Identification of FTY720 and COH29 as novel topoisomerase I catalytic inhibitors by experimental and computational studies.

The development of novel topoisomerase I (TOP1) inhibitors is crucial for overcoming the drawbacks and limitations of current TOP1 poisons. Here, we identified two potential TOP1 inhibitors, namely, FTY720 (a sphingosine 1-phosphate antagonist) and COH29 (a ribonucleotide reductase inhibitor), through experimental screening of known active compounds. Biological experiments verified that FTY720 and COH29 were nonintercalative TOP1 catalytic inhibitors that did not induce the formation of DNA-TOP1 covalent complexes. Molecular docking revealed that FTY720 and COH29 interacted favorably with TOP1. Molecular dynamics simulations revealed that FTY720 and COH29 could affect the catalytic domain of TOP1, thus resulting in altered DNA-binding cavity size. The alanine scanning and interaction entropy identified Arg536 as a hotspot residue. In addition, the bioinformatics analysis predicted that FTY720 and COH29 could be effective in treating malignant breast tumors. Biological experiments verified their antitumor activities using MCF-7 breast cancer cells. Their combinatory effects with TOP1 poisons were also investigated. Further, FTY720 and COH29 were found to cause less DNA damage compared with TOP1 poisons. The findings provide reliable lead compounds for the development of novel TOP1 catalytic inhibitors and offer new insights into the potential clinical applications of FTY720 and COH29 in targeting TOP1.

Activation of STING by the novel liposomal TLC388 enhances the therapeutic response to anti-PD-1 antibodies in combination with radiotherapy.

Current immune checkpoint inhibiters (ICIs) have contrasting clinical results in poorly immunogenic cancers such as microsatellite-stable colorectal cancer (MSS-CRC). Therefore, understanding and developing the combinational therapeutics for ICI-unresponsive cancers is critical. Here, we demonstrated that the novel topoisomerase I inhibitor TLC388 can reshape the tumor immune landscape, corroborating their antitumor effects combined with radiotherapy as well as immunotherapy. We found that TLC388 significantly triggered cytosolic single-stranded DNA (ssDNA) accumulation for STING activation, leading to type I interferons (IFN-Is) production for increased cancer immunogenicity to enhance antitumor immunity. TLC388-treated tumors were infiltrated by a vast number of dendritic cells, immune cells, and costimulatory molecules, contributing to the favorable antitumor immune response within the tumor microenvironment. The infiltration of cytotoxic T and NK cells were more profoundly existed within tumors in combination with radiotherapy and ICIs, leading to superior therapeutic efficacy in poorly immunogenic MSS-CRC. Taken together, these results showed that the novel topoisomerase I inhibitor TLC388 increased cancer immunogenicity by ssDNA/STING-mediated IFN-I production, enhancing antitumor immunity for better therapeutic efficacy in combination with radiotherapy and ICIs for poorly immunogenic cancer.

Minocycline and photodynamic priming significantly improve chemotherapy efficacy in heterotypic spheroids of pancreatic ductal adenocarcinoma.

The prognosis for patients with advanced-stage pancreatic ductal adenocarcinoma (PDAC) remains dismal. It is generally accepted that combination cancer therapies offer the most promise, such as Folforinox, despite their associated high toxicity. This study addresses the issue of chemoresistance by introducing a complementary dual priming approach to attenuate the DNA repair mechanism and to improve the efficacy of a type 1 topoisomerase (Top1) inhibitor. The result is a regimen that integrates drug-repurposing and nanotechnology using 3 clinically relevant FDA-approved agents (1) Top1 inhibitor (irinotecan) at subcytotoxic doses (2) benzoporphyrin derivative (BPD) as a photoactive molecule for photodynamic priming (PDP) to improve the delivery of irinotecan within the cancer cell and (3) minocycline priming (MNP) to modulate DNA repair enzyme Tdp1 (tyrosyl-DNA phosphodiesterase) activity. We demonstrate in heterotypic 3D cancer models that incorporate cancer cells and pancreatic cancer-associated fibroblasts that simultaneous targeting of Tdp1 and Top1 were significantly more effective by employing MNP and photoactivatable multi-inhibitor liposomes encapsulating BPD and irinotecan compared to monotherapies or a cocktail of dual or triple-agents. These data are encouraging and warrant further work in appropriate animal models to evolve improved therapeutic regimens.

