A novel 3D porous hydrogel model, mimicking the tumor microenvironment (TME), was developed as a physiologically relevant platform to investigate the role of extrachromosomal DNA (ecDNA) in colorectal cancer. We fabricated a tailorable collagen-based hydrogel that overcomes the limitation of 2D cultures by enabling crucial cell-cell and cell-matrix interactions. Methods:First, we validated whether the selected COLO320 cell lines were suitable for investigation of ecDNA in 3D tumor model and confirmed that ecDNA structures were stably maintained under 3D culture conditions by whole genome sequencing (WGS). Additionally, to provide an appropriate environment for colorectal cancer cells, we fabricated collagen-based porous hydrogels using a whipping process that requires no surfactants or sacrificial materials. During this process, we optimized the bioink formulation to achieve extracellular matrix (ECM) stiffness favorable for colorectal cancer cell proliferation, aggregation, stem-like behavior, and epithelial-mesenchymal transition (EMT)-related gene expression. Results:By optimizing the porous structure for enhanced nutrient diffusion and cell infiltration, we successfully maintained ecDNA structures in COLO320 cells. Our optimized porous platform significantly enhanced cellular proliferation, aggregation, and metabolic activity compared to conventional bulk model. We also observed elevated expressions of key oncogenes like MYC and activation of mechanotransduction pathways associated with aggressive tumor phenotypes. Conclusion:This reproducible and effective model accurately reflects ecDNA-driven biological behaviors, making it ideal for long-term ecDNA research and future TME-related studies.

Abstract Background: Extrachromosomal DNA (ecDNA) is increasingly recognized as a key driver of oncogene amplification, intratumoral heterogeneity, and poor clinical outcomes across solid tumors. However, its significance in multiple myeloma remains largely unexplored. Multiple myeloma is characterized by marked genomic instability including hyperdiploidy or IgH translocations, and widespread structural rearrangements that underlie biological heterogeneity and therapy resistance. Defining whether ecDNA contributes an additional layer of genomic complexity in this disease therefore represents an important unmet need. Methods: Whole-genome sequencing (WGS) BAM files of patient tumor samples from the Multiple Myeloma Research Foundation (MMRF) CoMMpass study were analyzed using the AmpliconSuite pipeline to identify circularized DNA amplicons indicative of ecDNA. Matched transcriptome (RNA-seq) data and clinical metadata from the same cohort were integrated to evaluate the prognostic impact of gene expression associated with ecDNA-harboring loci. Gene Set Enrichment Analysis (GSEA) was performed using the clusterProfiler R package to assess pathway-level biological signatures linked to ecDNA-positive tumors. Results: We identified ecDNA in 28 of 904 patients (3.1%) from the MMRF CoMMpass dataset. Despite the low prevalence, ecDNA-positive myeloma patients exhibited significantly inferior survival compared with patients without ecDNA. Frequently amplified ecDNA loci contained oncogenes such as MYC, TNFRSF17 (BCMA), and SNX29, as well as pro-tumoral lncRNAs including CASC11 and PVT1. GSEA revealed strong enrichment of MYC and E2F targets, oxidative phosphorylation, and PI3K-AKT-mTOR and mTORC1 signaling in ecDNA-positive tumors, highlighting transcriptional addiction and metabolic activation. These pathways align with known mechanisms of proliferation, metabolic rewiring, and treatment resistance in multiple myeloma. Conclusions: ecDNA-positive myeloma represents a biologically aggressive subset characterized by oncogene-enriched circular amplicons and hyperactivated proliferative and metabolic programs. Myeloma patients with ecDNA showed significantly worse survival, supporting ecDNA as a previously underrecognized high-risk feature. These findings highlight ecDNA as potential therapeutic vulnerabilities in myeloma and warrant further investigation. Citation Format: Hi Eun Jung, Saeam Shin, Yu Ri Kim, Ja Min Byun, Youngil Koh, Junshik Hong, Dong-Yeop Shin, Inho Kim, Hoon Kim, Hyunsoo Cho. Extrachromosomal DNA shapes aggressive transcriptional states in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5354.

