Methylation is a crucial biochemical reaction involved in a wide range of processes including gene regulation, signal transduction, epigenetics, metabolism and detoxification. A number of methyltransferases (MTases) catalyze transfer of methyl groups from S-adenosyl-L-methionine (AdoMet or SAM) to nucleic acids, proteins, and small molecules, affecting chromatin structure, RNA function, and metabolic pathways. MTase dysregulation is associated with maladies such as cancer, neurodevelopmental disorders, and metabolic syndromes. Advancements in bioinformatics and high-throughput genomics have resulted in identification of 200 human MTase genes, and most of the encoded proteins have now been characterized biochemically. Here, we have classified the human MTases into nine structural homology groups, including a distinct category of methyltransferases with unique structures. Major groups include the versatile seven-β-strand (7BS) MTases, the SET domain MTases which mainly mediate protein lysine methylation, and the SPOUT MTases involved in RNA modification. In addition, we categorized the MTases based on substrate specificity (e.g., nucleic acids, protein, and small-molecule MTases). This article provides a comprehensive classification and structural overview of human MTases, integrating recent nomenclature updates from the HUGO Gene Nomenclature Committee (HGNC). The evolutionary relationships and diversification of methyltransferases are also discussed in the context of structural classification and functional specialization. Emphasis is placed on biological functions, disease associations, and emerging therapeutic potential of the human MTases, particularly in oncology and neurodegenerative research. Despite significant progress, the biological function of many MTases remainselusive, necessitating further research to elucidate their enzymatic mechanisms and potential as drug targets. Understanding the MTase landscape is crucial for advancing biomedical research and developing targeted therapies for methylation-related disorders.

Significant loss of pigmentation can increase visual disability, skin cancer risk, and psychosocial stress. Tyrosinase (TYR) catalyzes the first and rate-limiting step of melanin synthesis. Inhibitors of TYR are well established and are currently used in clinical settings; however, there is a dearth of direct activators of TYR. Here, using a human TYR construct, we developed high-throughput screening methods, in cell confirmatory assays employing 13 C-tyrosine tracing, and computational analysis techniques, and identified ampyrone (4-aminoantipyrine) as a TYR activator. Ampyrone increased the in vitro catalytic activity of the human recombinant intramelanosomal domain of TYR (hTYR) and its hypomorphic variant, Pro406Leu (P406L), a cause of oculocutaneous albinism type 1B (OCA1B). Moreover, ampyrone induced melanin synthesis in both WT and OCA1B human melanocytes, mouse OCA2 melanocytes, as well as 3-dimensional (3D) human skin cultures. Computational studies provided additional insight into the effects of direct TYR agonists on enzyme activity. Our results identify ampyrone as a lead candidate for TYR activation, potentially supporting the development of therapies for patients with genetic and acquired diseases of hypopigmentation.

Metals and metalloids are widely used in industrial applications, and increasing experimental and epidemiological evidence has linkded metal exposure to adverse health outcomes. However, the underlying mechanisms for these effects have not been fully understood. As part of the Toxicology in 21st century (Tox21) program, we have screened more than 150 metal-containing compounds and their salt forms across over 90 biological endpoints. In this study, we analyzed the comprehensive toxicity of metal compounds using Tox21 screening data to enhance the understanding of their mechanism and molecular pathways involved in molecular initiating events. Integrated data analysis and in vitro confirmation experiments identified three potential novel targets of metal compounds (i.e., sonic hedgehog pathway, thyroid-stimulating hormone receptor, and thyrotropin-releasing hormone receptor). We also found that mercury- and tin-containing substances were highly bioactive. Furthermore, cell painting analysis uncovered metal-induced bioactivity could be classified into two patterns depending on their respective associations to mitochondrial-related morphology changes. Our results provide a comprehensive analysis of metals-association bioactivity data within Tox21 assays, which can be applied to estimate the potency ranges for metal-induced bioactivity that support risk assessment efforts for metal and metalloid exposures. These findings identify previously undercharacterized molecular targets of metal compounds, offering new insights into mechanisms of metal toxicity and informing improved risk assessment methodologies.

