TOP2-targeting Drugs Research Articles

Regulation of topoisomerase II stability and activity by ubiquitination and SUMOylation: clinical implications for cancer chemotherapy.

DNA topoisomerases II (TOP2) are peculiar enzymes (TOP2α and TOP2β) that modulate the conformation of DNA by momentarily breaking double-stranded DNA to allow another strand to pass through, and then rejoins the DNA phosphodiester backbone. TOP2α and TOP2β play vital roles in nearly all events involving DNA metabolism, including DNA transcription, replication, repair, and chromatin remodeling. Beyond these vital functions, TOP2 enzymes are therapeutic targets for various anticancer drugs, termed TOP2 poisons, such as teniposide, etoposide, and doxorubicin. These drugs exert their antitumor activity by inhibiting the activity of TOP2-DNA cleavage complexes (TOP2ccs) containing DNA double-strand breaks (DSBs), subsequently leading to the degradation of TOP2 by the 26S proteasome, thereby exposing the DSBs and eliciting a DNA damage response. Failure of the DSBs to be appropriately repaired leads to genomic instability. Due to this mechanism, patients treated with TOP2-based drugs have a high incidence of secondary malignancies and cardiotoxicity. While the cytotoxicity associated with TOP2 poisons appears to be TOP2α-dependent, the DNA sequence rearrangements and formation of DSBs appear to be mediated primarily through TOP2β inhibition, likely due to the differential degradation patterns of TOP2α and TOP2β. Research over the past few decades has shown that under various conditions, the ubiquitin-proteasome system (UPS) and the SUMOylation pathway are primarily responsible for regulating the stability and activity of TOP2 and are therefore critical regulators of the therapeutic effect of TOP2-targeting drugs. In this review, we summarize the current progress on the regulation of TOP2α and TOP2β by ubiquitination and SUMOylation. By fully elucidating the basic biology of these essential and complex molecular mechanisms, better strategies may be developed to improve the therapeutic efficacy of TOP2 poisons and minimize the risks of therapy-related secondary malignancy.

Intercalating TOP2 Poisons Attenuate Topoisomerase Action at Higher Concentrations.

Topoisomerase II (TOP2) poisons are effective cytotoxic anticancer agents that stabilize the normally transient TOP2-DNA covalent complexes formed during the enzyme reaction cycle. These drugs include etoposide, mitoxantrone, and the anthracyclines doxorubicin and epirubicin. Anthracyclines also exert cell-killing activity via TOP2-independent mechanisms, including DNA adduct formation, redox activity, and lipid peroxidation. Here, we show that anthracyclines and another intercalating TOP2 poison, mitoxantrone, stabilize TOP2-DNA covalent complexes less efficiently than etoposide, and at higher concentrations they suppress the formation of TOP2-DNA covalent complexes, thus behaving as TOP2 poisons at low concentration and inhibitors at high concentration. We used induced pluripotent stem cell (iPSC)-derived human cardiomyocytes as a model to study anthracycline-induced damage in cardiac cells. Using immunofluorescence, our study is the first to demonstrate the presence of topoisomerase IIβ (TOP2B) as the only TOP2 isoform in iPSC-derived cardiomyocytes. In these cells, etoposide robustly induced TOP2B covalent complexes, but we could not detect doxorubicin-induced TOP2-DNA complexes, and doxorubicin suppressed etoposide-induced TOP2-DNA complexes. In vitro, etoposide-stabilized DNA cleavage was attenuated by doxorubicin, epirubicin, or mitoxantrone. Clinical use of anthracyclines is associated with cardiotoxicity. The observations in this study have potentially important clinical consequences regarding the effectiveness of anticancer treatment regimens when TOP2-targeting drugs are used in combination. These observations suggest that inhibition of TOP2B activity, rather than DNA damage resulting from TOP2 poisoning, may play a role in doxorubicin cardiotoxicity. SIGNIFICANCE STATEMENT: We show that anthracyclines and mitoxantrone act as topoisomerase II (TOP2) poisons at low concentration but attenuate TOP2 activity at higher concentration, both in cells and in in vitro cleavage experiments. Inhibition of type II topoisomerases suppresses the action of other drugs that poison TOP2. Thus, combinations containing anthracyclines or mitoxantrone and etoposide may reduce the activity of etoposide as a TOP2 poison and thus reduce the efficacy of drug combinations.

