Top1 Cleavage Complexes Research Articles

Nuclear-penetrating scleroderma autoantibody inhibits topoisomerase 1 cleavage complex formation

The contributions of anti-Topoisomerase 1 (Top1) autoantibodies to the pathophysiology of diffuse cutaneous systemic sclerosis (dcSSc), the most aggressive scleroderma subtype, are unknown. Top1 catalyzes DNA relaxation and unwinding in cell nuclei, a site previously considered inaccessible to antibodies. The discovery of autoantibodies in systemic lupus erythematosus that penetrate nuclei and inhibit DNA repair raised the possibility that nuclear-penetrating autoantibodies contribute to mechanisms of autoimmunity. Here we show that an anti-Top1 autoantibody produced by a single B cell clone from a patient with dcSSc penetrates live cells and localizes into nuclei. Functionally, the autoantibody inhibits formation of the Top1 cleavage complex necessary for DNA nicking, which distinguishes it from cytotoxic camptothecin Top1 inhibitors used in cancer therapy that trap the cleavage complex rather than preventing its formation. Discovery of a patient-derived cell-penetrating scleroderma anti-Top1 autoantibody that inhibits Top1 cleavage complex formation supports the hypothesis that anti-Top1 autoantibodies contribute to cellular dysfunction in dcSSc and offers a valuable antibody reagent resource for future studies on anti-Top1 autoantibody contributions to scleroderma pathophysiology.

RAD54L2-mediated DNA damage avoidance pathway specifically preserves genome integrity in response to topoisomerase 2 poisons.

Type II topoisomerases (TOP2) form transient TOP2 cleavage complexes (TOP2ccs) during their catalytic cycle to relieve topological stress. TOP2ccs are covalently linked TOP2-DNA intermediates that are reversible but can be trapped by TOP2 poisons. Trapped TOP2ccs block transactions on DNA and generate genotoxic stress, which are the mechanisms of action of TOP2 poisons. How cells avoid TOP2cc accumulation remains largely unknown. In this study, we uncovered RAD54 like 2 (RAD54L2) as a key factor that mediates a TOP2-specific DNA damage avoidance pathway. RAD54L2 deficiency conferred unique sensitivity to treatment with TOP2 poisons. RAD54L2 interacted with TOP2A/TOP2B and ZATT/ZNF451 and promoted the turnover of TOP2 from DNA with or without TOP2 poisons. Additionally, inhibition of proteasome activity enhanced the chromatin binding of RAD54L2, which in turn led to the removal of TOP2 from chromatin. In conclusion, we propose that RAD54L2-mediated TOP2 turnover is critically important for the avoidance of potential TOP2-linked DNA damage under physiological conditions and in response to TOP2 poisons.

Abstract C120: Antitumor effect of HER3-DXd, an antibody-drug conjugate targeting HER3, in gastric cancer cell lines

