Topoisomerases and cancer chemotherapy: recent advances and unanswered questions

DNA topoisomerases are enzymes that catalyze changes in the torsional and flexural strain of DNA molecules. Earlier studies implicated these enzymes in a variety of processes in both prokaryotes and eukaryotes, including DNA replication, transcription, recombination, and chromosome segregation. Studies performed over the past 3 years have provided new insight into the roles of various topoisomerases in maintaining eukaryotic chromosome structure and facilitating the decatenation of daughter chromosomes at cell division. In addition, recent studies have demonstrated that the incorporation of ribonucleotides into DNA results in trapping of topoisomerase I (TOP1)–DNA covalent complexes during aborted ribonucleotide removal. Importantly, such trapped TOP1–DNA covalent complexes, formed either during ribonucleotide removal or as a consequence of drug action, activate several repair processes, including processes involving the recently described nuclear proteases SPARTAN and GCNA-1. A variety of new TOP1 inhibitors and formulations, including antibody–drug conjugates and PEGylated complexes, exert their anticancer effects by also trapping these TOP1–DNA covalent complexes. Here we review recent developments and identify further questions raised by these new findings.


Introduction
The helical structure of duplex DNA provides a physical basis for the faithful duplication and deciphering of genetic information while also ensuring DNA strand integrity. The intertwining of the two complementary polynucleotide strands is stabilized by hydrogen bonding and stacking interactions between the hydrophobic bases. Yet these features also impose topological constraints during processes involving DNA 1-4 . For example, during DNA replication, each strand serves as a template for polymerization of a complementary strand. However, the progressive unwinding of antiparallel DNA strands may cause overwinding (positive supercoiling) ahead of the replication fork and intertwining of daughter DNA molecules (precatenanes) behind the fork. Similar topological considerations apply to transcription, which induces local unwinding (negative supercoiling) of the DNA helix behind the advancing RNA polymerase complex and positive supercoiling ahead of it.
The ability of cells to resolve local domains of DNA supercoiling and separate multiply intertwined DNA molecules is critical for gene expression, recombination, DNA replication, and chromosome segregation, yet it must be achieved while still maintaining chromosomal integrity. Solutions to these problems involve a family of enzymes called DNA topoisomerases, which catalyze changes in the linkage of DNA strands (or helices) by nicking one or both strands of the DNA duplex and, at the same time, becoming covalently linked to one end of the cleaved DNA through a phosphotyrosyl bond. After another DNA strand (or duplex) is passed through the protein-linked break(s) to produce a change in DNA topology, the original phosphodiester bond is religated to restore integrity of the DNA backbone.
As detailed in Table 1, topoisomerases perform critical functions in all kingdoms of life and can be divided into five subfamilies (type IA, IB, IC, IIA, and IIB) based on the number of DNA strands cleaved (one or two, for type I or II, respectively), the nature of the covalent phosphotyrosyl intermediate formed (5' or 3' linkage), and other aspects of enzyme structure and catalysis (see Figure 1). Nevertheless, these enzymes all share a common mechanism of transient breakage and rejoining of DNA strand(s).
Topoisomerase-linked DNA breaks (topoisomerase-cleavage complexes or TOPccs) are integral to topoisomerase-mediated changes in DNA topology but also pose potential threats to genome integrity. For example, trapping of a TOPcc in advance of the replication machinery or during chromosome segregation, where interwound (or catenated) DNA helices are unlinked by topoisomerases, can have dire effects on genome stability and cell viability. Indeed, topoisomerases are the cellular targets of a wide spectrum of antimicrobial and anticancer agents, which either act to stabilize TOPccs (termed poisons) or otherwise inhibit enzyme catalysis to induce DNA damage [5][6][7][8] . This difference between poisons and inhibitors is illustrated in Figure 2. In eukaryotes, topoisomerase poisons include camptothecins (topotecan and SN-38, the active metabolite of the drug irinotecan), which stabilize TOP1ccs, and doxorubicin or etoposide, which stabilize TOP2ccs. In addition to these drugs, DNA modifications themselves, such as lesions induced by oxidative damage or ribonucleotides mistakenly incorporated into DNA, may also stabilize TOPccs. Although topoisomerases provide critical solutions to the topological problems imposed by the helical structure of duplex DNA, the hallmark of these activities-the formation of a covalent enzyme-DNA intermediate-constitutes an inherent threat to genome integrity.
