Topoisomerase Inhibitors Research Articles

Discovery of Indolo[3,2-c]isoquinoline Derivatives as Novel Top1/2 Dual Inhibitors with Orally Efficacious Antitumor Activity and Low Toxicity.

Topoisomerase (Top) inhibitors used in clinical cancer treatments are limited because of their toxicity and severe side effects. Noteworthily, Top1/2 dual inhibitors overcome the compensatory effect between Top1 and 2 inhibitors to exhibit stronger antitumor efficacies. In this study, a series of indolo[3,2-c]isoquinoline derivatives were designed as Top1/2 dual inhibitors possessing apparent antiproliferative activities. Mechanistic studies indicated that the optimal compounds 23 and 31 with increasing reactive oxygen species levels damage DNA, inducing both cancer cell apoptosis and cycle arrest. Importantly, the results of the toxicity studies showed that compounds 23 and 31 possessed good oral safety profiles. In xenograft models, compound 23 exhibited remarkable antitumor potency, which was superior to the clinical Top inhibitors irinotecan and etoposide. Overall, this work highlights the therapeutic potential and safety profile of compound 23 as a Top1/2 dual inhibitor in tumor therapy and provides valuable lead compounds for further development of Top inhibitors.

A DXd/TLR7-Agonist Dual-Conjugate Anti-HER2 ADC Exerts Robust Antitumor Activity Through Tumor Cell Killing and Immune Activation.

The emergence of trastuzumab deruxtecan (T-DXd), a new-generation antibody-drug conjugate (ADC), has profoundly altered the therapeutic paradigm for HER2-positive solid tumors, demonstrating remarkable clinical benefits. However, the combined outcomes of T-DXd with immunotherapy agents remain ambiguous. In this study, we introduce Tras-DXd-MTL1, an innovative HER2 targeting ADC that integrates the topoisomerase inhibitor DXd and a toll like receptor 7 (TLR7) agonist MTT5, linked to trastuzumab via a GGFG tetrapeptide linker. Mechanistically, Tras-DXd-MTL1 retains the DNA-damaging and cell-killing properties of topoisomerase inhibitors while simultaneously enhancing the immune response within the tumor microenvironment. This is achieved by promoting immune cell infiltration and activating dendritic cells and CD8+T cells via MTT5. In vivo evaluation of Tras-DXd-MTL1's antitumor potency revealed a notably superior performance compared with the T-DXd (Tras-DXd) or Tras-MTL1 in immunocompetent mice with trastuzumab-resistant EMT6-HER2 tumor and immunodeficient mice with JIMT-1 tumor. This improved efficacy is primarily attributed to its dual functions of immune stimulation and cytotoxicity. Our findings highlight the potential of incorporating immunostimulatory agents into ADC design to potentiate antitumor effects and establish durable immune memory, thereby reducing tumor recurrence risks. Therefore, our study offers a novel strategy for the design of immune-activating ADCs and provides a potential approach for targeting solid tumors with different levels of HER2 expression.

Computational study of phytochemical derived bioactive compounds of Vernonia amygdalina as a potential inhibitor of Topoisomerase IV in pneumonia

Several of the bacteria responsible for pneumonia had become resistant to available antibiotics. According to the WHO, the resistance of Klebsiella pneumonia to Ciprofloxacin is 79.4%, making infections such as pneumonia and several diseases more difficult, if not impossible to treat. The prevalence of antimicrobial resistance is a global problem and among the 10 top global health threats facing humanity. With the projection of 10 million deaths per year by 2050 due to bacterial infections associated with antibiotic resistance according to WHO, there is an urgent need to identify an alternative approach to curb the incessant antimicrobial resistance and a complementary approach is to employ the use of naturally occurring compounds with potent antibacterial activities. We employed structural bioinformatics and theoretical chemistry techniques via molecular docking, and pharmacokinetic study, to identify novel S. pneumoniae topoisomerase IV inhibitors. The stringent molecular docking identified Arg140, Arg81, Glu55, Pro84, Met83, and Asn51, as principal amino acid residues for topoisomerase IV ligand interactions. Ten of the bioactive compounds found in Vernonia amygdalina showed a higher binding energy when compared to the reference compound (Ciprofloxacin). The overall analysis of MD results and binding free energy calculations reveal that Luteolin and 7-O-methylwogonin displayed stable trajectories with acceptable RMSD values and sufficient high negative energies throughout the MD simulation run of 100ns. These identified bioactive compounds in this study can be taken further for in vitro and In vivo studies to examine their efficacy against Pneumonia.