Abstract Background: HER3, one of the HER family members, is expressed across various solid tumors and known to be an important oncogenic driver. HER3-DXd is an antibody-drug conjugate (ADC) that combines patritumab, an anti-HER3 antibody, and deruxtecan, a topoisomerase 1 inhibitor payload through peptide cleavable linker system. HER3-DXd has been shown to have anti-tumor effects in breast, non-small cell lung, and colorectal cancers, in vitro and in vivo, and is being evaluated in phase 2 and phase 3 clinical trials. However, the effect of HER3-DXd in gastric cancer and the mechanism of sensitivity are largely unknown. Methods: Seven gastric cancer cell lines were used (NUGC-4, NCI-N87, SNU-601, SNU-216, SNU-638, SNU-668, and MKN-45). Colony formation assay (CFA) was performed with increasing concentration of HER3-DXd (doses range: 0-100 nM) for 14 days. Cell cycle change and internalization of HER3-DXd bound to HER3 protein in the cell membrane were seen by flow cytometry analysis. Apoptotic cell death was assessed by Annexin-V assay. DNA strand breaks were examined using alkaline comet assay. DNA damage accumulation and TOP1-cleavage complexes (TOP1ccs) were identified using immunofluorescence assay. Results: Seven gastric cancer cell lines showed heterogeneous mRNA expression of HER2 and HER3, which correlated with protein expression. Three cell lines with different sensitivities to HER3-DXd were selected based on CFA. SNU-601 was sensitive to U3-1402 (IC50 value: 11.01 nM), SNU-638 was less sensitive (IC50 value: 50.04 nM), and SNU-668 was insensitive (IC50 value >100 nM). HER3-DXd increased the Annexin-V positive population and the sub-G1 population in SNU-601 and SNU-638 cells. Moreover, cleavage of PARP and caspase-3 were increased in SNU-601 and SNU-638 cells, but not in SNU-668 cells. HER3-DXd was efficiently internalized within 1 hour in all three cells, regardless of their sensitivities to HER3-DXd. Signal transduction through the HER2 and HER3 receptors, which mediates a PI3K-AKT signaling pathway, was inhibited in SNU-601 cells. In SNU-601 and SNU-638 cells, HER3-DXd increased the expression of γ-H2AX and length of the comet tails, which indicate DNA damage. HER3-DXd also induced topoisomerase 1 cleavage complexes (TOP1ccs) in SNU-601 and SNU-638 cells. In contrast, TOP1cc was not observed in resistant SNU-668 cells. Conclusion: HER3-DXd induced formation of TOP1ccs, subsequent DNA damage, and apoptotic cell death in sensitive cell lines, but not in an insensitive cell line. These data indicate that internalized HER3-DXd causes accumulation of DNA damage through TOP1cc formation, which leads to apoptotic cell death in gastric cancer. Citation Format: Hae Min Hwang, So Hyeon Kim, Sujin Ham, Minyoung Lee, Youlim Noh, Yu-Jin Kim, Changyun Lee, Jinyoung Kim, Dae-Won Lee, Kyung-Hun Lee, Seock-Ah Im. Antitumor effect of HER3-DXd, an antibody-drug conjugate targeting HER3, in gastric cancer cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C120.

NEDDylated Cullin 3 mediates the adaptive response to topoisomerase 1 inhibitors.

DNA topoisomerase 1 (TOP11) inhibitors are mainstays of anticancer therapy. These drugs trap TOP1 on DNA, stabilizing the TOP1-cleavage complex (TOP1-cc). The accumulation of TOP1-ccs perturbs DNA replication fork progression, leading to DNA breaks and cell death. By analyzing the genomic occupancy and activity of TOP1, we show that cells adapt to treatment with multiple doses of TOP1 inhibitor by promoting the degradation of TOP1-ccs, allowing cells to better tolerate subsequent doses of TOP1 inhibitor. The E3-RING Cullin 3 ligase in complex with the BTBD1 and BTBD2 adaptor proteins promotes TOP1-cc ubiquitination and subsequent proteasomal degradation. NEDDylation of Cullin 3 activates this pathway, and inhibition of protein NEDDylation or depletion of Cullin 3 sensitizes cancer cells to TOP1 inhibitors. Collectively, our data uncover a previously unidentified NEDD8-Cullin 3 pathway involved in the adaptive response to TOP1 inhibitors, which can be targeted to improve the efficacy of TOP1 drugs in cancer therapy.

Mitochondrial topoisomerase 1 inhibition induces topological DNA damage and T cell dysfunction in patients with chronic viral infection.