In this review, we focus on recent advances in our understanding of topoisomerase function in eukaryotic cells, the therapeutic targeting of topoisomerases in cancer, and the repair pathways that resolve the resulting drug-induced lesions. While highlighting these advances, we also identify unanswered questions that these new findings raise.

Roles of topoisomerases in nuclear organization and genomic stability
The distinct biological functions of individual topoisomerases and the physiological consequences of altering their activity have been extensively studied (for reviews, see [1][2][3][4]9). Nevertheless, the technical challenges of assessing local changes in DNA topology in live cells leave perplexing questions regarding topoisomerase function in maintaining chromosome architecture and genome stability. In this section, we summarize recent studies that highlight surprising aspects of eukaryal topoisomerase function.
Type IB topoisomerases: maintenance of nuclear and mitochondrial genome stability In eukaryotes, nuclear TOP1 catalyzes the relaxation of local domains of positive and negative supercoils during DNA replication, recombination, transcription, and possibly chromosome condensation 4,5,10 . Stabilization of TOP1ccs by camptothecins during replication is an effective strategy for treating solid tumors and hematologic malignances, as discussed below. During transcription, the phosphorylated C-terminal domain of the catalytic subunit of RNA polymerase II binds and activates TOP1, effectively tethering TOP1 to the transcriptional machinery 11,12 . TOP1 then relaxes positive supercoils, which are generated ahead of the transcription complex and could otherwise impede its progress, as well as negative supercoils behind the transcription complex.
In the absence of TOP1, local accumulation of negative supercoils facilitates the formation of R-loops, stable hybrid RNA-DNA duplexes of the nascent RNA transcripts and template strands. R-loops also allow the formation of secondary structures, such as G-quadraplexes and hairpins, in the singlestranded non-template strand. RNase H1 and H2 can degrade RNA in these RNA-DNA heteroduplexes. While genome-wide R-loop mapping indicates context-dependent gains and losses in R-loops when TOP1 is depleted 13 , it is the increased levels of R-loops and G-quadraplexes that are associated with dysregulation of transcription and replication as well as genome instability.
The misincorporation of ribonucleotides into DNA, at rates approaching 10 6 ribonucleotides per genome per replication cycle 17 , can also lead to replication stress, single-and doublestrand breaks, and small deletions [18][19][20] . Ordinarily these ribonucleotides are removed by the concerted action of RNAse H2, DNA polymerase δ, FLAP endonuclease, and DNA ligase 1 17 .
However, if ribonucleotides are not removed, TOP1 cleavage of the strand immediately 3' to the ribonucleotide results in the nucleophilic attack of the 2'OH of the ribonucleotide on the TOP1cc to generate a 2',3' cyclic phosphate at the 3' DNA end and release of TOP1. A second, upstream TOP1 cleavage event can then liberate a short oligo with the modified   In the topoisomerase I cleavage complex (TOP1cc) (top), the 3' DNA end is covalently linked to the active site tyrosine (Y). Changes in the linkage of DNA strands occur through a mechanism of strand rotation, where the untethered 5' DNA end of the cleaved strand swivels about the noncleaved DNA strand. TOP2 (middle) and TOP3 (bottom) both involve mechanisms of DNA strand transfer. In the case of TOP2cc, the G segment of duplex DNA is cleaved by the two active sites of the homodimer, following capture of the T segment by the closure of the N-terminal ATPase domains. The T segment DNA is then successively passed through the double-strand break in the G segment and out through the bottom dimer interface. For type IA enzymes, depicted for TOP3cc, a single strand of negatively supercoiled DNA is cleaved to form a 5' phosphotyrosyl bond, while the 3'OH end is held by the enzyme. A conformational change in the protein then allows the intact complementary strand to be passed through the protein-linked break, followed by religation of the cleaved DNA. The same principles apply to TOP2, although, in these instances, the dimeric enzymes produce two enzyme-linked DNA breaks staggered by 4 bp. (B) Based on these distinct modes of action, increased topoisomerase levels in an isogenic cell line would induce opposing effects on drug sensitivity: resistance to an inhibitor versus increased sensitivity to a poison. Shown in this diagram are the dose response curves for killing that result from an increase in topoisomerase levels relative to cells that yield the black curve.
