Topoisomerase Research Articles

Abstract B016: Liquid biopsy biomarker analysis of a phase II trial of sacituzumab govitecan in castrate resistant metastatic prostate cancer

Abstract Background: Sacituzumab Govitecan (SG) is an antibody-drug conjugate targeting Trop-2 and has been approved for use in breast and bladder cancers. Trop-2 has been shown to be overexpressed in multiple solid tumors including prostate cancer. Here we report an interim analysis of longitudinal liquid biopsies collected in a phase II clinical trial of SG to identify biomarkers of response and resistance. Methods: We collected longitudinal blood samples from 20 patients for circulating tumor cell (CTC) and cell-free DNA (cfDNA) analysis as part of an open label Phase II trial of SG in patients with mCRPC (ClinicalTrials.gov Identifier: NCT03725761). Eligibility criteria for the trial included rising PSA and radiographic progression on abiraterone acetate or enzalutamide in the mCRPC setting. Liquid biopsies were collected at baseline, C1D8, and then every 3 cycles until disease progression. cfDNA was sequenced using a custom 822 gene targeted capture panel (IDT). CTCs were isolated with antibodies to either EpCAM or Trop-2 followed by parallel enumeration, EpCAM and Trop-2 protein phenotyping and RNA extraction for AR/NEPC marker transcriptional profiling. Results: Although no PSA50 responses were observed, the 6-month rPFS rate was 45% (64% in chemotherapy naïve patients). For this analysis, these patients will be termed responding patients. The number of Trop-2-postive CTCs at baseline was significantly higher in responding (24.3 ±13.4 per 7.5 mL blood) than non-responding (10.0±3.8) patients. During treatment the number of Trop-2 positive CTCs in responders significantly decreased by C3D1 to 10.0 ±3.8. Interestingly, while the number of Trop-2 positive CTCs continued to decline to 2.8±1.99 cells/7.5 mL, EpCAM-positive CTCs increased from 12.5±10.52 cells/7.5 mL to 23.2±19.49 at end of trial. Gene expression analysis demonstrated high expression of genes specific to adenocarcinoma including AR-splice variants, TMPRSS2, KLK2 and KLK3 in the majority of patients, with conversion to AR-variant negative CTCs identified in responders. Preliminary cfDNA analysis identified one patient with a mutation acquired in the gene encoding topoisomerase 1 (TOP1) upon progression on SG, with additional analysis ongoing. Conclusions: Patients who responded to SG had a more Trop-2 positive CTCs with adenocarcinoma characteristics with expression of AR pathway genes. Longitudinal analysis of liquid biopsy biomarkers identified multiple mechanisms of acquired SG resistance. Decline in Trop2-CTC population with parallel expansion of EpCAM-CTC population in multiple patients suggests loss of Trop2 surface protein expression as resistance mechanism, while identification of a cfDNA TOP1 mutation in one patient at time of progression is consistent with acquired resistance to the topoisomerase inhibitor payload as has been reported in metastatic triple negative breast cancer. Citation Format: Jamie M. Sperger, Amy K. Taylor, Yue Shi, Charlotte N. Stahlfeld, Kyle Helzer, Matthew Bootsma, Katherine R. Kaufmann, Marina N. Sharifi, Christos E. Kyriakopoulos, Susan Slovin, Scott Tagawa, Scott Dehm, Shuang G. Zhao, Joshua M. Lang. Liquid biopsy biomarker analysis of a phase II trial of sacituzumab govitecan in castrate resistant metastatic prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B016.

Phase 1 study of indenoisoquinoline LMP744 in adults with relapsed solid tumors and lymphomas.