T cells are crucial for controlling viral infections; however, the mechanisms that dampen their responses during viral infections remain incompletely understood. Here, we studied the role and mechanisms of mitochondrial topoisomerase 1 (Top1mt) inhibition in mitochondrial dysfunction and T cell dysregulation using CD4 T cells from patients infected with HCV or HIV and compared it with CD4 T cells from healthy individuals following treatment with Top1 inhibitor - camptothecin (CPT). We found that Top1mt protein levels and enzymatic activity are significantly decreased, along with Top1 cleavage complex (Top1cc) formation, in mitochondria of CD4 T cells from HCV- and HIV-infected patients. Notably, treatment of healthy CD4 T cells with CPT caused similar changes, including inhibition of Top1mt, accumulation of Top1cc in mitochondria, increase in PARP1 cleavage, and decrease in mtDNA copy numbers. These molecular changes resulted in mitochondrial dysfunction, T cell dysregulation, and programmed cell death through multiple signaling pathways, recapitulating the phenotype we detected in CD4 T cells from HCV- and HIV-infected patients. Moreover, treatment of CD4 T cells from HCV or HIV patients with CPT further increased cellular and mitochondrial reactive oxygen species (ROS) production and cell apoptosis, demonstrating a critical role for Top1 in preventing mtDNA damage and cell death. These results provide new insights into the molecular mechanisms underlying immune dysregulation during viral infection and indicate that Top1 inhibition during chronic HCV or HIV infection can induce mtDNA damage and T cell dysfunction. Thus, reconstituting Top1mt protein may restore the mtDNA topology and T cell functions in humans with chronic viral infection.

The Potential of Topoisomerase Inhibitor-Based Antibody-Drug Conjugates.

DNA topoisomerases are essential enzymes that stabilize DNA supercoiling and resolve entanglements. Topoisomerase inhibitors have been widely used as anti-cancer drugs for the past 20 years. Due to their selectivity as topoisomerase I (TOP1) inhibitors that trap TOP1 cleavage complexes, camptothecin and its derivatives are promising anti-cancer drugs. To increase accumulation of TOP1 inhibitors in cancer cells through the targeting of tumors, TOP1 inhibitor antibody–drug conjugates (TOP1-ADC) have been developed and marketed. Some TOP1-ADCs have shown enhanced therapeutic efficacy compared to prototypical anti-cancer ADCs, such as T-DM1. Here, we review various types of camptothecin-based TOP1 inhibitors and recent developments in TOP1-ADCs. We then propose key points for the design and construction of TOP1-ADCs. Finally, we discuss promising combinatorial strategies, including newly developed approaches to maximizing the therapeutic potential of TOP1-ADCs.

TDP1-independent pathways in the process and repair of TOP1-induced DNA damage

Anticancer drugs, such as camptothecin (CPT), trap topoisomerase I (TOP1) on DNA and form TOP1 cleavage complexes (TOP1cc). Alternative repair pathways have been suggested in the repair of TOP1cc. However, how these pathways work with TDP1, a key repair enzyme that specifically hydrolyze the covalent bond between TOP1 catalytic tyrosine and the 3’-end of DNA and contribute to the repair of TOP1cc is poorly understood. Here, using unbiased whole-genome CRISPR screens and generation of co-deficient cells with TDP1 and other genes, we demonstrate that MUS81 is an important factor that mediates the generation of excess double-strand breaks (DSBs) in TDP1 KO cells. APEX1/2 are synthetic lethal with TDP1. However, deficiency of APEX1/2 does not reduce DSB formation in TDP1 KO cells. Together, our data suggest that TOP1cc can be either resolved directly by TDP1 or be converted into DSBs and repaired further by the Homologous Recombination (HR) pathway.

Abstract 2638: Sacituzumab Govitecan, combination with PARP inhibitor, Talazoparib, in metastatic triple-negative breast cancer (TNBC): Translational investigation

