Biology subjects

Lee, O. W.

Publications and source records attributed to Lee, O. W..

11 recordsLinked to original sources

bioRxiv2026

Ablation of glypican-3 enhances radiosensitivity in liver cancer by prolonging G2/M arrest and activating the ATM/CHK2 pathway

Glypican-3 (GPC3) is an oncofetal protein widely being explored as a diagnostic and therapeutic target in hepatocellular carcinoma (HCC). Given that radiotherapy in the form of external beam and radioembolization are standard-of-care treatments for HCC, we aimed to determine whether there was any relationship between GPC3 and response to radiotherapy. Here, we demonstrate that GPC3 expression confers radioresistance in liver cancer through integrated in vitro, in vivo, and patient-level clinical analyses. Stable GPC3-knockout in liver cancer cell lines (HepG2, Hep3B, Huh7) and ectopic GPC3 expression in GPC3-negative liver cancer cells (SNU449), as well as in non-hepatic A431 cells, demonstrated that GPC3-mediated radioresistance is not restricted to hepatic lineage. Following irradiation, GPC3-deficient cells exhibited reduced proliferation, impaired clonogenic survival, persistent DNA damage, prolonged G2/M arrest, and increased apoptosis. Transcriptomic profiling demonstrated enrichment of cell-cycle and DNA damage response pathways in irradiated GPC3-deficient cells compared with GPC3-positive cells, and protein analyses confirmed sustained activation of the ATM/CHK2 axis. In vivo, GPC3 deletion markedly enhanced radiation-induced tumor growth delay in both HepG2 and A431 xenograft models. Consistent with these findings, high GPC3 expression was associated with inferior clinical outcomes in patients with HCC undergoing external-beam radiotherapy or radioembolization. Together, these findings identify GPC3 as a determinant of radioresistance in liver cancer and suggest its potential utility as a biomarker to guide radiotherapeutic strategies. Significance statementRadiotherapy is an important treatment option for HCC, but biomarkers that predict tumor response remain limited. GPC3 is highly expressed in most HCCs and is being investigated as an important biomarker for diagnosis and treatment of this disease, yet its relationship, if any, on radiosensitivity has not been previously reported. Here, we identify GPC3 as a modulator of radioresistance. GPC3 loss enhances radiosensitivity and is associated with persistent unresolved DNA damage, prolonged G2/M arrest, and sustained activation of the ATM/CHK2 pathway, resulting in delayed tumor growth after irradiation. In a clinical cohort of patients treated with radiotherapy, high GPC3 expression was associated with poorer overall survival. These findings suggest that GPC3 expressing tumors may necessitate either more dose-intense radiotherapy, radiobioligically ablative and/or combined with other modalities, or alternative therapeutic modalities to adequately treat HCC.

cancer biology↗

bioRxiv2025

Revealing the Drivers Underlying Distinct Evolutionary Trajectories in Lung Adenocarcinoma

Elucidating the evolution of cancers allows us to understand their key events, and the order in which they occur. To chart and interpret these evolutionary trajectories, we leverage whole-genome sequencing of lung tumours, including those from the largest cohort to date of lung cancers in subjects who have never smoked. Through ordering frequent genomic alterations, we discover three distinct evolutionary paths taken by lung adenocarcinomas; two dominated by tumours from people who have never smoked (NS-LUAD), and one followed by the vast majority of those who have smoked (S-LUAD). However, one in six NS-LUAD follow the smoking-dominant trajectory. These tumours, surprisingly, have fewer somatic alterations than the other NS-LUAD, and have shorter latency. They are strongly enriched for KRAS mutations. Our results suggest that gaining KRAS mutations allows these tumours to evolve more rapidly, acquiring a set of smoking-associated key alterations, with less need for genomic instability to progress. These tumours are three times more frequent in subjects of European vs. East Asian ancestry. These findings could shape clinical management strategies for lung adenocarcinoma patients, particularly for tumours driven by smoking-like evolutionary trajectories.