3022 Background: Indenoisoquinolines (IIQ) are non-camptothecin inhibitors of topoisomerase 1 (TOP1) that have improved characteristics over camptothecin TOP1 inhibitors. IIQs demonstrate better chemical stability, produce more persistent DNA breaks induced by trapping DNA-TOP1 cleavage complexes, induce DNA breaks at different sites than camptothecins, and are not substrates for cellular export proteins. We present data from a trial of LMP744 (NSC 706744), which was the most efficacious IIQ in a canine lymphoma trial, designed to establish the safety, efficacy, and maximum tolerated dose (MTD) of LMP744. Methods: We conducted a multicenter phase 1 trial in adult patients (pts) with dose escalations following design 3 of the Simon accelerated titration designs. LMP744 was administered intravenously daily on d1-5 in 28-day cycles (C), starting at dose level (DL) 1 (6 mg/m2/d). Responses and adverse events (AEs) were defined by RECIST 1.1 and CTCAE v4.0, respectively. Plasma concentrations were determined by LC-MS/MS with a validated assay over a range of 1-3,000 ng/mL. Paired biopsies (baseline and C1D2) were analyzed for DNA damage response markers (γH2AX, pNBS1, and Rad51) by IFA. Results: A total of 36 pts were enrolled, median age was 61 years (range 35-81), median prior lines of systemic treatment was 5 (range 2-10) and 26 pts (72%) had received prior TOP1 inhibitor therapy. One pt on DL6 (190 mg/m2/d) experienced a dose-limiting toxicity (DLT) of grade 3 hypokalemia during C1, reverting the study to a 3+3 design. MTD was established at DL6. Of 24 patients evaluable for response, 1 pt with small cell lung cancer on DL6 achieved a partial response (PR), remaining on treatment for 7C. 17 pts had stable disease (including 1 pt with an unconfirmed PR), and 6 had progressive disease. Median duration of response among pts with SD was 4.5C (range 1-28). A total of 6 pts on DL6 with colorectal cancer (n=4, all had received prior irinotecan), mesothelioma (n=1), and low-grade appendiceal mucinous neoplasm (n=1) remained on study for >6C. Treatment-related grade 3/4 AEs included leukopenia (n=9), anemia (n=4), dehydration (n=2), neutropenia (n=2) and nausea (n=2). Pharmacokinetic (PK) data for the first 17 patients suggests dose-linear PK, best fit to a 2-compartment model. Approximate half-life, clearance, central and peripheral volume values were 30 h, 90 L/h, 200 L, and 2500 L, respectively. Induction of γH2AX and Rad51 was measured in the pt with PR. Conclusions: The MTD of LMP744 was established at 190 mg/m2/d on d1-5 in a 28d C. The single agent activity of LMP744 in this heavily pretreated population was limited; however, 4 colorectal cancer pts had prolonged stabilization of disease with prior progression on irinotecan. Analyses of secondary and exploratory correlatives are ongoing. Clinical trial information: NCT03030417 .

CBX-12-101: A first-in-human study of CBX-12, an alphalex peptide drug conjugate (PDC) in patients (pts) with advanced or metastatic solid tumors.

3087 Background: Tumor-targeted drug delivery technologies are urgently needed to overcome conventional chemotherapy’s lack of tumor selectivity. Alphalex conjugates, which contain a pH-low insertion peptide (pHLIP), a linker and a payload, are designed to overcome this lack of selectivity. Unlike antibody drug conjugates, alphalex PDCs target tumors in an antigen-agnostic manner. At pH ≥7.0, the peptide is unstructured. In the low-pH tumor microenvironment the peptide forms an alpha helix and inserts directionally into the tumor cell membrane delivering the linker and payload intracellularly where the linker is cleaved releasing the payload in the cytosol. CBX-12 consists of the pH-sensitive peptide, a self-immolating linker, and the topoisomerase 1 (TOP1) inhibitor exatecan. Methods: In this Phase 1 trial, cohorts of pts were treated with escalating doses of CBX-12 in a 3 + 3 design on three dosing schedules: daily x 5 every 3 weeks, daily x 3 every 3 weeks or once weekly. The primary objectives are safety, tolerability and to determine the MTD and/or RP2D. Secondary and exploratory objectives include evaluation of plasma PK of CBX-12 and exatecan, anti-tumor activity per RECIST v1.1, and measurement of anti-drug antibodies. Results: As of 26 Jan 2023, 42 pts have been treated on three schedules. The most frequent treatment-related AEs (TRAEs) were anaemia (42.9%), nausea (38.1%), fatigue (33.3%), ANC and WBC decreased (33.3% each), vomiting (26.2%), diarrhoea (23.8%) and thrombocytopenia (19.0%). The most frequent Gr3/4 TRAEs were ANC decreased (28.6%), anemia (23.8%), WBC decrease (16.7%), thrombocytopenia (14.3%). Dose limiting toxicity was observed in 6 pts: myelosuppression (6 pts) febrile neutropenia (2 pts), and sepsis (2 pts). Responses were observed in 2 of 9 pts with ovarian cancer and 2 of 8 with breast cancer. The MTD on the daily x 3 schedule is 45mg/m2. Dose evaluation continues on the weekly schedule. Conclusions: In this FIH study of a pH-targeting alphalex PDC, CBX-12 demonstrated single-agent antitumor activity including 4 responses with the dominant toxicity of myelosuppression. Phase 2 expansion cohorts are planned in pts with platinum-resistant ovarian cancer and hormone-receptor positive, HER2 negative breast cancer. Clinical trial information: NCT04902872 .