Inhibition of DNA Topoisomerase Ι by Flavonoids and Polyacetylenes Isolated from Bidens pilosa L.

Human DNA topoisomerase I (Topo I) is an essential enzyme in regulating DNA supercoiling during transcription and replication, and it is an important therapeutic target for anti-tumor agents. Bidens pilosa L. is a medicinal herb that is used as a folk medicine for cancers in China. A new flavonoid (1) and a new polyacetylene (20), along with eighteen flavonoids (2-19) and nine polyacetylenes (21-29), were isolated and identified from the methanol extract of the whole plant of B. pilosa, and some of the compounds (4, 5, 6 and 7) exhibited potent cytotoxicity against a panel of five human cancer cell lines. The DNA relaxation assay revealed that some flavonoids and polyacetylenes exerted inhibitory activities on human DNA Topo I, among them compounds 1, 2, 5, 6, 7, 8, 15, 19, 20, 22, and 24 were the most active ones, with IC50 values of 393.5, 328.98, 145.57, 239.27, 224.38, 189.84, 89.91, 47.5, 301.32, 178.03, and 218.27 μM, respectively. The structure-activity analysis of flavonoids was performed according to the results from the Topo I inhibition assay. The DNA content analysis revealed that 5, 6, and 7 potently arrested cell cycle at the G1/S and G2/M phases in human colon cancer cell DLD-1 depending on the concentration of the inhibitors. The levels of protein expression related to the G1/S and G2/M cell cycle checkpoints were in accordance with the results from the DNA content analysis. These findings suggest that flavonoids are one of the key active ingredients accounting for the anti-tumor effect of B. pilosa.

Discovery of aminopiperidine based potent & novel topoisomerase inhibitor with broad spectrum anti-bacterial activity

Bacterial DNA gyrase and topoisomerase IV inhibition has emerged as a promising strategy for the cure of infections caused by antibiotic-resistant bacteria. The Novel Bacterial Topoisomerase Inhibitors (NBTIs) bind to a different site from that of the quinolones with novel mechanism of action. This evades the existing target-mediated bacterial resistance associated with quinolones. This article presents our efforts to identify in vitro potent and broad-spectrum antibacterial agent 4l.

Selagibenzophenone B and Its Derivatives: SelB-1, a Dual Topoisomerase I/II Inhibitor Identified through In Vitro and In Silico Analyses.

The development of multitargeted drugs represents an innovative approach to cancer treatment, aiming to enhance drug effectiveness while minimizing side effects. Herein, we sought to elucidate the inhibitory effect of selagibenzophenone B derivatives on the survival of cancer cells and dual topoisomerase I/II enzyme activity. Results demonstrated that among the compounds, SelB-1 selectively inhibited the proliferation and migration of prostate cancer cells while exhibiting minimal effects on healthy cells. Furthermore, SelB-1 showed a dual inhibitory effect on topoisomerases. Computational analyses mirrored the results from enzyme inhibition assays, demonstrating the compound's strong binding affinity to the catalytic sites of the topoisomerases. To our surprise, SelB-1 did not induce apoptosis in prostate cancer cells; instead, it induced autophagic gene expression and lipid peroxidation while reducing GSH levels, which might be associated with ferroptotic death mechanisms. To summarize, the findings suggest that SelB-1 possesses the potential to serve as a dual topoisomerase inhibitor and can be further developed as a promising candidate for prostate cancer treatment.

Targeting DNA junction sites by bis-intercalators induces topological changes with potent antitumor effects.

Targeting inter-duplex junctions in catenated DNA with bidirectional bis-intercalators is a potential strategy for enhancing anticancer effects. In this study, we used d(CGTATACG)2, which forms a tetraplex base-pair junction that resembles the DNA-DNA contact structure, as a model target for two alkyl-linked diaminoacridine bis-intercalators, DA4 and DA5. Cross-linking of the junction site by the bis-intercalators induced substantial structural changes in the DNA, transforming it from a B-form helical end-to-end junction to an over-wounded side-by-side inter-duplex conformation with A-DNA characteristics and curvature. These structural perturbations facilitated the angled intercalation of DA4 and DA5 with propeller geometry into two adjacent duplexes. The addition of a single carbon to the DA5 linker caused a bend that aligned its chromophores with CpG sites, enabling continuous stacking and specific water-mediated interactions at the inter-duplex contacts. Furthermore, we have shown that the different topological changes induced by DA4 and DA5 lead to the inhibition of topoisomerase 2 activities, which may account for their antitumor effects. Thus, this study lays the foundations for bis-intercalators targeting biologically relevant DNA-DNA contact structures for anticancer drug development.