ETHNOPHARMACOLOGICAL RELEVANCE:Broussonetia papyrifera (BP) has been used for traditional medicine in amelioration of cognitive decline. Accumulation of β-amyloid (Aβ) plaques majorly contributed to the pathogenesis of Alzheimer's disease (AD). AIM OF THE STUDY:This study aimed to investigate the role of broussochalcone A (BCA), a bioactive constituent of BP, in alleviation of cognitive impairment via Aβ aggregation inhibition in model mice. METHODS:We screened a potent Aβ aggregation inhibitor from a herbal library and analyzed its effect on improving cognitive functions in model mice and principal protein expression. In addition, we compared the metabolite profiling of blood and tissues. We used the aggregation inhibition assay to screen 960 herbal compounds using Aβ42-, and a leading compound was selected based on drug-like properties. Animal behavioral tests including the Morris water maze were performed using scopolamine (SCO)-treated mice. Western blotting and histopathological analysis were performed. Key compounds in the blood, hippocampus, and cortex were analyzed to compare the metabolic profiles. RESULTS:BCA was a potent Aβ aggregation inhibitor (IC50 = 1.75 ± 0.021 μM) with a predicted binding energy of -6.405 kcal/mol, and nontoxic to MDCK and SH-SY5Y cells. Molecular dynamics simulation revealed that the atomic contact numbers of BCA with Aβ were highly fluctuated during 100 ns; however, the transient contacts might prevent the aggregation. Cognitive function was significantly improved in BCA-treated mice in behavioral tests. Western blotting and histopathological analysis demonstrated that BCA treatment attenuated apoptosis, preserved hippocampal pyramidal neuron integrity, and alleviated SCO-induced spatial memory impairment. Metabolite profiling demonstrated that BCA modulated the metabolic pathways related to energy metabolism, redox homeostasis, amino acid turnover, and lipid metabolism in the serum and brain tissues, partially attenuating SCO-associated metabolic alterations. CONCLUSIONS:BCA is a potent Aβ aggregation inhibitor and exhibits significant cognitive improvement, as well as neuroprotective effects, decreasing inflammation, and retaining neuron structures. In addition, BCA induced distinct metabolic alterations in the serum and brain tissues compared to SCO. These results strongly support the use of BCA as a promising candidate for the amelioration of cognitive impairment and application to AD therapeutics.

A library of twenty-seven piperidine-based chalcones (PBCs) was designed, synthesised via microwave-assisted methodology, and subsequently evaluated for their inhibitory activity against monoamine oxidases (MAO-A/B), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). The PBCs exhibited potent and selective inhibition of MAO-B, with IC50 values ranging from 0.014 to >40 μM, while activity towards MAO-A was negligible (>40 μM). Among them, PBC21 was the most active (IC50 = 0.014 ± 0.0026 μM), whereas PBC1, PBC2, and PBC5 showed comparable potencies around 0.5-0.6 μM. Kinetic and reversibility studies identified PBC21 as a mixed, reversible MAO-B inhibitor (Kᵢ = 6.0 ± 2.8 nM). In contrast, cholinesterase inhibition was modest, with AChE activity ranging from 9.36 to >40 μM (best: PBC7, IC50 = 9.36 ± 0.15 μM) and BChE activity from 16.63 to >40 μM (best: PBC3, IC50 = 16.63 ± 1.94 μM). Selected sub-micromolar MAO-B inhibitors were further evaluated for in vitro metabolic stability and blood-brain barrier permeability. Among them, PBC21 exhibited a favourable microsomal stability profile in both rat and human liver microsomes, indicating improved metabolic robustness compared with the reference compound. Although experimental permeability assays were limited by compound adsorption to assay membranes, in silico prediction suggested that PBC21 possesses adequate oral bioavailability and the capacity to penetrate the blood-brain barrier. The most potent and selective MAO-B inhibitor, PBC21, was further evaluated in an MPTP-induced rat model of Parkinson's disease. Behavioural assessments including open field, pole, bar, rotarod, and forced swim tests demonstrated significant improvement in locomotor activity and motor coordination compared with the MPTP-treated group. Notably, PBC21 exhibited efficacy comparable to the reference drug selegiline in several parameters, highlighting its potential as a promising antiparkinsonian lead candidate. Molecular docking and molecular dynamics simulations were performed to elucidate the binding mode of PBC21 towards hMAO isoforms. The results indicated stable accommodation of PBC21 within the hMAO-B catalytic cavity through hydrophobic interactions and transient engagement with aromatic cage residues, while weaker interactions were observed with hMAO-A, consistent with the experimentally observed selectivity. Based on the results obtained, it is proposed that PBC21 is a potent and selective MAO-B inhibitor. These findings position PBC21 as a promising lead compound in the treatment of neurodegenerative diseases such as Parkinson's disease.

