The role of DNA topoisomerase 1α (AtTOP1α) in regulating arabidopsis meiotic recombination and chromosome segregation.

Meiosis is a critical process in sexual reproduction, and errors during this cell division can significantly impact fertility. Successful meiosis relies on the coordinated action of numerous genes involved in DNA replication, strand breaks, and subsequent rejoining. DNA topoisomerase enzymes play a vital role by regulating DNA topology, alleviating tension during replication and transcription. To elucidate the specific function of DNA topoisomerase 1α ( A t T O P 1 α ) in male reproductive development of Arabidopsis thaliana, we investigated meiotic cell division in Arabidopsis flower buds. Combining cytological and biochemical techniques, we aimed to reveal the novel contribution of A t T O P 1 α to meiosis. Our results demonstrate that the absence of A t T O P 1 α leads to aberrant chromatin behavior during meiotic division. Specifically, the top1α1 mutant displayed altered heterochromatin distribution and clustered centromere signals at early meiotic stages. Additionally, this mutant exhibited disruptions in the distribution of 45s rDNA signals and a reduced frequency of chiasma formation during metaphase I, a crucial stage for genetic exchange. Furthermore, the atm-2×top1α1 double mutant displayed even more severe meiotic defects, including incomplete synapsis, DNA fragmentation, and the presence of polyads. These observations collectively suggest that A t T O P 1 α plays a critical role in ensuring accurate meiotic progression, promoting homologous chromosome crossover formation, and potentially functioning in a shared DNA repair pathway with ATAXIA TELANGIECTASIA MUTATED (ATM) in Arabidopsis microspore mother cells.

TDP1 phosphorylation by CDK1 in mitosis promotes MUS81-dependent repair of trapped Top1-DNA covalent complexes.

Topoisomerase 1 (Top1) controls DNA topology, relieves DNA supercoiling during replication and transcription, and is critical for mitotic progression to the G1 phase. Tyrosyl-DNA phosphodiesterase 1 (TDP1) mediates the removal of trapped Top1-DNA covalent complexes (Top1cc). Here, we identify CDK1-dependent phosphorylation of TDP1 at residue S61 during mitosis. A TDP1 variant defective for S61 phosphorylation (TDP1-S61A) is trapped on the mitotic chromosomes, triggering DNA damage and mitotic defects. Moreover, we show that Top1cc repair in mitosis occurs via a MUS81-dependent DNA repair mechanism. Replication stress induced by camptothecin or aphidicolin leads to TDP1-S61A enrichment at common fragile sites, which over-stimulates MUS81-dependent chromatid breaks, anaphase bridges, and micronuclei, ultimately culminating in the formation of 53BP1 nuclear bodies during G1 phase. Our findings provide new insights into the cell cycle-dependent regulation of TDP1 dynamics for the repair of trapped Top1-DNA covalent complexes during mitosis that prevents genomic instability following replication stress.

Transcriptomic analysis of HEK293A cells with a CRISPR/Cas9-mediated TDP1 knockout.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a human DNA repair protein. It is a member of the phospholipase D family based on structural similarity. TDP1 is a key enzyme of the repair of stalled topoisomerase 1 (TOP1)-DNA complexes. Previously, with the CRISPR/Cas9 method, we obtained HEK293A cells with a homozygous knockout of the TDP1 gene and used the TDP1 knockout cells as a cellular model for studying mechanisms of action of an anticancer therapy. In the present work, we hypothesized that the TDP1 knockout would alter the expression of DNA repair-related genes. By transcriptomic analysis, we investigated for the first time the effect of the TDP1 gene knockout on genes' expression changes in the human HEK293A cell line. We obtained original data implying a role of TDP1 in other processes besides the repair of the DNA-TOP1 complex. Differentially expressed gene analysis revealed that TDP1 may participate in cell adhesion and communication, spermatogenesis, mitochondrial function, neurodegeneration, a cytokine response, and the MAPK signaling pathway.

Neighboring macrophage-induced alteration in the phenotype of colorectal cancer cells in the tumor budding area.