DB-1310, an ADC comprised of a novel anti-HER3 antibody conjugated to a DNA topoisomerase I inhibitor, is highly effective for the treatment of HER3-positive solid tumors.

BACKGROUND: HER3 (ErbB3), a member of the human epidermal growth factor receptor family, is frequently overexpressed in various cancers. Multiple HER3-targeting antibodies and antibody-drug conjugates (ADCs) were developed for the solid tumor treatment, however none of HER3-targeting agent has been approved for tumor therapy yet. We developed DB-1310, a HER3 ADC composed of a novel humanized anti-HER3 monoclonal antibody covalently linked to a proprietary DNA topoisomerase I inhibitor payload (P1021), and evaluate the efficacy and safety of DB-1310 in preclinical models. METHODS: The binding of DB-1310 to Her3 and other HER families were measured by ELISA and SPR. The competition of binding epitope for DB-1310 and patritumab was tested by FACS. The sensitivity of breast, lung, prostate and colon cancer cell lines to DB-1310 was evaluated by in vitro cell killing assay. In vivo growth inhibition study evaluated the sensitivity of DB-1310 to Her3 + breast, lung, colon and prostate cancer xenograft models. The safety profile was also measured in cynomolgus monkey. RESULTS: DB-1310 binds HER3 via a novel epitope with high affinity and internalization capacity. In vitro, DB-1310 exhibited cytotoxicity in numerous HER3 + breast, lung, prostate and colon cancer cell lines. In vivo studies in HER3 + HCC1569 breast cancer, NCI-H441 lung cancer and Colo205 colon cancer xenograft models showed DB-1310 to have dose-dependent tumoricidal activity. Tumor suppression was also observed in HER3 + non-small cell lung cancer (NSCLC) and prostate cancer patient-derived xenograft (PDX) models. Moreover, DB-1310 showed stronger tumor growth-inhibitory activity than patritumab deruxtecan (HER3-DXd), which is another HER3 ADC in clinical development at the same dose. The tumor-suppressive activity of DB-1310 synergized with that of EGFR tyrosine kinase inhibitor, osimertinib, and exerted efficacy also in osimertinib-resistant PDX model. The preclinical assessment of safety in cynomolgus monkeys further revealed DB-1310 to have a good safety profile with a highest non severely toxic dose (HNSTD) of 45 mg/kg. CONCLUSIONS: These finding demonstrated that DB-1310 exerted potent antitumor activities against HER3 + tumors in in vitro and in vivo models, and showed acceptable safety profiles in nonclinical species. Therefore, DB-1310 may be effective for the clinical treatment of HER3 + solid tumors.

Mechanisms and emerging strategies for irinotecan-induced diarrhea.

Irinotecan (also known as CPT-11) is a topoisomerase I inhibitor first approved for clinical use as an anticancer agent in 1996. Over the past more than two decades, it has been widely used for combination regimens to treat various malignancies, especially in gastrointestinal and lung cancers. However, severe dose-limiting toxicities, especially gastrointestinal toxicity such as late-onset diarrhea, were frequently observed in irinotecan-based therapy, thus largely limiting the clinical application of this agent. Current knowledge regarding the pathogenesis of irinotecan-induced diarrhea is characterized by the complicated metabolism of irinotecan to its active metabolite SN-38 and inactive metabolite SN-38G. A series of enzymes and transporters were involved in these metabolic processes, including UGT1A1 and CYP3A4. Genetic polymorphisms of these metabolizing enzymes were significantly associated with the occurrence of irinotecan-induced diarrhea. Recent discoveries and progress made on the detailed mechanisms enable the identification of potential biomarkers for predicting diarrhea and as such guiding the proper patient selection with a better range of tolerant dosages. In this review, we introduce the metabolic process of irinotecan and describe the pathogenic mechanisms underlying irinotecan-induced diarrhea. Based on the mechanisms, we further outline the potential biomarkers for predicting the severity of diarrhea. Finally, based on the current experimental evidence in preclinical and clinical studies, we discuss and prospect the current and emerging strategies for the prevention of irinotecan-induced diarrhea.