3' end, trapping the TOP1cc across a gap. If DNA strand realignments juxtapose the free 5'OH from the first cleavage and the TOP1cc, enzyme-mediated ligation can produce short deletions. In highly transcribed genes, this TOP1-mediated mutagenesis can be exacerbated by the tethering of TOP1 to RNA polII 21 .
A recent genome-wide CRISPR screen showed that interruption of genes encoding the three subunits of RNase H2 enhances human cell line sensitivity to the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib 22 . Further studies attribute this olaparib hypersensitivity to increased ribonucleotide-dependent stabilization of TOP1ccs, which can serve as PARP1 substrates. These observations provide a compelling rationale for inhibiting PARP in order to trigger TOP1cc-initiated killing in cancers with deleted or mutated RNASEH2B.
In vertebrates, a second nuclear-encoded type IB topoisomerase (TOP1MT) selectively localizes to mitochondria and catalyzes the relaxation of circular mitochondrial DNA 23 . Despite its similarity to nuclear TOP1, TOP1MT does not contribute to camptothecin-induced toxicity. Instead, TOP1MT physically associates with mitochondrial ribosome subunits to promote mitochondrial translation, which is critical for hepatocellular carcinoma cell growth 24 . These findings suggest that inhibition of TOP1MT activity, rather than stabilization of TOP1MTccs, might be an effective strategy for targeting this enzyme to treat some cancers.
Evolving understanding of eukaryal topoisomerase IIIα and β (TOP3α and β) Distinct from the swivelase activity ascribed to type IB enzymes, type IA topoisomerases exhibit a mechanism of enzymebridged strand passage (Figure 1) 25 . As with bacterial TOPA, eukaryotic TOP3α enzymes (including yeast TOP3) preferentially relax highly negatively supercoiled DNA and decatenate duplex DNA molecules tethered by single-stranded DNA interlinks or hemicatenanes 26 . Differential splicing produces nuclear and mitochondrial isoforms of vertebrate TOP3α.
Nuclear TOP3α forms a complex with the BLM helicase and RMI1 and RMI2 proteins 27 to resolve double Holliday junctions during recombination 28 . In contrast, mitochondrial TOP3α decatenates newly replicated mtDNA circles, which are linked by a hemicatenane formed at the origin of replication, to allow segregation of replicated mitochondrial genomes 29 . Accordingly, TOP3α dysregulation results in human mitochondrial disease. Contribution of topoisomerase II to chromosome architecture and genomic stability In eukaryotes, TOP2 is a homodimeric enzyme that relaxes positively or negatively supercoiled DNA and catenates or decatenates duplex DNA via transient breakage of both DNA strands ( Figure 1). Yeast encode a single TOP2, while human cells express TOP2α and TOP2β enzymes, encoded by the TOP2A and TOP2B genes, respectively. Although human TOP2 enzymes exhibit structural and mechanistic similarities, TOP2α decatenates sister chromatids during chromosome segregation, whereas TOP2β has been implicated in transcription. Several recent studies further define distinct roles of these enzymes in chromosome dynamics.