TOP2B Enzymatic Activity on Promoters and Introns Modulates Multiple Oncogenes in Human Gliomas

Oncogene expression varies across gliomas. We discovered that TOP2B modulates transcription and splicing of multiple oncogenes in Drosophila brain tumors and human gliomas. TOP2B regulated transcription at sites where it was active, but not where found natively binding. TOP2B activity localized in enhancers, promoters and introns of PDGFRA and MYC, facilitating their expression. TOP2B levels and genomic localization was associated with PDGFR and MYC expression across gliomas specimens. Susceptibility to MST-16, a TOP2 inhibitor drug, correlated with baseline PDGRA expression across glioma cell lines. Our results indicate that TOP2B activity exerts a pleiotropic role in transcriptional regulation of oncogenes in a subset of gliomas. Modulation of oncogenes by TOP2-targeting drugs is the basis for a personalized approach for cancer therapy.

Abstract 4724: Regulation of DNA cleavage by the amino and carboxyl domains of human Top2α

Abstract DNA topoisomerases are involved in processing DNA structures such as knots and supercoils formed during cellular processes. hTop2α functions by creating transient DNA double-strand breaks, through which a second DNA molecule can be passed. DNA breakage by hTop2α involves formation of a transient covalent DNA-protein intermediate that can be stabilized by small molecules like etoposide and doxorubicin. Therefore, these agents convert Top2 into a DNA damaging agent. The overall architecture of type II topoisomerases has been defined by a combination of biochemical and structural studies. The homodimeric eukaryotic Top2 includes an N-terminal domain that includes an ATP dependent dimerization and ATPase activity, a domain that connects the ATPase domain to the catalytic core, termed the transducer domain, a central breakage/reunion core that carries out metal ion dependent formation of the protein/DNA covalent complex and a C-terminal domain that includes a second dimerization domain. Since drug resistance to Top2 targeting drugs can occur by mutations that greatly reduce enzyme activity, they are of limited usefulness in understanding detailed mechanisms of drug action. We carried out a large-scale screen to identify etoposide hypersensitive mutations in hTop2α. We developed novel approaches to target small regions of the hTop2α coding sequence. As anticipated, we identified several domains of hTop2α that were hotspots for changes leading to etoposide hypersensitivity. These regions included the N-terminal region of the protein (transducer and ATPase domain), the breakage/reunion catalytic core of the enzyme, and C-terminal dimerization domains. For example, we identified a prominent class of mutations including Asp374Gly, Asp374Glu, Asn445Asp, Val415Leu and Thr377Ile in the transducer domain, which confer hypersensitivity to etoposide. We purified and characterized the Asp374Gly mutant protein. In addition to an increased in vitro sensitivity to etoposide, Top2α (Asp374Gly) had a decreased requirement for ATP compared to the wild-type enzyme. The decreased requirement for ATP is unexpected and may suggest an alteration in the communication between the ATPase domain and the catalytic core. In addition, we also isolated etoposide hypersensitive alleles close to the C-terminal dimerization domain. These mutations included Asp1130Asn, Ala1052Gly and Gln1109Lys. Our results suggest at least two distinct controlling modules for Top2 cleavage: the ATPase/transducer module, and the C-terminal dimerization domain. Our results provide additional insight into how etoposide inhibits Top2α and may suggest new domains of the protein suitable for drug targeting. Citation Format: Abhishek Dilip Deshpande, Matthew A. Gilbertson, Hannah N. Miles, Karin C. Nitiss, John L. Nitiss. Regulation of DNA cleavage by the amino and carboxyl domains of human Top2α [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4724.

Abstract 4727: Role of SUMOylating enzymes in repair of Topoisomerase II mediated DNA damage