BACKGROUND: A higher number of tumor buds in the invasive front of colorectal cancer (CRC) specimens has been shown to contribute to a poor prognosis in CRC patients. Because macrophages (Mφs) have been demonstrated to alter the phenotype of cancer cells, we hypothesized that the phenotype of CRC cells in the tumor budding (TB) area might be changed by the interaction between CRC cells and Mφs. METHODS: We assessed the expression of topoisomerase 1 in CRC cells to estimate the acquisition of chemoresistance in CRC. To demonstrate the tumor-stromal interaction between CRC cells and Mφs, we assessed two histological findings, the number of Mφs per single CRC cell and the proximity between CRC cells and Mφs by histological spatial analysis using HALO software. RESULTS: The expression levels of topoisomerase 1 in CRC cells were decreased in deeper areas, especially in the TB area, compared to the surface area. Our histological spatial analysis revealed that 2.6 Mφs located within 60 µm of a single CRC cell were required to alter the phenotype of the CRC cell. Double-immunofluorescence staining revealed that higher Mφs were positive for interleukin-6 (IL-6) in the TB area and that AE1/AE3-positive CRC cells were also positive for phospho-STAT3 (pSTAT3) in the TB area; thus, the IL-6 receptor (IL-6R)/STAT3 signaling pathway in CRC cells was upregulated by IL-6 derived from neighboring Mφs. CONCLUSION: IL-6 secreted from the neighboring Mφs would alter the phenotype of CRC cells via IL-6R/STAT3 signaling pathway.

[High anti-topoisomerase-1 autoantibodies levels are associated with the extension of skin fibrosis and vascular progression in patients with systemic sclerosis]. / Des taux élevés d'auto-anticorps anti-topo-isomérase-1 sont associés à l'extension de la fibrose cutanée et à la progression vasculaire chez les patients atteints de sclérodermie systémique.

BACKGROUND: Anti-centromere antibodies, anti-topoisomerase-1 antibodies (ATA), and anti-RNA-polymerase III antibodies are three Systemic Sclerosis (SSc)-specific autoantibodies. Their detection is helpful in determining the prognosis. We aimed to evaluate whether ATA levels were associated with disease severity at diagnosis or disease progression during follow-up in ATA positive patients. METHODS: We conducted a single-centre French retrospective observational study, between 2014 and 2021. ATA positive patients fulfilling the ACR/EULAR 2013 classification criteria for SSc with a minimal follow-up of 1 year and 2 ATA dosages were included. SSc patients with high IgG ATA levels at baseline (>240IU/mL) were compared with SSc patients with low levels (≤240IU/mL), at inclusion and at 1 and 3 years. A variation of at least 30 % of ATA levels was considered significant. RESULTS: Fifty-nine SSc patients were included and analysed. There was a predominance of women and of patients with diffuse interstitial lung disease. Patients with high ATA levels exhibited a higher skin sclerosis assessed by the modified Rodnan skin score (P=0.0480). They had a lower carbon monoxide transfer coefficient (P=0.0457), a lower forced vital capacity (FVC) (P=0.0427) and more frequently had a FVC under 80 %, when compared to patients with low ATA levels (P=0.0423). Initial high ATA levels were associated with vascular progression at one year (21.95 % vs. 0 %; P=0.0495). CONCLUSION: ATA levels are associated with skin sclerosis and vascular progression in SSc. Beyond the detection of ATA, quantifying this autoantibody might be of interest in predicting disease severity and prognosis in SSc.

Condensation of the N-terminal domain of human topoisomerase 1 is driven by electrostatic interactions and tuned by its charge distribution.