Development of Novel Bacterial Topoisomerase Inhibitors Assisted by Computational Screening.

Multidrug-resistant bacterial infections pose an ever-evolving threat to public health. Since the outset of the antibacterial age, bacteria have developed a multitude of diverse resistance mechanisms that suppress the effectiveness of current therapies. New drug entities, such as Novel Bacterial Topoisomerase Inhibitors (NBTIs), can circumvent this major issue. A computational docking model was employed to predict the binding to DNA gyrase of atypical NBTIs with novel pharmacophores. Synthesis of NBTIs based on computational docking and subsequent antibacterial evaluation against both Gram-positive and Gram-negative bacteria yielded congeners with outstanding anti-staphylococcal activity and varying activity against select Gram-negative pathogens.

Antibacterial and Cytotoxicity of Extracts and Isolated Compounds from Artemisia abyssinica: A Combined Experimental and Computational Study.

Artemisia abyssinica is a widely cultivated hedge plant in Ethiopia. Traditionally, they have been used to treat a variety of health conditions, including intestinal problems, infectious diseases, tonsillitis, and leishmaniasis. Silica gel chromatographic separation of the methanol and ethyl acetate extracts of the leaves, roots, and stem barks of A. abyssinica led to the isolation of 12 compounds, labeled as 1-12. Among these, compounds 1, 3, 4, 5, and 7-11 are reported as new to the genus Artemisia. The extracts and isolated compounds from A. abyssinica were evaluated for their in vitro antibacterial activity against four bacterial strains: Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, using the disc diffusion assay. All of the extracts displayed weak antibacterial activity, with inhibition zone diameters (IZDs) ranging from 6.10 ± 0.3 to 9.30 ± 0.20 mm. The isolated compounds, on the other hand, exhibited weak to moderate antibacterial activity, with IZDs ranging from 6.00 ± 0.300 to 13.50 ± 0.50 mm. The most potent antibacterial activity was observed for compound 6, which showed an IZD of 13.30 ± 0.50 mm against E. coli and 13.50 ± 0.50 mm against P. aeruginosa. This activity was comparable to that of the positive control ceftriaxone, which had IZDs of 14.1 ± 0.3 and 13.8 ± 0.5 mm against E. coli and P. aeruginosa, respectively. The in silico molecular docking analysis against DNA gyrase B revealed that compound 5 showed a higher binding affinity (-6.9 kcal/mol), followed by compound 10 (-6.7 kcal/mol) and compound 12 (-6.3 kcal/mol), whereas ciprofloxacin showed -7.3 kcal/mol. The binding affinities of compounds 5, 11, 10, and 9 were found to be -5.0, -4.3, -4.2, and -4.0 kcal/mol against S. aureus Pyruvate kinase, respectively, whereas ciprofloxacin showed a binding affinity of -4.9 kcal/mol, suggesting that compound 5 had a better binding affinity compared with ciprofloxacin. The effect of extracts of A. abyssinica was evaluated for cytotoxic activity against the breast cancer cell line (MCF-7) by the MTT assay. The extracts induced a decrease in cell viability and exerted a cytotoxic effect at a concentration of 20 μg/mL. The highest percent cell viability was observed for the methanol extract of the stem (92.9%), whereas the least was observed for the methanol extract of the root (34.5%). The result of the latter was significant compared with the positive control. The binding affinities of the isolated compounds were also assessed against human topoisomerase inhibitors IIβ. Results showed that compound 5 showed a binding affinity of -6.0 kcal/mol, followed by 11 (-5.4 kcal/mol), 10 (-5.0 kcal/mol), and 11 (-4.9 kcal/mol). Similar to ciprofloxacin, compounds 4, 5, 6, 9, 10, and 12 comply with Lipinski's rule of five. Overall, the comprehensive investigation of the chemical constituents and their biological activities reinforces the traditional medicinal applications of A. abyssinica and warrants further exploration of this plant as a source of novel therapeutic agents.