Abstract Topoisomerase 2 (Top2) enzymes regulate DNA topology for efficient DNA replication and transcription by introducing transient double strand breaks (DSBs) in the DNA. To generate the DSBs, Top2 forms a covalent intermediate with the DNA termed as the Top2 DNA covalent complex (Top2cc). Top2 is a target of clinically used anticancer agents such as etoposide and doxorubicin. Mammalian cells have two Top2 isozymes, Top2α and Top2β. Top2α is primarily expressed in proliferating cells and essential for cell division. Top2β is expressed in all cells and has been shown to play key roles in transcription. Top2 targeting agents generate anticancer activity by trapping both Top2αcc and Top2βcc. Factors that affect repair of Top2cc are likely to be a key determinant of the clinical efficacy of Top2 targeting agents. Identifying factors that play a role in repair of Top2cc offers a potential strategy for increasing clinical efficacy of Top2 targeting agents. Previous work has demonstrated that an important pathway for removal of trapped Top2 from DNA is initiated through proteolytic degradation of Top2 by the proteasome. Other proteases may also participate in degradation of the protein covalently bound to DNA. Proteasomal inhibition increased Top2cc, especially Top2β, and enhanced the cytotoxicity of Top2 targeting drugs. To study steps involved in proteasomal degradation of Top2cc, we developed a method to purify genomic DNA under conditions that preserve proteins covalently bound to DNA. Levels of trapped topoisomerases can be measured using Top2 isoform specific antibodies. Alternately, after micrococcal nuclease degradation of DNA, the proteins covalently bound to DNA can be characterized for post-translational modifications (PTMs) using antibodies specific for the modifying agents. Etoposide treatment induced SUMOylation of both Top2α and Top2β covalently bound to DNA. Blocking SUMOylation of Top2 by siRNA knockdown of the SUMO E2 conjugating enzyme UBC9 increased levels of both Top2α and Top2β covalent complexes, indicating that SUMOylation is likely involved in the repair of both Top2α and Top2β covalent complexes. We have extended this approach to identify SUMO ligases that directly act on Top2 isoforms. We found that depletion of the SUMO E3 PIAS4 resulted in elevated levels of Top2αcc with minimal effect on levels of Top2βcc. This result suggest that PIAS4 participates in a repair pathway that is specific for repairing Top2α covalent complexes. We have constructed PIAS4 knockouts using CRISPR/Cas9 and are currently examining the roles of other SUMO E3 ligases that may participate in the repair of Top2 damage. Our current results suggest that some pathways for repair of Top2 covalent complexes are likely isoform specific. This allows targeting of specific Top2 isoforms without the need to identify agents that discriminate between Top2 isoforms, and may help alleviate some of the undesirable side effects of Top2 targeting agents. Citation Format: Bhargavi Ramesh, Yilun Sun, John L. Nitiss, Jay Anand, Karin C. Nitiss. Role of SUMOylating enzymes in repair of Topoisomerase II mediated DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4727.

Characterization of DNA topoisomerase I in three SN-38 resistant human colon cancer cell lines reveals a new pair of resistance-associated mutations.

BackgroundDNA topoisomerase I (Top1) is a DNA unwinding protein and the specific target of the camptothecin class of chemotherapeutic drugs. One of these, irinotecan, acting through its active metabolite SN-38, is used in the treatment of metastatic colorectal cancer. However, resistance to irinotecan represents a major clinical problem. Since molecular alterations in Top1 may result in resistance to irinotecan, we characterized Top1 in three human colon cancer cell lines with acquired resistance to SN-38.MethodsThree SN-38 resistant (20–67 fold increased resistance) cell lines were generated and compared to wild-type parental cells with regards to: TOP1 gene copy number and gene sequence, Top1 expression (mRNA and protein), Top1 enzymatic activity in the absence and presence of drug, and Top1-DNA cleavage complexes in drug treated cells. TOP1 mutations were validated by PCR using mutant specific primers. Furthermore, cross-resistance to two indenoisoquinoline Top1-targeting drugs (NSC 725776 and NSC 743400) and two Top2-targeting drugs (epirubicin and etoposide) was investigated.ResultsTwo of three SN-38 resistant cell lines carried TOP1 gene copy number aberrations: A TOP1 gene copy gain and a loss of chromosome 20, respectively. One resistant cell line harbored a pair of yet unreported TOP1 mutations (R364K and G717R) in close proximity to the drug binding site. Mutant TOP1 was expressed at a markedly higher level than wild-type TOP1. None or very small reductions were observed in Top1 expression or Top1 activity in the absence of drug. In all three SN-38 resistant cell lines Top1 activity was maintained in the presence of high concentrations of SN-38. None or only partial cross-resistance were observed for etoposide and epirubicin, respectively. SN-38 resistant cells with wild-type TOP1 remained sensitive to NSC 743400, while cells with mutant TOP1 was fully cross-resistant to both indenoisoquinolines. Top1-DNA cleavage complex formation following drug treatment supported the other findings.ConclusionsThis study adds to the growing knowledge about resistance mechanisms for Top1-targeting chemotherapeutic drugs. Importantly, two yet unreported TOP1 mutations were identified, and it was underlined that cross-resistance to the new indenoisoquinoline drugs depends on the specific underlying molecular mechanism of resistance to SN-38.