Design, Synthesis, and Investigation of the Pharmacokinetics and Anticancer Activities of Indenoisoquinoline Derivatives That Stabilize the G-Quadruplex in the MYC Promoter and Inhibit Topoisomerase I.

G-quadruplexes are noncanonical four-stranded DNA secondary structures. MYC is a master oncogene and the G-quadruplex formed in the MYC promoter functions as a transcriptional silencer and can be stabilized by small molecules. We have previously revealed a novel mechanism of action for indenoisoquinoline anticancer drugs, dual-downregulation of MYC and inhibition of topoisomerase I. Herein, we report the design and synthesis of novel 7-aza-8,9-methylenedioxyindenoisoquinolines based on desirable substituents and π-π stacking interactions. These compounds stabilize the MYC promoter G-quadruplex, significantly lower MYC levels in cancer cells, and inhibit topoisomerase I. MYC targeting was demonstrated by differential activities in Raji vs CA-46 cells and cytotoxicity in MYC-dependent cell lines. Cytotoxicities in the NCI-60 panel of human cancer cell lines were investigated. Favorable pharmacokinetics were established, and in vivo anticancer activities were demonstrated in xenograft mouse models. Furthermore, favorable brain penetration, brain pharmacokinetics, and anticancer activity in an orthotopic glioblastoma mouse model were demonstrated.

Design, synthesis, and biological evaluation of novel benzo[6,7]indolo[3,4-c]isoquinolines as anticancer agents with topoisomerase I inhibition.

A novel series of benzo[6,7]indolo[3,4-c]isoquinolines 3a-3f was designed by scaffold hopping of topoisomerase I inhibitor benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-ones (BBPIs), which were developed by structural modification of the natural marine product lamellarin. The unconventional pentacycle was constructed by Bischler-Napieralski-type condensation of amide 11 and subsequent intramolecular Heck reaction. In vitro anticancer activity of the synthesized benzo[6,7]indolo[3,4-c]isoquinolines was evaluated on a panel of 39 human cancer cell lines (JFCR39). Among the compounds tested, N-(3-morpholinopropyl) derivative 3e showed the most potent antiproliferative activity, with a mean GI50 value of 39 nM. This compound inhibited topoisomerase I activity by stabilizing the enzyme-DNA complex.

Quantification of Unencapsulated Drug in Target Tissues Demonstrates Pharmacological Properties and Therapeutic Effects of Liposomal Topotecan (FF-10850).

PURPOSE: Quantifying unencapsulated drug concentrations in tissues is crucial for understanding the mechanisms underlying the efficacy and safety of liposomal drugs; however, the methodology for this has not been fully established. Herein, we aimed to investigate the enhanced therapeutic potential of a pegylated liposomal formulation of topotecan (FF-10850) by analyzing the concentrations of the unencapsulated drug in target tissues, to guide the improvement of its dosing regimen. METHODS: We developed a method for measuring unencapsulated topotecan concentrations in tumor and bone marrow interstitial fluid (BM-ISF) and applied this method to pharmacokinetic assessments. The ratios of the area under the concentration-time curves (AUCs) between tumor and BM-ISF were calculated for total and unencapsulated topotecan. DNA damage and antitumor effects of FF-10850 or non-liposomal topotecan (TPT) were evaluated in an ES-2 mice xenograft model. RESULTS: FF-10850 exhibited a much larger AUC ratio between tumor and BM-ISF for unencapsulated topotecan (2.96), but not for total topotecan (0.752), than TPT (0.833). FF-10850 promoted milder DNA damage in the bone marrow than TPT; however, FF-10850 and TPT elicited comparable DNA damage in the tumor. These findings highlight the greater tumor exposure to unencapsulated topotecan and lower bone marrow exposure to FF-10850 than TPT. The dosing regimen was successfully improved based on the kinetics of unencapsulated topotecan and DNA damage. CONCLUSIONS: Tissue pharmacokinetics of unencapsulated topotecan elucidated the favorable pharmacological properties of FF-10850. Evaluation of tissue exposure to an unencapsulated drug with appropriate pharmacodynamic markers can be valuable in optimizing liposomal drugs and dosing regimens.

Unleashing the Potential of Camptothecin: Exploring Innovative Strategies for Structural Modification and Therapeutic Advancements.

Camptothecin (CPT) is a potent anti-cancer agent targeting topoisomerase I (TOP1). However, CPT has poor pharmacokinetic properties, causes toxicities, and leads to drug resistance, which limit its clinical use. In this paper, to review the current state of CPT research. We first briefly explain CPT's TOP1 inhibition mechanism and the key hurdles in CPT drug development. Then we examine strategies to overcome CPT's limitations through structural modifications and advanced delivery systems. Though modifications alone seem insufficient to fully enhance CPT's therapeutic potential, structure-activity relationship analysis provides insights to guide optimization of CPT analogs. In comparison, advanced delivery systems integrating controlled release, imaging capabilities, and combination therapies via stimulus-responsive linkers and targeting moieties show great promise for improving CPT's pharmacological profile. Looking forward, multifaceted approaches combining selective CPT derivatives with advanced delivery systems, informed by emerging biological insights, hold promise for fully unleashing CPT's anti-cancer potential.

G-quadruplexes on chromosomal DNA negatively regulates topoisomerase 1 activity.

Human DNA topoisomerase 1 (Top1) is a crucial enzyme responsible for alleviating torsional stress on DNA during transcription and replication, thereby maintaining genome stability. Previous researches had found that non-working Top1 interacted extensively with chromosomal DNA in human cells. However, the reason for its retention on chromosomal DNA remained unclear. In this study, we discovered a close association between Top1 and chromosomal DNA, specifically linked to the presence of G-quadruplex (G4) structures. G4 structures, formed during transcription, trap Top1 and hinder its ability to relax neighboring DNAs. Disruption of the Top1-G4 interaction using G4 ligand relieved the inhibitory effect of G4 on Top1 activity, resulting in a further reduction of R-loop levels in cells. Additionally, the activation of Top1 through the use of a G4 ligand enhanced the toxicity of Top1 inhibitors towards cancer cells. Our study uncovers a negative regulation mechanism of human Top1 and highlights a novel pathway for activating Top1.

Tapcin, an In Vivo Active Dual Topoisomerase I/II Inhibitor Discovered by Synthetic Bioinformatic Natural Product (Syn-BNP)-Coupled Metagenomics.

DNA topoisomerases are attractive targets for anticancer agents. Dual topoisomerase I/II inhibitors are particularly appealing due to their reduced rates of resistance. A number of therapeutically relevant topoisomerase inhibitors are bacterial natural products. Mining the untapped chemical diversity encoded by soil microbiomes presents an opportunity to identify additional natural topoisomerase inhibitors. Here we couple metagenome mining, bioinformatic structure prediction algorithms, and chemical synthesis to produce the dual topoisomerase inhibitor tapcin. Tapcin is a mixed p-aminobenzoic acid (PABA)-thiazole with a rare tri-thiazole substructure and picomolar antiproliferative activity. Tapcin reduced colorectal adenocarcinoma HT-29 cell proliferation and tumor volume in mouse hollow fiber and xenograft models, respectively. In both studies it showed similar activity to the clinically used topoisomerase I inhibitor irinotecan. The study suggests that the interrogation of soil microbiomes using synthetic bioinformatic natural product methods has the potential to be a rewarding strategy for identifying potent, biomedically relevant, antiproliferative agents.

Potent antitumor activity of anti-HER2 antibody-topoisomerase I inhibitor conjugate based on self-immolative dendritic dimeric-linker.

Antibody-drug conjugates (ADCs) are a rapidly expanding class of anticancer therapeutics, with 14 ADCs already approved worldwide. We developed unique linker technologies for the bioconjugation of drug molecules with controlled-release applications. We synthesized cathepsin-cleavable ADCs using a dimeric prodrug system based on a self-immolative dendritic scaffold, resulting in a high drug-antibody ratio (DAR) with the potential to reach 16 payloads due to its dendritic structure, increased stability in the circulation and efficient release profile of a highly cytotoxic payload at the targeted site. Using our novel cleavable linker technologies, we conjugated the anti-human epidermal growth factor receptor 2 (anti-HER2) antibody, trastuzumab, with topoisomerase I inhibitors, exatecan or belotecan. The newly synthesized ADCs were tested in vitro on mammary carcinoma cells overexpressing human HER2, demonstrating a substantial inhibitory effect on the proliferation of HER2-positive cells. Importantly, a single dose of our trastuzumab-based ADCs administered in vivo to mice bearing HER2-positive tumors, showed a dose-dependent inhibition of tumor growth and survival benefit, with the most potent antitumor effects observed at 10 mg/kg, which resulted in complete tumor regression and survival of 100% of the mice. Overall, our novel dendritic technologies using the protease-cleavable Val-Cit linker present an opportunity for the development of highly selective and potent controlled-released therapeutic payloads. This strategy could potentially lead to the development of novel and effective ADC technologies for patients diagnosed with HER2-positive cancers. Moreover, our proposed ADC linker technology can be implemented in additional medical conditions such as other malignancies as well as autoimmune diseases that overexpress targets, other than HER2.

Camptothecin structure simplification elaborated new imidazo[2,1-b]quinazoline derivative as a human topoisomerase I inhibitor with efficacy against bone cancer cells and colon adenocarcinoma.

Camptothecin is a pentacyclic natural alkaloid that inhibits the hTop1 enzyme involved in DNA transcription and cancer cell growth. Camptothecin structure pitfalls prompted us to design new congeners using a structure simplification strategy to reduce the ring extension number from pentacyclic to tetracyclic while maintaining potential stacking of the new compounds with the DNA base pairs at the Top1-mediated cleavage complex and aqueous solubility, as well as minimizing compound-liver toxicity. The principal axis of this study was the verification of hTop1 inhibiting activity as a possible mechanism of action and the elaboration of new simplified inhibitors with improved pharmacodynamic and pharmacokinetic profiling using three structure panels (A-C) of (isoquinolinoimidazoquinazoline), (imidazoquinazoline), and (imidazoisoquinoline), respectively. DNA relaxation assay identified five compounds as hTop1 inhibitors belonging to the imidazoisoquinolines 3a,b, the imidazoquinazolines 12, and the isoquinolinoimidazoquinazolines 7a,b. In an MTT cytotoxicity assay against different cancer cell lines, compound 12 was the most potent against HOS bone cancer cells (IC50 = 1.47 µM). At the same time, the other inhibitors had no detectable activity against any cancer cell type. Compound (12) demonstrated great penetrating power in the HOS cancer cells' 3D-multicellular tumor spheroid model. Bioinformatics research of the hTop1 gene revealed that the TP53 cell proliferative gene is in the network of hTop1. The finding is confirmed empirically using the gene expression assay that proved the increase in p53 expression. The impact of structure simplification on compound 12 profile, characterized by the absence of acute oral liver toxicity when compared to Doxorubicin as a standard inhibitor, the lethal dose measured on Swiss Albino female mice and reported at LD50 = 250 mg/kg, and therapeutic significance in reducing colon adenocarcinoma tumor volume by 75.36 % after five weeks of treatment with compound 12. The molecular docking solutions of the active CPT-based derivative 12 and the inactive congener 14 into the active site of hTop1 and the activity cliffing of such MMP directed us to recommend the addition of HBD and HBA variables to compound 12 imidazoquinazoline core scaffold to enhance the potency via hydrogen bond formation with the major groove amino acids (Asp533, Lys532) as well as maintaining the hydrogen bond with the minor groove amino acid Arg364.

Raludotatug Deruxtecan, a CDH6-Targeting Antibody-Drug Conjugate with a DNA Topoisomerase I Inhibitor DXd, Is Efficacious in Human Ovarian and Kidney Cancer Models.

Cadherin-6 (CDH6) is expressed in several cancer types, but no CDH6-targeted therapy is currently clinically available. Here, we generated raludotatug deruxtecan (R-DXd; DS-6000), a novel CDH6-targeting antibody-drug conjugate with a potent DNA topoisomerase I inhibitor, and evaluated its properties, pharmacologic activities, and safety profile. In vitro pharmacologic activities and the mechanisms of action of R-DXd were assessed in serous-type ovarian cancer and renal cell carcinoma cell lines. In vivo pharmacologic activities were evaluated with several human cancer cell lines and patient-derived xenograft mouse models. The safety profile in cynomolgus monkeys was also assessed. R-DXd exhibited CDH6 expression-dependent cell growth-inhibitory activity and induced tumor regression in xenograft models. In this process, R-DXd specifically bound to CDH6, was internalized into cancer cells, and then translocated to the lysosome. The DXd released from R-DXd induced the phosphorylation of Chk1, a DNA damage marker, and cleaved caspase-3, an apoptosis marker, in cancer cells. It was also confirmed that the DXd payload had a bystander effect, passing through the cell membrane and impacting surrounding cells. The safety profile of R-DXd was favorable and the highest non-severely toxic dose was 30 mg/kg in cynomolgus monkeys. R-DXd demonstrated potent antitumor activity against CDH6-expressing tumors in mice and an acceptable safety profile in monkeys. These findings indicate the potential of R-DXd as a new treatment option for patients with CDH6-expressing serous-type ovarian cancer and renal cell carcinoma in a clinical setting.

Design and synthesis of luotonin A-derived topoisomerase targeting scaffold with potent antitumor effect and low genotoxicity.

Conventional topoisomerase (Topo) inhibitors typically usually exert their cytotoxicity by damaging the DNAs, which exhibit high toxicity and tend to result in secondary carcinogenesis risk. Molecules that have potent topoisomerase inhibitory activity but involve less DNA damage provide more desirable scaffolds for developing novel chemotherapeutic agents. In this work, we broke the rigid pentacyclic system of luotonin A and synthesized thirty-three compounds as potential Topo inhibitors based on the devised molecular motif. Further investigation disclose that two compounds with the highest antiproliferation activity against cancer cells, 5aA and 5dD, had a distinct Topo I inhibitory mechanism different from those of the classic Topo I inhibitors CPT or luteolin, and were able to obviate the obvious cellular DNA damage typically associated with clinically available Topo inhibitors. The animal model experiments demonstrated that even in mice treated with a high dosage of 50 mg/kg 5aA, there were no obvious signs of toxicity or loss of body weight. The tumor growth inhibition (TGI) rate was 54.3 % when 20 mg/kg 5aA was given to the T24 xenograft mouse model, and 5aA targeted the cancer tissue precisely without causing damage to the liver and other major organs.

Current Role of Topoisomerase I Inhibitors for the Treatment of Mesenchymal Malignancies and Their Potential Future Use as Payload of Sarcoma-Specific Antibody-Drug Conjugates.

BACKGROUND: Topoisomerase I is an enzyme that plays a crucial part in DNA replication and transcription by the relaxation of supercoiled double-stranded DNA. Topoisomerase I inhibitors bind to the topoisomerase I cleavage complex, thereby stabilizing it and preventing the religation of the DNA strands, leading to DNA damage, cell cycle arrest, and apoptosis. Various topoisomerase I inhibitors have been evaluated in solid tumors, and irinotecan and topotecan have been approved for the treatment of epithelial malignancies. None of them have been approved for sarcoma, a diverse group of rare solid tumors with an unmet need for effective treatments. SUMMARY: Topoisomerase I inhibitors have been evaluated in preclinical studies as single agents or in combination in solid tumors, some of which have included sarcomas where activity was observed. Clinical trials evaluating topoisomerase I inhibitors for the treatment of sarcoma have shown limited efficacy as monotherapy. In combination with other cytotoxic agents, topoisomerase I inhibitors have become part of clinical routine in selected sarcoma subtypes. Regimens such as irinotecan/vincristine/temozolomide are used in relapsed rhabdomyosarcoma, irinotecan/temozolomide and vincristine/topotecan/cyclophosphamide are commonly given in refractory Ewing sarcoma, and topotecan/carboplatin showed some activity in advanced soft tissue sarcoma. This review provides an overview of key studies with topoisomerase I inhibitors for the treatment of sarcoma. Topoisomerase I inhibitors are currently also being assessed as "payloads" for antibody-drug conjugates (ADCs), allowing for the targeting of specific antigen-expressing tumor cells and the delivery of the inhibitor directly to the tumor cells with the potential of enhancing therapeutic efficacy while minimizing systemic toxicity. Here, we also provide a brief overview on topoisomerase I-ADCs. KEY MESSAGE: Topoisomerase I inhibitors are an important component of some systemic therapies for selected sarcomas and have potent cytotoxic properties and pharmacological characteristics that make them relevant candidates as payloads for the development of sarcoma-specific ADCs. ADCs are antibody-based targeted agents allowing for efficient and specific delivery of a given drug to the tumor cell. Topoisomerase I-ADCs are a novel targeted delivery approach which may have the potential to improve the therapeutic index of topoisomerase I inhibitors in the treatment of sarcoma and warrants investigation in a broad variety of mesenchymal malignancies.

Design, synthesis, and biological evaluation of harmine derivatives as topoisomerase I inhibitors for cancer treatment.

A series of ß-carboline derivatives were designed and synthesized by introducing the chalcone moiety into the harmine. The synthesized derivatives were evaluated their anti-proliferative activities against six human cancer cell lines (MCF-7, MDA-MB-231, HepG2, HT29, A549, and PC-3) and one normal cell line (L02). Among them, compound G11 exhibited the potent anti-proliferative activity against MCF-7 cell line, with an IC50 value of 0.34 µM. Further biological studies revealed that compound G11 inhibited colony formation of MCF-7 cells, suppressed MCF-7 cell migration by downregulating migration-associated protein MMP-2. In addition, it could induce apoptosis of MCF-7 cells by downregulating Bcl-2 and upregulating Cleaved-PARP, Bax, and phosphorylated Bim proteins. Furthermore, compound G11 can act as a Topo I inhibitor, affecting DNA synthesis and transcription, thereby inhibiting cancer cell proliferation. Moreover, compound G11 inhibited tumor growth in 4T1 syngeneic transplant mice with an inhibition rate of 43.19 % at a dose of 10 mg/kg, and 63.87 % at 20 mg/kg, without causing significant toxicity to the mice or their organs, achieving the goal of reduced toxicity and increased efficacy. All these results indicate of G11 has enormous potential as an anti-tumor agent and merits further investigation.

Reduction of HIF-1α/PD-L1 by Catalytic Topoisomerase Inhibitor Induces Cell Death Through Caspase Activation in Cancer Cells Under Hypoxia.

BACKGROUND/AIM: Under severe hypoxia, cellular apoptosis is induced through hypoxia-inducible factor 1, alpha subunit (HIF-1α)-dependent P53 accumulation and P53 phosphorylation via ataxia telangiectasia mutated and ataxia telangiectasia and RAD3-related (ATR) activation via replication stress-induced DNA damage response (DDR) activation. We previously demonstrated that the topoisomerase I catalytic inhibitor, 3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol (3EZ,20Ac-ingenol), induced apoptosis in Jeko-1 and Panc-1 cells, both of which show cyclin D1 overexpression. After progression to the S phase facilitated by nuclear cyclin D1, an intra S phase checkpoint was induced in the presence of 3EZ,20Ac-ingenol, by ATR activation in response to replication stress-induced DDR. MATERIALS AND METHODS: In this study, we examined whether 3-O-(2'E,4'E-decadienoyl)-20-O-acetylingenol (3EE,20Ac-ingenol) might induce a higher degree of P53 phosphorylation and additional HIF-1α and P53 accumulation in response to replication stress-induced DDR activation under hypoxic conditions than under normoxic conditions, by controlling ATR activation. RESULTS: In the Panc-1 cells, 3EE,20Ac-ingenol induced P53 activation and HIF-1α-dependent P53 accumulation through cooperative ATR activation via hypoxia-induced DDR activation. Jeko-1 cells showed slight HIF-1α accumulation under hypoxia, but HIF-1α-dependent 53 accumulation was not observed in the presence of 3EE,20Ac-ingenol, so that the cells remained resistant to hypoxia. CONCLUSION: 3EE,20Ac-ingenol induces an intricate interplay between P53 and HIF-1α accumulation via ATR activation that results in a high P53 accumulation, which promoted transient expression and early disappearance of HIF-1α, accelerating cell death. Strong P53 accumulation and consequent phosphatase and tensin homolog deleted on chromosome 10 activation in Panc-1 cells also reduced HIF-1α accumulation and programmed death-ligand 1 expression, which resulted in intense apoptosis.