Inhibition of topoisomerase 2 catalytic activity impacts the integrity of heterochromatin and repetitive DNA and leads to interlinks between clustered repeats

DNA replication and transcription generate DNA supercoiling, which can cause topological stress and intertwining of daughter chromatin fibers, posing challenges to the completion of DNA replication and chromosome segregation. Type II topoisomerases (Top2s) are enzymes that relieve DNA supercoiling and decatenate braided sister chromatids. How Top2 complexes deal with the topological challenges in different chromatin contexts, and whether all chromosomal contexts are subjected equally to torsional stress and require Top2 activity is unknown. Here we show that catalytic inhibition of the Top2 complex in interphase has a profound effect on the stability of heterochromatin and repetitive DNA elements. Mechanistically, we find that catalytically inactive Top2 is trapped around heterochromatin leading to DNA breaks and unresolved catenates, which necessitate the recruitment of the structure specific endonuclease, Ercc1-XPF, in an SLX4- and SUMO-dependent manner. Our data are consistent with a model in which Top2 complex resolves not only catenates between sister chromatids but also inter-chromosomal catenates between clustered repetitive elements.

Ube3a unsilencer for the potential treatment of Angelman syndrome

Deletion of the maternal UBE3A allele causes Angelman syndrome (AS); because paternal UBE3A is epigenetically silenced by a long non-coding antisense (UBE3A-ATS) in neurons, this nearly eliminates UBE3A protein in the brain. Reactivating paternal UBE3A holds promise for treating AS. We previously showed topoisomerase inhibitors can reactivate paternal UBE3A, but their therapeutic challenges prompted our search for small molecule unsilencers with a different mechanism of action. Here, we found that (S)-PHA533533 acts through a novel mechanism to significantly increase paternal Ube3a mRNA and UBE3A protein levels while downregulating Ube3a-ATS in primary neurons derived from AS model mice. Furthermore, peripheral delivery of (S)-PHA533533 in AS model mice induces widespread neuronal UBE3A expression. Finally, we show that (S)-PHA533533 unsilences paternal UBE3A in AS patient-derived neurons, highlighting its translational potential. Our findings provide a lead for developing a small molecule treatment for AS that could be safe, non-invasively delivered, and capable of brain-wide unsilencing of paternal UBE3A.

Camptothecin and its Analogues as Putative Bruton Tyrosine Kinase (BTK) Inhibitors in Cancer Therapy - Biophysical Simulations of Wild Type and C481S Mutation

Camptothecin (CPT) and its derivatives are extracted from the Chinese tree Camptotheca acuminata and have been long known for their significant antitumor activity. The widely reported primary mechanism of their anti-cancer activity is through inhibition of topoisomerase I (Topo I), a key anticancer target. However, there is a lack of research on other possible mechanisms of action of CPT and its analogues (CPT/analogues). In this report, we investigated the potential inhibitory mechanism of CPT/derivatives against Bruton’s tyrosine kinase (BTK) ‒ a crucial protein for regulating various cellular functions and is essential for B-cell growth and cyclical division. The binding mechanism of CPT and its analogues (Irinotecan, Diflomotecan, and Topotecan) against BTK was investigated using molecular docking, molecular dynamics simulations and thermodynamic binding free energy calculations. NRX-0492, a potent orally active degrader of both wild-type and mutant BTK, was used as a reference/control inhibitor. We also included a set of active binders (actives) and non-binders (inactives) to distinguish between effective binders and false positives to ensure the validity of our results. Binding affinity analysis suggested that CPT and its analogues could potentially bind to BTK comparably to NRX-0492. The studied compounds demonstrated stable binding with the protein throughout the simulation time via hydrogen bonds, π-π interactions, van der Waals forces and π-sulfur interactions. Irinotecan exhibited a binding affinity of ΔGbinding = –42.4±5.3 kcal/mol, closely matching that of NRX-0492 (ΔGbinding = –48.8±3.4 kcal/mol). Furthermore, the activity of CPT/derivatives was examined against the C481S mutant. Although the activity was slightly decreased due to the mutation, the compounds retained a promising binding affinity when compared to the results of NRX-0492. The findings of this study provide foundation for future experimental investigation of unexplored potential anti-cancer mechanism of CPT/derivatives and offer a new perspective on repurposing of natural products in cancer therapy.