cancer biology↗

bioRxiv2025

A prognostic signature for lung adenocarcinoma in people who have never smoked

Knowledge of tumor cell dynamics can inform prognosis and treatment yet is largely lacking for lung adenocarcinoma in people who have never smoked (NS-LUAD). With RNA-seq data from 684 NS-LUAD and validation in an independent dataset, we identified three subtypes with distinct phenotypic traits and cell compositions. Additional genomic and histological data further characterized the subtypes. Steady, marked by low proliferation, high alveolar cell fraction, moderate-to-well differentiation, and fewer driver genes alterations, is linked to prolonged survival and low immune evasion. Proliferative shows high proliferation markers, TP53 mutations, and gene fusions. Chaotic, with high epithelial-to-mesenchymal transition markers, has the worst prognosis even within stage I tumors. Lacking known molecular or histological characteristics, this aggressive subtype is solely identified by transcriptomic data. A 60-gene signature recapitulates the overall classification and strongly predicts survival even within subgroups based on tumor stage or known genomic features, emphasizing its potential for improving NS-LUAD prognostication in clinical settings. SignificanceThe transcriptome of 684 lung adenocarcinomas in people who have never smoked (NS-LUAD) identifies three subtypes with different cellular dynamics, and genomic and morphologic features. A 60-gene signature accurately stratifies subjects for mortality risk, even in stage I, offering a clinically applicable tool for treatment decision making in NS-LUAD patients.

genomics↗

bioRxiv2025

Examining the Role of Extrachromosomal DNA in 1,216 Lung Cancers

The role of extrachromosomal DNA (ecDNA) in lung cancer, particularly in subjects who never smoked (LCINS), remains unclear. Examination of over 1200 whole-genome-sequenced lung cancers identified ecDNA in 18.9% of patients. Recurrent amplification of MDM2 and other oncogenes via ecDNA possibly drives a LCINS subset. Tumors harboring ecDNA showed worse overall survival than tumors harboring other focal amplifications. A strong association with whole-genome doubling suggests most ecDNA reflects genomic instability in treatment-naive lung cancer.

bioinformatics↗

bioRxiv2025

Deciphering lung adenocarcinoma evolution and the role of LINE-1 retrotransposition

Understanding lung cancer evolution can identify tools for intercepting its growth. In a landscape analysis of 1024 lung adenocarcinomas (LUAD) with deep whole-genome sequencing integrated with multiomic data, we identified 542 LUAD that displayed diverse clonal architecture. In this group, we observed an interplay between mobile elements, endogenous and exogenous mutational processes, distinct driver genes, and epidemiological features. Our results revealed divergent evolutionary trajectories based on tobacco smoking exposure, ancestry, and sex. LUAD from smokers showed an abundance of tobacco-related C:G>A:T driver mutations in KRAS plus short subclonal diversification. LUAD in never smokers showed early occurrence of copy number alterations and EGFR mutations associated with SBS5 and SBS40a mutational signatures. Tumors harboring EGFR mutations exhibited long latency, particularly in females of European-ancestry (EU_N). In EU_N, EGFR mutations preceded the occurrence of other driver genes, including TP53 and RBM10. Tumors from Asian never smokers showed a short clonal evolution and presented with heterogeneous repetitive patterns for the inferred mutational order. Importantly, we found that the mutational signature ID2 is a marker of a previously unrecognized mechanism for LUAD evolution. Tumors with ID2 showed short latency and high L1 retrotransposon activity linked to L1 promoter demethylation. These tumors exhibited an aggressive phenotype, characterized by increased genomic instability, elevated hypoxia scores, low burden of neoantigens, propensity to develop metastasis, and poor overall survival. Re-activated L1 retrotransposition-induced mutagenesis can contribute to the origin of the mutational signature ID2, including through the regulation of the transcriptional factor ZNF695, a member of the KZFP family. The complex nature of LUAD evolution creates both challenges and opportunities for screening and treatment plans.

genomics↗

bioRxiv2021

Characterization and tissue localization of zebrafish homologs of the human ABCB1 multidrug transporter