Lack of topoisomerase copy number changes in patients with de novo and relapsed diffuse large B-cell lymphoma

Topoisomerase (TOP) gene copy number changes may predict response to treatment with TOP-targeting drugs in cancer treatment. This was first described in patients with breast cancer and is currently being investigated in other malignant diseases. TOP-targeting drugs may induce TOP gene copy number changes at relapse, with possible implications for relapse therapy efficacy. TOP gene alterations in lymphoma are poorly investigated. In this study, TOP1 and TOP2A gene alterations were investigated in patients with de novo diffuse large B-cell lymphoma (DLBCL) (n = 33) and relapsed DLBCL treated with chemotherapy regimens including TOP2-targeting drugs (n = 16). No TOP1 or TOP2A copy number changes were found. Polysomy of chromosomes 20 and 17 was seen in 3 of 25 patients (12%) and 2 of 32 patients (6%) with de novo DLBCL. Among relapsed patients, chromosome polysomy was more frequently observed in 5 of 13 patients (38%) and 4 of 16 patients (25%) harboring chromosome 20 and 17 polysomy, respectively; however, these differences only tended to be significant (p = 0.09 and p = 0.09, respectively). The results suggest that TOP gene copy number changes are very infrequent in DLBCL and not likely induced by TOP2-targeting drugs. Increased polyploidy of chromosomes 17 and 20 among patients with relapsed DLBCL may reflect genetic compensation in the tumor cells after TOP2 inhibition, but is more likely due to the increased genetic instability often seen in progressed cancers. Therefore, it is unlikely that TOP1 and TOP2A gene alterations can be used as predictive markers for response to treatment with TOP2-targeting drugs in patients with DLBCL.

Anthracenedione–methionine conjugates are novel topoisomerase II-targeting anticancer agents with favorable drug resistance profiles

Structure-associated drug resistance and DNA-unwinding abilities have greatly limited the clinical usage of anthracenediones, including mitoxantrone (MX) and ametantrone (AT), which intercalate into DNA and induce topoisomerase II (TOP2)-mediated DNA break. We studied a series of 1,4-bis(2-amino-ethylamino) MX- and AT-amino acid conjugates (M/AACs) and showed that abilities in cancer cell killing correlate with the amounts of chromosomal DNA breaks induced by M/AACs. Notably, the 1,4-bis-L/l-methionine-conjugated MAC (L/LMet-MAC) exhibits DNA-breaking, cancer cell-killing and anti-tumor activities rivaling those of MX. Interestingly, l- and d-form Met-M/AACs unwind DNA poorly compared to MX and AT. The roles of the two human TOP2 isozymes (hTOP2α and 2β) in the L/LMet-MAC-induced DNA breakage and cancer cell-killing were suggested by the following observations: (i) M/AAC-induced DNA breakage, cytotoxicity and apoptosis are greatly reduced in various TOP2-deficient conditions; (ii) DNA breaks induced by MACs are highly reversible and effectively antagonized by the TOP2 catalytic inhibitors; (iii) MACs induced differential TOP2-mediated DNA cleavage in vitro using recombinant hTOP2α proteins and the formation of hTOP2α/βcc in the cell culture system. Interestingly, d-aa-conjugated MACs often caused a lower level in hTOP2-mediated DNA breaks and cell-killing than the corresponding l-form ones indicating a steric-specific effect of MACs. Together, our results suggest that both enzyme- and DNA-drug interactions might contribute to TOP2-targeting by M/AACs. Furthermore, Met-MACs are poor substrates for the MDR1 transporter. Therefore, L/LMet-MAC represents a promising class of TOP2-targeting drugs with favorable drug resistance profiles.

Abstract CN08-02: Repair of topoisomerase II mediated DNA damage: A potential mechanism for targeting specific Top2 isoforms

Abstract CN08-02: Repair of topoisomerase II mediated DNA damage: A potential mechanism for targeting specific Top2 isoforms

Isolation and Characterization of mAMSA-hypersensitive Mutants: CYTOTOXICITY OF Top2 COVALENT COMPLEXES CONTAINING DNA SINGLE STRAND BREAKS