Preclinical antitumor activity of an anti-HER2/Trop-2 bispecific antibody-drug conjugate with a new DNA topoisomerase I inhibitor.

e15013 Background: In recent years, antibody-drug conjugates (ADCs) have made significant progress in cancer therapy and show a promising future. Among the approved ADCs, Enhertu, the HER2 directed ADC and Trodelvy, the Trop-2 directed ADC, have shown considerable clinical benefits. Bispecific antibody-drug conjugate represents a fast-growing class of next generation ADC due to its potentials in enhancing specificity, improving efficacy, and reducing toxicity. Additionally, targeting more than one molecule can be useful to avoid resistance to treatment and broaden the patient population. Methods: We generated a novel bispecific ADC (BIO-201) co-targeting HER2 and Trop-2 which are the two clinically validated tumor-associated antigens that are expressed on a wide variety of tumors. BIO-201 is consisted of a novel bispecific anti-HER2/Trop-2 antibody conjugated with a potent DNA topoisomerase I inhibitor (TOPO Ii) via a cleavable linker. TOPO Ii is a proprietary payload platform developed in house that exhibits similar characteristics including in vitro and in vivo anti-tumor activities and safety profile to DXd. The bispecific antibody was fully characterized and in vitro and in vivo anti-tumor activity of BIO-201 was assessed using preclinical models. Results: The bispecific antibody showed comparable or increased cell binding and internalization compared with the parental antibodies in various tumor cells that express different surface levels of HER2 and Trop-2. In the in vitro cytotoxicity assay, the bispecific ADC BIO-201 effectively killed cancer cells with IC50 in the sub nM range with BIO-201 being most potent against cancer cells that co-express HER2 and Trop-2. The anti-tumor activity of BIO-201 was further demonstrated in vivo in several tumor xenograft models. Treatment of BIO-201 induced significant tumor regression in HER2 or Trop-2 positive tumors, suggesting broader coverage of tumor types than conventional HER2 and Trop-2 ADCs. BIO-201 also showed similar anti-tumor activity to Herceptin-DXd. Conclusions: Taken together, these findings suggest that anti-HER2/Trop-2 bispecific ADC has potential as an effective therapy for a variety of cancers that co-express HER2/Trop-2 or express either of the targets. BIO-201 may be expected to have a similar clinical activity to Enhertu or Trodelvy but treat more patient sub-populations and indications.

MiR-30c-2-3p suppresses the proliferation of human renal cell carcinoma cells by targeting TOP2A.

The ambiguity of renal cell carcinoma (RCC) symptoms hinders early diagnosis, thereby contributing to high mortality rates. By attaching to the 3'-untranslated region (UTR) of the target gene, microRNAs (miRNAs) exert significant control over the expression of genes. To investigate the influence of miR-30c-2-3p and DNA topoisomerase II alpha (TOP2A) on RCC growth and the mechanisms underlying the regulation of its expression. The expression of miRNA-30c-2-3p and TOP2A in RCC cells was examined using quantitative real-time polymerase chain reaction (qRT-PCR). MiR-30c-2-3p mimics, its inhibitors, and controls, as well as TOP2A short hairpin RNA (shRNA) and controls, were used to transfect the human RCC cell lines 786-O, Caki-1, and ACHN. Additionally, the roles of miRNA-30c-2-3p and TOP2A in the growth of RCC were evaluated using the cell counting kit (CCK)-8 test, colony formation assay, apoptosis analysis, and Western blotting. Meanwhile, binding of miRNA-30c-2-3p and TOP2A was verified using dual-luciferase reporter assays and Western blotting. miR-30c-2-p is underexpressed in RCC cells. Overexpression of miR-30c-2-p promotes apoptosis and inhibits proliferation of ACHN, Caki-1, and 786-O cells. miR-30c-2-3p targets TOP2A, which is elevated in RCC tissues and cells, whereas TOP2A silencing inhibits the proliferation ability of RCC cells. The miRNA-30c-2-3p inhibitor compromises TOP2A shRNA-induced apoptosis of RCC. RCC cells cotransfected with miRNA-30c-2-3p inhibitors and TOP2A shRNAs have a higher proliferation rate than those transfected with only TOP2A shRNAs. Collectively, our results verify that miRNA-30c-2-3p has a tumor suppressor property. miRNA-30c-2-3p inhibits the proliferation of RCC through regulation of TOP2A. The data provide a viable therapeutic target for RCC.