Novel antipyrine substituted 4-thiazolidinones: Synthesis, DNA binding and topoisomerase inhibition activities, and in-silico studies

In this study, we report the synthesis and characterization of novel 4-thiazolidinones containing antipyrine (3a-e) derived from the starting compounds of Schiff bases (2a-e). The Schiff bases were synthesized using aldehydes of natural origin, such as citronellal, cuminaldehyde, 2-thiazolecarboxaldehyde, 5-methylfurfural, and syringaldehyde. Their FTIR spectra revealed the enol- tautomeric solid-state structures of compounds 3b and 3d. The pUC18 plasmid DNA binding specificity and capacity of the compounds to catalytically generate topoisomerase I activity were also examined using agarose gel electrophoresis. The compounds induced changes in DNA mobility and showed a topoisomerase I inhibitory effect at all doses. 4-Thiazolidinones had a higher affinity in DNA and topoisomerase I than the Schiff bases. DNA binding and topoisomerase I activities were most effective at low concentration (6 μM) for the compounds of Schiff bases 2a, d, and 4-thiazolidinones 3a, d. Furthermore, this study presents computational methods for addressing the molecules’ drug-like properties. 2a, d, and 3a, d Compounds were used as representative of the most bioactive among the newly synthesized molecules in in-silico studies. The web tools of ADMETlab, SwissADME, OSIRIS, and the BOILED-Egg method were used to conduct in-silico biological studies involving ADME prediction, BBB penetration, gastrointestinal absorption, and toxicity studies. The ADME profile of the query compounds gets within the range of applicability and is at an appropriate level. In-silico studies revealed that compounds 2a, d, and 3a, d are neither hERG blockers nor AMES toxic. However, compounds 2a and 3a have hepatotoxicity.

A Review on Indole as a Cardinal Scaffold for Anticancer Drugs Development

Abstract: Chemotherapy is the mainstay of therapeutic cancer therapy; however, the development of resistance typically makes it less effective. There are continuous efforts by researchers to find novel lead compounds with potent anti-cancer activity. Generally, synthetic or natural heterocyclic compounds have been investigated in detail as a scaffold for cancer therapeutics. Among them, indole, owing to its unique physiochemical and biological properties, provides a promising platform for the development of pharmacophores for drug development against cancer, acting via various mechanisms. Till now, several indole-based derivatives have been identified as anti-cancer agents, which are either being used in clinics or are in various phases of clinical trials, suggesting their importance in anti-cancer drug development. These anti-cancer drugs have been classified into different classes depending on their mechanism of action. For example, histone deacetylase inhibitors (HDAC inhibitors), silent mating type information regulation 2 homolog (SIRT) inhibitors, tubulin inhibitors, proviral insertion site in Moloney murine leukemia virus (Pim) inhibitors, DNA Topoisomerase inhibitors, and kinase inhibitors. In this review, the author's approach is to compile the recent developments on indole-based anti-cancer drugs and provide insight into the respective structureactivity relationships (SARs) of the compounds. We hope the review will provide a thorough understanding to the reader and guide to developing novel and potent indole-based anticancer agents against drug-sensitive and drug-resistant cancer in the future.

Chemoradiotherapy and nanomedicine: Drug mechanisms and delivery systems.

Radiotherapy is an invaluable tool in the treatment of cancer. However, when used as a monotherapy, it fails to provide curative outcomes. Chemotherapy drugs are often included to boost the effects of radiation. Key classes of radiosensitizing drugs include platinum compounds, anthracyclines, antimetabolites, taxanes, topoisomerase inhibitors, alkylating agents, and DNA damage repair inhibitors. Chemoradiotherapy suffers from not only systemic toxicities from chemotherapy drugs but also synergistic radiation toxicity as well. It is critical to deliver radiosensitizing molecules to tumor cells while avoiding adjacent healthy tissues. Currently, nanomedicine provides an avenue for tumor specific delivery of radiosensitizers. Nanoscale delivery vehicles can be synthesized from lipids, polymers, or inorganic materials. Additionally, nanomedicine encompasses stimuli responsive particles including prodrug formulation for tumor specific activation. Clinically, nanomedicine and radiotherapy are intertwined with approved formulation including DOXIL and Abraxane. Though many challenges remain, the ongoing progress evidences a promising future for both nanomedicine and chemoradiotherapy. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Therapeutic Approaches and Drug Discovery > Nanomedicine for Cardiovascular Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies.

An update of the molecular mechanisms underlying anthracycline induced cardiotoxicity.

Anthracycline drugs mainly include doxorubicin, epirubicin, pirarubicin, and aclamycin, which are widely used to treat a variety of malignant tumors, such as breast cancer, gastrointestinal tumors, lymphoma, etc. With the accumulation of anthracycline drugs in the body, they can induce serious heart damage, limiting their clinical application. The mechanism by which anthracycline drugs cause cardiotoxicity is not yet clear. This review provides an overview of the different types of cardiac damage induced by anthracycline-class drugs and delves into the molecular mechanisms behind these injuries. Cardiac damage primarily involves alterations in myocardial cell function and pathological cell death, encompassing mitochondrial dysfunction, topoisomerase inhibition, disruptions in iron ion metabolism, myofibril degradation, and oxidative stress. Mechanisms of uptake and transport in anthracycline-induced cardiotoxicity are emphasized, as well as the role and breakthroughs of iPSC in cardiotoxicity studies. Selected novel cardioprotective therapies and mechanisms are updated. Mechanisms and protective strategies associated with anthracycline cardiotoxicity in animal experiments are examined, and the definition of drug damage in humans and animal models is discussed. Understanding these molecular mechanisms is of paramount importance in mitigating anthracycline-induced cardiac toxicity and guiding the development of safer approaches in cancer treatment.

PEMF Potentiates Doxorubicin-induced Late G2 Arrest in MDA-MB-231 Breast Cancer Cells.

Pulsed electromagnetic field (PEMF) stimulation enhances the efficacy of several anticancer drugs. Doxorubicin is an anticancer drug used to treat various types of cancer, including breast cancer. However, the effect of PEMF stimulation on the efficacy of doxorubicin and the underlying mechanisms remain unclear. Thus, this study aimed to investigate the effect of PEMF stimulation on the anticancer activity of doxorubicin in MDA-MB-231 human breast cancer cells. MDA-MB-231 cells were seeded and allowed to incubate for 48 h. The cells were treated with doxorubicin, cisplatin, 5-fluorouracil, or paclitaxel for 48 h. Subsequently, the cells were stimulated with a 60-min PEMF session thrice a day (with an interval of 4 h between each session) for 24 or 48 h. Cell viability was assessed by trypan blue dye exclusion assay and cell-cycle analysis was analyzed by flow cytometry. Molecular mechanisms involved in late G2 arrest were confirmed by a western blot assay and confocal microscopy. MDA-MB-231 cells treated with a combination of doxorubicin and PEMF had remarkably lower viability than those treated with doxorubicin alone. PEMF stimulation increased doxorubicin-induced cell-cycle arrest in the late G2 phase by suppressing cyclin-dependent kinase 1 (CDK1) activity through the enhancement of myelin transcription factor 1 (MYT1) expression, cell division cycle 25C (CDC25C) phosphorylation, and stratifin (14-3-3σ) expression. PEMF also increased doxorubicin-induced DNA damage by inhibiting DNA topoisomerase II alpha (TOP2A). These findings support the use of PEMF stimulation as an adjuvant to strengthen the antiproliferative effect of doxorubicin on breast cancer cells.

Topoisomerase Inhibitors and PIM1 Kinase Inhibitors Improve Gene Editing Efficiency Mediated by CRISPR-Cas9 and Homology-Directed Repair.

The CRISPR-Cas9 system has emerged as the most prevalent gene editing technology due to its simplicity, high efficiency, and low cost. However, the homology-directed repair (HDR)-mediated gene knock-in in this system suffers from low efficiency, which limits its application in animal model preparation, gene therapy, and agricultural genetic improvement. Here, we report the design and optimization of a simple and efficient reporter-based assay to visualize and quantify HDR efficiency. Through random screening of a small molecule compound library, two groups of compounds, including the topoisomerase inhibitors and PIM1 kinase inhibitors, have been identified to promote HDR. Two representative compounds, etoposide and quercetagetin, also significantly enhance the efficiency of CRISPR-Cas9 and HDR-mediated gene knock-in in mouse embryos. Our study not only provides an assay to screen compounds that may facilitate HDR but also identifies useful tool compounds to facilitate the construction of genetically modified animal models with the CRISPR-Cas9 system.