Monoamine oxidase (MAO) inhibitors are widely used to treat neurological disorders, including Parkinson's disease and depression, by increasing and prolonging dopamine activity. In this study, fourteen isatin-based derivatives from phenyl substituted thiosemicarbazone (IT) and semicarbazone (ISZ) sub-series were synthesized and evaluated for MAO inhibition. Most compounds showed higher MAO-B selectivity, with ISZ9 being the most potent MAO-B inhibitor (IC50 = 0.016 mu M), surpassing safinamide (IC50 =0.021 mu M) and pargyline (IC50 =0.14 mu M). In the IT series, IT20 (IC50 = 0.162 mu M) also exhibited strong MAO-B inhibition. For MAO-A, ISZ12 (IC50 = 0.17 mu M) was the most potent inhibitor. ISZ8 had the highest selectivity index (SI = 962.33). Kinetic studies confirmed competitive and reversible inhibition, with ISZ9 and IT20 targeting MAO-B, while ISZ12 inhibited MAO-A. ISZ9 showed blood-brain barrier permeability, was non-toxic, and improved behavioral parameters and dopamine levels in rotenone-treated Drosophila. Docking and molecular dynamics studies revealed that ISZ9 stably binds MAO-B via hydrogen bonds (Tyr326, Ile198) and it-it stacking (Phe343), supporting its potential for neuroprotective therapy.

A new series of quinazolinone-triazole hybrids (QTHs, 5a-n) was designed, synthesized, and evaluated as potential multi-target-directed ligands for Alzheimer's disease. All synthesized compounds were screened against human monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B). The obtained results revealed pronounced selectivity toward MAO-B, with IC50 values ranging from 0.65 to 7.51 µM, while exhibiting negligible MAO-A inhibition (IC50 > 40 µM). Compounds 5a, 5d, 5g, 5h, and 5m emerged as the most potent and selective MAO-B inhibitors and were subsequently evaluated for inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Among them, compound 5h displayed the most favorable biological profile, showing potent inhibition of MAO-B (IC50 = 0.65 µM), AChE (IC50 = 0.084 µM), and BuChE (IC50 = 0.667 µM). In silico ADMET analysis indicated acceptable drug-like properties for the lead compounds. Furthermore, molecular docking studies against MAO-B and AChE revealed favorable binding interactions, while a 500 ns molecular dynamics simulation confirmed the stability of the 5h-MAO-B complex. Collectively, the present findings identify compound 5h as a promising lead candidate and highlight quinazolinone-triazole hybrids as attractive scaffolds for the development of selective MAO-B inhibitors with additional cholinesterase inhibitory activity for Alzheimer's disease treatment.

7555 Background: Extrachromosomal DNA (ecDNA) is increasingly recognized as a key driver of oncogene amplification, intratumoral heterogeneity, and poor clinical outcomes across solid tumors. However, its significance in multiple myeloma remains largely unexplored. Multiple myeloma is characterized by marked genomic instability including hyperdiploidy or IgH translocations, and widespread structural rearrangements that underlie biological heterogeneity and therapy resistance. Defining whether ecDNA contributes an additional layer of genomic complexity in this disease therefore represents an important unmet need. Methods: Whole-genome sequencing (WGS) BAM files of primary myeloma cells from the Multiple Myeloma Research Foundation (MMRF) CoMMpass study were analyzed using the AmpliconSuite pipeline to identify circularized DNA amplicons indicative of ecDNA. Matched transcriptome (RNA-seq) data and clinical metadata from the same cohort were integrated to evaluate the prognostic impact of gene expression associated with ecDNA-harboring loci. Gene Set Enrichment Analysis (GSEA) was performed using the clusterProfiler R package to assess pathway-level biological signatures linked to ecDNA-positive tumors. Results: We identified ecDNA in 28 of 904 patients (3.1%) from the MMRF CoMMpass dataset. Despite the low prevalence, ecDNA-positive myeloma patients exhibited significantly inferior survival compared with patients without ecDNA. Frequently amplified ecDNA loci contained oncogenes such as MYC, TNFRSF17 (BCMA), and SNX29, as well as pro-tumoral lncRNAs including CASC11 and PVT1. GSEA revealed strong enrichment of MYC and E2F targets, oxidative phosphorylation, and PI3K–AKT–mTOR and mTORC1 signaling in ecDNA-positive tumors, highlighting transcriptional addiction and metabolic activation. These pathways align with known mechanisms of proliferation, metabolic rewiring, and treatment resistance in multiple myeloma. Conclusions: ecDNA-positive myeloma represents a biologically aggressive subset characterized by oncogene-enriched circular amplicons and hyperactivated proliferative and metabolic programs. Myeloma patients with ecDNA showed significantly worse survival, supporting ecDNA as a previously underrecognized high-risk feature. These findings highlight ecDNA as potential therapeutic vulnerabilities in myeloma and warrants further investigation.