Fluoroquinolone resistance does not facilitate phage Φ13 integration or excision in Staphylococcus aureus.

Prophages of the ΦSa3int family are commonly found in human-associated strains of Staphylococcus aureus where they encode factors for evading the human innate immune system. In contrast, they are usually absent in livestock-associated methicillin-resistant S. aureus (LA-MRSA) strains where the phage attachment site is mutated compared to the human strains. However, ΦSa3int phages have been found in a subset of LA-MRSA strains belonging to clonal complex 398 (CC398), including a lineage that is widespread in pig farms in Northern Jutland, Denmark. This lineage contains amino acid changes in the DNA topoisomerase IV and the DNA gyrase encoded by grlA and gyrA, respectively, which have been associated with fluoroquinolone (FQ) resistance. As both of these enzymes are involved in DNA supercoiling, we speculated that the mutations might impact recombination between the ΦSa3int phage and the bacterial chromosome. To examine this, we introduced the FQ resistance mutations into S. aureus 8325-4attBLA that carry the mutated CC398-like bacterial attachment site for ΦSa3int phages. When monitoring phage integration and release of Φ13, a well-described representative of the ΦSa3int phage family, we did not observe any significant differences between the FQ-resistant mutant and the wild-type strain. Thus our results suggest that mutations in grlA and gyrA do not contribute to the presence of the ΦSa3int phages in LA-MRSA CC398.

Safety and efficacy of DB-1303 in patients with advanced/metastatic solid tumors: A multicenter, open-label, first-in-human, phase 1/2a study.

3023 Background: DB-1303 is an antibody-drug conjugate (ADC) consisting of a humanized anti-HER2 IgG1 monoclonal antibody, covalently linked to proprietary DNA topoisomerase I inhibitor (P1003) via a maleimide tetrapeptide-based cleavable linker, with a high drug-to-antibody ratio (~8). Methods: This is a global first-in-human, dose-escalation and -expansion study in patients (pts) with advanced/metastatic solid tumors. Pts (ECOG 0-1) with HER2 – (high or low) expressing or mutant cancers who failed previously systemic therapies were recruited and received DB-1303 intravenously Q3W as monotherapy. The objectives were safety, tolerability, maximum tolerated dose (MTD) or recommended phase 2 dose (PR2D), pharmacokinetics (PK), and preliminary antitumor activity. Here we report the results from dose-escalation. Results: As of Jan 13, 2023, 85 pts received DB-1303 at 6 dose levels (2.2, 4.4, 6.0, 7.0, 8.0, and 10.0 mg/kg) and received a median of 7 (range, 1-27) prior lines of therapy, including previous anti-HER2 ADC therapy in 32.9% of pts. The median duration of treatment was 63.0 (range, 21-211) days, and 68 pts (80.0%) remained on treatment. Treatment-emergent adverse events (TEAEs) and ≥ grade (G) 3 TEAEs occurred in 74 pts (87.1%) and 18 pts (21.2%), respectively; the most common TEAEs were nausea (51.8%, 3.5% ≥ G3), vomiting (43.5%, 1.2% ≥ G3), platelet count decreased (35.3%, 3.5% ≥ G3), and anemia (29.4%, 5.9% ≥ G3). Few pts experienced neutropenia (10 [11.8%]) and alopecia (3 [3.5%]). There was no DLT or TEAEs leading to death. Interstitial lung disease occurred in 2 pts (2.4%, G1), without any ≥ G2. The exposure parameters (Cmax and AUC) of DB-1303 ADC increased with ascending doses from 2.2 to 10.0 mg/kg. The half-life of DB-1303 ADC is approximately 6-7 days for 6.0-8.0 mg/kg dose cohorts. The exposure of serum release payload was magnitudes lower than that of DB-1303 ADC, demonstrating stability of the ADC in systemic circulation. A total of 52 pts had undergone at least one post-baseline tumor scan. Twenty-three pts (44.2%, 23/52) had objective partial tumor response per RECIST 1.1: Thirteen HER2+ breast cancer (BC) (50.0%, 13/26, including 5 pts with brain metastases [55.6%, 5/9]), 5 HER2 low BC (38.5%, 5/13), 2 colorectal cancer (66.7%, 2/3), 1 endometrial carcinoma (33.3%, 1/3), 1 esophageal cancer (50.0%, 1/2), and 1 ovarian cancer (50.0%, 1/2). Among all the pts, the DCR was 88.5% (46/52); for pts with HER2+ BC and HER2-low BC, the DCRs were 96.2% (25/26) and 84.6% (11/13), respectively. Conclusions: DB-1303 was well tolerated with encouraging preliminary antitumor activity in heavily pretreated pts with advanced/metastatic solid tumors, especially in pts with HER2+ BC and brain metastasis as well as in HER2-low BC. Expansion is ongoing in selected tumor pts treated at the RP2D. Clinical trial information: NCT05150691 .