Liquid-liquid phase separation (LLPS) is pivotal in forming biomolecular condensates, which are crucial in several biological processes. Intrinsically disordered regions (IDRs) are typically responsible for driving LLPS due to their multivalency and high content of charged residues that enable the establishment of electrostatic interactions. In our study, we examined the role of charge distribution in the condensation of the disordered N-terminal domain of human topoisomerase I (hNTD). hNTD is densely charged with oppositely charged residues evenly distributed along the sequence. Its LLPS behavior was compared with that of charge permutants exhibiting varying degrees of charge segregation. At low salt concentrations, hNTD undergoes LLPS. However, LLPS is inhibited by high concentrations of salt and RNA, disrupting electrostatic interactions. Our findings show that, in hNTD, moderate charge segregation promotes the formation of liquid condensates that are sensitive to salt and RNA, whereas marked charge segregation results in the formation of aberrant condensates. Although our study is based on a limited set of protein variants, it supports the applicability of the "stickers-and-spacers" model to biomolecular condensates involving highly charged IDRs. These results may help generate reliable models of the overall LLPS behavior of supercharged polypeptides.

Synthesis and antitumor activity of cyclopentane-fused anthraquinone derivatives.

In our pursuit of developing novel analogs of anthracyclines with enhanced antitumor efficacy and safety, we have designed a synthesis scheme for 4,11-dihydroxy-5,10-dioxocyclopenta[b]anthracene-2-carboxamides. These newly synthesized compounds exhibit remarkable antiproliferative potency against various mammalian tumor cell lines, including those expressing activated mechanisms of multidrug resistance. The structure of the diamine moiety in the carboxamide side chain emerges as a critical determinant for anticancer activity and interaction with key targets such as DNA, topoisomerase 1, and ROS induction. Notably, the introduced modification to the doxorubicin structure results in significantly increased lipophilicity, cellular uptake, and preferential distribution in lysosomes. Consequently, while maintaining an impact on anthracyclines targets, these novel derivatives also demonstrate the potential to induce cytotoxicity through pathways associated with lysosomes. In summary, derivatives of cyclic diamines, particularly 3-aminopyrrolidine, can be considered a superior choice compared to aminosugars for incorporation into natural and semi-synthetic anthracyclines or new anthraquinone derivatives, aiming to circumvent efflux-mediated drug resistance.

Mining and analysis of ADE signals of two camptothecin topoisomerase 1 inhibitors / 中国药房

OBJECTIVE To mine and analyze the adverse drug events （ADE） signals of two camptothecin topoisomerase 1 inhibitors， i.e. irinotecan and topotecan， and to provide reference for clinical medication safety. METHODS Based on the U.S. Food and Drug Administration Adverse Event Reporting System （FAERS） database， ADE report data for the aforementioned two drugs were extracted from January 1， 2004 to March 31， 2023. After processing the data， signal mining was conducted by using the reporting odds ratio in conjunction with the Bayesian confidence propagation neural network， followed by analysis. RESULTS A total of 14 738 relevant ADE reports were screened， among which 11 483 were associated with irinotecan and 3 255 with topotecan. The ADE reports for irinotecan were predominantly male， whereas for topotecan， they were predominantly female； the age of patients using the two drugs mainly concentrated in 45-＜75 years old. A total of 847 signals were detected， involving 24 system organ classes （SOCs）. Among them， 565 signals of irinotecan were detected， involving 24 SOCs， primarily concentrating on gastrointestinal disorders， general disorders and administration site conditions， blood and lymphatic system disorders； the most frequently reported ADE was diarrhea， and the ADE with the strongest signal intensity was cholinergic syndrome. A total of 282 signals of topotecan were detected， involving 22 SOCs， primarily concentrating on general disorders and administration site conditions， investigations， blood and lymphatic system disorders， and gastrointestinal disorders； the most frequently reported ADEs were death and anemia， and the ADE with the strongest signal intensity was febrile bone marrow aplasia. ADE signals for irinotecan such as metastatic colorectal cancer， peripheral sensory neuropathy， steatohepatitis， and those for topotecan such as iris atrophy， retinal degeneration， vitreous hemorrhage， were not documented in their respective drug instruction. CONCLUSIONS ADEs of irinotecan and topotecan primarily involve the digestive and hematologic systems， warranting close clinical monitoring. Cholinergic syndrome caused by irinotecan should be concerned. In addition， patients receiving irinotecan should also be monitored for ADE such as metastatic colorectal cancer， peripheral sensory neuropathy， steatohepatitis， and proteinuria； for patients using topotecan， enhanced surveillance of ocular diseases is recommended to ensure medication safety.