TOP2β plays a surprising and important role in interphase chromatin organization. High-resolution whole-genome chromatin conformation capture (Hi-C), or in situ Hi-C with DNA-DNA proximity ligation, allows chromatin fragments in close proximity to be identified. These techniques have determined that chromosomes are organized into topologically associated domains (TADs) of ~200 kb to 1 Mb, typically bound by chromatin enriched in transcriptionally active genes. According to current models, DNA is actively extruded through one or paired cohesin rings to generate TADs until DNA bound by the CCCTC binding factor (CTCF) is encountered 36,37 . Recent studies suggest that CTCF becomes associated with loop anchors and unidirectionally halts DNA extrusion. TOP2β is then recruited to loop anchors to alleviate the positive supercoils induced by cohesin-derived DNA extrusion. The resulting TOP2β-induced breaks are transcription independent but correlate with cohesin 38 . At a low frequency, unresolved TOP2βccs at these loop anchors can also lead to DNA breakage and translocations 39 . Thus, TOP2β involvement in topological dynamics associated with chromosome organization contributes somewhat unexpectedly to chromosome breakage and rearrangements.
During chromosome segregation, intertwined DNA duplexes (catenanes) are resolved or decatenated by TOP2 in yeast and TOP2α in human cells. TOP2 enzymes can also readily catenate DNA duplexes in close proximity. Yet increased positive supercoiling drives decatenation, based in part on an intrinsic enzyme bias towards decatenation. A persistent question, then, has been the source of this positive supercoiling to drive decatenation. In yeast, condensin-mediated positive DNA supercoiling increases as cells enter mitosis 40 .
In human cells, we now know that this positive supercoiling reflects the action of TOP3α, which (as part of the TRR complex with RMI1 and RMI2) associates with the Plk1-interacting checkpoint helicase (PICH) to produce extremely high-density positive supercoils 41 . Subsequent relaxation of negative supercoils by TOP3α results in the accumulation of positive supercoils, which drives decatenation by TOP2α. These studies provide the first evidence for topoisomerase-induced stable domains of positive supercoils in eukaryotic cells and illustrate how DNA extrusion can be locally harnessed to drive chromosome disjunction.

Recognition and resolution of TOPccs
During their catalytic cycles, all topoisomerases transiently form covalent linkages between active site tyrosines and DNA [42][43][44][45] . While the vast majority of these TOPccs are normally resolved by completion of the catalytic cycle, there is increasing interest in the question of what happens when the TOP1 or TOP2 catalytic cycle is slowed or impaired. These issues are particularly critical in the context of anticancer drugs (Table 2) and endogenous DNA lesions (abasic sites, oxidized nucleotides, and alkylated bases), which stabilize or trap TOPccs 46-50 . Thus, the way in which cells deal with TOPccs has biological and pharmacological implications.
TOP1cc removal: multiple pathways and unanswered questions DNA-protein crosslinks (DPCs) include not only TOP1ccs, but also crosslinks induced by aldehyde products of demethylation reactions, cisplatin, UV light or ionizing radiation, and trapping of DNA methyltransferases covalently bound to 5-aza-cytosine (reviewed in 51-53). Distinct repair pathways have evolved to resolve these DPCs; however, TOPccs present unique challenges because they also involve protein-linked DNA breaks. Recent studies have provided new insight into the action of tyrosyl-DNA phosphodiesterases 1 and 2 (TDP1 and TDP2, respectively) and DNA-dependent proteases such as SPARTAN (also known as SPRTN) that recognize and reverse persistent TOP1ccs.
