, proteasome inhibition reduces the appearance of etoposide‐induced γH2AX, a phosphorylated form of the histone variant H2AX, which is a well‐known DSB marker (Rogakou et al. 1998; Paull et al. 2000; Mao et al. 2001; Zhang et al. 2006; Sciascia et al. 2020; Swan et al. 2020). Additionally, proteasome inhibition suppresses the activation of ataxia–telangiectasia mutated (ATM) and ATM‐ and Rad3‐related (ATR) proteins, which function as DNA damage sensors and key regulators of the DNA damage response (DDR), along with their downstream signaling pathways (Zhang et al. 2006; Robison et al. 2007; Fan et al. 2008). These findings suggest that proteasome activity is necessary for converting “low‐reactive DSBs” concealed by trapped TOP2 into “high‐reactive naked DSBs” capable of inducing DSB responses.

Etoposide has been reported to cause the accumulation of phosphorylated forms of RNA polymerase II including Ser2 phosphorylation on its C‐terminal domain (Ban et al. 2013), which results from the arrest of transcription elongation. This suggests that TOP2–DPCs induce transcriptional stress through collisions between TOP2–DPCs and the transcription machinery. Proteasomal processing of TOP2–DPCs is thought to be transcription‐dependent; the collision of the transcription machinery with TOP2–DPCs may act as a trigger to induce TOP2 degradation (Mao et al. 2001; Xiao et al. 2003; Zhang et al. 2006; Fan et al. 2008; Tammaro et al. 2013). Indeed, genome‐wide analyses have revealed that TOP2–DPC resolution is accelerated at transcriptionally active loci (Canela et al. 2019). By contrast, DNA replication has not been shown to affect TOP2 degradation (Fan et al. 2008), suggesting that TOP2–DPC resolution is not associated with DNA replication. However, as DNA replication affects etoposide sensitivity and the processing of DSBs, it may influence chemotherapy sensitivity through a mechanism distinct from transcription‐dependent TOP2–DPC resolution (Fan et al. 2008; Tammaro et al. 2013).

Valosin‐containing protein (VCP) (also known as p97) is a molecular chaperone of the proteasome with AAA+ ATPase activity. VCP utilizes the energy from ATP hydrolysis to induce conformational changes in ubiquitinated substrate proteins, thereby contributing to protein degradation and the regulation of protein–protein interactions (Meyer et al. 2012). VCP has also been reported to play a role in the DDR by detaching specific DNA repair proteins, including Ku70/80 and DNA‐PK, from chromatin (Acs et al. 2011; Bergink et al. 2013; Jiang et al. 2013; van den Boom et al. 2016). In budding yeast cells with compromised Cdc48 (the yeast homolog of VCP/p97), accumulation of ubiquitinated TOP2 has been reported (Wei et al. 2017), suggesting that TOP2 degradation is regulated by VCP. Similar to the effects of proteasome inhibition, inhibition of VCP activity results in the delayed removal of TOP2–DPC after etoposide treatment and reduces ATM and H2AX phosphorylation in human cells (Swan et al. 2021; Sakasai et al. 2025). In addition, DNA‐PK activation is strongly inhibited by proteasome or VCP inhibition. Consistent with this, DSB repair efficiency after etoposide treatment is also suppressed by VCP inhibition (Sakasai et al. 2025). As DNA‐PK is activated by binding to DNA ends in a Ku protein‐dependent manner (Dvir et al. 1992; Gottlieb and Jackson 1993), DNA‐PK is considered to be significantly affected by trapped TOP2 at the DSB ends. The reduction in DSB repair efficiency may result from a combination of physical obstructions in DNA‐PK end‐binding and rejoining reactions by trapped TOP2. Furthermore, the conformational changes in trapped TOP2 by VCP are thought to play an important role not only in TOP2 degradation but also in the cleanup step for a protein adduct–free DSB ends, as discussed in Section 5.

For the degradation of trapped TOP2 via the proteasome and VCP, the trapped TOP2 must undergo ubiquitination. BMI1 and RING1A, which form a ubiquitin ligase for the polycomb repressive complex 1 (PRC1), are reported to be involved in TOP2α degradation induced by TOP2 poisons (Alchanati et al. 2009; Swan et al. 2020). BMI1 and RING1A directly ubiquitinate TOP2α. Additionally, the SCF (SKP1–Cul1–F‐box) complex containing β‐TrCP has also been reported to ubiquitinate trapped TOP2β. After ATM activation in response to the TOP2 poison teniposide, casein kinase 1‐mediated TOP2β phosphorylation leads to SCF^β‐TrCP‐mediated TOP2β ubiquitination and degradation (Shu et al. 2020). Furthermore, ZATT (ZNF451) and PIAS4, both SUMO ligases, SUMOylate TOP2. The SUMOylated TOP2 is then ubiquitinated by the sumoylation‐dependent ubiquitin ligase RNF4, which promotes its degradation by the proteasome (Schellenberg et al. 2017; Sun et al. 2020) (Figure 2).

In addition, ubiquitination factors not involved in TOP2–DPC resolution have also been identified. APC/C‐Cdh1 controls the stability of TOP2α by ubiquitinating TOP2α (Eguren et al. 2014). Additionally, BRCA1 and RNF168 are known to regulate decatenation activity by adding a ubiquitin chain to TOP2α via lysine 63, which is different from the lysine 48‐linked ubiquitin chains related to protein degradation (Guturi et al. 2016).

SPRTN is a DNA‐binding metalloprotease that is reported to be involved in resolving DPCs induced by formaldehyde or the TOP1 poison camptothecin (Lopez‐Mosqueda et al. 2016). SPRTN is also thought to promote the removal of DPCs at replication forks by its recruitment to DPCs through the interactions with proteins in the replication machinery (Vaz et al. 2016; Morocz et al. 2017; Larsen et al. 2019). SPRTN deficiency sensitizes cells to etoposide and increases the level of etoposide‐induced TOP2–DPCs. Additionally, SPRTN binds to TOP2 and degrades it in the presence of ubiquitin in vitro (Lopez‐Mosqueda et al. 2016). SPRTN is known to be ubiquitinated, and its ubiquitination state regulates chromatin binding (Stingele et al. 2016). The ubiquitination of target proteins is reported to enhance SPRTN‐mediated proteolysis (Durauer et al. 2025). These findings imply that SPRTN‐mediated TOP2–DPC resolution is regulated by the ubiquitination state of both SPRTN and TOP2 (Figure 2). However, the relationship between SPRTN and replication during TOP2–DPC resolution is not well understood. RNF4 is also involved in SPRTN‐mediated DPC degradation via DPC SUMOylation. In RNF4‐deficient cells, etoposide‐induced SPRTN activation depends on DNA replication (Weickert et al. 2023). This suggests that, under certain conditions, TOP2–DPC degradation by SPRTN may be coupled with DNA replication.

5. DSB End Cleanup for DSB Repair

After TOP2 trapping and subsequent degradation by the proteasome or SPRTN, a peptide containing the TOP2 tyrosine residue remains at the DSB end. This degradation increases the accessibility of Tyrosyl DNA phosphodiesterase 2 (TDP2) to the DSB ends (Gao et al. 2014; Lee et al. 2018), and TDP2 cleaves the covalent bond between this peptide and DNA (Cortes Ledesma et al. 2009; Zeng et al. 2011). Through the reaction by TDP2, the DSB bearing TOP2–DPC is converted into a free DSB end that can be joined by NHEJ (Schellenberg et al. 2016) (Figure 3). As TDP2 deficiency induces etoposide sensitivity, removing the peptide from the DSB end is thought to be crucial for DSB repair (Zeng et al. 2011; Gomez‐Herreros et al. 2013). However, TDP2 deficiency does not significantly impact the sensing or signaling of DSBs in response to etoposide (Sakasai et al. 2025), suggesting that as long as TOP2 is degraded, DDR can be activated regardless of the presence of the residual peptide. Recently, Saha et al. reported that SPRTN targets TOP3α–DPC and has an epistatic relationship with TDP2 (Saha et al. 2023), suggesting that TDP2 removes the remaining peptides after SPRTN degrades TOP3α. In TOP2–DPC, TDP2 may work in coordination with SPRTN through a similar mechanism to TOP3α–DPC resolution (Figure 2).

FIGURE 3.

Pathways for end cleanup of DSBs bearing TOP2. TOP2–DPCs undergo ubiquitination followed by proteasomal degradation; however, the peptide containing the TOP2 tyrosine residue remains at the DSB end. This residual peptide is removed by TDP2, making the DSB end repairable (central pathway). ZATT‐dependent SUMOylation can also facilitate end processing by TDP2, independent of proteasomal degradation of TOP2 (left pathway). Alternatively, the DSB end bearing TOP2 is resected by MRN (MRE11–RAD50–NBS1)/CtIP nucleases, generating a repairable DSB through excision of the TOP2–DPC (right pathway).

The cleanup of DSB ends by TDP2 is promoted by TOP2 degradation. However, recent reports have shown that ZATT promotes the direct removal of TOP2–DPC by TDP2 through a proteasome‐independent mechanism (Schellenberg et al. 2017). SUMOylation of trapped TOP2 by ZATT is thought to induce conformational changes in TOP2, enabling TDP2 to access the binding site of TOP2 and facilitate cleavage of TOP2–DPC (Figure 3). Recently, RAD54L2 was identified as a factor that binds to SUMOylated TOP2. D'Alessandro et al. and Zhang et al. showed that RAD54L2, using its ATPase activity, is involved in a ZATT‐dependent, proteasome‐independent TOP2–DPC removal pathway (D'Alessandro et al. 2023; Zhang et al. 2023). A novel RAD54L2‐mediated pathway has been proposed to dissociate TOP2 from chromatin in a TDP2‐independent manner.

Another pathway for DSB end cleanup is the nucleolytic pathway. In this pathway, TOP2 proteins are removed by nucleases such as CtIP and MRE11 (Figure 3). These nucleases are involved in DNA‐end resection during DSB repair (Sartori et al. 2007; Nicolette et al. 2010; Garcia et al. 2011). DNA‐end resection is an early stage of DSB repair via homologous recombination (HR). However, DNA‐end resection has been reported to be involved in the repair of TOP2 poison–induced DSB via NHEJ in the G1 and G0 phases, in which HR does not function (Quennet et al. 2011; Akagawa et al. 2020). Increases in TOP2 poison–induced TOP2–DPCs and cytotoxicity have been reported in cells depleted of MRE11 and/or CtIP (Nakamura et al. 2010; Lee et al. 2012; Aparicio et al. 2016; Hoa et al. 2016). In addition, MRE11 dysfunction has been reported to cause TOP2–DPC accumulation, even in the absence of TOP2 poisons, suggesting the importance of MRE11 in removing naturally generated TOP2ccs (Hoa et al. 2016). MRE11 is a well‐known nuclease that functions as a heterotrimer with NBS1 and RAD50 during DNA‐end resection (Paull 2018). Similar to that in MRE11‐depleted cells, TOP2–DPC accumulation was observed in NBS1‐depleted cells, suggesting that the MRE11–RAD50–NBS1 (MRN) complex functions in the nucleolytic removal pathway of TOP2‐DPC (Sun et al. 2022). MRE11‐mediated removal of TOP2–DPC is BRCA1‐dependent, and the interaction between BRCA1 and CtIP has been shown to be important for this pathway (Nakamura et al. 2010; Aparicio et al. 2016; Sasanuma et al. 2018). In the repair of DSB induced by TOP2 poisons, MRE11 and TDP2 function via separate pathways, suggesting that the nucleolytic and proteolytic pathways additively promote NHEJ (Hoa et al. 2016). By contrast, VCP has been suggested to play an important role in both pathways. Overexpression of MRE11 can remove TOP2–DPCs induced by etoposide, even in the presence of proteasome inhibitors, but not in the presence of VCP inhibitors (Sun et al. 2022). Thus, the VCP‐mediated conformational change in TOP2 is considered necessary for both the proteolytic pathway mediated by the proteasome/SPRTN and the nucleolytic pathway mediated by the MRN complex/CtIP.