Topoisomerase 1 Activity Is Reduced in Response to Thermal Stress in Fruit Flies and in Human HeLa Cells.

In the modern world with climate changes and increasing pollution, different types of stress are becoming an increasing challenge. Hence, the identification of reliable biomarkers of stress and accessible sensors to measure such biomarkers are attracting increasing attention. In the current study, we demonstrate that the activity, but not the expression, of the ubiquitous enzyme topoisomerase 1 (TOP1), as measured in crude cell extracts by the REEAD sensor system, is markedly reduced in response to thermal stress in both fruit flies (Drosophila melanogaster) and cultivated human cells. This effect was observed in response to both mild-to-moderate long-term heat stress and more severe short-term heat stress in D. melanogaster. In cultivated HeLa cells a reduced TOP1 activity was observed in response to both cold and heat stress. The reduced TOP1 activity appeared dependent on one or more cellular pathways since the activity of purified TOP1 was unaffected by the utilized stress temperatures. We demonstrate successful quantitative measurement of TOP1 activity using an easily accessible chemiluminescence readout for REEAD pointing towards a sensor system suitable for point-of-care assessment of stress responses based on TOP1 as a biomarker.

"Cholinergic side Effect-Free anticancer drugs: paving the way for safer and more effective cancer treatment".

Cancer is a leading cause of mortality worldwide, and various anticancer medications have been developed that target different biological pathways involved in cancer growth and progression. Topoisomerase 1 (Top1) is an essential enzyme involved in unwinding supercoiled DNA, and it serves as a key target for several anti-cancer drugs. Irinotecan (FDA approved drug), a semi-synthetic camptothecin derivative, is an effective Top1 toxin that eliminates human cancer cells. Cancer patients suffer from the cholinergic syndrome caused by irinotecan and other Top1 inhibitors. Irinotecan-treated patients have developed cholinergic syndrome due to acetylcholinesterase (AChE) enzyme inhibition. It appears that irinotecan or its metabolites directly interact with AChE and inhibit its role of converting acetylcholine to choline, leading to an accumulation of acetylcholine and subsequent symptoms of the cholinergic syndrome. The phytochemicals present in Phyllanthus emblica, commonly referred to as amla, have been studied to determine their therapeutic effects. As an alternative treatment for cancer, this study explores the potential of phytochemicals found in amla to target and inhibit the Top1 protein. Additionally, the study aims to identify a non-inhibitor for AChE. Molecular docking studies assessed phytochemical binding affinities to Top1 and AChE enzymes, and ADME analyses were performed to assess their drug-likeness properties. Subsequently, molecular dynamic simulation was employed to assess the stability of these compounds. The results suggest that new anticancer medications that do not inhibit AChE or fresh Top1 inhibitors that use the camptothecin scaffold may alleviate some of the irinotecan's side effects.Communicated by Ramaswamy H. Sarma.

Anti-topoisomerase 1 Antibody Level Changes after B Cell Depletion Therapy in Systemic Sclerosis.

The aim of our study was to assess the relationship between the changes of antinuclear autoantibodies (ANAs) and autoantibodies to topoisomerase 1 (anti-Topo 1) in systemic sclerosis (SSs) patients on rituximab (RTX) therapy. The prospective study included 88 patients (73 women) with a mean age of 47 (17-71) years. The mean disease duration was 5.9 ± 4.8 years. The mean follow-up period was more than 2 years (27 (12-42) months). We documented a statistically significant change in skin score, the disease activity index, improvement of pulmonary function and reduction of mean dose of prednisolone after RTX treatment. There was a significant decrease in the number of patients with high levels of ANA and overall decrease of the ANA and anti-Topo 1 levels. A moderate positive statistically significant correlation was found between ANA and anti-Topo 1 (r = 0.403). In the group of patients positive for anti-Topo 1 there were a more pronounced depletion of B lymphocytes, significantly higher increase in forced vital capacity and diffusion capacity, decrease in the disease activity index, compared with patients negative for anti-Topo 1. We observed the decline in the level of ANA and anti-Topo 1 in SSc patients after RTX therapy, and it was correlated by an improvement of the main outcome parameters of the disease. Therefore, anti-Topo 1 positivity could be considered as a predictor of a better response to RTX treatment, especially in SSc patients with hyperproduction of anti-Topo 1.