O-GlcNAcylation promotes topoisomerase IIα catalytic activity in breast cancer chemoresistance.

DNA topoisomerase IIα (TOP2A) plays a vital role in replication and cell division by catalytically altering DNA topology. It is a prominent target for anticancer drugs, but clinical efficacy is often compromised due to chemoresistance. In this study, we investigate the role of TOP2A O-GlcNAcylation in breast cancer cells and patient tumor tissues. Our results demonstrate that elevated TOP2A, especially its O-GlcNAcylation, promotes breast cancer malignant progression and resistance to adriamycin (Adm). O-GlcNAcylation at Ser1469 enhances TOP2A chromatin DNA binding and catalytic activity, leading to resistance to Adm in breast cancer cells and xenograft models. Mechanistically, O-GlcNAcylation-modulated interactions between TOP2A and cell cycle regulators influence downstream gene expression and contribute to breast cancer drug resistance. These results reveal a previously unrecognized mechanistic role for TOP2A O-GlcNAcylation in breast cancer chemotherapy resistance and provide support for targeting TOP2A O-GlcNAcylation in cancer therapy.

Synthesis, Characterization and in vitro Anticancer Evaluation of 5-Sulfinyl(sulfonyl)-4-arylsulfonyl-Substituted 1,3-Oxazoles.

A novel series of 5-sulfinyl(sulfonyl)-4-arylsulfonyl-substituted 1,3-oxazoles has been synthesized, characterized and subjected to NCI in vitro assessment. Cancer cell lines of all subpanels were most sensitive to 2-{[4-[(4-fluorophenyl)sulfonyl]-2-(2-furyl)-1,3-oxazol-5-yl]sulfinyl}acetamide (3 l). Its antiproliferative and cytotoxic activity averaged over each subpanel was manifested in a very narrow range of concentrations (GI50 : 1.64-1.86 μM, TGI: 3.16-3.81 μM and LC50 : 5.53-7.27 μM), i. e. practically did not depend on the origin of the cancer cell line. The COMPARE matrix using TGI vector showed a high positive correlation of 3 l (r=0.88) with the intercalating agent aclarubicin, which inhibits topoisomerases. The absence in the database of standard agents that have a high correlation with the cytotoxicity of this compound suggests that it may have a unique mechanism of action. According to the results of the docking analysis, the most promising anticancer target for compound 3 l is DNA topoisomerase IIβ. The obtained results indicate the anticancer activity of 5-sulfinyl(sulfonyl)-4-arylsulfonyl-substituted 1,3-oxazoles, which may be useful for the development of new anticancer drugs. 2-{[4-[(4-Fluorophenyl)sulfonyl]-2-(2-furyl)-1,3-oxazol-5-yl]sulfinyl}acetamide (3 l), as the most active, can be recommended for further in-depth studies.

Exploring Alternative Pathways to Target Bacterial Type II Topoisomerases Using NBTI Antibacterials: Beyond Halogen-Bonding Interactions.

Novel bacterial topoisomerase inhibitors (NBTIs) are a new class of antibacterial agents that target bacterial type II topoisomerases (DNA gyrase and topoisomerase IV). Our recently disclosed crystal structure of an NBTI ligand in complex with DNA gyrase and DNA revealed that the halogen atom in the para position of the phenyl right hand side (RHS) moiety is able to establish strong symmetrical bifurcated halogen bonds with the enzyme; these are responsible for the excellent enzyme inhibitory potency and antibacterial activity of these NBTIs. To further assess the possibility of any alternative interactions (e.g., hydrogen-bonding and/or hydrophobic interactions), we introduced various non-halogen groups at the p-position of the phenyl RHS moiety. Considering the hydrophobic nature of amino acid residues delineating the NBTI's binding pocket in bacterial topoisomerases, we demonstrated that designed NBTIs cannot establish any hydrogen-bonding interactions with the enzyme; hydrophobic interactions are feasible in all respects, while halogen-bonding interactions are apparently the most preferred.