ETMR-18. ADVANCING ETMR RESEARCH: NOVELIN VITRO ANDIN VIVO PRECLINICAL MODELS FOR DEVELOPING MORE EFFECTIVE TREATMENTS

Abstract BACKGROUND Embryonal Tumor with Multilayered Rosettes (ETMR) is a rare and aggressive tumor that occurs mainly in infants under three years of age. Genetically, ETMRs are characterized by amplification and fusion of a microRNA cluster on chromosome 19 (C19MC) with TTYH1, present in ~90% of the tumors. ETMR patients still have a poor prognosis without any recent advances in treatment. A major limitation to develop more effective treatment strategies is the lack of sufficient preclinical models. METHODS Establishment of ETMR patient-derived xenograft (PDX) models and tumoroid lines, molecular characterization, high throughput in vitro drug screens, targeted drug treatments in vitro and in vivo. RESULTS We have established four new tumoroid and five new PDX ETMR models. These models faithfully recapitulate ETMR patients’ tumors based on their histological, transcriptomic, and methylome signatures. The in vitro models were derived from two patients and two PDX models and included three C19MC-amplified cell lines, of which two were a primary-relapse pair, and one a primary C19MC non-amplified model. In vivo we established the same models as PDX models subcutaneously plus an additional C19MC-amplified relapse model. High-throughput drug screening assays, using drug libraries with 225 clinically approved drugs and other small molecules, were conducted across the panel of in vitro models. Potential therapeutic candidates with promising activity in ETMR tumoroid lines identified from these screens include FGFR, XPO1, MTOR, MDM2 and topoisomerase inhibitors. CONCLUSIONS The establishment of novel preclinical models represents a significant advancement in ETMR research, offering a platform that captures the molecular complexity of the disease. The identification of several inhibitors shows the potential of these models for drug discovery and preclinical testing. Ongoing in vitro and in vivo experiments with these drugs and combinations aim to further validate their activity, moving towards the development of more effective therapies for ETMR patients.

MDB-61. STARVING TUMORS OF POLYAMINES: A NOVEL APPROACH IN TREATING AGGRESSIVE PEDIATRIC MEDULLOBLASTOMA

Abstract BACKGROUND Polyamines, essential intracellular polycations, govern fundamental cell processes and drive oncogenesis. Our research unveiled the dependency of pediatric Diffuse Midline Gliomas (DMG) on the polyamine pathway (Khan et al.,2021). Disruption of this pathway with the synthesis inhibitor DFMO, and the transport inhibitor AMXT1501, potently inhibited DMG tumors, yielding unprecedented survival in-vivo. We hypothesized that dual polyamine pathway targeting could similarly suppress growth in other aggressive pediatric brain tumors. METHODS/RESULTS We analyzed the expression of polyamine pathway regulators in high-risk pediatric brain cancers ZERO precision medicine platform and found elevated expression in medulloblastoma. ODC1, a polyamine synthesis driver, was found to have significant correlation in high-grade gliomas with four key functional tumor states—stemness, cell cycle, hypoxia, and angiogenesis. Medulloblastoma cells were sensitive to treatment with DFMO but showed a compensatory increase in polyamine transporter expression. Conversely, treatment with AMXT1501 increased ODC1 expression. Dual targeting of the polyamine pathway potently and synergistically inhibited cell proliferation and clonogenic potential in Group-3 medulloblastoma cells. Flow cytometric analysis of Annexin V-stained cells showed that treatment with DFMO and AMXT1501 in combination led to induction of apoptosis. Critically, dual polyamine inhibition potently sensitized medulloblastoma cells to the topoisomerase-I inhibitor, SN-38, leading to complete eradication of medulloblastoma cell proliferation and colony formation at nanomolar chemotherapeutic concentrations, with the effect most pronounced in Group-3 medulloblastoma. In triple combination-treated cells, over-representation analysis revealed heightened mitotic DNA damage, macroautophagy, and apoptosis, coupled with diminished neuron/synapse development, glycosylation, and cellular signaling pathways. We are currently testing this treatment strategy in aggressive orthotopic medulloblastoma models. CONCLUSION These findings suggest targeting the polyamine pathway as a promising strategy to sensitize aggressive pediatric medulloblastoma tumors to chemotherapy. DFMO/AMXT1501 is in Phase I evaluation for adults (NCT05500508) and advancing to a Phase I/II trial for children through the CONNECT consortium.