Repair of topoisomerase 1-induced DNA damage by tyrosyl-DNA phosphodiesterase 2 (TDP2) is dependent on its magnesium binding.

Topoisomerases are enzymes that relax DNA supercoiling during replication and transcription. Camptothecin, a topoisomerase 1 (TOP1) inhibitor, and its analogs trap TOP1 at the 3'-end of DNA as a DNA-bound intermediate, resulting in DNA damage that can kill cells. Drugs with this mechanism of action are widely used to treat cancers. It has previously been shown that tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs TOP1-induced DNA damage generated by camptothecin. In addition, tyrosyl-DNA phosphodiesterase 2 (TDP2) plays critical roles in repairing topoisomerase 2 (TOP2)-induced DNA damage at the 5'-end of DNA and in promoting the repair of TOP1-induced DNA damage in the absence of TDP1. However, the catalytic mechanism by which TDP2 processes TOP1-induced DNA damage has not been elucidated. In this study, we found that a similar catalytic mechanism underlies the repair of TOP1- and TOP2-induced DNA damage by TDP2, with Mg2+-TDP2 binding playing a role in both repair mechanisms. We show chain-terminating nucleoside analogs are incorporated into DNA at the 3'-end and abort DNA replication to kill cells. Furthermore, we found that Mg2+-TDP2 binding also contributes to the repair of incorporated chain-terminating nucleoside analogs. Overall, these findings reveal the role played by Mg2+-TDP2 binding in the repair of both 3'- and 5'-blocking DNA damage.

Design, synthesis and development of a dual inhibitor of Topoisomerase 1 and poly (ADP-ribose) polymerase 1 for efficient killing of cancer cells.

Combinatorial inhibition of Topoisomerase 1 (TOP1) and Poly (ADP-ribose) polymerase 1 (PARP1) is an attractive therapeutic strategy which is under active investigation to address chemoresistance to TOP1 inhibitors. However, this combinatorial regimen suffers from severe dose limiting toxicities. Dual inhibitors often offer significant advantages over combinatorial therapies involving individual agents by minimizing toxicity and providing conducive pharmacokinetic profiles. In this study, we have designed, synthesized and evaluated a library of 11 candidate conjugated dual inhibitors for PARP1 and TOP1, named as DiPT-1 to DiPT-11. Our extensive screening showed that one of the hits i.e.DiPT-4 has promising cytotoxicity profile against multiple cancers with limited toxicities towards normal cells. DiPT-4 induces extensive DNA double stand breaks (DSBs), cell cycle arrest and apoptosis in cancer cells. Mechanistically, DiPT-4 has the propensity to bind catalytic pockets of TOP1 and PARP1, leading to significant inhibition of both TOP1 and PARP1 at in vitro and cellular level. Interestingly, DiPT-4 causes extensive stabilization of TOP1-DNA covalent complex (TOP1cc), a key lethal intermediate associated with induction of DSBs and cell death. Moreover, DiPT-4 inhibited poly (ADP-ribosylation) i.e. PARylation of TOP1cc, leading to long lived TOP1cc with a slower kinetics of degradation. This is one of the important molecular processes which helps in overcoming resistance in cancer in response to TOP1 inhibitors. Together, our investigation showed DiPT-4 as a promising dual inhibitor of TOP1 and PARP1, which may have the potential to offer significant advantages over combinatorial therapy in clinical settings.

Protective effects of hsa_circ_0072568 on interleukin­1ß­stimulated human chondrocytes are mediated via the miR­382­5p/TOP1 axis.

