Biology subjects

Wong, S.

Publications and source records attributed to Wong, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Novel Target Combinations in Lung Squamous Cell Carcinoma proposed by the Emet AI Research Environment and supported by discovery stage experimentation

Early drug discovery is frequently bottlenecked by target identification, a challenge that becomes particularly difficult in complex diseases driven by overlapping, redundant pathways rather than a single dominant driver. Lung squamous cell carcinoma (LUSC) is one such disease: mutationally complex, lacking a dominant actionable target, and marked by a long series of failed single-agent targeted trials. We hypothesized a research environment, capable of reasoning across interconnected datasets, would be well-suited to generate novel, mechanistically grounded therapeutic hypotheses for LUSC. Emet, an AI research environment utilizing a biomedical knowledge graph of more than 1.5 billion triples connected to over 100 specialised biological databases, was paired with an Agentic Research Director that pursues each hypothesis through a graph-of-thoughts search, invoking scoped link-prediction and retrieval tools and committing every round of findings to persistent memory. The top-ranked hypotheses were two-target combinations rather than a single target. The four highest-ranked combinations were advanced to dose-matrix testing in NCI-H520 and SK-MES-1 cells, and two produced combination effects exceeding either single agent; to our knowledge neither of these two pairings had previously been evaluated in LUSC. Dual inhibition of CDC7 (TAK-931) and PKMYT1 (RP-6306) produced statistically significant synergy in both lines that strengthened from day 5 to day 7 (HSA 19.24 to 24.19 in NCI-H520; 11.07 to 13.73 in SK-MES-1). Dual inhibition of USP13 (spautin-1) and PI3K (alpelisib) produced an additive, cell-line-dependent effect, and immunoblotting confirmed the predicted mechanism: time-dependent depletion of MCL-1, c-Myc and SOX-2 driven by the USP13 arm. Agentic reasoning over multi-domain biomedical evidence can therefore nominate testable, mechanism-bearing combination hypotheses that survive experimental scrutiny.

bioinformatics↗

bioRxiv2026

Integrative single cell analysis of CD8+ T-cells across early and advanced oral cancers reveals signatures of anti-tumour activity

Tumour-targeting CD8 T cells drive responses to every major form of cancer immunotherapy. Identifying them, however, remains an unsolved problem in solid tumours. The antigens they recognize are rarely defined and almost never shared between patients. We profiled 51,459 CD8+ T cells by paired single-cell RNA and T-cell receptor sequencing across 28 samples from 17 HPV-negative oral cancers spanning primary tumours, draining lymph nodes, metastases, and pembrolizumab-treated recurrences. We found that clonotypes that were expanded and shared across anatomical sites and timepoints were enriched within tumours and progressively selected over disease evolution and checkpoint blockade. Designating these shared-expanded clones as putative tumour-targeting cells, we trained a machine learning classifier that identifies them from transcriptome data alone. This 108-feature random forest signature recapitulated programmes of tumour reactivity and generalized to an integrated atlas of 89,318 CD8+ T cells from independent cohorts, showing progressive enrichment from normal to malignant tissue, and localized to tumour-proximal niches in spatial transcriptomics. By demonstrating that clonal behaviour across space and time encodes tumour reactivity in the transcriptome, this work establishes a generalizable framework for mapping tumour-engaged immunity without knowledge of the underlying antigen.

cancer biology↗

bioRxiv2026

Cryo-EM reveals patient- versus organ-specific structural diversity and bound ligands in lambda-6 light chain amyloids

Immunoglobulin light chain (LC) amyloidosis is a debilitating multiorgan disease with limited treatment options. Sequence and structural variability make LC amyloids particularly challenging for therapeutic targeting. We report four cryo-EM structures of lambda6-LC amyloid fibrils from four organs of two patients. Fibrils from different patients show different N-terminal conformations expanding known repertoire of lambda6-LC amyloid folds. These folds contain a planar beta-arch with a flexible linker containing the complementarity-determining region 2, flanked by N- and C-terminal segments in variable patient-specific conformations. The surface location of the structurally frustrated charged segment may contribute to the overrepresentation of the lambda6-LC family in amyloidosis. These and other lambda6-LC amyloid structures from different patients show different side chain packing. Conversely, cardiac, renal and splenic amyloids from the same patient exhibit similar structures with small peripheral organ-specific variations. Moreover, they show similar orphan densities, suggesting collagen-like triple helices bound to a tyrosine ladder along the fibril spine. Mass spectrometry detects collagen type-VI in tissue-extracted amyloids. Molecular dynamics simulations suggest amyloid binds collagen-VI triple helices via mixed interactions facilitated by the geometric complementarity between the layered amyloid structure and the triple helix. Similar interactions may drive formation of other amyloid-collagen complexes, influencing biological properties of amyloids.

biophysics↗

bioRxiv2026

Complete mitochondrial genomes of Arizona West Nile virus vectors, Culex quinquefasciatus and Culex tarsalis

Here we report a newly developed method utilizing long-range PCR and long-read Pacific Biosciences HiFi sequencing that successfully obtained two full-length and annotated mitochondrial genomes from Culex quinquefasciatus Say, 1823 and Culex tarsalis Coquillett, 1896, both from Maricopa County, Arizona, USA. Given the substantial burden of West Nile virus in Maricopa County over the past decade, and that these vectors are primarily responsible for spillover to human populations in the county, it is critical to better understand their distribution over time and space. This study begins to approach this need by contributing a novel approach that has resulted in the first West Nile virus vector mitochondrial genomes from Arizona. Our circular Cx. quinquefasciatus mitogenome is 15,587 bp in length, making it the first USA-based mitogenome sequenced through the AT-rich control region. The Cx. tarsalis mitochondrial genome is 16,416 bp long, longer than recently published California-based CTarK1 and Texas-based PQ585801 mitogenomes. The increased length of the Cx. tarsalis mitogenome is a result of a 905 bp insertion in the AT-rich control region, not present in the species publicly available mitogenomes. A maximum likelihood-based phylogenetic reconstruction supports the species designation of these newly-sequenced mitogenomes. The newly developed methodology offers a unique approach to study medically-important vector species around the globe, providing a solution to study populations through pooled vector pathogen surveillance programs.

genomics↗

bioRxiv2026

Sequence-dependent Stability and the Apparent Two-state Thermal Transition of Extended Collagen Triple Helices

The thermal stability of collagen triple helices is strongly influenced by the amino acid sequence of the repeating Gly-X-Y tripeptides, yet how these residue-specific interactions are integrated within an extended triple helix to determine thermal behavior remains poorly understood. Here, we addressed this question using recombinant collagen mimetic peptides (rCMPs) containing extended native sequences from the 1(I) and 2(I) chains of human type I collagen. Triple-helix formation was nucleated by a C-terminal foldon domain and further stabilized by interchain disulfide crosslinking, allowing the apparent melting temperature (T) to reflect interactions within the triple-helical domain independent of nucleation. The stabilizing effects of Pro and Y-position Arg identified in host-guest peptides were largely preserved in extended triple helices, whereas the proposed Lys-Gly-Glu (KGE) interchain salt bridge produced little measurable stabilization, demonstrating the influence of sequence context. Remarkably, identical triple-helical sequences exhibited markedly different thermal behavior when unfolding was initiated under different conditions. Nevertheless, extended triple helices differing substantially in sequence and length retained an apparently two-state thermal transition. These findings support a mechanism in which unfolding is preferentially initiated within regions of lower intrinsic stability, while the continuity of the triple helix couples neighboring regions into a cooperative unfolding process throughout the helix. This mechanism provides a plausible explanation for the longstanding paradox that extended collagen triple helices exhibit persistent sequence-dependent thermodynamic heterogeneity despite a two-state thermal transition, and a framework for investigating how sequence-dependent stability contributes to the structure and function of collagen molecules. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/741348v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@64f65borg.highwire.dtl.DTLVardef@69f999org.highwire.dtl.DTLVardef@181b647org.highwire.dtl.DTLVardef@e60ccf_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Differential efficiency of sampling devices in the measurement of microbial diversity of Yellowstone National Park hot springs

Metagenomic characterization of low-biomass Yellowstone National Park (YNP) hot spring waters remains challenging because microbial recovery is influenced by filtration methodology, sample preservation, DNA extraction, and sequencing strategy. We characterized thermophilic microbial communities in alkaline YNP hot spring waters (62-90.5{degrees}C) using three high-temperature-compatible filtration systems (Sterivex, Supor, and polycarbonate membranes), automated onsite DNA extraction ({micro}Titan), and shotgun metagenomic sequencing with Illumina short-read and Oxford Nanopore Technologies (ONT) long-read platforms. Across all filtration systems and sequencing workflows, microbial communities were consistently dominated by Bacteria ([~]90% of reads), whereas Archaea represented <10% of recovered sequences. Dominant microbial populations were reproducibly recovered across all approaches; however, recovery of lower-abundance taxa varied among methods. This variability was most evident in polycarbonate-filtered samples, which exhibited greater replicate-to-replicate variation and less consistent detection of microbial species. Thermocrinis ruber and related Aquificae-associated thermophiles dominated the hottest waters (78.5-90.5{degrees}C), whereas warmer effluent-channel waters (63.5-66.5{degrees}C) contained T. ruber together with photosynthetic taxa, including Synechococcus spp. and Candidatus Thermochlorobacter aerophilum. Archaeal communities were primarily represented by Pyrobaculum- and Thermoproteus-related taxa. Non-metric multidimensional scaling analyses indicated that overall community structure was largely unaffected by filtration or sequencing methodology, whereas alpha-diversity metrics showed that filter selection influenced richness and diversity estimates. These findings identify field-deployable workflows for metagenomic characterization of low-biomass thermophilic aquatic systems and demonstrate the importance of integrating filtration and sequencing strategies for studying extremophile microbiomes under remote sampling conditions. IMPORTANCEAccurate characterization of low-biomass geothermal water microbiomes remains challenging because microbial recovery is strongly influenced by sample handling, filtration efficiency, DNA extraction chemistry, and sequencing methodology. This study demonstrated that Yellowstone National Park alkaline hot spring water microbiomes were consistently dominated by Bacteria (>90% of recovered reads), whereas Archaea represented <10% of community abundance across all filtration systems. Although dominant microbial populations were reproducibly recovered, filtration-device selection influenced the recovery of microbial diversity and low-abundance taxa. By integrating field-deployable onsite DNA extraction with ONT shotgun metagenomic sequencing, this work evaluates practical workflows for studying thermophilic planktonic microbial communities under remote field conditions. These findings are relevant, not only to geothermal microbiology, but also to low-biomass environments in medical, pharmaceutical, and aerospace industries, where rapid onsite processing and contamination-aware workflows are essential for preserving authentic microbial signatures in extreme environments.

ecology↗

bioRxiv2026

A Murine Model of Mycobacterium abscessus Encapsulated in Alginate-Beads: Advancing Toward a Chronic Infection Model

BackgroundThe increasing incidence of Mycobacterium abscessus (M. abscessus) lung infections, together with its intrinsic multidrug resistance, highlights the need for new therapeutic regimens. However, the lack of a reliable chronic infection model in immunocompetent mice limits preclinical evaluation. MethodsTo mimic the bronchial environment of infected patients, we evaluated the effect of encapsulating M. abscessus in alginate beads on infection progression in BALB/cJRJ mice following intratracheal inoculation, compared with intranasal infection using non-encapsulated bacteria. The impact of dexamethasone treatment (DEX) was also assessed. Bacterial loads in lungs, spleen, liver, and kidneys were quantified over time in untreated and antibiotic-treated mice. Lung inflammation was evaluated by measuring IFN-{gamma} and TNF- levels. In vitro, the activity of imipenem and bedaquiline was assessed against free or alginate-encapsulated M. abscessus. ResultsCompared with intranasal infection, intratracheal infection with alginate-encapsulated bacteria resulted in slower pulmonary clearance and greater extrapulmonary dissemination. DEX further enhanced these features, reducing lung clearance, increasing dissemination, and amplifying lung inflammation. Bedaquiline showed no effect, whereas imipenem efficacy depended on treatment timing. For both drugs, alginate encapsulation reduced in vitro antibacterial activity. ConclusionThis model represents a step toward a chronic M. abscessus infection model characterized by moderate lungs clearance, extrapulmonary dissemination, and pronounced inflammatory responses. Reduced antibiotic activity against alginate-encapsulated bacteria may more accurately predict treatment efficacy in humans than activity measured against free bacteria.

microbiology↗

bioRxiv2026

Tonic REM sleep EEG components predict better mood, cognition and reduce cortical excitability overnight

Rapid Eye Movement (REM) sleep makes up approximately 20% of sleep in the adult human and is altered in psychiatric and neurodegenerative conditions. REM sleep comprises two substates, during which eye movements do (phasic REM) and do not (tonic REM) occur. Tonic REM makes up 70-90% of REM sleep but its role in regulating brain function, mood and cognition remain underexplored. We investigated how seven nights of insufficient sleep (6 h time in bed), compared to sufficient sleep, alter periodic and aperiodic components of the phasic and tonic REM sleep electroencephalography (EEG), in 542 sleep recordings of 36 young adults. Associations between phasic and tonic REM sleep EEG and mood, cognitive performance, and overnight changes in cortical excitability as indexed by 1/f spectral slopes were assessed. Insufficient sleep predominantly affected tonic REM EEG components, specifically the density of theta, the amplitude, density, and frequency of alpha oscillations and the 1/f slope in the 30 to 45 Hz range. These changes associated with mood and cognitive performances, and with overnight reductions in cortical excitability. These results provide evidence for a role of tonic REM sleep in regulating mood and counteracting cognitive deterioration and excitability changes associated with insufficient sleep.

neuroscience↗

bioRxiv2025

Cryo-EM of cardiac AL-224L amyloid reveals shared features in {lambda}6 light chain fibril folds

In amyloid light chain (AL) amyloidosis, aberrant monoclonal antibody light chains (LCs) deposit in vital organs causing organ damage. Each AL patient features a unique LC. Previous cryogenic electron microscopy (cryo-EM) studies revealed different amyloid structures in different AL patients. How LC mutations influence amyloid structures remains unclear. We report a cryo-EM structure of cardiac AL-224L amyloid (2.92 [A] resolution) from {lambda}6-LC family, which is overrepresented in amyloidosis. Comparison with {lambda}6-LC structures from two other patients reveals similarities in amyloid folds. Mutation-induced structural differences in AL-224L include altered C-terminal conformation with an exposed ligand-binding surface; an enlarged hydrophilic pore with orphan density; and altered steric zipper registry with backbone flipping, which likely represent general adaptive mechanisms in amyloids. The results suggest shared features in {lambda}6-LC amyloid folds and reveal how mutation-induced structural changes influence amyloid-ligand interactions in a patient-specific manner.

biophysics↗

bioRxiv2025

TidyMass2: Advancing LC-MS Untargeted Metabolomics Through Metabolite Origin Inference and Metabolic Feature-based Functional Module Analysis

Untargeted metabolomics provides a direct window into biochemical activities but faces critical challenges in determining metabolite origins and interpreting unannotated metabolic features. Here, we present TidyMass2, an enhanced computational framework for Liquid Chromatography-Mass Spectrometry (LC-MS) untargeted metabolomics that addresses these limitations. TidyMass2 introduces three major innovations compared its predecessor, TidyMass: (1) a comprehensive metabolite origin inference capability that traces metabolites to human, microbial, dietary, pharmaceutical, and environmental sources through integration of 11 metabolite databases containing 532,488 metabolites with source information; (2) a metabolic feature-based functional module analysis approach that bypasses the annotation bottleneck by leveraging metabolic network topology to extract biological insights from unannotated metabolic features; and (3) an intuitive graphical interface that makes advanced metabolomics analyses accessible to researchers without programming expertise. Applied to longitudinal urine metabolomics data from human pregnancy, TidyMass2 identified diverse metabolites originating from human, microbiome, and environment, and uncovered 27 dysregulated metabolic modules. It increased the proportion of biologically interpretable metabolic features from 5.8% to 58.8%, revealing coordinated changes in steroid hormone biosynthesis, carbohydrate metabolism, and amino acid processing. By expanding biological interpretation beyond conventionally MS2 spectra-based annotated metabolites, TidyMass2 enables more comprehensive metabolic phenotyping while upholding strict open-source principles of reproducibility, traceability, and transparency.