Abstract Background: Sacituzumab Govitecan (SG), the first antibody-drug conjugate approved for metastatic TNBC (mTNBC), is comprised of SN-38 (active metabolite of irinotecan), a topoisomerase I (TOP1) inhibitor, coupled via a hydrolyzable linker to monoclonal antibody targeting trophoblast cell surface antigen 2 (Trop-2), an antigen overexpressed in mTNBC. Poly (ADP-ribose) polymerase inhibitors (PARPi) block resolution of TOP1 cleavage complexes (TOP1CCs) induced by TOP1 inhibitors, thus unmasking the inability of remaining pathways to repair DNA damage. However, previous clinical trials combining PARPi with standard TOP1 inhibitors (irinotecan, topotecan) were terminated early due to dose-limiting myelosuppression. We evaluated the combination of SG with PARP inhibitor in both pre-clinical models and phase 1b clinical trial. Methods and Results: In pre-clinical models we demonstrated that the targeted antibody-based delivery of SN-38 increased the ratio of tumor-to-normal cell SN-38, resulting in stabilized TOP1CCs, enhanced DNA damage and increased cytotoxicity with the combination, selectively in tumor cells but not normal cells, despite temporal separation of SG and PARPi exposure. To validate the hypothesis, we conducted a phase 1b investigator-initiated clinical trial combining SG with PARPi (talazoparib) in patients with mTNBC (NCT04039230). Inclusion criteria included female patients ≥ 18 years of age with mTNBC (per ASCO/CAP guidelines) and previous treatment with at least one prior therapeutic regimen for mTNBC. Clinical outcomes were assessed by Objective Response Rate per RECIST v1.1. In the phase 1b clinical trial (SG day 18, every 21 days with talazoparib), the staggered schedule with supportive therapy was relatively well-tolerated without DLTs, as predicted by the pre-clinical models. Furthermore, the staggered schedule demonstrated promising clinical activity. Molecular analysis of paired pre-treatment and on-treatment specimens demonstrated γ-H2AX accumulation, confirming pharmacodynamic inhibition with combination therapy. The dose-escalation portion of clinical trial successfully completed enrollment with a recommended phase-2 dose (R2PD) of sequential SG (10 mg/kg on days 1,8) with talazoparib (1 mg on days 15-21), every 21 days. Conclusion: Staggered dosing of SG and PARPi, leveraging the selective drug delivery mechanism of SG to minimize toxicity while maintaining efficacy, was feasible and demonstrated encouraging evidence of clinical activity with objective responses among patients with mTNBC. The translational study highlights how mechanistic insights and innovative scheduling could be utilized to develop promising drug combinations, including previously rejected combinations, for patients with mTNBC. Citation Format: Aditya Bardia, James T. Coates, Laura Spring, Sheng Sun, Dejan Juric, Nayana Thimmiah, Andrzej Niemierko, Phoebe Ryan, Ann Partridge, Jeffrey Peppercorn, Heather Parsons, Seth Wander, Kelsey Pierce, Victoria Attaya, Donna Fitzgerald, Brenda Lormil, Maria Shellock, Aiko Nagayama, Veerle Bossuyt, Bev Moy, Sara Tolaney, Leif Ellisen. Sacituzumab Govitecan, combination with PARP inhibitor, Talazoparib, in metastatic triple-negative breast cancer (TNBC): Translational investigation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2638.

TOP1-DNA Trapping by Exatecan and Combination Therapy with ATR Inhibitor.

Exatecan and deruxtecan are antineoplastic camptothecin derivatives in development as tumor-targeted-delivery warheads in various formulations including peptides, liposomes, polyethylene glycol nanoparticles, and antibody-drug conjugates. Here, we report the molecular pharmacology of exatecan compared with the clinically approved topoisomerase I (TOP1) inhibitors and preclinical models for validating biomarkers and the combination of exatecan with ataxia telangiectasia and Rad3-related kinase (ATR) inhibitors. Modeling exatecan binding at the interface of a TOP1 cleavage complex suggests two novel molecular interactions with the flanking DNA base and the TOP1 residue N352, in addition to the three known interactions of camptothecins with the TOP1 residues R364, D533, and N722. Accordingly, exatecan showed much stronger TOP1 trapping, higher DNA damage, and apoptotic cell death than the classical TOP1 inhibitors used clinically. We demonstrate the value of SLFN11 expression and homologous recombination (HR) deficiency (HRD) as predictive biomarkers of response to exatecan. We also show that exatecan kills cancer cells synergistically with the clinical ATR inhibitor ceralasertib (AZD6738). To establish the translational potential of this combination, we tested CBX-12, a clinically developed pH-sensitive peptide-exatecan conjugate that selectively targets cancer cells and is currently in clinical trials. The combination of CBX-12 with ceralasertib significantly suppressed tumor growth in mouse xenografts. Collectively, our results demonstrate the potency of exatecan as a TOP1 inhibitor and its clinical potential in combination with ATR inhibitors, using SLFN11 and HRD as predictive biomarkers.