Several lines of evidence implicate TDP1 in TOP1cc removal. TDP1 can de-esterify peptidic tyrosine-phosphoesters 54,55 , and TDP1 knockdown results in increased foci containing the TOP1 active site peptide covalently bound to DNA 56 . Earlier studies suggested that TPD1 efficiently removes short TOP1 peptides from DNA but is less efficient at removing longer peptides or full-length TOP1 57 . However, recent studies of TDP1 mutants suggest that full-length TOP1 can, in fact, be released from chromatin-bound TOP1ccs in yeast and human cells 58,59 .
The observation that TDP1 knockdown or knockout has little impact on yeast or mammalian cell sensitivity to camptothecins 55,60 suggested early on that there must be redundant or overlapping repair pathways. In the absence of TDP1, the 5'-tyrosyl phosphodiesterase TDP2 73,74 and a pathway involving the repair proteins XPF and ERCC1 75 participate in TOP1cc removal. An additional pathway involves cleavage of the adducted DNA by the nuclease MUS81 followed by polymerization and ligation across the resulting gap 76 .
Conditions that promote the use of one pathway over another are still being elucidated. Poly(ADP-ribosyl)ation of TDP1 appears to influence this choice 75 . In addition, the deubiquitylase UCHL3 was recently shown to regulate TDP1 proteostasis 77 , implicating ubiquitin-dependent regulation of TDP1 in the repair of camptothecin-induced TOP1ccs.
Emerging results also suggest a role for proteases in the removal of TOP1ccs. Although early studies implicated the proteasome in this process [78][79][80][81]   impact sensitivity to drug-stabilized TOP1ccs, these observations support the notion that other, as-yet-uncharacterized metalloproteases may regulate cellular responses to topoisomerase-mediated DNA damage via distinct ubiquitin-like protein modifications. The potential therapeutic implications of these recently recognized repair pathways remain to be more fully investigated.
Extending the paradigm to TOP2cc The machinery responsible for removing trapped TOP2ccs is even less clearly defined. Proteasomal degradation of TOP2 after teniposide treatment has been reported 101 , contributing to a model in which collisions between advancing transcription complexes and TOP2ccs result in irreversibly trapped TOP2-DNA complexes, which are marked by ubiquitylation and degraded by the proteasome. Two recent studies further suggest that coordination of SUMOand Ub-dependent TOP2 modifications may be critical for genomic stability. In etoposide-treated fission yeast, the DNA translocase Rrp2 binds to SUMOylated TOP2ccs and prevents recruitment of the SUMO-dependent E3 ubiquitin ligase STUbL, thereby preventing STUbL-mediated TOP2 ubiquitinylation and degradation 108 . Instead, Rrp2 facilitates the eviction of intact TOP2 from the DNA and concomitant DNA resealing, thereby increasing genomic stability and etoposide resistance. In other studies, the Smurf2 E3 ubiquitin ligase was shown to switch the pattern of TOP2α modification from K48 polyubiquitylation that promotes proteasomal degradation to monoubiquitylation, which leads to increased TOP2α protein levels, suppression of anaphase bridge formation, and etoposide resistance 109 .
In summary, although multiple pathways have been implicated in the reversal of trapped TOP2ccs, other studies suggest that protecting TOP2ccs from proteolytic degradation is also critical for maintaining genome stability. Further studies are required to assess whether distinct pathways are called into play in response to different levels of DNA damage, as appears to be the case with TOP1, or whether current inconsistencies reflect differential expression of pathway components in different cell types.

Translating biological knowledge into improved therapy
The TOP1-and TOP2-targeted drugs in Table 2 all have activity in the clinical setting, albeit with narrow therapeutic windows. Accordingly, recent efforts to develop topoisomerase poisons 66 into more effective antineoplastic agents have tried to address a series of issues.