Given its similarities with mammalian systems, the zebrafish has emerged as a potential model to study the blood-brain barrier (BBB). Capillary endothelial cells at the human BBB express high levels of P-glycoprotein (P-gp, encoded by the ABCB1 gene) and ABCG2 (encoded by the ABCG2 gene). However, little information has been available about ATP-binding cassette transporters expressed at the zebrafish BBB. In this study, we focus on the characterization and tissue localization of two genes that are similar to human ABCB1, zebrafish abcb4 and abcb5. Cytotoxicity assays with stably-transfected cell lines revealed that zebrafish Abcb5 cannot efficiently transport the substrates doxorubicin and mitoxantrone compared to human P-gp and zebrafish Abcb4. Additionally, zebrafish Abcb5 did not transport the fluorescent probes BODIPY-ethylenediamine or LDS 751, while they were readily transported by Abcb4 and P-gp. A high-throughput screen conducted with 90 human P-gp substrates confirmed that zebrafish Abcb4 has overlapping substrate specificity with P-gp. Basal ATPase activity of zebrafish Abcb4 and Abcb5 was comparable to that of human P-gp. In the brain vasculature, RNAscope probes to detect abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. Zebrafish abcb4 also colocalized with claudin-5 expression in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, consistent with different functions. The data suggest that zebrafish Abcb4 most closely phenocopies P-gp and that the zebrafish may be a viable model to study the role of the multidrug transporter P-gp at the BBB.

pharmacology and toxicology↗

bioRxiv2019

Identification of activators of human fumarate hydratase by quantitative high-throughput screening

Fumarate hydratase (FH) is a metabolic enzyme that is part of the Krebs-cycle, and reversibly catalyzes the hydration of fumarate to malate. Mutations of the FH gene have been associated with fumarate hydratase deficiency (FHD), hereditary leiomyomatosis, renal cell cancer (HLRCC), and other diseases. Currently there are no high-quality small molecule probes for studying human fumarate hydratase. To address this, we developed a quantitative high throughput screening (qHTS) FH assay and screened a total of 57,037 compounds from in-house libraries in dose-response. While no inhibitors of FH were confirmed, a series of phenyl-pyrrolo-pyrimidine-diones were identified as activators of human fumarate hydratase. These compounds were not substrates of fumarate hydratase, were inactive in a malate dehydrogenase counter screen, and showed no detectable reduction-oxidation activity. The binding of two compounds from the series to human fumarate hydratase was confirmed by microscale thermophoresis. The low hit rate in this screening campaign confirmed that FH is a tough target to modulate, and the small molecule activators of human fumarate hydratase reported here may serve as a starting point for further optimization and development into cellular probes of human FH and potential drug candidates.

biochemistry↗

bioRxiv2019

A High-Throughput Screen of a Library of Therapeutics Identifies Substrates of P-glycoprotein

The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to limit brain penetration of many chemotherapy drugs. Although Food and Drug Administration guidelines require that potential interactions of investigational drugs with P-gp be explored, often this information does not enter into the literature. As such, we developed a high-throughput screen (HTS) to identify substrates of P-gp from a series of chemical libraries, testing a total of 10,804 compounds, most of which have known mechanisms of action. We used the CellTiter-Glo viability assay to test library compounds against parental KB-3-1 human cervical adenocarcinoma cells and the colchicine-selected sub-line KB-8-5-11 that over-expresses P-gp. KB-8-5-11 cells were also tested in the presence of a P-gp inhibitor (tariquidar) to assess reversability of transporter-mediated resistance. Of the tested compounds, a total of 90 P-gp substrates were identified including 55 newly identified P-gp substrates. Substrates were confirmed using an orthogonal killing assay against HEK-293 cells transfected with P-gp. We confirmed that AT7159 (cyclin-dependent kinase inhibitor); AT9283, (Janus kinase 2/3 inhibitor); ispinesib (kinesin spindle protein inhibitor); gedatolisib (PKI-587, phosphoinositide 3-kinase/mammalian target of rampamycin inhibitor); GSK-690693 (AKT inhibitor); and KW-2478 (heat shock protein 90 inhibitor) were substrates, and direct ATPase stimulation was assessed. ABCG2 was also found to confer high levels of resistance to AT9283, GSK-690693 and gedatolisib, while ispinesib, AT7519 and KW-2478 were weaker substrates. Combinations of P-gp substrates and inhibitors were assessed to demonstrate on-target synergistic cell killing. This data will be of use in determining understanding how chemotherapeutic agents will cross the blood-brain barrier.

pharmacology and toxicology↗

bioRxiv2018

Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening

Cell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaigns are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for downstream evaluation can influence the future direction of projects, so cytotoxicity profiling of screening libraries at an early stage is essential for increasing the likelihood of candidate success. In this study, we assessed cell-based cytotoxicity of nearly 10,000 compounds in NCATS annotated libraries, and over 100,000 compounds in a diversity library, against four normal cell lines (HEK 293, NIH 3T3, CRL-7250 and HaCat) and one cancer cell line (KB 3-1, a HeLa subline). This large-scale library profiling was analyzed for overall screening outcomes, hit rates, pan-activity and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile a large screening collection (>100,000 compounds) for cytotoxicity in both normal and cancer cell lines. The results generated here constitutes a valuable resource for the scientific community and provides insight on the extent of cytotoxic compounds in screening libraries, identifying and avoiding compounds with cytotoxicity during high-throughput screening campaigns.

cell biology↗

bioRxiv2017

Small Molecule Inhibitors of the Human Histone Lysine Methyltransferase NSD2 / WHSC1 / MMSET Identified from a Quantitative High-Throughput Screen with Nucleosome Substrate

The activity of the histone lysine methyltransferase NSD2 is thought to play a driving role in oncogenesis. Both overexpression of NSD2 and point mutations that increase its catalytic activity are associated with a variety of human cancers. While NSD2 is an attractive therapeutic target, no potent, selective and cell-active inhibitors have been reported to date, possibly due to the challenging nature of developing high-throughput assays for NSD2. To establish a platform for the discovery and development of selective NSD2 inhibitors, multiple assays were optimized and implemented. Quantitative high-throughput screening was performed with full-length wild-type NSD2 and a nucleosome substrate against a diverse collection of known bioactives comprising 16,251 compounds. Actives from the primary screen were further interrogated with orthogonal and counter assays, as well as activity assays with the clinically relevant NSD2 mutants E1099K and T1150A. Five confirmed inhibitors were selected for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. The identification of NSD2 inhibitors that bind the catalytic SET domain and demonstrate activity in cells validates the workflow, providing a template for identifying selective NSD2 inhibitors.

cancer biology↗

bioRxiv2017

Assessing Inhibitors Of Mutant Isocitrate Dehydrogenase Using A Suite Of Pre-Clinical Discovery Assays

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that are mutated in a variety of cancers to confer a gain-of-function activity resulting in the accumulation and secretion of an oncometabolite, D-2-hydroxyglutarate (2-HG). Accumulation of 2-HG can result in epigenetic dysregulation and a block in cellular differentiation, suggesting these mutations play a role in neoplasia. Based on its potential as a cancer target, a number of small molecule inhibitors have been developed to specifically inhibit mutant forms of IDH (mIDH1 and mIDH2). Here, a panel of mIDH inhibitors were systematically profiled using biochemical, cell-based, and tier-one ADME techniques. We quantified the biochemical effect of each inhibitor on mIDH1 (R132H and R132C) and mIDH2 (R172Q). The effect of these inhibitors on 2-HG concentrations in seven cell lines representing five different IDH1 mutations in both 2D and 3D cell cultures was assessed. Target engagement of these inhibitors was analyzed utilizing cellular thermal shift assays (CETSA), the effects of inhibitors on reversing 2-HG-induced block on leukemic cellular differentiation. We conclude from our mIDH1 assay panel that AG-120 and a Novartis inhibitor exhibited excellent activity in all biochemical and most cellular assays. While AG-120 has superior DMPK properties, it lacks efficacy a leukemic differentiation model. In conclusion, we present a comprehensive suite of in vitro preclinical drug development assays that can be used as a tool-box to identify lead compounds for mIDH drug discovery programs, as well as what we believe is the most comprehensive publically available dataset on the top mIDH inhibitors.

cancer biology↗