EXTH-03. COMBINED TOP1 AND PARP INHIBITION ENFORCES GENOMIC INSTABILITY AND CELL DEATH IN PTEN-DEFICIENT GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma is an aggressive brain tumor with high mortality. The development of new therapies is critical for improving patient outcomes. LMP400, a novel topoisomerase I (TOP1) inhibitor, traps TOP1 cleavage complexes, thereby generating DNA damage. Poly(ADP-ribose) polymerase (PARP) is involved in DNA repair responses triggered by TOP1 inhibition. Niraparib is a potent PARP inhibitor that can cross the blood-brain barrier. Loss of phosphatase and tensin homolog (PTEN) occurs in 40% of GBM patients and is known to promote DNA damage repair deficiency. Here, we hypothesize that PTEN loss presents a vulnerability to a combined induction of DNA damage and inhibition of repair mechanisms. METHODS Human glioblastoma cells (U251, SNB-75, SF-295, LN18) and patient-derived glioblastoma stem cells (GSC923 and GSC827) were treated with LMP400 and/or Niraparib. Cell viability and apoptosis were examined using Celigo image cytometer and Annexin V/PI assay at 72h after treatment. Single clones after PTEN knockdown using shRNA were isolated after puromycin selection. For planned studies of PTEN knockout, sgRNA plasmids targeting PTEN will be transiently transfected and GFP-positive single KO clones will be isolated. PTEN will be restored in PTEN-null cells using lentiviral transduction. RESULTS CRISPR-Cas9 KO screening in GSC923 cells suggests that LMP400 is unlikely a substrate for ABC transporters. LMP400 and Niraparib synergistically induced cytotoxic effects in U251, SF-295, GSC923, GSC827 cells lacking PTEN expression. Combined LMP400/Niraparib led to increased expression of gamma-H2AX, cleaved caspase 3 and PARP, indicative of enhanced DNA damage and cell death. CONCLUSION LMP400 and Niraparib act synergistically to target PTEN-deficient glioblastoma by inducing DNA damage and cell death. These results will be further verified in isogenic cells in vitro as well as in vivo in a mouse model driven by PTEN deletion which would strongly support a novel therapeutic strategy in a subset of glioblastoma with PTEN loss.

The influence of an enamine usnic acid derivative (a tyrosyl-DNA phosphodiesterase 1 inhibitor) on the therapeutic effect of topotecan against transplanted tumors in vivo.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a repair enzyme for 3'-end DNA lesions, predominantly stalled DNA-topoisomerase 1 (Top1) cleavage complexes. Tdp1 is a promising target for anticancer therapy based on DNA damage caused by Top1 poisoning. Earlier, we have reported about usnic acid enamine derivatives that are Tdp1 inhibitors sensitizing tumor cells to the action of Top1 poison (Zakharenko in J Nat Prod 79:2961-2967, 2016). In the present work, we showed a sensitizing effect of an enamine derivative of usnic acid (when administered intragastrically) on Lewis lung carcinoma in mice in combination with topotecan (TPT, Top1 poison used in the clinic). In the presence of the usnic acid derivative, both the volume of the primary tumor and the number of metastases significantly diminished. The absence of acute toxicity of this compound was demonstrated, as was the importance of the method of its administration for the manifestation of the sensitizing properties.

Improved Antitumor Activity of the Fluoropyrimidine Polymer CF10 in Preclinical Colorectal Cancer Models through Distinct Mechanistic and Pharmacologic Properties.