Can the delivery and efficacy of topoisomerase poisons be improved? Consistent with observations that TOP1 and TOP2 poisons are preferentially toxic during S phase, classic studies demonstrated that the administration of irinotecan on a five-times-daily schedule for 2 weeks is more active against human cancer xenografts than less-protracted schedules 110 . Etoposide administered every other day for three doses is likewise more effective against L1210 murine leukemia than a higher dose administered once. In the clinical setting, these observations have been translated into protracted schedules of both irinotecan 111 and etoposide 112 . Because these prolonged schedules can be inconvenient and toxic 111 , there has been an ongoing search for alternatives, including new topoisomerase poisons, drug formulations that extend the half-life of TOPccs, and strategies to increase tumor-selective drug delivery (Table 3) 113 .
Among the new classes of TOP1 or TOP2 poisons, TOP1directed indenoisoquinolines 114,115 are furthest along in development. These agents, which lack a lactone ring and, in contrast to camptothecin derivatives, do not exist in equilibrium between an active agent and inactive derivative 115 , exhibit promising activity against canine lymphomas 116 . Assessments of their activity in humans are awaited with interest.
An alternative approach involves new formulations that extend tumor exposure. MM398, a nanoliposomal irinotecan formulation 117 , gained FDA approval in combination with 5-fluorouracil and leucovorin for gemcitabine-resistant pancreatic cancer 64,65 . In contrast, NKTR-102, a PEGylated irinotecan, exhibited disappointing activity in breast and ovarian cancer 118,119 . Whether the different outcomes for these two sustained-release irinotecan formulations reflect differences in pharmacokinetics, intratumoral accumulation, or simply choice of tumors studied is not clear.
Santi and coworkers developed an ultra-long-acting Prolynx PEG~SN-38 that accumulates in tumors and delivers active SN-38 rather than the prodrug irinotecan 120 . Liposomal topotecan formulations are also being developed 121 . Whether the promising preclinical activity seen in experimental tumors, which is thought to reflect enhanced permeability and retention of nanoformulations 122 , can be translated into increased clinical efficacy remains to be determined.
Antibody-drug conjugates (Table 3) also hold the promise of more selectively delivering TOP1 poisons to tumor cells. DS-8201 123,124 , a conjugate of the TOP1 poison deruxtecan with the anti-HER2 antibody trastuzumab, is currently undergoing extensive preclinical and early clinical testing (www.Clinical-Trials.gov). Promising clinical activity has been observed in trastuzumab-resistant breast and gastric cancers 125,126 . An immunoconjugate of SN-38 and antibody to human trophoblast cell Table 3. Emerging inhibitors of mammalian TOP1 or TOP2 a .

New TOP1 inhibitors Compound
Unique features and references STA-8666 1. Covalent fusion of STA-8663 (HSP90 inhibitor) and SN-38 through a cleavable chemical linker 2. Prolonged tumor exposure relative to irinotecan in vivo 3. Very active against small cell lung cancer and sarcoma xenografts 127,128 Indenoisoquinolines 1. Stabilize TOP1-DNA covalent complexes but lack the lactone ring of camptothecin and its derivatives 2. As a consequence, the TOP1-DNA covalent complexes do not peak and then decrease as they do with camptothecins 115,129 7-aza-indenoisoquinolines 1. These non-camptothecin agents lack the lactone ring that is part of the camptothecin backbone 130 Fluoroindenoisoquinolines 1. These indenoisoquinoline derivatives contain fluorine in place of methoxy side chains and are more potent than the parent compounds 131,132 Novel formulations of TOP1 inhibitors  (Table 4).
Most started with myelosuppressive topotecan or irinotecan regimens 170 . Because PARPis also suppress bone marrow function 171 , it is not surprising that profound myelosuppression occurs with these combinations, limiting drug doses that can be safely administered together (Table 4). In contrast, by starting with a less myelosuppressive weekly topotecan regimen 172 and only administering PARPi for 72 hours around each topotecan dose to maximize the synergy, Wahner Hendrickson and coworkers were able to escalate topotecan and veliparib to three-quarters of the single-agent MTDs 173 . Whether the approach of i) using a less myelosuppressive TOP1-directed regimen and/or ii) giving intermittent PARPi timed to coincide with maximal TOP1cc stabilization will be an effective way forward with TOP1 poison/PARPi combinations remains to be further assessed.  185 . More recent studies have examined ATR inhibitors (e.g. M6620 and AZD6738) 186,187 with TOP1 poisons. Reportedly, an M6620/topotecan combination was well tolerated, except for myelosuppression, and induced partial responses in two out of 21 (9.5%) patients 188 .