High-resolution NMR analysis of heterogeneous, insoluble lignocellulosic materials is a significant challenge because poor magnetic field homogeneity and restricted molecular mobility often hinder the acquisition of reproducible solution-state NMR spectra. In studies of lignocellulosic biomass and plant cell walls, gel-state NMR methods have enabled important advances. However, practical limitations in sample preparation, shimming stability, and spectral broadening have constrained method robustness and routine use. Here, we present a finely pulverized low-moisture (FPL) gel-state NMR method that enables the reliable acquisition of high-quality two-dimensional (2D) HSQC spectra from intact whole plant cell walls without chemical pretreatment. Systematic evaluation of drying protocols, ball-milling efficiency, and solvent composition shows that moisture control and particle fineness are the primary factors influencing gel homogeneity and magnetic field shimming performance. Use of a DMSO-d6/DMF-d7 (4:1, v/v) solvent system produces gels with reproducible chemical shifts, narrow linewidths, and well-resolved heteronuclear correlations. This stability allows standard automated shimming and acquisition protocols to be applied reliably. The method is validated across diverse types of biomass, establishing a robust NMR sample-preparation framework for whole plant cell wall analysis and extending the practical reach of solution-state NMR methodologies.

Abstract Glioblastoma (GBM) remains lethal despite maximal therapy. The adult subventricular zone (SVZ), a neural stem-cell niche, has been implicated as a potential site of origin, yet the identity and functional properties of putative GBM origin-like cells (GBM-OCs) within the SVZ remain unclear. An SVZ-restricted somatic mutation mouse model (Cre-induced EGFRvIII expression with Trp53 and Pten disruption) was established and mouse SVZ-derived cells were prospectively isolated for functional and molecular profiling. Self-renewal, multipotency, invasive potential and tumour-initiating capacity were assessed relative to control SVZ cells and matched tumour-derived tumourspheres. Whole-genome and RNA sequencing defined genomic and transcriptional alterations during early progression. Mouse GBM-OCs exhibited self-renewal and multilineage differentiation and initiated tumours only after re-implantation into the SVZ (11/29, 38%), whereas direct striatal implantation failed (0/25, 0%), indicating context-dependent tumorigenic potential associated with the SVZ microenvironment. In contrast, tumour-derived tumourspheres retained tumorigenic capacity upon implantation into both the SVZ and the striatum. During progression from mouse GBM-OCs to tumours, whole-chromosome and arm-level aneuploidies accumulated. In patients with GBM, multi-region single-nucleus RNA sequencing of tumour-free SVZ, matched tumours and tumour-free cortex identified rare neural stem cell-like, astrocyte-like and oligodendrocyte precursor-like SVZ populations transcriptionally aligned with GBM programmes. These cells showed single-nucleus RNA-inferred chromosome 7 gain and/or chromosome 10 loss signals, with concordant low-frequency copy-number alterations in the SVZ detected by exome sequencing and enriched in matched tumours. Together, these findings support the presence of SVZ-resident stem or progenitor-like populations with early GBM-associated features, consistent with putative GBM-OCs, and highlight the SVZ niche as a potential target for early detection and niche-informed therapeutic strategies.

Accumulation of amyloid-β (Aβ) plaques is an important cause of Alzheimer's disease (AD) pathogenesis. In this study, we evaluated Aβ aggregation inhibitory activity of synthesized naphthoquinone derivatives as well as improvement in cognitive functions and metabolite profiling of brain tissues using scopolamine (SCO)-induced mice. Compound 888 (2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)naphthalene-1,4-dione, [TPN]) showed the highest Aβ aggregation inhibitory activity (IC50 = 0.14 μM), and was more potent than the reference compound curcumin (IC50 = 1.63 μM). Compound TPN showed effective monoamine oxidase (MAO)-A, MAO-B, acetylcholinesterase, and butyrylcholinesterase inhibitions at 10 μM, likely as candidates for multitarget-directed ligands. TPN was permeable through the blood-brain barrier, and non-toxic to MDCK and SH-SY5Y cells. TPN displayed prolonged and stable interactions with Aβ42 during molecular dynamics simulations, in contrast to the short-lived contacts observed for curcumin. Cognitive impairment was significantly improved by TPN-treatment in behavioral tests. TPN treatment attenuated Aβ-related protein expression, inflammatory responses, oxidative stress-related changes, and apoptosis-related alterations, while preserving hippocampal pyramidal neurons and their typical morphology. In metabolite profiling, TPN modulated a narrower set of pathways mainly related to amino acid and kynurenine metabolism, whereas donepezil induced broader adjustments involving amino acid, mitochondrial/energy, and lipid-related pathways compared to those in the serum and cortex of the SCO group, in contrast to those in the hippocampus. Collectively, a potent Aβ aggregation inhibitor TPN showed significant cognitive improvement, accompanying by neuroprotective effects, decreasing inflammation, and retaining neuron structures, exhibiting changed metabolic profiles compared to the control treatments. These findings suggest that TPN has cognitive-protective and neuroprotective potential under scopolamine-induced impairment conditions and warrants further validation in AD-relevant models.

Pharmaceutical process impurities, even at trace levels, can exhibit unexpected biological activities that remain undetected during conventional safety evaluations. In the present study, we investigated the off target neuropharmacological liability of the aminomethyl process impurity of rivaroxaban using an integrated computational and experimental framework. In silico toxicity prediction using the ProTox 3.0 platform identified a high probability of off target neuropharmacological activity with monoamine oxidase (MAO) enzymes predicted as potential molecular targets. Experimental validation demonstrated selective inhibition of human MAO-B by the impurity (IC₅₀ = 27.90 ± 2.91 µM), whereas rivaroxaban itself showed no significant MAO inhibition, indicating that the impurity possesses distinct neuroactive properties absent in the parent drug. Molecular docking studies revealed stronger binding affinity of the impurity toward MAO-B (−8.88 kcal/mol) compared to rivaroxaban (−7.72 kcal/mol), supported by unique interactions including a salt bridge with Glu84. Furthermore, 100 ns molecular dynamics simulations confirmed the structural stability and persistence of key protein–ligand interactions within the MAO-B active site. Collectively, these findings provide the first mechanistic evidence that the aminomethyl process impurity of rivaroxaban exhibits measurable MAO-B inhibitory activity and potential neurotoxic behavior under experimental conditions. Although the impurity exists at trace levels unlikely to pose immediate clinical risk, this study highlights the growing importance of integrating predictive toxicology, molecular modelling, and biochemical validation into impurity risk assessment workflows for modern pharmaceutical development.

Abstract Extrachromosomal DNA (ecDNA) drives oncogene amplification and therapeutic resistance in multiple cancers, but its contribution to acquired resistance to EGFR inhibitors (EGFRi) in EGFR-mutant non-small cell lung cancer (NSCLC) is not well defined. While EGFR TKI resistance frequently arises through acquisition of the EGFR T790M mutation, many cases remain unexplained. We therefore aimed to determine whether ecDNA represents an additional mechanism associated with EGFR TKI resistance. We analyzed whole-genome sequencing datasets from TCGA, PCAWG, and the Hartwig Medical Foundation, encompassing 536 NSCLC tumors, and detected ecDNA structures using AmpliconArchitect. Across these public cohorts, ecDNA prevalence was markedly elevated in EGFR TKI-treated tumors (50%, 10/20) compared with primary tumors (14%, 20/147; OR≈6.35, p<0.001) and advanced untreated tumors (25%, 92/369; OR≈3.01, p=0.02), indicating a significant association between EGFR TKI exposure and increased ecDNA prevalence. Notably, all ecDNAs detected in the EGFR TKI-treated group harbored oncogenic drivers, underscoring their potential functional relevance. To investigate whether this enrichment reflects resistance-associated events, we established an isogenic PC9 resistance model by generating WGS and RNA-seq data from resistant subclones derived from a single EGFR TKI-sensitive clone following long-term erlotinib exposure. Analysis of 25 resistant PC9 samples revealed ecDNA acquisition in approximately 20% of clones (5/25), all of which contained oncogenes. These ecDNA-positive clones were mutually exclusive with EGFR T790M-positive samples (also 20%, 5/25), suggesting that ecDNA emergence represents an alternative resistance route rather than a secondary event downstream of T790M. Functional analyses demonstrated activation of EGFR downstream signaling pathways in ecDNA-positive resistant cells. For example, in one resistant subclone harboring a newly formed RAF1 ecDNA (copy number ∼24), we observed over 100-fold RAF1 overexpression and marked MAPK/ERK pathway activation, as supported by GSEA (NES = 1.39, p < 0.01). Functional RAF1-inhibition experiments further confirmed that suppressing RAF1-driven signaling restored erlotinib sensitivity, providing strong evidence that ecDNA-mediated RAF1 amplification leads to EGFR-independent MAPK pathway activation. EcDNA may promote the development of EGFR TKI resistance by enabling alternative signaling pathways that bypass EGFR activity. EcDNA-mediated oncogene amplification represents an important feature of EGFR TKI-resistant NSCLC and a potential therapeutic vulnerability, providing a foundation for strategies aimed at overcoming ecDNA-driven resistance. Citation Format: Boyoon Kim, Sujin Kim, Jeonghee Cho, Hoon Kim. EcDNA-mediated oncogene amplification underlies EGFR TKI resistance in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7036.