Single-cell transcriptome analysis reveals development atlas of mouse molar pulp cells

Objective: Single-cell RNA sequencing (scRNA-seq) was used to analyze the developing mouse molars, in order to construct a spatiotemporal development atlas of pulp cells, and further to reveal the developmental process and regulatory mechanism of tooth development. Methods: Ten mandibular first molars from C57BL/6 mice in postnatal day (PN) 0 and 3 were respectively dissected and digested to obtain single-cell suspensions. scRNA-seq was performed on 10× Genomics platform. PN 7 mouse molar scRNA-seq data were obtained from our previous study. PN 0, 3, and 7 scRNA-seq data were integrated for following analysis. The initial quality control, mapping and single cell expression matrix construction were performed by Cell Ranger. Quality control, standardization, dimensional reduction and cluster analysis were performed by using Seurat. Monocle was used to generate the pseudotime trajectory. Scillus was used to perform gene ontology analysis. In order to detect the spatiotemporal change of different population of pulp cells, the marker genes of each cluster were demonstrated by RNAscope in situ hybridization. Results: There were twenty-six cell clusters within mouse molars, which were identified as eight different cell types, including dental pulp cells, dental follicle cells, epithelial cells, immune cells, endothelial cells, perivascular cells, glial cells and erythrocytes. We further re-clustered and analyzed dental pulp cells. Cluster 0 were mature pulp cells, which located at the upper portion of crown. The main functions of cluster 0 were osteogenesis and extracellular structure organization. Cluster 1 were apical papilla cells, which located at the apical part of roots, whose main functions were extracellular structure organization and organ development. Cluster 2 were cycling cells, which were actively proliferated, resided in the lower portion of the crown. Cluster 3 and 4 were preodontoblasts and odontoblasts, respectively. Their functions were closely related to biomineralization. The proportion of mature pulp cells increased with the development process, while the proportion of cycling cells and odontoblast lineage decreased. According to the expression pattern of marker genes of each cluster, we constructed a cell atlas of dental pulp. Pseudotime trajectory analysis found there were two development trajectories within dental pulp. They both started from SPARC related modular calcium binding 2 (Smoc2)+ dental papilla cells, then went through DNA topoisomerase Ⅱ alpha (Top2a)+ cycling cells, and finally divided into coxsackie virus and adenovirus receptor (Cxadr)+ mature pulp cells or dentin sialophosphoprotein (Dspp)+ odontoblasts two lineages. Conclusions: scRNA-seq could fully discover the intercellular heterogeneity of cells on transcriptome level, which provides a powerful tool to study the process and regulatory mechanism of organ development.

Recent Developments in Combination Chemotherapy for Colorectal and Breast Cancers with Topoisomerase Inhibitors.

DNA topoisomerases are important enzymes that stabilize DNA supercoiling and resolve entanglements. There are two main types of topoisomerases in all cells: type I, which causes single-stranded DNA breaks, and type II, which cuts double-stranded DNA. Topoisomerase activity is particularly increased in rapidly dividing cells, such as cancer cells. Topoisomerase inhibitors have been an effective chemotherapeutic option for the treatment of several cancers. In addition, combination cancer therapy with topoisomerase inhibitors may increase therapeutic efficacy and decrease resistance or side effects. Topoisomerase inhibitors are currently being used worldwide, including in the United States, and clinical trials on the combination of topoisomerase inhibitors with other drugs are currently underway. The primary objective of this review was to comprehensively analyze the current clinical landscape concerning the combined application of irinotecan, an extensively investigated type I topoisomerase inhibitor for colorectal cancer, and doxorubicin, an extensively researched type II topoisomerase inhibitor for breast cancer, while presenting a novel approach for cancer therapy.

