Biology subjects

Lee, T. D.

Publications and source records attributed to Lee, T. D..

9 recordsLinked to original sources

bioRxiv2026

Early dynamics of a neutrophil interferon program drive immunotherapy resistance and limit durable anti-tumor immunity

Despite the remarkable success of immune checkpoint blockade (ICB), durable clinical benefit remains limited to a subset of patients, highlighting the need for predictive biomarkers and new therapeutic targets. Here, we investigated the early immune dynamics associated with anti-PD1 responsiveness using longitudinal single-cell transcriptomics, spatial transcriptomics, and neutrophil-specific genetic perturbations in preclinical models of triple-negative breast cancer. We identified a conserved neutrophil interferon-stimulated gene (ISG) program that emerges before overt tumor divergence and predicts resistance to anti-PD1 therapy. Successful treatment was associated with an early reduction of IFN-responsive neutrophils in both blood and tumor tissues, whereas persistent neutrophil IFN signaling correlated with therapeutic resistance. Mechanistically, genetic disruption of either type I or type II interferon signaling in neutrophils enhanced anti-PD1 efficacy and promoted durable anti-tumor immune memory. Spatial analyses further revealed an inverse association between IFN-responsive neutrophils and tertiary lymphoid structure-enriched regions during effective therapy. Together, these findings identify IFN-responsive neutrophils as early determinants of immunotherapy outcome and establish dynamic neutrophil interferon programs as candidate blood-based biomarkers and therapeutic targets for cancer immunotherapy.

cancer biology↗

bioRxiv2023

Conditional lethality profiling reveals anticancer mechanisms of action and drug-nutrient interactions

Chemical screening studies have identified drug sensitivities across hundreds of cancer cell lines but most putative therapeutics fail to translate. Discovery and development of drug candidates in models that more accurately reflect nutrient availability in human biofluids may help in addressing this major challenge. Here we performed high-throughput screens in conventional versus Human Plasma-Like Medium (HPLM). Sets of conditional anticancer compounds span phases of clinical development and include non-oncology drugs. Among these, we characterize a unique dual-mechanism of action for brivudine, an agent otherwise approved for antiviral treatment. Using an integrative approach, we find that brivudine affects two independent targets in folate metabolism. We also traced conditional phenotypes for several drugs to the availability of nucleotide salvage pathway substrates and verified others for compounds that seemingly elicit off-target anticancer effects. Our findings establish generalizable strategies for exploiting conditional lethality in HPLM to reveal therapeutic candidates and mechanisms of action.

cancer biology↗

bioRxiv2023

Artificial intelligence-driven morphology-based enrichment of malignant cells from body fluid

Cell morphology is a fundamental feature used to evaluate patient specimens in pathological analysis. However, traditional cytopathology analysis of patient effusion samples is limited by low tumor cell abundance coupled with high background of non-malignant cells, restricting the ability for downstream molecular and functional analyses to identify actionable therapeutic targets. We applied the Deepcell platform that combines microfluidic sorting, brightfield imaging, and real-time deep learning interpretations based on multi-dimensional morphology to enrich carcinoma cells from malignant effusions without cell staining or labels. Carcinoma cell enrichment was validated with whole genome sequencing and targeted mutation analysis, which showed higher sensitivity for detection of tumor fractions and critical somatic variant mutations that were initially at low-levels or undetectable in pre-sort patient samples. Combined, our study demonstrates the feasibility and added value of supplementing traditional morphology-based cytology with deep learning, multi-dimensional morphology analysis, and microfluidic sorting.

pathology↗

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

Keeping it clean: the cell culture quality control experience at the National Center for Advancing Translational Sciences

Quality control monitoring of cell lines utilized in biomedical research is of critical importance, and is critical for reproducibility of data. Two key pitfalls in tissue culture are 1) cell line authenticity, and 2) mycoplasma contamination. As a collaborative research institute, the National Center for Advancing Translational Sciences receives cell lines from a range of commercial and academic sources, that are adapted for high-throughput screening. Here, we describe the implementation of a routine NCATS-wide mycoplasma testing and short-tandem repeat (STR) testing for cell lines. Initial testing identified a >10% mycoplasma contamination rate, but the implementation of clearly defined protocols that included immediate destruction of contaminated cell lines wherever possible has led to a much-reduced mycoplasma contamination rate, and data for >2,000 cell line samples tested over 3 years, and case studies are provided. STR testing of 170 cell lines with established STR profiles revealed only 5 mis-identified cell lines received from external labs. The data collected over the three years since implementation of this systematic testing demonstrates the importance of continual vigilance for rapid identification of problem cell lines, for ensuring reproducible data in translational science research.

cell biology↗

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↗

bioRxiv2018

Discovery and optimization of piperazine-1-thiourea-based human phosphoglycerate dehydrogenase inhibitors

Proliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate dehydrogenase (PHGDH) controls flux through the biosynthetic pathway for this important amino acid and represents a putative target in oncology. To discover inhibitors of PHGDH, a coupled biochemical assay was developed and optimized to enable high-throughput screening for inhibitors of human PHGDH. Feedback inhibition was minimized by coupling PHGDH activity to two downstream enzymes (PSAT1 and PSPH), providing a significant improvement in enzymatic turnover. Further coupling of NADH to a diaphorase/resazurin system enabled a red-shifted detection readout, minimizing interference due to compound autofluorescence. With this protocol, over 400,000 small molecules were screened for PHGDH inhibition, and following hit validation and triage work, a piperazine-1-thiourea was identified. Following rounds of medicinal chemistry and SAR exploration, two probes (NCT-502 and NCT-503) were identified. These molecules demonstrated improved target activity and encouraging ADME properties, enabling both in vitro and in vivo assessment of the biological importance of PHGDH, and its role in the fate of serine in PHGDH-dependent cancer cells.

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↗