G-protein-coupled receptors (GPCRs) are a diverse family of seven-transmembrane domain receptors that play pivotal roles in various physiological and neurological processes by mediating extracellular signals through G proteins. Notable GPCRs such as ADRB2, CHRM1, DRD2, and HTR2A are important therapeutic targets linked to conditions ranging from asthma to schizophrenia. The human ether-à-go-go-related gene (hERG), encoding the Kv11.1 potassium channel, is critical for cardiac repolarization, the inhibition of which can lead to prolonged QT intervals and an increased risk of arrhythmias. Consequently, assessing hERG-GPCR interactions is essential during drug development to enhance safety and ensure regulatory compliance. In this study, we utilized quantitative high-throughput screening (qHTS) to identify GPCR agonists and inhibitors in the Tox21 10K compound library. We applied machine-learning (ML)-based quantitative structure-activity relationship (QSAR) models to predict selective GPCR-targeting compounds with reduced hERG liability, employing different data processing sequences. Our models trained on the Tox21 10K library screening data were subsequently validated by using the Library of Pharmacologically Active Compounds (LOPAC). Furthermore, the models were applied to virtually screen approximately 360 K diverse compounds, with the top predictions experimentally validated, revealing new GPCR modulators with minimal hERG liability. The findings provide efficient strategies for the development of lead compounds targeting GPCRs while minimizing the cardiac risks associated with hERG inhibition.

Aim: Antibody-drug conjugates (ADCs) feature an antibody recognizing a specific protein joined to a potent toxic payload. Numerous ADCs have received U.S. Food and Drug Administration (FDA) approval; however, clinical resistance arises. Resistance mechanisms include decreased expression or mutation of the antibody target, impaired payload release, or increased expression of adenosine triphosphate (ATP)-binding cassette (ABC) efflux transporters associated with multidrug resistance. We therefore sought to characterize the interactions of ABC multidrug transporters with ADC payloads. Methods: We performed a high-throughput screen with 27 common ADC payloads using cell lines expressing ABC transporters P-glycoprotein [P-gp, encoded by ABC subfamily B member 1 (ABCB1)] or ABC subfamily B member G2 (ABCG2, encoded by ABCG2). Confirmatory assays were also performed using cells transfected to express P-gp, ABCG2, or multidrug resistance-associated protein 1 (MRP1, encoded by ABCC1). Results: Several commonly used ADC payloads were substrates of P-gp, including calicheamicin γ1, monomethyl auristatin E, mertansine (DM1), and ravtansine (DM4). All the pyrrolobenzodiazepines tested - SJG136, SGD-1882, SG2057, and SG3199 - were substrates of P-gp, ABCG2, and MRP1. The modified anthracyclines nemorubicin and its metabolite PNU-159682 were poorly transported by both ABCB1 and ABCG2 and displayed nanomolar to picomolar toxicity. Further, we found that the efficacy of the FDA-approved ADC mirvetuximab soravtansine, with DM4 as the toxic payload, was decreased in cell lines expressing P-gp. In contrast, Duocarmycin DM and PNU-159682 were exquisitely toxic to a panel of 99 cancer cell lines of varying origins. Conclusion: Several commonly used ADC payloads can be transported by ABC transporters, potentially leading to transporter-mediated drug resistance in patients. Future ADCs should be developed using payloads that are not ABC transporter substrates.

Ewing sarcoma is a highly aggressive solid malignancy affecting children and young adults. Ewing sarcoma is driven primarily by EWSR1::FLI1, a fusion oncoprotein that has been notoriously difficult to target with traditional pharmacologic agents. There are numerous examples of promising preclinical combinations of small molecules that are never tested in pediatric clinical trials because agents fail to reach the market due to limited efficacy for common adult cancers. Moreover, the effectiveness of single-agent therapies for cancer treatment is often limited. To address these limitations, we selected 28 compounds that were FDA approved at the time of the study or in late stages of clinical development and known to regulate important pathways in Ewing sarcoma. We performed a drug screen in Ewing sarcoma cell lines with 180 combinations of tyrosine kinase inhibitors, cell cycle inhibitors, and conventional chemotherapy. The results of the screen revealed that a PI3K inhibitor, copanlisib, combined with a CDK4/6 inhibitor, ribociclib, exhibited strong synergistic anti-Ewing sarcoma activity. Using proteomic methods such as a reverse-phase protein array and western immunoblotting, we demonstrated that this combination induced a downregulation of the PI3K/AKT pathway as well as proteins involved in cell cycle regulation. We further confirmed these in vitro data using bulk RNA-sequencing. To evaluate the phenotypic effect of the PI3K/CDK4/6 inhibition in Ewing sarcoma lines, we performed apoptosis and cell cycle analyses using flow cytometry and demonstrated that ribociclib primarily induced a G0/G1 arrest with minimal effect on Ewing cell viability but significantly enhanced the apoptotic effect of copanlisib treatment. In two xenograft models of Ewing sarcoma, we demonstrated that the combination significantly prolonged survival compared to treatment with either vehicle or single-agent therapy alone. Our findings identify a new candidate therapy combination for Ewing sarcoma and provide a resource of additional potential synergistic combinations for future validation.

RNA seq data and RUNX1-PDGF-BB geneset

Primers for qRT-PCR, Chip-PCR and O-GlcNAc mutant sites

Cyclometallated gold(III) compounds were evaluated for their chemoselective capability to promote C-Se coupling reactions under biocompatible conditions. Competitive reactions with selenium and sulfur substrates highlighted the preference for selenium, and this selectivity was further confirmed in selenopeptide models mimicking the GPx active site. Given that thioredoxin reductase (TXNRD1) is a canonical target for gold compounds, we confirmed that our complexes also inhibit this enzyme, with the two six-membered metallacycles exhibiting a higher potency than auranofin. Expanding beyond TXNRD1, the compounds were further investigated as inhibitors of other selenoenzymes, specifically glutathione peroxidase isoenzymes (GPx1, GPx4). The metallacycles were potent inhibitors of GPx1, while in vitro GPx4 inhibition was overall less pronounced, with LC/MS studies identifying selenocysteine (Sec51) as the primary arylation site on GPx1. We demonstrated that this chemoselectivity could be translated to an intracellular setting. The selectivity towards Sec over Cys was further explored using A375 GPx4 WT and A375 GPx4 U46C mutant cell lines, where proliferation assays showed a greater effect in the GPx4 WT cells. By integrating structural and functional insights across selenoenzyme families, this study reveals glutathione peroxidases as pivotal molecular targets of cyclometallated gold(III) compounds and lays the groundwork for designing selective Sec-targeting metallodrugs, an approach with untapped potential in anticancer therapy.

Supplementary Figures associated with each Main Figure

Biliary tract cancers (BTC) are aggressive malignancies encompassing intrahepatic and extrahepatic cholangiocarcinoma, gallbladder carcinoma, and ampullary carcinoma. Here, we report integrative analysis of 63 BTC cell lines via multi-omics and genome-scale CRISPR screens. We identify widespread EGFR dependency in BTC, alongside dependencies selective to anatomic subtypes. Additionally, we delineate strategies to overcome therapeutic resistance, with combined EGFR inhibition potentiating targeting of KRAS-mutant and FGFR2 fusion-driven models and SHP2 inhibition effective in the latter context. Clustering RNA/protein expression and dependencies data revealed functional relationships transcending single-gene alterations, with biliary, squamous, or dual biliary/hepatocyte lineage signatures stratifying BTC models. These subtypes exhibit distinct dependency profiles-including cell fate transcription factors GRHL2, TP63, and HNF1B, respectively-and demonstrate prognostic significance in patient samples. Potential subtype-specific targetable vulnerabilities include integrinα3 and the detoxification enzyme UXS1. This cell line atlas reveals therapeutic targets in molecularly defined BTCs, unveils disease subtypes, and provides a resource for therapeutic development. SIGNIFICANCE:This integrative analysis of BTC cell lines defines the landscape of vulnerabilities across BTCs, stratifying distinct subtypes, and provides a key resource for studying disease heterogeneity. The findings highlight strategies for targeting BTCs with specific genomic alterations, as well as broader approaches based on shared molecular programs and essential pathways.

Excel file containing all data corresponding to Figure 7

Excel file containing all data corresponding to Figure 2

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer. Most MCCs contain Merkel cell polyomavirus (virus-positive MCC; VP-MCC), and the remaining are virus-negative (VN-MCC). Immune checkpoint inhibitors are the first-line treatment for metastatic MCC, but durable responses are achieved in less than 50% of patients. To identify new treatments, we screen ~4,000 compounds for their ability to reduce MCC viability and demonstrate that VP-MCC and VN-MCC exhibit distinct response profiles. Aurora kinase inhibitors selectively reduce VP-MCC viability, with RNAi screening independently identifying AURKB as an essential gene for MCC survival, especially in VP-MCC. AZD2811, a selective AURKB inhibitor, induces mitotic dysregulation and apoptosis in MCC cells, with greater efficacy in VP-MCC. In mice, AZD2811 nanoparticles inhibit tumor growth and increase survival in both VP-MCC and VN-MCC xenograft models. Overall, our unbiased screens identify AURKB as a promising therapeutic target and AZD2811NP as a potential treatment for MCC.

Mechanism of decreased PTEN expression in RD