Topoisomerase 1 Inhibition in MYC-Driven Cancer Promotes Aberrant R-Loop Accumulation to Induce Synthetic Lethality.

MYC is a central regulator of gene transcription and is frequently dysregulated in human cancers. As targeting MYC directly is challenging, an alternative strategy is to identify specific proteins or processes required for MYC to function as a potent cancer driver that can be targeted to result in synthetic lethality. To identify potential targets in MYC-driven cancers, we performed a genome-wide CRISPR knockout screen using an isogenic pair of breast cancer cell lines in which MYC dysregulation is the switch from benign to transformed tumor growth. Proteins that regulate R-loops were identified as a potential class of synthetic lethal targets. Dysregulated MYC elevated global transcription and coincident R-loop accumulation. Topoisomerase 1 (TOP1), a regulator of R-loops by DNA topology, was validated to be a vulnerability in cells with high MYC activity. Genetic knockdown of TOP1 in MYC-transformed cells resulted in reduced colony formation compared with control cells, demonstrating synthetic lethality. Overexpression of RNaseH1, a riboendonuclease that specifically degrades R-loops, rescued the reduction in clonogenicity induced by TOP1 deficiency, demonstrating that this vulnerability is driven by aberrant R-loop accumulation. Genetic and pharmacologic TOP1 inhibition selectively reduced the fitness of MYC-transformed tumors in vivo. Finally, drug response to TOP1 inhibitors (i.e., topotecan) significantly correlated with MYC levels and activity across panels of breast cancer cell lines and patient-derived organoids. Together, these results highlight TOP1 as a promising target for MYC-driven cancers. SIGNIFICANCE: CRISPR screening reveals topoisomerase 1 as an immediately actionable vulnerability in cancers harboring MYC as a driver oncoprotein that can be targeted with clinically approved inhibitors.

Synthesis and Biological Activity of a New Indenoisoquinoline Copper Derivative as a Topoisomerase I Inhibitor.

Topoisomerases are interesting targets in cancer chemotherapy. Here, we describe the design and synthesis of a novel copper(II) indenoisoquinoline complex, WN198. The new organometallic compound exhibits a cytotoxic effect on five adenocarcinoma cell lines (MCF-7, MDA-MB-231, HeLa, HT-29, and DU-145) with the lowest IC50 (0.37 ± 0.04 µM) for the triple-negative MDA-MB-231 breast cancer cell line. Below 5 µM, WN198 was ineffective on non-tumorigenic epithelial breast MCF-10A cells and Xenopus oocyte G2/M transition or embryonic development. Moreover, cancer cell lines showed autophagy markers including Beclin-1 accumulation and LC3-II formation. The DNA interaction of this new compound was evaluated and the dose-dependent topoisomerase I activity starting at 1 µM was confirmed using in vitro tests and has intercalation properties into DNA shown by melting curves and fluorescence measurements. Molecular modeling showed that the main interaction occurs with the aromatic ring but copper stabilizes the molecule before binding and so can putatively increase the potency as well. In this way, copper-derived indenoisoquinoline topoisomerase I inhibitor WN198 is a promising antitumorigenic agent for the development of future DNA-damaging treatments.