Topoisomerase II (Top2) is the primary target for active anti-cancer agents. We developed an efficient approach for identifying hypersensitive Top2 mutants and isolated a panel of mutants in yeast Top2 conferring hypersensitivity to the intercalator N-[4-(9-acridinylamino)-3-methoxyphenyl]methanesulphonanilide (mAMSA). Some mutants conferred hypersensitivity to etoposide as well as mAMSA, whereas other mutants exhibited hypersensitivity only to mAMSA. Two mutants in Top2, changing Pro(473) to Leu and Gly(737) to Val, conferred extraordinary hypersensitivity to mAMSA and were chosen for further characterization. The mutant proteins were purified, and their biochemical activities were assessed. Both mutants encode enzymes that are hypersensitive to inhibition by mAMSA and other intercalating agents and exhibited elevated levels of mAMSA-induced Top2:DNA covalent complexes. While Gly(737) --> Val Top2p generated elevated levels of Top2-mediated double strand breaks in vitro, the Pro(473) --> Leu mutant protein showed only a modest increase in Top2-mediated double strand breaks but much higher levels of Top2-mediated single strand breaks. In addition, the Pro(473) --> Leu mutant protein also generated high levels of mAMSA-stabilized covalent complexes in the absence of ATP. We tested the role of single strand cleavage in cell killing with alleles of Top2 that could generate single strand breaks, but not double strand breaks. Expression in yeast of a Pro(473) --> Leu mutant that could only generate single strand breaks conferred hypersensitivity to mAMSA. These results indicate that generation of single strand breaks by Top2-targeting agents can be an important component of cell killing by Top2-targeting drugs.

Sequential topoisomerase targeting and analysis of mechanisms of resistance to topotecan in patients with acute myelogenous leukemia

Resistance to topoisomerase I (TOP1)-targeting drugs such as topotecan often involves upregulation of topoisomerase II (TOP2), with accompanying increased sensitivity to TOP2-targeting drugs such as etoposide. This trial was designed to investigate sequential topoisomerase targeting in the treatment of patients with high-risk acute myelogenous leukemia. An initial cohort of patients received topotecan and cytosine arabinoside daily for 5 days. Serial samples of circulating mononuclear cells were examined to evaluate peak elevations of TOP2-alpha protein expression. In subsequent cohorts, etoposide was administered daily for 3 days, beginning 6 h after initiation of the topotecan infusion. The etoposide dose was escalated to determine a maximum-tolerated dose. Circulating mononuclear cells were analyzed for TOP1 mutations and ABCG2 protein expression. In addition, systemic and intracellular topotecan concentrations were measured. Thirty-one patients were enrolled. On the basis of TOP1-alpha protein levels in three patients with peripheral blast counts greater than 50%, etoposide administration began 6 h after initiation of the topotecan/cytosine arabinoside infusion. Using this schedule of administration, the maximum-tolerated dose of etoposide was 90 mg/m. No TOP1 mutations were identified, but increases in ABCG2 expression during the infusion were observed in mononuclear cells from two of four evaluable patients. Administration of etoposide 6 h after initiation of a topotecan/cytosine arabinoside infusion is feasible and is associated with clinical activity. Analysis of TOP2-alpha protein levels in this small number of patients indicated that peak increases occurred earlier than expected based on earlier publications. Upregulation of ABCG2 was detected in circulating cells and may represent an inducible form of drug resistance that should be investigated further.

Tyrosyl-DNA phosphodiesterase (Tdp1) participates in the repair of Top2-mediated DNA damage

Agents targeting topoisomerases are active against a wide range of human tumors. Stabilization of covalent complexes, converting topoisomerases into DNA-damaging agents, is an essential aspect of cell killing by these drugs. A unique aspect of the repair of topoisomerase-mediated DNA damage is the requirement for pathways that can remove protein covalently bound to DNA. Tyrosyl-DNA phosphodiesterase (Tdp1) is an enzyme that removes phosphotyrosyl moieties bound to the 3' end of DNA. Cells lacking Tdp1 are hypersensitive to camptothecin, consistent with a role for Tdp1 in processing 3' phosphotyrosyl protein-DNA covalent complexes. Because Top2p forms a 5' phosphotyrosyl linkage with DNA, previous work predicted that Tdp1p would not be active against lesions involving Top2p. We found that deletion of the TDP1 gene in yeast confers hypersensitivity to Top2 targeting agents. Combining tdp1 mutations with deletions of genes involved in nonhomologous end joining, excision repair, or postreplication repair enhanced sensitivity to Top2 targeting drugs over the level seen with single mutants, suggesting that Tdp1 may function in collaboration with multiple pathways involved in strand break repair. tdp1 mutations can sensitize yeast cells to drugs targeting Top2 even when TOP1 is deleted. Finally, bacterially expressed yeast Tdp1p is able to remove a peptide derived from yTop2 that is covalently bound to DNA by a 5' phosphotyrosyl linkage. Our results show that Tdp1 plays more general roles in DNA repair than repair of Top1 mediated DNA damage, and may participate in repairing many types of base damage to DNA.