Combinations with ATR and CHK1 inhibitors.
A phase II trial of this combination in small cell lung cancer (ClinicalTrials.gov identifier: NCT02487095) and a phase I trial of an irinotecan/M6620 combination (NCT02595931) are ongoing.

TOP1 poison/immune checkpoint inhibitor combinations.
While immune checkpoint blockade is highly active in certain solid tumors 189,190 , many common cancers respond poorly. However, recent studies suggest that DNA damage can stimulate immune responses through multiple mechanisms. First, release of DNA to the cytosol after DNA damage 191 activates the stimulator of interferon genes (STING) pathway 192,193 , leading to the production of pro-inflammatory cytokines. Second, DNA damage-induced release of tumor cell microvesicles can increase immune activation 194,195 . Third, DNA damage increases antigen presentation on tumor cell MHC class I molecules, leading to enhanced dendritic cell activation and T cell responses 196 . Importantly, these changes have been observed after treatment with TOP1 poisons, potentially contributing to the synergy observed when irinotecan or DS-8201a is combined with anti-PD-1 in vivo 133,197,198 . Clinical trials are also evaluating TOP2 poisons in combination with PD-1 antibodies (www.ClinicalTrials.gov).
Predicting response to topoisomerase poisons Given the toxicities of topoisomerase poisons, the ability to predict responses and avoid treatment of patients unlikely to benefit would represent a major advance. In isogenic yeast 199,200 or mammalian cells 201,202 , elevated TOP1 or TOP2 levels are associated with increased killing by topoisomerase poisons (Figure 2). Additional studies indicate that high TOP1 expression correlates with improved colorectal cancer response to irinotecan [203][204][205][206] and TOP2 gene amplification is associated with improved breast cancer response to doxorubicin 207,208 . However, expression and response are not so tightly correlated that outcomes of individual patients can be predicted from expression data alone.
The frequent occurrence of transport-mediated resistance raises the possibility that responses might be better predicted by assaying TOPccs after the first dose of therapy. While earlier techniques for measuring TOPccs were labor intensive and nonspecific, a recently described antibody to TOP1ccs 209 opens the possibility of specific, quantitative assays to address the relationship between TOP1ccs and response to TOP1 poisons. Unfortunately, similar reagents to assess TOP2ccs are not currently available.  combination with PARPi 173 . Thus, HR status might need to be considered in predictive algorithms, but the impact of HR status might also vary by drug class.

Challenges for the coming decade
As indicated above, recent advances bring into focus a number of topics for future investigation. First, the cellular functions of topoisomerases are incompletely understood, in part because DNA topology still cannot be visualized in intact cells. Second, based on provocative examples, cooperation between various topoisomerases and other enzymes requires further study. Third, when TOPccs are trapped, we still have only rudimentary understanding of the processes that reverse these DPCs and limited insight into the factors that dictate choice between overlapping repair pathways. Finally, even though topoisomerase-directed drugs exhibit anti-neoplastic properties, patients would benefit from more efficacious schedules, more selective delivery of active agents to tumor cells, and potentially bioassays that accurately predict responses to topoisomerase-directed therapy. Editorial Note on the Review Process are written by members of the prestigious . They are commissioned and F1000 Faculty Reviews F1000 Faculty are peer reviewed before publication to ensure that the final, published version is comprehensive and accessible. The reviewers who approved the final version are listed with their names and affiliations.
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Anthony Maxwell
Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK No competing interests were disclosed. Competing Interests: