Biology subjects

Nguyen, J. V.

Publications and source records attributed to Nguyen, J. V..

10 recordsLinked to original sources

bioRxiv2026

Spatially resolved multimodal hallmarks of response to neoadjuvant immunotherapies in the melanoma ecosystem in 2D and 3D

Neoadjuvant immunotherapy has transformed cancer treatment, yet the spatial molecular architecture governing response and resistance across distinct immune checkpoint blockade (ICB) regimens remains poorly defined. We assembled the largest neoadjuvant ICB (NICB) spatial multi-omics cohort to date, profiling over 112 million cells at single-cell resolution across three melanoma ICB regimens using MERFISH spatial transcriptomics, multiplexed immunofluorescence, and scRNA-sequencing. These analyses revealed the full multicellular spatial architecture of the NICB tumor microenvironment, including mature TLS with germinal centers, TCF7+ stem-like T cells, myeloid cells organized into spatially distinct cellular neighborhoods with unique intercellular signaling circuits, and CCL19/CCL21-expressing fibroblasts as a previously unrecognized stromal scaffold sustaining these immune hubs. We developed three purpose-built computational tools that together enabled comprehensive quantification of this microenvironment for the first time: SCIRA for whole-slide single-cell receptor-ligand quantification, GCSCAN for molecularly grounded TLS and germinal center structural delineation, and PathNet-TLS for automated TLS detection on H&E images. Applying these tools across the cohort, we defined the immune and stromal composition and cellular neighborhood organization distinguishing responders from non-responders. We also quantified cell-cell interactions and regimen-specific immune architectures, including a markedly stronger mature TLS/germinal center response with IPI-NIVO than NIVO-RELA. Importantly, GCSCAN-quantified TLS and germinal center density each stratified disease-free survival, with responders that lack germinal centers having an elevated risk of relapse. Open-top light-sheet imaging and CODA-based 3D reconstruction further uncovered interconnected germinal center-TLS tunnels invisible to standard 2D histopathology. These findings establish a discovery-to-tool paradigm linking single-cell tumor microenvironment interrogation to clinically deployable computational pathology for biomarker-driven NICB assessment across cancer types.

cancer biology↗

bioRxiv2025

Shared and distinct sequence-function signatures define different modes of human TpoR activation

The human thrombopoietin receptor (hTpoR) exists primarily as JAK2-associated monomers that become activated when converted to dimeric forms that support JAK trans-phosphorylation. This can be achieved by several different modes of stimuli, including the natural ligand Tpo, biologic agonists that bind the same site as Tpo, small-molecule drugs that bind the transmembrane (TM) domain, oncogenic mutations in and near the TM domain, and by association with constitutively active JAK V617F or a mutant form of the chaperone protein calreticulin. It is unclear how the dimeric structures induced by synthetic agonists and mutations relate to one another, and whether any of these induce the same active structure as the native ligand Tpo, yet this has important implications both for fundamental cytokine receptor biology and for development of targeted interventions for hTpoR-driven myeloproliferative diseases. Here we used deep mutational scanning (DMS) across the TM and juxtamembrane (JM) regions of hTpoR to extract feature-rich sequence-function signatures across a variety of different activating contexts. While each displayed some unique features, synthetic agonists and activating mutations all exhibited strong dependence on a common TM interface that is consistent with previous models of a left-handed, near-parallel helix dimer with H499 facing lipid. In contrast, Tpo-mediated activation was broadly insensitive to TM-JM substitutions, indicating that it does not rely on the same interface. Modeling with AlphaFold 3 (AF3) consistently yielded a right-handed, "splayed" helix dimer that is close at the extracellular face, contains H499 in the interface and diverges toward the cytosolic face, resting on an intracellular amphipathic JM helix that lies parallel to the membrane, which is also observed in a DMS/AF3 analysis of human erythropoietin receptor. This splayed Tpo-bound dimer could be stably inserted into a lipid bilayer with associated JAK2 using molecular dynamics and is supported by experiments showing that most or all of the TM domain can be replaced by poly-valine, with little effect on Tpo-driven activation but catastrophic effects on responses to synthetic ligands. Our data support at least two different structural modes of hTpoR activation that reconcile prior biochemical models, rationalize patient variants, and inform mechanism-based agonist and antagonist design.

biochemistry↗

bioRxiv2025

Comparison of QuPath and HALO platforms for analysis of the tumor microenvironment in prostate cancer

QuPath, an open-source digital pathology platform, has gained widespread use for image analysis in biomedical research since its release in 2016. However, its reproducibility and reliability compared to commercial software, such as HALO, requires further validation, particularly for multiplex immunofluorescence (mIF) analysis. In this study, we performed a direct comparison of QuPath and HALO using a mIF-stained prostate cancer tissue microarray (TMA) inclusive of 192 unique cores. We evaluated performance across three key analytical modules: immune cell phenotyping, tumor infiltration with immune cells, and nearest neighbor analysis. Furthermore, we integrated QuPath with CytoMap, an open-source spatial analysis tool, to perform unsupervised clustering of immune cell infiltration--a feature not available in HALO. Our results demonstrated high concordance between two platforms, with correlation coefficients exceeding 0.89 for immune cell density, distance and pattern of cell organization in tumor microenvironment (TME). A neighborhood analysis using CytoMap was further performed and provided a more detailed spatial analysis of immune cell distribution across different prostate cancer grades. A significant increase of CD103+ T cell infiltration into TME was observed in prostate cancer, which might be associated with the expression level of its ligand (E-cadherin) in the tumor region. In conclusion, our findings validate QuPath as a robust and reproducible alternative to commercial platforms for fluorescence-based digital pathology. By demonstrating QuPaths capability to perform high-quality quantitative analysis with additional flexibility for integration with external tools, our study underscores its potential for advancing tumor microenvironment research in translational oncology.

pathology↗

bioRxiv2024

Mutational Profiling of SARS-CoV-2 PLpro in human cells reveals requirements for function, structure, and drug escape

SARS-CoV-2, the causative agent of COVID-19, is responsible for the recent global pandemic and remains a major source of mortality. Papain-like protease (PLpro) is a target for SARS-CoV-2 inhibitor development, as it is not only essential for viral replication through cleavage of the viral poly-proteins pp1a and pp1ab, but also has de-ubiquitylation and de-ISGylation activities, which can affect innate immune responses. To understand the features of PLpro that dictate activity and anticipate how emerging PLpro variants will affect function, we employed Deep Mutational Scanning to evaluate the mutational effects on enzymatic activity and protein stability in mammalian cells. We confirm features of the active site and identify all mutations in neighboring residues that support or ablate activity. We characterize residues responsible for substrate binding and demonstrate that although the blocking loop is remarkably tolerant to nearly all mutations, its flexibility is important for enzymatic function. We additionally find a connected network of mutations affecting function but not structure that extends far from the active site. Using our DMS libraries we were able to identify drug-escape variants to a common PLpro inhibitor scaffold and predict that plasticity in both the S4 pocket and blocking loop sequence should be considered during the drug design process.

biochemistry↗

bioRxiv2023

Increased spatial coupling of integrin and collagen IV in the immunoresistant clear cell renal cell carcinoma tumor microenvironment

BackgroundImmunotherapy (IO) has improved survival for patients with advanced clear cell renal cell carcinoma (ccRCC), but resistance to therapy develops in most patients. We use cellular-resolution spatial transcriptomics in patients with IO naive and IO exposed primary ccRCC tumors to better understand IO resistance. Spatial molecular imaging (SMI) was obtained for tumor and adjacent stroma samples. Spatial gene set enrichment analysis (GSEA) and autocorrelation (coupling with high expression) of ligand-receptor transcript pairs were assessed. Multiplex immunofluorescence (mIF) validation was used for significant autocorrelative findings and the cancer genome atlas (TCGA) and the clinical proteomic tumor analysis consortium (CPTAC) databases were queried to assess bulk RNA expression and proteomic correlates. Results21 patient samples underwent SMI. Viable tumors following IO harbored more stromal CD8+ T cells and neutrophils than IO naive tumors. YES1 was significantly upregulated in IO exposed tumor cells. The epithelial-mesenchymal transition pathway was enriched on spatial GSEA and the associated transcript pair COL4A1-ITGAV had significantly higher autocorrelation in the stroma. Fibroblasts, tumor cells, and endothelium had the relative highest expression. More integrin V+ cells were seen in IO exposed stroma on mIF validation. Compared to other cancers in TCGA, ccRCC tumors have the highest expression of both COL4A1 and ITGAV. In CPTAC, collagen IV protein was more abundant in advanced stages of disease. ConclusionsOn spatial transcriptomics, COL4A1 and ITGAV were more autocorrelated in IO-exposed stroma compared to IO-naive tumors, with high expression amongst fibroblasts, tumor cells, and endothelium. Integrin represents a potential therapeutic target in IO treated ccRCC.

cancer biology↗

bioRxiv2022

Mechanism of Lys6 poly-ubiquitin specificity by the L. pneumophila deubiquitinase LotA

The versatility of ubiquitination to impose control over vast domains of eukaryotic biology is due, in part, to diversification through differently-linked poly-ubiquitin chains. Deciphering the signaling roles for some poly-ubiquitin chain types, including those linked via K6, has been stymied by a lack of stringent linkage specificity among the implicated regulatory proteins. Forged through strong evolutionary pressures, pathogenic bacteria have evolved intricate mechanisms to regulate host ubiquitin, and in some cases even with exquisite specificity for distinct poly-ubiquitin signals. Herein, we identify and characterize a deubiquitinase domain of the secreted effector protein LotA from Legionella pneumophila that specifically regulates K6-linked poly-ubiquitin during infection. We demonstrate the utility of LotA as a tool for studying K6 poly-ubiquitin. By determining apo and diUb-bound structures, we identify the mechanism of LotA activation and K6 poly-ubiquitin specificity, and identify a novel ubiquitin-binding domain utilized among bacterial deubiquitinases.

biochemistry↗

bioRxiv2022

A conserved signaling pathway activates bacterial CBASS immune signaling in response to DNA damage

To protect themselves from the constant threat of bacteriophage (phage) infection, bacteria have evolved diverse immune systems including restriction/modification, CRISPR/Cas, and many others. Here we describe the discovery of a two-protein transcriptional regulator module associated with hundreds of CBASS (Cyclic oligonucleotide Based Anti-phage Signaling System) immune systems, and demonstrate that this module drives expression of its associated CBASS system in response to DNA damage. We show that the helix-turn-helix transcriptional repressor CapH binds the promoter region of its associated CBASS system to repress transcription until it is cleaved by the metallopeptidase CapP. CapP is inactive except in the presence of single-stranded DNA, and CapP activity in cells is stimulated by DNA-damaging drugs. Together, CapH and CapP drive increased expression of their associated CBASS system in response to DNA damage. In both their structures and mechanisms, CapH and CapP resemble regulators that drive increased expression of DNA damage response genes in radiation-resistant Deinococcus, and control the mobilization of prophages and mobile elements in response to DNA damage. We also identify CapH and CapP-related proteins associated with diverse known and putative bacterial immune systems, including DISARM and two uncharacterized operons encoding proteins related to eukaryotic ubiquitin signaling pathways. Overall, our data highlight a mechanism by which bacterial immune systems can sense and respond to a universal stress signal, potentially enabling multiple immune systems to mount a coordinated defensive effort against an invading pathogen.

biochemistry↗

bioRxiv2022

Control of bacterial anti-phage signaling by a WYL domain transcription factor

Bacteria use diverse immune systems to defend themselves from ubiquitous viruses termed bacteriophages (phages). Many anti-phage systems function by abortive infection to kill a phage-infected cell, raising the question of how they are regulated to avoid cell killing outside the context of infection. Here, we identify a transcription factor associated with the widespread CBASS bacterial immune system, that we term CapW. CapW forms a homodimer and binds a palindromic DNA sequence in the CBASS promoter region. Two crystal structures of CapW suggest that the protein switches from an unliganded, DNA binding-competent state to a ligand-bound state unable to bind DNA. We show that CapW strongly represses CBASS gene expression in uninfected cells, and that phage infection causes increased CBASS expression in a CapW-dependent manner. Unexpectedly, this CapW-dependent increase in CBASS expression is not required for robust anti-phage activity, suggesting that CapW may mediate CBASS activation and cell death in response to a signal other than phage infection. Our results parallel concurrent reports on the structure and activity of BrxR, a transcription factor associated with the BREX anti-phage system, suggesting that CapW and BrxR are members of a family of universal defense signaling proteins.

biophysics↗

bioRxiv2020

De novo designed receptor transmembrane domains enhance CAR-T cell cytotoxicity and attenuate cytokine release

De novo designed receptor transmembrane domains (TMDs) present opportunities for precise control of cellular receptor functions. We developed a de novo design strategy for generating programmed membrane proteins (proMPs): single-pass -helical TMDs that self-assemble through computationally defined and crystallographically validated interfaces. We used these proMPs to program specific oligomeric interactions into a chimeric antigen receptor (CAR) and found that both in vitro CAR T cell cytokine release and in vivo antitumor activity scaled linearly with the oligomeric state encoded by the receptor TMD, from monomers up to tetramers. All programmed CARs (proCARs) stimulated substantially lower T cell cytokine release relative to the commonly used CD28 TMD, which we show elevated cytokine release through lateral recruitment of the endogenous T cell costimulatory receptor CD28. Precise design using orthogonal and modular TMDs thus provides a new way to program receptor structure and predictably tune activity for basic or applied synthetic biology.

synthetic biology↗

bioRxiv2020

Identification and characterization of diverse OTU deubiquitinases in bacteria

Manipulation of host ubiquitin signaling is becoming an increasingly apparent evolutionary strategy among bacterial and viral pathogens. By removing host ubiquitin signals, for example, invading pathogens can inactivate immune response pathways and evade detection. The Ovarian Tumor (OTU) family of deubiquitinases regulates diverse ubiquitin signals in humans. Viral pathogens have also extensively co-opted the OTU fold to subvert host signaling, but the extent to which bacteria utilize the OTU fold was unknown. We have predicted and validated a set of OTU deubiquitinases encoded by several classes of pathogenic bacteria. Biochemical assays highlight the ubiquitin and polyubiquitin linkage specificities of these bacterial deubiquitinases. By determining the ubiquitin-bound structures of two examples, we demonstrate the novel strategies that have evolved to both thread an OTU fold and to recognize a ubiquitin substrate. With these new examples, we perform the first cross-kingdom structural analysis of the OTU fold that highlights commonalities among distantly-related OTU deubiquitinases.

biochemistry↗