6. Top2‐Targeting Chemotherapy and Carcinogenesis

Ectopic rejoining of DSBs causes chromosomal abnormalities such as chromosome translocation. This is a well‐known side effect of TOP2 poisons and is associated with secondary leukemia (Felix 1998; Mistry et al. 2005; Pendleton et al. 2014). For example, approximately 2%–9% of patients treated with etoposide develop acute myeloid leukemia (t‐AML) (Winick et al. 1993), which is associated with translocations involving the MLL (KMT2A) gene located at 11q23 and multiple fusion partner genes, such as ENL, AF4, AF6, and AF9 (Felix 1998; Cowell et al. 2012). Additionally, many cases of primary infant leukemia exhibit the same chromosomal translocations as those observed in patients with TOP2 poison–induced t‐AML (Pendleton et al. 2014). The infant leukemia may also be caused by a similar mechanism mediated by spontaneous TOP2ccs.

Chromatin fibers form loop structures which are considered fundamental structural features for transcriptional regulation. At the base of the loops, distal chromosomal regions become proximate, increasing the proximity of genes to the distal chromosomal regions within the loop. Furthermore, multiple genes in close proximity are cooperatively transcribed. Such transcriptionally active sites, concentrated in specific nuclear regions, have been proposed as a transcription factory model. The concept of transcriptional concentrates, in which transcription domains aggregate via liquid–liquid phase separation, has also been accepted (Jha et al. 2022; Qi et al. 2025). Cowell et al. suggested that MLL and fusion partner genes are transcribed by a common transcription machinery, which may increase the proximity between MLL and fusion partner genes during transcription (Cowell et al. 2012). The proximity of genomic structures mediated by transcription underlies chromosomal translocations, and Meaburn et al. observed that gene fusions are more likely to occur when spatial proximity increases (Meaburn et al. 2007). Similar to the chromosomal rearrangements observed in leukemia, DSBs mediated by TOP2β are associated with the translocations of androgen‐responsive genes in prostate cancer. Genes such as TMPRSS2 and ERG, which are well‐known fusion genes in prostate cancer, are thought to be transcribed by a common transcription apparatus that exhibits high spatial proximity. This situation creates an environment in which they are prone to chromosomal translocations (Kumar‐Sinha et al. 2008; Mani et al. 2009; Haffner et al. 2010). Spatial proximity depends on chromosome structure, such as transcription‐associated chromosome loops, which are formed in a cohesin‐ and CCCTC‐binding factor (CTCF)‐dependent manner. TOP2β is also known to colocalize with cohesin and CTCF in the loop structure (Uuskula‐Reimand et al. 2016; Canela et al. 2017). In fact, DSBs are frequently induced by etoposide in these regions (Canela et al. 2017; Gothe et al. 2019). As treatment with TOP2 poisons can induce DSBs when distal genes are adjacent, it can increase the risk of chromosomal rearrangements (Canela et al. 2019; Gothe et al. 2019).