bioinformatics↗

bioRxiv2025

Diploid genome assembly of human fibroblast cell lines enables clone specific variant calling, improved read mapping and accurate phasing

Human cell lines are fundamental tools in biomedical research and are widely used in disease modeling, drug development, and many other domains. Here, we present chromosome-level, phased diploid genome assemblies of two popular human cell lines: the BJ foreskin fibroblast line and the IMR-90 fetal lung fibroblast line. Our high-quality assemblies, generated using long-read and Hi-C sequencing data, reveal substantial structural variation, including more than 50,000 insertions, deletions, duplications, and inversions compared to the recent T2T-CHM13v2.0 reference. Our assemblies provide detailed maps of genetic variation, enabling more accurate variant calling and the ability to phase reads when using newly generated or historical sequencing data on these cell lines or their derivatives. All assemblies and associated data have been made available as a resource for the research community. We envision that diploid genome assembly will become a cornerstone approach for personalized medicine in the near future.

genomics↗

bioRxiv2025

Alterations in Lipid Saturation Trigger Remodeling of the Outer Mitochondrial Membrane

Lipid saturation is a key determinant of membrane function and organelle health, with changes in saturation triggering adaptive quality control mechanisms to maintain membrane integrity. Among cellular membranes, the mitochondrial outer membrane (OMM) is an important interface for many cellular functions, but how lipid saturation impacts OMM function remains unclear. Here, we show that increased intracellular unsaturated fatty acids (UFAs) remodel the OMM by promoting the formation of multilamellar mitochondrial-derived compartments (MDCs), which sequester proteins and lipids from the OMM. These effects depend on the incorporation of UFAs into membrane phospholipids, suggesting that changes in membrane bilayer composition mediate this process. Furthermore, elevated UFAs impair the assembly of the OMM protein translocase (TOM) complex, with unassembled TOM components captured into MDCs. Collectively, these findings suggest that alterations in phospholipid saturation may destabilize OMM protein complexes and trigger an adaptive response to sequester excess membrane proteins through MDC formation. Significance StatementO_LIMitochondrial-derived compartments are multilamellar structures that sequester protein and lipids of the outer mitochondrial membrane in response to metabolic and membrane perturbations, but it is largely unknown how membrane fluidity influences this pathway. C_LIO_LIIncreased levels of unsaturated phospholipids may disrupt the TOM complex, a large multi-subunit complex on the outer mitochondrial membrane, to promote the formation of mitochondrial-derived compartments, while increased levels of saturated phospholipids inhibits formation of mitochondrial-derived compartments. C_LIO_LIThese findings reveal a link between phospholipid composition and protein stress in driving mitochondrial-derived compartment biogenesis, and thus mitochondrial quality control. C_LI

cell biology↗

bioRxiv2025

Genetically Defined Organoid Models Reveal Mechanisms Driving Squamous Cell Neoplastic Evolution and Identify Potential Therapeutic Vulnerabilities