Circular RNA (circRNA) dysregulation has been linked to osteoarthritis (OA). The present study investigated the involvement of hsa_circ_0072568 (circ0072568) in OA. The expression of circ0072568 was detected in OA tissues and interleukin (IL)-1ß-stimulated human chondrocytes. After performing dual-luciferase reporter and RNA immunoprecipitation assays, MTT, enzyme-linked immunosorbent assay and western blot analysis were used to assess the functions of circ0072568 in IL-1ß-induced inflammation in chondrocytes in vitro. Circ0072568 was inhibited in OA tissues and the cell model in vitro. Circ0072568 overexpression protected the chondrocytes against IL-1ß-induced inflammation and extracellular matrix (ECM) breakdown. Circ0072568 directly attached to microRNA (miR)-382-5p and enhanced the production of topoisomerase 1 (TOP1). Furthermore, miR-382-5p overexpression or TOP1 knockdown attenuated the effects of circ0072568 in IL-1ß-stimulated human chondrocytes. On the whole, the present study demonstrates that the Circ0072568/miR-382-5p/TOP1 axis is involved in inflammation and ECM degradation in OA. These findings may contribute to the development of potential therapeutic strategies for OA.

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.

MUS81 cleaves TOP1-derived lesions and other DNA-protein cross-links.

BACKGROUND: DNA-protein cross-links (DPCs) are one of the most deleterious DNA lesions, originating from various sources, including enzymatic activity. For instance, topoisomerases, which play a fundamental role in DNA metabolic processes such as replication and transcription, can be trapped and remain covalently bound to DNA in the presence of poisons or nearby DNA damage. Given the complexity of individual DPCs, numerous repair pathways have been described. The protein tyrosyl-DNA phosphodiesterase 1 (Tdp1) has been demonstrated to be responsible for removing topoisomerase 1 (Top1). Nevertheless, studies in budding yeast have indicated that alternative pathways involving Mus81, a structure-specific DNA endonuclease, could also remove Top1 and other DPCs. RESULTS: This study shows that MUS81 can efficiently cleave various DNA substrates modified by fluorescein, streptavidin or proteolytically processed topoisomerase. Furthermore, the inability of MUS81 to cleave substrates bearing native TOP1 suggests that TOP1 must be either dislodged or partially degraded prior to MUS81 cleavage. We demonstrated that MUS81 could cleave a model DPC in nuclear extracts and that depletion of TDP1 in MUS81-KO cells induces sensitivity to the TOP1 poison camptothecin (CPT) and affects cell proliferation. This sensitivity is only partially suppressed by TOP1 depletion, indicating that other DPCs might require the MUS81 activity for cell proliferation. CONCLUSIONS: Our data indicate that MUS81 and TDP1 play independent roles in the repair of CPT-induced lesions, thus representing new therapeutic targets for cancer cell sensitisation in combination with TOP1 inhibitors.

Compartmentalization of the SUMO/RNF4 pathway by SLX4 drives DNA repair.

SLX4, disabled in the Fanconi anemia group P, is a scaffolding protein that coordinates the action of structure-specific endonucleases and other proteins involved in the replication-coupled repair of DNA interstrand cross-links. Here, we show that SLX4 dimerization and SUMO-SIM interactions drive the assembly of SLX4 membraneless compartments in the nucleus called condensates. Super-resolution microscopy reveals that SLX4 forms chromatin-bound clusters of nanocondensates. We report that SLX4 compartmentalizes the SUMO-RNF4 signaling pathway. SENP6 and RNF4 regulate the assembly and disassembly of SLX4 condensates, respectively. SLX4 condensation per se triggers the selective modification of proteins by SUMO and ubiquitin. Specifically, SLX4 condensation induces ubiquitylation and chromatin extraction of topoisomerase 1 DNA-protein cross-links. SLX4 condensation also induces the nucleolytic degradation of newly replicated DNA. We propose that the compartmentalization of proteins by SLX4 through site-specific interactions ensures the spatiotemporal control of protein modifications and nucleolytic reactions during DNA repair.

Transcriptomic Analysis of CRISPR/Cas9-Mediated PARP1-Knockout Cells under the Influence of Topotecan and TDP1 Inhibitor.