Abstract The amplification of oncogenes on extrachromosomal DNA (ecDNA) enables aggressive, rapidly evolving tumors. Its defiance of Mendelian segregation enables extreme copy number amplifications that escape chromosomal regulatory constraints. Our AmpliconSuite toolset is the most widely used tool for ecDNA analysis in whole genome sequencing data, now deployed on over 43,000 tumor samples. Here, we present novel insights into ecDNA biology enabled through large-scale integrative analysis with these methods. We analyzed 6,366 whole-genome sequenced tumors from combined ICGC and Hartwig Medical Foundation datasets, identifying 2,366 distinct ecDNA capturing >11,000 different genes. Our systematic gene co-amplification analysis revealed striking patterns of gene selection on ecDNA. CDK4-MDM2 co-amplification occurred predominantly via ecDNA (75% of co-amplifications). These loci, normally separated by >11Mbp on chromosome 12, preferentially assembled onto the same ecDNA molecule 93% of the time as revealed by structural analysis of the ecDNA. This suggests selective pressure for maintaining cell cycle and p53 pathway regulators on the same inheritable unit. Our analysis of frequent co-amplifications also revealed that ecDNA preferentially packages chromatin remodelers (NSD3, RSF1) alongside driver oncogenes, as well as including genes that support transcription and translation (INTS4, BRF2), creating self-contained oncogene “support” hubs. Structural analysis revealed cancer type-specific patterns to ecDNA structures, with EGFR ecDNA showing simple architectures in glioblastoma versus complex rearrangements in lung and breast cancers, suggesting distinct formation histories in different cancers. Through AmpliconRepository.org, we provide public access to these ecDNA predictions and co-amplification analysis across major cancer cohorts, currently hosting 16,000+ analyzed samples and 5,000+ characterized ecDNA amplifications. Uniquely open to community contributions, this resource enables researchers to explore patterns across datasets and validate findings. These findings reveal fundamental principles governing ecDNA formation and selection, with implications for understanding tumor heterogeneity, therapeutic resistance, and dependencies which underlie ecDNA-targeted therapies. Citation Format: Jens Luebeck, Ted Liefeld, Edwin Huang, Forrest Kim, Bhargavi Dameracharla, Michael A. Chan, Dhruv Khatri, Kyra Fetter, Kaiyuan Zhu, Thorin Tabor, Soyeon Kim, Hoon Kim, Roel Verhaak, Michael M. Reich, Paul S. Mischel, Jill P. Mesirov, Vineet Bafna. Gene co-amplification and structural patterns reveal principles of extrachromosomal DNA in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 66.

Ten halogenated benzofuran derivatives (BF1-BF10) were synthesized and evaluated for their inhibitory activity against monoamine oxidase isoforms. All compounds showed stronger inhibition of MAO-B compared to MAO-A. Among them, BF2 exhibited the most potent MAO-B inhibition (IC₅₀ = 0.082 µM), followed by BF5 (IC₅₀ = 0.095 µM) and BF1 (IC₅₀ = 0.16 µM), with high selectivity indices, particularly BF2 (SI = 1038.05). Kinetic studies revealed that BF2 acts as a competitive inhibitor with a Ki value of 0.083 ± 0.0056 µM. Dialysis experiments confirmed its reversible inhibition profile, comparable to the reference drug Safinamide. Both BF2and BF5 were non-cytotoxic toward L929 cells and demonstrated moderate blood-brain barrier permeability in PAMPA assay. Molecular docking showed stabilization of MAO-B via π-π stacking interactions with Tyr326 and Tyr398, and molecular dynamics simulations further confirmed stable binding of BF2 within the MAO-B active site. Overall, BF2 emerged as a potent, selective, and reversible MAO-B inhibitor, warranting for further investigation.

Abstract Gastric cancer (GC) is the fifth most prevalent cancer and the fourth leading cause of cancer mortality. However, clinical strategies targeting these amplifications in GC have been unsuccessful, often leading to treatment resistance and poor prognosis. Extrachromosomal DNA (ecDNA) has emerged as a major mechanism associated with oncogene focal amplification and adverse outcomes. In this study, we performed whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) on paired tumor-normal samples from 76 Korean GC patients collected through Seoul National University Bundang Hospital to understand the prevalence of ecDNAs and their clinical relevance in GC patients. Focal amplification regions amplicons were identified and classified using AmpliconArchitect (AA) and Amplicon Classifier into ecDNA (circular amplification) and non-ecDNA (Chromosomal Amplicons, ChAmps). Focal amplifications were highly frequent and showed a strong association with the chromosomal instability (CIN) subtype (P = 2.37e-09). Of the 76 patients, 17 (22.4%) were classified as "ecDNA positive patients". Notably, 75% of CIN subtype patients carried one or more ecDNA amplicons. Genomic analysis revealed that ecDNA amplicons were significantly larger (P = 0.00056) and more structurally complex than ChAmps, exhibiting a higher frequency of structural variants. ecDNA amplicons also harbored significantly more canonical cancer genes (P = 3.90e-03) and displayed significantly higher copy numbers of these genes compared to ChAmps (P = 6.50e-04). Furthermore, ecDNA regions were significantly enriched with putative transcriptional regulatory elements (P = 1.20e-04) and GC-specific accessible chromatin regions. In WTS data, genes within ecDNA exhibited significantly higher expression compared to in ChAmp (P = 3.59e-05). Gene Set Enrichment Analysis (GSEA) revealed that ecDNA cohorts displayed a significantly more pronounced immunosuppressive phenotype—characterized by downregulation of immune response gene sets—compared to ChAmp patients in both SNUBH and TCGA cohorts. Clinically, the presence of ecDNA conferred a significantly worse Overall Survival (OS) rate compared to ChAmp cohorts (Log-rank test, P = 0.012). Multivariate Cox proportional hazards analysis confirmed that ecDNA status acts as an independent risk factor for OS (HR = 14.4, P = 0.001)Our findings demonstrate that ecDNA amplification is frequent in GC, particularly within the CIN subtype, and is associated with distinct genomic complexity, higher oncogene burden, unique transcriptional consequences (immune suppression), and poor patient prognosis. The presence of ecDNA amplification may serve as a critical prognostic factor in GC, highlighting the need for personalized treatment, including the development of ecDNA-targeted therapies to improve treatment outcomes. Citation Format: Jieun Lee, Donghyeok Seol, Seunghyun Kang, Chanmi Bang, Mira Yoo, Soyeon Kim, Hyeongjin Cho, So Hyun Kang, Young Suk Park, Sang-Hoon Ahn, Hyung-Ho Kim, Eunhee YI, Sanghyun Kim, Hoon Kim, Yun-Suhk Suh. Extrachromosomal DNA amplification defines a high-risk subgroup and unique molecular features in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1411.

Twenty semicarbazone and thiosemicarbazone derivatives (T1-T20) were synthesized and evaluated for their inhibition of monoamine oxidases and cholinesterases. Most compounds showed stronger inhibition of MAO-B than MAO-A. Among the synthesized compounds, T6 most strongly inhibited MAO-B (IC50 = 6.45 µM), followed by T17 (IC50 = 9.46 µM). T6 and T17 exhibited the highest selectivity index values (3.60 and >4.23, respectively) for MAO-B as compared with MAO-A. T6 was a competitive MAO-B inhibitor. Regarding structure-activity relationship, the thiosemicarbazone T1 (IC50 = 20.74 µM) showed >1.93 times better MAO-B inhibition than the semicarbazone T5 (IC50 >40 µM); a similar pattern was observed for T6 vs. T10, T11 vs. T15, and T16 vs. T20, indicating that the thiosemicarbazone derivatives exhibited better MAO-B inhibition than the semicarbazone derivatives when the terminal -NH2 group was free. All the compounds showed less than 50% inhibition of acetylcholinesterase at 10 µM. Comparatively, compounds T3 and T12 exhibited 70.65 and 61.94% inhibition of butyrylcholinesterase at 10 µM, respectively. Docking studies of the MAO-B inhibition of the most promising molecules were performed using MzDOCK. Collectively, thiosemicarbazone derivatives, specially T6 and T17, warrant further investigation as the promising candidates for the development of novel therapeutics targeting MAO-B-related disorders.