Chemotherapy regimens that include 5-fluorouracil (5-FU) are central to colorectal cancer treatment; however, risk/benefit concerns limit 5-FU's use, necessitating development of improved fluoropyrimidine (FP) drugs. In our study, we evaluated a second-generation nanoscale FP polymer, CF10, for improved antitumor activity. CF10 was more potent than the prototype FP polymer F10 and much more potent than 5-FU in multiple colorectal cancer cell lines including HCT-116, LS174T, SW480, and T84D. CF10 displayed improved stability to exonuclease degradation relative to F10 and reduced susceptibility to thymidine antagonism due to extension of the polymer with arabinosyl cytidine. In colorectal cancer cells, CF10 strongly inhibited thymidylate synthase (TS), induced Top1 cleavage complex formation and caused replication stress, while similar concentrations of 5-FU were ineffective. CF10 was well tolerated in vivo and invoked a reduced inflammatory response relative to 5-FU. Blood chemistry parameters in CF10-treated mice were within normal limits. In vivo, CF10 displayed antitumor activity in several colorectal cancer flank tumor models including HCT-116, HT-29, and CT-26. CF10's antitumor activity was associated with increased plasma levels of FP deoxynucleotide metabolites relative to 5-FU. CF10 significantly reduced tumor growth and improved survival (84.5 days vs. 32 days; P < 0.0001) relative to 5-FU in an orthotopic HCT-116-luc colorectal cancer model that spontaneously metastasized to liver. Improved survival in the orthotopic model correlated with localization of a fluorescent CF10 conjugate to tumor. Together, our preclinical data support an early-phase clinical trial of CF10 for treatment of colorectal cancer.

AraC-FdUMP[10] Is a Next-Generation Fluoropyrimidine with Potent Antitumor Activity in PDAC and Synergy with PARG Inhibition.

AraC-FdUMP[10] (CF10) is a second-generation polymeric fluoropyrimidine that targets both thymidylate synthase (TS), the target of 5-fluorouracil (5-FU), and DNA topoisomerase 1 (Top1), the target of irinotecan, two drugs that are key components of FOLFIRNOX, a standard-of-care regimen for pancreatic ductal adenocarcinoma (PDAC). We demonstrated that F10 and CF10 are potent inhibitors of PDAC cell survival (in multiple cell lines including patient-derived lines) with IC50s in the nanomolar range and are nearly 1,000-fold more potent than 5-FU. The increased potency of CF10 relative to 5-FU correlated with enhanced TS inhibition and strong Top1 cleavage complex formation. Furthermore, CF10 displayed single-agent activity in PDAC murine xenografts without inducing weight loss. Through a focused drug synergy screen, we identified that combining CF10 with targeting the DNA repair enzyme, poly (ADP-ribose) glycohydrolase, induces substantial DNA damage and apoptosis. This work moves CF10 closer to a clinical trial for the treatment of PDAC. IMPLICATIONS: CF10 is a promising polymeric fluoropyrimidine with dual mechanisms of action (i.e., TS and Top1 inhibition) for the treatment of PDAC and synergizes with targeting of DNA repair. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/4/565/F1.large.jpg.

Top1-PARP1 association and beyond: from DNA topology to break repair.

Selective trapping of human topoisomerase 1 (Top1) on the DNA (Top1 cleavage complexes; Top1cc) by specific Top1-poisons triggers DNA breaks and cell death. Poly(ADP-ribose) polymerase 1 (PARP1) is an early nick sensor for trapped Top1cc. New mechanistic insights have been developed in recent years to rationalize the importance of PARP1 beyond the repair of Top1-induced DNA breaks. This review summarizes the progress in the molecular mechanisms of trapped Top1cc-induced DNA damage, PARP1 activation at DNA damage sites, PAR-dependent regulation of Top1 nuclear dynamics, and PARP1-associated molecular network for Top1cc repair. Finally, we have discussed the rationale behind the synergy between the combination of Top1 poison and PARP inhibitors in cancer chemotherapies, which is independent of the ‘PARP trapping’ phenomenon.