Topoisomerase 1 (TOP1) is an enzyme that regulates DNA topology and is essential for replication, recombination, and other processes. The normal TOP1 catalytic cycle involves the formation of a short-lived covalent complex with the 3' end of DNA (TOP1 cleavage complex, TOP1cc), which can be stabilized, resulting in cell death. This fact substantiates the effectiveness of anticancer drugs-TOP1 poisons, such as topotecan, that block the relegation of DNA and fix TOP1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is able to eliminate TOP1cc. Thus, TDP1 interferes with the action of topotecan. Poly(ADP-ribose) polymerase 1 (PARP1) is a key regulator of many processes in the cell, such as maintaining the integrity of the genome, regulation of the cell cycle, cell death, and others. PARP1 also controls the repair of TOP1cc. We performed a transcriptomic analysis of wild type and PARP1 knockout HEK293A cells treated with topotecan and TDP1 inhibitor OL9-119 alone and in combination. The largest number of differentially expressed genes (DEGs, about 4000 both up- and down-regulated genes) was found in knockout cells. Topotecan and OL9-119 treatment elicited significantly fewer DEGs in WT cells and negligible DEGs in PARP1-KO cells. A significant part of the changes caused by PARP1-KO affected the synthesis and processing of proteins. Differences under the action of treatment with TOP1 or TDP1 inhibitors alone were found in the signaling pathways for the development of cancer, DNA repair, and the proteasome. The drug combination resulted in DEGs in the ribosome, proteasome, spliceosome, and oxidative phosphorylation pathways.

The Interactions of the 70 kDa Fragment of Cell Adhesion Molecule L1 with Topoisomerase 1, Peroxisome Proliferator-Activated Receptor γ and NADH Dehydrogenase (Ubiquinone) Flavoprotein 2 Are Involved in Gene Expression and Neuronal L1-Dependent Functions.

The cell adhesion molecule L1 is essential not only for neural development, but also for synaptic functions and regeneration after trauma in adulthood. Abnormalities in L1 functions cause developmental and degenerative disorders. L1's functions critically depend on proteolysis which underlies dynamic cell interactions and signal transduction. We showed that a 70 kDa fragment (L1-70) supports mitochondrial functions and gene transcription. To gain further insights into L1-70's functions, we investigated several binding partners. Here we show that L1-70 interacts with topoisomerase 1 (TOP1), peroxisome proliferator-activated receptor γ (PPARγ) and NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2). TOP1, PPARγ and NDUFV2 siRNAs reduced L1-dependent neurite outgrowth, and the topoisomerase inhibitors topotecan and irinotecan inhibited L1-dependent neurite outgrowth, neuronal survival and migration. In cultured neurons, L1 siRNA reduces the expression levels of the long autism genes neurexin-1 (Nrxn1) and neuroligin-1 (Nlgn1) and of the mitochondrially encoded gene NADH:ubiquinone oxidoreductase core subunit 2 (ND2). In mutant mice lacking L1-70, Nrxn1 and Nlgn1, but not ND2, mRNA levels are reduced. Since L1-70's interactions with TOP1, PPARγ and NDUFV2 contribute to the expression of two essential long autism genes and regulate important neuronal functions, we propose that L1 may not only ameliorate neurological problems, but also psychiatric dysfunctions.

Payload diversification: a key step in the development of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) is a fast moving class of targeted biotherapeutics that currently combines the selectivity of monoclonal antibodies with the potency of a payload consisting of cytotoxic agents. For many years microtubule targeting and DNA-intercalating agents were at the forefront of ADC development. The recent approval and clinical success of trastuzumab deruxtecan (Enhertu®) and sacituzumab govitecan (Trodelvy®), two topoisomerase 1 inhibitor-based ADCs, has shown the potential of conjugating unconventional payloads with differentiated mechanisms of action. Among future developments in the ADC field, payload diversification is expected to play a key role as illustrated by a growing number of preclinical and clinical stage unconventional payload-conjugated ADCs. This review presents a comprehensive overview of validated, forgotten and newly developed payloads with different mechanisms of action.