Neurodegenerative disorders such as Parkinson's disease are closely associated with dysregulated activity of monoamine oxidase-B (MAO-B), which leads to dopamine depletion and oxidative stress. Despite the availability of numerous monoamine oxidase (MAO) inhibitors on the market, their irreversibility and associated side effects necessitate the development of more effective and reversible MAO-B inhibitors. In this study, a series of twenty-one indole-based derivatives (PSH1-PSH21), collectively designated as PSH, was synthesised and evaluated for inhibitory activity against MAO isoforms. Most synthesised compounds showed higher inhibitory activity toward MAO-B than MAO-A, indicating selectivity for MAO-B. Among the PSH derivatives, PSH18 exhibited the most potent MAO-B inhibitory activity (IC50 = 0.95 ± 0.02 µM), followed by PSH6 (IC50 = 1.79 ± 0.40 µM) and PSH2 (IC50 = 1.96 ± 0.08 µM). Compound PSH18 showed the highest selectivity index value of 42.11, followed by PSH6 (22.35) and PSH2 (20.41). Additionally, PSH18 was confirmed to be a competitive and reversible inhibitor of MAO-B with an inhibition constant value of 0.89 ± 0.035 µM. Notably, PSH18 exhibited good permeability across the blood-brain barrier in parallel artificial membrane permeability assay experiments, along with acceptable absorption, distribution, metabolism, excretion, and toxicity parameters predicted through in silico modelling, suggesting its potential as a central nervous system-targeted molecule. Molecular docking and a 200 ns molecular dynamics simulation demonstrated stable binding of the ligand to the MAO-B active-site pocket, driven by hydrophobic and π-π stacking interactions with key amino acid residues lining the aromatic cage. Moreover, the calculated binding energy indicated strong ligand-protein interactions. Overall, these results indicate that the PSH scaffold could serve as a promising lead structure for the development of potent and selective MAO-B inhibitors. These compounds may have therapeutic potential for the treatment of neurodegenerative diseases.

A focused library of 19 donepezil-linked chalcones (DLCs) was efficiently synthesised through microwave-assisted Claisen-Schmidt condensation and subsequently profiled for their inhibitory activities against cholinesterases (AChE and BuChE) as well as monoamine oxidases (MAO-A and MAO-B). The DLCs exhibited potent and selective inhibition of MAO-B, with IC50 values ranging from 0.019 to 18.98 μM, whereas activity toward MAO-A was moderate to low (IC50 = 0.81 to > 20 μM). Among the tested DLCs, DLC9 and DLC14 showed the highest MAO-B inhibitory potential with IC50 values of 0.054 ± 0.004 μM and 0.019 ± 0.0015 μM, respectively, and high selectivity indexes (> 370 and > 1052, respectively), whereas DLC12 displayed notable MAO-A inhibition (IC50 = 0.81 ± 0.035 μM). Kinetic and reversibility studies revealed that the selected two lead DLCs (DLC9 and DLC14) acted as mixed-type reversible MAO-B inhibitors, with Kᵢ values of 20.0 ± 2.83 nM and 10.0 ± 2.82 nM, respectively. Furthermore, IC50 values of AChE inhibitory activities ranged from 5.40 to > 40 µM, whereas those of BuChE inhibitory activity range from 4.30 to > 40 µM. DLC6 showed the best AChE inhibitory potential with IC50 values of 5.40 ± 0.29 µM, while DLC13 revealed effective BuChE inhibitory potential with an IC50 value of 4.30 ± 0.89 µM. Molecular docking studies performed on hMAO-A and hMAO-B revealed that DLC14 establishes favourable π-π stacking within the aromatic cage of hMAO-B and maintains complementary hydrophobic contacts along the substrate cavity, whereas DLC6 lacks this key interaction due to steric interference of the ethoxy substituent. Among the three most potent MAO-B inhibitors (DLC2, DLC9, and DLC14), DLC2 exhibited the most favourable microsomal stability with the longest half-life and lowest intrinsic clearance, whereas DLC14 showed comparable metabolic profiles in rat and human liver microsomes. Experimental BBB permeability assays were hindered by compound-membrane interactions; however, in silico predictions indicated satisfactory oral bioavailability and brain penetration for all three candidates. In the MPTP-induced rat model of parkinsonism, the selective MAO-B inhibitor DLC14 and the non-selective inhibitor DLC6 significantly improved motor deficits and behavioural impairments across open field, pole, bar, rotarod, and forced swim tests, with progressive improvements observed up to Day 28. Notably, DLC14 consistently outperformed DLC6 and demonstrated an efficacy profile comparable to that of Selegiline, highlighting its therapeutic potential as an antiparkinsonian agent. These results indicate that DLC14 is potent and selective MAO-B inhibitor and could serve as promising candidate for the treatment of neurodegenerative disorders, such as Parkinson's disease.