4-Benzylideneisoquinoline-1,3(2H,4H)-diones as tyrosyl DNA phosphodiesterase 2 (TDP2) inhibitors.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs topoisomerase II (Top2) mediated DNA damages, including double-strand breaks (DSBs) that underpin the anticancer mechanism of clinical TOP2 poisons such as etoposide (ETP). Inhibition of TDP2 could sensitize cancer cells toward TOP2 poisons by increasing Top2 cleavage complex. We have previously identified isoquinoline-1,3-dione as a selective inhibitor type of TDP2. However, the reported structure-activity relationship (SAR) was limited to simple substitutions on the isoquinoline-1,3-dione core. Herein, we report the extended SAR consisting of the synthesis and testing of a total of 50 analogs featuring N-2 and C-4 modifications. Major SAR observations include the loss of potency upon N-2 substitution, the lack of inhibition with C-4 enamine analogs (subtype 11), or any other C-4 modifications (subtypes 13-15) except for the benzylidene substitution (subtype 12), where eight analogs showed low micromolar potency. The best analog, 12q, inhibited TDP2 with an IC50 of 4.8 μM. Molecular modeling was performed to help understand the observed SAR trends. Overall, these SAR observations which could significantly benefit future work on the design of improved TDP2 inhibitors.

The Indenoisoquinoline LMP517: A Novel Antitumor Agent Targeting both TOP1 and TOP2.

The camptothecin derivatives topoisomerase I (TOP1) inhibitors, irinotecan and topotecan, are FDA approved for the treatment of colorectal, ovarian, lung and breast cancers. Because of the chemical instability of camptothecins, short plasma half-life, drug efflux by the multidrug-resistance ABC transporters, and the severe diarrhea produced by irinotecan, indenoisoquinoline TOP1 inhibitors (LMP400, LMP776, and LMP744), which overcome these limitations, have been developed and are in clinical development. Further modifications of the indenoisoquinolines led to the fluoroindenoisoquinolines, one of which, LMP517, is the focus of this study. LMP517 showed better antitumor activity than its parent compound LMP744 against H82 (small cell lung cancer) xenografts. Genetic analyses in DT40 cells showed a dual TOP1 and TOP2 signature with selectivity of LMP517 for DNA repair-deficient tyrosyl DNA phosphodiesterase 2 (TDP2)- and Ku70-knockout cells. RADAR assays revealed that LMP517, and to a lesser extent LMP744, induce TOP2 cleavage complexes (TOP2cc) in addition to TOP1ccs. Histone γH2AX detection showed that, unlike classical TOP1 inhibitors, LMP517 targets cells independently of their position in the cell cycle. Our study establishes LMP517 as a dual TOP1 and TOP2 inhibitor with therapeutic potential.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs topoisomerase 1 DNA-protein crosslinks and 3′-blocking lesions in the absence of tyrosyl-DNA phosphodiesterase 1 (TDP1)

Topoisomerase I (TOP1) resolves DNA topology during replication and transcription. The enzyme forms an intermediate TOP1 cleavage complex (TOP1cc) through transient TOP1–DNA-protein crosslinks. Camptothecin is a frontline anticancer agent that freezes this reaction intermediate, leading to the generation of irreversible TOP1ccs that act as 3′-blocking lesions. It is widely accepted that TOP1cc is repaired via a two-step pathway involving proteasomal degradation of TOP1cc to the crosslinked peptide, followed by removal of the TOP1cc-derived peptide from DNA by tyrosyl-DNA phosphodiesterase 1 (TDP1). In the present study, we developed an assay system to estimate repair kinetics of TOP1cc separately in the first and second steps, using monoclonal antibodies against the TOP1 protein and the TOP1 catalytic site peptide–DNA complex, respectively. Although TDP1-deficient (TDP1−/−) TK6 cells had normal kinetics of the first step, a delay in the kinetics of the second step was observed relative to that in wild-type cells. Tyrosyl-DNA phosphodiesterase 2 (TDP2) reportedly promotes the repair of TOP1-induced DNA damage in the absence of TDP1. The present assays additionally demonstrated that TDP2 promotes the second, but not the first, step of TOP1cc repair in the absence of TDP1. We also analyzed sensitivities of TK6 cells with deficiencies in TDP1 and/or TDP2 to agents that produce 3′ -blocking lesions. These experiments showed that TDP1−/−TDP2−/− cells were more sensitive to the agents Azidothymidine (zidovudine), Cytarabine, Abacavir, Gemcitabine, and Trifluridine than TDP1−/− or TDP2−/− cells. Taken together, our findings confirm the roles of TDP2 in the repair of 3′-blocking lesions.