Proteasome inhibitors, such as bortezomib, are clinically used as anticancer agents for multiple myeloma, mantle cell lymphoma, and other conditions (Manasanch and Orlowski 2017; Sin and Man 2021). VCP inhibitors are also currently being studied in clinical trials as potential anticancer agents (Kilgas and Ramadan 2023). Because proteasome and VCP inhibition suppress DDR and DSB repair caused by TOP2 poisons, it might reasonably be concluded that TOP2 poisons could enhance antitumor effects by cotreatment with proteasome or VCP inhibitors. Indeed, the combination of proteasome inhibitors with TOP2 poisons has been clinically evaluated for use in chemotherapy (Cowell and Austin 2012; Manasanch and Orlowski 2017; Dittus et al. 2018). Previous studies have shown that this combination enhances cell death (Ceruti et al. 2006; von Metzler et al. 2009; Aras and Yerlikaya 2016). However, recent studies at the cultured cell level have shown seemingly contradictory results, in which the combination of proteasome inhibitors and etoposide restores the survival of cells treated with etoposide alone (Lee et al. 2016; Sciascia et al. 2020; Sakasai et al. 2025). Sciascia et al. have shown that the timing of proteasome inhibition significantly affects the cytotoxicity of TOP2 poisons (Sciascia et al. 2020). They reported that TOP2 degradation is suppressed under preinhibited proteasome conditions, and that TOP2 enzymatic activity recovers after TOP2 poison removal, allowing the rejoining of DSBs and TOP2 dissociation from chromosomes. Therefore, although TOP2–DPC may accumulate temporarily in the presence of proteasome inhibitors, the undegraded TOP2 is reactivated and dissociates from the chromosome after TOP2 poison removal, thereby potentially leading to a reduction in the total amount of DSBs and cytotoxicity. Therefore, the development of effective combination therapies using proteasome inhibitors requires further investigation. Future studies into the toxicity of TOP2 poison–induced DSBs themselves, as well as their impact on chromosomal rearrangements as a side effect, may lead to new chemotherapy strategies.

7. Future Perspectives

The removal pathway of TOP2–DPCs caused by TOP2 poisons is controlled by several complementary pathways. This is crucial for maintaining genomic stability and serves as an effective defense mechanism against naturally occurring TOP2ccs. However, the complex relationship between these pathways makes it challenging to understand the effects of TOP2 poisons in cancer treatment. The existence of multiple repair pathways also provides a reliable defense mechanism for cancer cells. Clarifying the factors that affect pathway selection could lead to the development of combination therapies targeting these factors in conjunction with TOP2 poisons. However, the risk of secondary cancers caused by chromosomal rearrangements resulting from TOP2 poisoning remains a major issue. Further elucidation of the intracellular phenomena associated with TOP2–DPCs and verification of the appropriate balance between TOP2–DPC removal and DSB repair are necessary to improve chemotherapy using TOP2 poisons.

TOP1 poisons, such as irinotecan and topotecan, which are camptothecin derivatives, have also been used in chemotherapy. TOP1 poisons inhibit DNA replication and transcription by inducing TOP1–DPCs, and replication‐coupled DSBs are the cause of the cytotoxicity of TOP1 poisons (Venkatachalam and Kaufmann 2025). Similar to TOP2 poisons, proteasome inhibition suppresses the DSB response to TOP1 poisons (Sakasai et al. 2010). Distinct from TOP2 poisons, TOP1 poisons generate a single‐strand break (SSB) bearing TOP1 at its 3′ end. Therefore, although the SSB is converted to a DSB by collision with the replication fork, this DSB theoretically does not possess TOP1 at the end. However, in actual cellular conditions, the DSB end can somehow be covered by undegraded TOP1–DPC in the presence of a proteasome inhibitor. Various studies have been conducted on the combination of TOP1 poisons with proteasome inhibitors (Cusack et al. 2001; Ryan et al. 2006; Tang et al. 2014). Recently, research on TOP3 has progressed rapidly, and a TOP3β poison has also been identified (Wang et al. 2023). TOP3β is a type I topoisomerase and the only topoisomerase that acts on RNA. The newly identified TOP3β poison traps the reaction intermediates of TOP3β, primarily on RNA. Future research will likely clarify its biological responses and the specificity of its inhibitory properties. Therefore, it has potential as an anticancer or antiviral agent.

Topoisomerases are biologically intriguing enzymes with highly attractive activity directly involved in nucleic acid metabolism, which is a core cellular process. Therefore, topoisomerases still retain considerable potential as targets for anticancer and antiviral agents, and further research is required to expand their clinical application.

Conflicts of Interest

The authors declare no conflicts of interest.

Sakasai, R. , and Iwabuchi K.. 2026. “Processing of DNA Topoisomerase II–DNA–Protein Crosslinks Associated With Anticancer Drugs.” Genes to Cells 31, no. 1: e70076. 10.1111/gtc.70076.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.