DNA Sequence-Specific Ligands. XIX. Synthesis, Spectral Properties, Virological and Biochemical Studies of Fluorescent Dimeric Trisbenzimidazoles DB<sub>3</sub>(<i>n</i>)

In this work, we synthesized and characterized the properties of a series of new fluorescent narrow-groove ligands DB3(n). DB3(n) compounds based on dimeric trisbenzimidazoles have the ability to bind to the AT regions of DNA. The synthesis of DB3(n), trisbenzimidazole fragments of which are linked by oligomethylene linkers of different lengths (n = 1, 5, 9), is based on the condensation of monomeric trisbenzimidazole MB3 with α,ω-alkyldicarboxylic acids. DB3(n) proved to be effective inhibitors of the catalytic activity of HIV-1 integrase at submicromolar concentrations (0.20–0.30 µM). DB3(n) was found to inhibit the catalytic activity of DNA topoisomerase I at low micromolar concentrations.

Gel-Free Tools for Quick and Simple Screening of Anti-Topoisomerase 1 Compounds.

With the increasing need for effective compounds against cancer or pathogen-borne diseases, the development of new tools to investigate the enzymatic activity of biomarkers is necessary. Among these biomarkers are DNA topoisomerases, which are key enzymes that modify DNA and regulate DNA topology during cellular processes. Over the years, libraries of natural and synthetic small-molecule compounds have been extensively investigated as potential anti-cancer, anti-bacterial, or anti-parasitic drugs targeting topoisomerases. However, the current tools for measuring the potential inhibition of topoisomerase activity are time consuming and not easily adaptable outside specialized laboratories. Here, we present rolling circle amplification-based methods that provide fast and easy readouts for screening of compounds against type 1 topoisomerases. Specific assays for the investigation of the potential inhibition of eukaryotic, viral, or bacterial type 1 topoisomerase activity were developed, using human topoisomerase 1, Leishmania donovani topoisomerase 1, monkeypox virus topoisomerase 1, and Mycobacterium smegmatis topoisomerase 1 as model enzymes. The presented tools proved to be sensitive and directly quantitative, paving the way for new diagnostic and drug screening protocols in research and clinical settings.

An Insightful Evaluation of Evodiamine Analogs Effect as DNA Topoisomerase I Inhibitors Using QSAR Method

An Insightful Evaluation of Evodiamine Analogs Effect as DNA Topoisomerase I Inhibitors Using QSAR Method

Methylated histones on mitotic chromosomes promote topoisomerase IIα function for high fidelity chromosome segregation

DNA Topoisomerase IIα (TopoIIα) decatenates sister chromatids, allowing their segregation in mitosis. Without the TopoIIα Strand Passage Reaction (SPR), chromosome bridges and ultra-fine DNA bridges (UFBs) arise in anaphase. The TopoIIα C-terminal domain is dispensable for the SPR invitro but essential for mitotic functions invivo. Here, we present evidence that the Chromatin Tether (ChT) within the CTD interacts with specific methylated nucleosomes and is crucial for high-fidelity chromosome segregation. Mutation of individual αChT residues disrupts αChT-nucleosome interaction, induces loss of segregation fidelity and reduces association of TopoIIα with chromosomes. Specific methyltransferase inhibitors reducing histone H3 or H4 methylation decreased TopoIIα at centromeres and increased segregation errors. Methyltransferase inhibition did not further increase aberrant anaphases in the ChT mutants, indicating a functional connection. The evidence reveals novel cellular regulation whereby TopoIIα specifically interacts with methylated nucleosomes via the αChT to ensure high-fidelity chromosome segregation.

Preclinical validation of a novel therapeutic strategy for choroid plexus carcinoma

Choroid plexus carcinoma (CPC) is a rare infantile brain tumor with an aggressive clinical course that often leaves children with debilitating side effects due to aggressive and toxic chemotherapies. Development of novel therapeutical strategies for this disease have been extremely limited owing to the rarity of the disease and the paucity of biologically relevant substrates. We conducted the first high-throughput screen (HTS) on a human patient-derived CPC cell line (Children Cancer Hospital Egypt, CCHE-45) and identified 427 top hits highlighting key molecular targets in CPC. Furthermore, a combination screen with a wide variety of targets revealed multiple synergistic combinations that may pave the way for novel therapeutical strategies against CPC. Based on in vitro efficiency, central nervous system (CNS) penetrance ability and feasible translational potential, two combinations using a DNA alkylating or topoisomerase inhibitors in combination with an ataxia telangiectasia mutated and rad3 (ATR) inhibitor (topotecan/elimusertib and melphalan/elimusertib respectively) were validated in vitro and in vivo. Pharmacokinetic assays established increased brain penetrance with intra-arterial (IA) delivery over intra-venous (IV) delivery and demonstrated a higher CNS penetrance for the combination melphalan/elimusertib. The mechanisms of synergistic activity for melphalan/elimusertib were assessed through transcriptome analyses and showed dysregulation of key oncogenic pathways (e.g. MYC, mammalian target of rapamycin mTOR, p53) and activation of critical biological processes (e.g. DNA repair, apoptosis, hypoxia, interferon gamma). Importantly, IA administration of melphalan combined with elimusertib led to a significant increase in survival in a CPC genetic mouse model. In conclusion, this study is, to the best of our knowledge, the first that identifies multiple promising combinatorial therapeutics for CPC and emphasizes the potential of IA delivery for the treatment of CPC.