Suppressing proteasome mediated processing of topoisomerase II DNA-protein complexes preserves genome integrity.

Topoisomerase II (TOP2) relieves topological stress in DNA by introducing double-strand breaks (DSBs) via a transient, covalently linked TOP2 DNA-protein intermediate, termed TOP2 cleavage complex (TOP2cc). TOP2ccs are normally rapidly reversible, but can be stabilized by TOP2 poisons, such as the chemotherapeutic agent etoposide (ETO). TOP2 poisons have shown significant variability in their therapeutic effectiveness across different cancers for reasons that remain to be determined. One potential explanation for the differential cellular response to these drugs is in the manner by which cells process TOP2ccs. Cells are thought to remove TOP2ccs primarily by proteolytic degradation followed by DNA DSB repair. Here, we show that proteasome-mediated repair of TOP2cc is highly error-prone. Pre-treating primary splenic mouse B-cells with proteasome inhibitors prevented the proteolytic processing of trapped TOP2ccs, suppressed the DNA damage response (DDR) and completely protected cells from ETO-induced genome instability, thereby preserving cellular viability. When degradation of TOP2cc was suppressed, the TOP2 enzyme uncoupled itself from the DNA following ETO washout, in an error-free manner. This suggests a potential mechanism of developing resistance to topoisomerase poisons by ensuring rapid TOP2cc reversal.

Molecular mechanisms of topoisomerase 2 DNA-protein crosslink resolution.

The compaction of DNA and the continuous action of DNA transactions, including transcription and DNA replication, create complex DNA topologies that require Type IIA Topoisomerases, which resolve DNA topological strain and control genome dynamics. The human TOP2 enzymes catalyze their reactions via formation of a reversible covalent enzyme DNA-protein crosslink, the TOP2 cleavage complex (TOP2cc). Spurious interactions of TOP2 with DNA damage, environmental toxicants and chemotherapeutic "poisons" perturbs the TOP2 reaction cycle, leading to an accumulation of DNA-protein crosslinks, and ultimately, genomic instability and cell death. Emerging evidence shows that TOP2-DNA protein crosslink (DPC) repair entails multiple strand break repair activities, such as removal of the poisoned TOP2 protein and rejoining of the DNA ends through homologous recombination (HR) or non-homologous end joining (NHEJ). Herein, we discuss the molecular mechanisms of TOP2-DPC resolution, with specific emphasis on the recently uncovered ZATTZnf451-licensed TDP2-catalyzed TOP2-DPC reversal mechanism.

Targeting Topoisomerase I in the Era of Precision Medicine.

Irinotecan and topotecan have been widely used as anticancer drugs for the past 20 years. Because of their selectivity as topoisomerase I (TOP1) inhibitors that trap TOP1 cleavage complexes, camptothecins are also widely used to elucidate the DNA repair pathways associated with DNA-protein cross-links and replication stress. This review summarizes the basic molecular mechanisms of action of TOP1 inhibitors, their current use, and limitations as anticancer agents. We introduce new therapeutic strategies based on novel TOP1 inhibitor chemical scaffolds including the indenoisoquinolines LMP400 (indotecan), LMP776 (indimitecan), and LMP744, and on tumor-targeted delivery TOP1 inhibitors using liposome, PEGylation, and antibody-drug conjugates. We also address how tumor-specific determinants such as homologous recombination defects (HRD and BRCAness) and Schlafen 11 (SLFN11) expression can be used to guide clinical application of TOP1 inhibitors in combination with DNA damage response inhibitors including PARP, ATR, CHEK1, and ATM inhibitors.