Autoantibodies against DNA topoisomerase I promote renal allograft rejection by increasing alloreactive T cell responses

Antibodies reactive to self-antigens are an important component of posttransplant immune responses. The generation requirements and functions of autoantibodies, as well as the mechanisms of their influence on alloimmune responses, still remain to be determined. Our study investigated the contribution of autoimmunity during rejection of renal allografts. We have previously characterized a mouse model in which the acute rejection of a life-supporting kidney allograft is mediated by antibodies. At rejection, recipient sera screening against >4000 potential autoantigens revealed DNA topoisomerase I peptide 205-219 (TI-I205-219) as the most prominent epitope. Subsequent analysis showed TI-I205-219-reactive autoantibodies are induced in nonsensitized recipients of major histocompatibility complex–mismatched kidney allografts in a T cell–dependent manner. Immunization with TI-I205-219 broke self-tolerance, elicited TI-I205-219 immunoglobin G autoantibodies, and resulted in acute rejection of allogeneic but not syngeneic renal transplants. The graft loss was associated with increased priming of donor-reactive T cells but not with donor-specific alloantibodies elevation. Similarly, passive transfer of anti-TI-I205-219 sera following transplantation increased donor-reactive T cell activation with minimal effects on donor-specific alloantibody levels. The results identify DNA topoisomerase I as a novel self-antigen in transplant settings and demonstrate that autoantibodies enhance activation of donor-reactive T cells following renal transplantation.

Prognostic Significance of DNA Topoisomerase II Alpha (TOP2A) in Cholangiocarcinoma

Cholangiocarcinoma (CCA) is a malignant tumor with an increasing incidence worldwide. Although radiation therapy has improved the therapeutic efficiency of CCA treatment, differential expression of genes among cholangiocarcinoma subtypes has been revealed through precise sequencing. However, no specific molecular therapeutic targets or biomarkers have been figured out for use in precision medicine, and the exact mechanism by which antitumorigenic effects occur is still unclear. Therefore, it is necessary to conduct further studies on the development and mechanisms associated with CCA. We examined the clinical data and pathological features of patients with cholangiocarcinomas. We investigated the associations between DNA Topoisomerase II Alpha (TOP2A) expression and patient outcomes, such as metastasis-free survival (MFS) and disease-specific survival (DSS), as well as clinical characteristics and pathological results. TOP2A expression was shown to be upregulated in CCA tissue sections by immunohistochemistry staining and data mining. Moreover, we observed that the TOP2A expression correlated with clinical features, such as the primary tumor stage, histological variants, and patients with hepatitis. Furthermore, high expression of TOP2A was associated with worse survival outcomes in terms of the overall survival (p < 0.0001), disease-specific survival (p < 0.0001), and metastasis-free survival (p < 0.0001) compared with patients in the low TOP2A expression group. This indicates that a high level of TOP2A expression is related to an unfavorable prognosis. Our results show that TOP2A is highly expressed in CCA tissues, and its upregulation is correlated with the primary disease stage and poor prognosis significantly. Consequently, TOP2A is a prognostic biomarker and a novel therapeutic target for the treatment of CCA.

A simple, dual direct expression plasmid system in prokaryotic and mammalian cells.

We introduce a simple, dual direct cloning plasmid system (pgMAX-II) for gene expression analysis in both prokaryotic (Escherichia coli) and mammalian cells. This system, which uses a prokaryotic expression unit adapted from the pgMAX system and a mammalian promoter, is effective for subcloning using the DNA topoisomerase II toxin CcdB. Given that molecular biological cloning systems broadly rely on E. coli for rapid growth, the proposed concept may have wide applicability beyond mammalian cells.