Upper aerodigestive squamous cell carcinoma (UASCC) is an aggressive and lethal neoplasm, with its early neoplastic transformation mechanisms remaining poorly understood. Here, we characterize over 25 genetically-defined organoid models derived from murine and human oral/esophageal tissues harboring key driver mutations. Double knockout of TP53 and CDKN2A induced morphological dysplasia, hyperproliferation, loss of squamous differentiation, and tumorigenicity, which were further exacerbated by additional driver mutations (e.g., PIK3CA, NOTCH1, KMT2C). Single-cell analysis revealed an expansion of quiescent basal cells and proliferative squamous cells, alongside a loss of differentiated squamous cells during malignant transformation. A distinct senescence program, regulated by ANXA1, was markedly diminished during early neoplastic evolution. Mechanistically, the ANXA1-SMAD3-p27KIP1 pathway was identified as a critical regulator of this senescence program, acting to suppress neoplastic features in organoid models. Lastly, our high-throughput, single-organoid-resolution drug screens unexpectedly revealed PIK3CA-driven organoids exhibited sensitivity to Mitomycin C and Onalespib. This study provides novel mechanistic insights into early neoplastic evolution and underscores the value of genetically-defined organoid models for investigating cancer biology and identifying targeted therapies.

cancer biology↗

bioRxiv2024

A novel miniaturized filamentous phagemid as a gene delivery vehicle to target mammalian cells

The filamentous phage M13 is a single-stranded DNA phage with several attractive characteristics for gene delivery, including a capsid amenable to the display of foreign peptides, and a simple well-characterized genome that is easy to genetically modify. Previously, we constructed a DNA minivector based on M13 (a miniphagemid), which minimized the inflammatory bacterial and phage DNA content in the vector. In general, DNA minivectors, devoid of their prokaryotic components have shown improved gene transfer and safety. We examined the miniphagemids capacity for in vitro transgene delivery to target cells through phage display of epidermal growth factor to target its cognate receptor. The absence of the prokaryotic backbone and smaller vector size conferred by the miniphagemid was associated with improved transgene expression for purified single-stranded phagemid DNA and phagemid virion particles. We further engineered this system to enhance packaging of DNA minivectors by deletion of the packaging signal within the helper plasmid, used to produce miniphagemids, and observed improved phage-mediated gene expression in mammalian cells. Overall, here, we present a set of novel transgene delivery vectors that combine cell-targeting ligand display and vector minimization. This platform showcases the flexibility of M13 as a gene delivery tool with immense therapeutic potential.

molecular biology↗

bioRxiv2024

An updated AL-Base reveals ranked enrichment of immunoglobulin light chain variable genes in AL amyloidosis

BackgroundEach monoclonal antibody light chain associated with AL amyloidosis has a unique sequence. Defining how these sequences lead to amyloid deposition could facilitate faster diagnosis and lead to new treatments. MethodsLight chain sequences are collected in the Boston University AL-Base repository. Monoclonal sequences from AL amyloidosis, multiple myeloma and the healthy polyclonal immune repertoire were compared to identify differences in precursor gene use, mutation frequency and physicochemical properties. ResultsAL-Base now contains 2,193 monoclonal light chain sequences from plasma cell dyscrasias. Sixteen germline precursor genes were enriched in AL amyloidosis, relative to multiple myeloma and the polyclonal repertoire. Two genes, IGKV1-16 and IGLV1-36, were infrequently observed but highly enriched in AL amyloidosis. The number of mutations varied widely between light chains. AL-associated {kappa} light chains harbored significantly more mutations compared to multiple myeloma and polyclonal sequences, whereas AL-associated {lambda} light chains had fewer mutations. Machine learning tools designed to predict amyloid propensity were less accurate for new sequences than their original training data. ConclusionsRarely-observed light chain variable genes may carry a high risk of AL amyloidosis. New approaches are needed to define sequence-associated risk factors for AL amyloidosis. AL-Base is a foundational resource for such studies.

bioinformatics↗

bioRxiv2024

Novel Role of Endothelial CD45 in Regulating Endothelial-to-Mesenchymal Transition in Atherosclerosis

BackgroundThe protein tyrosine phosphatase CD45 is expressed in all nucleated cells of the hematopoietic system and in mitral valve endothelial cells (ECs) undergoing endothelial-to-mesenchymal transition (EndoMT). Our recent work indicated that activation of endogenous CD45 in human endothelial colony-forming cells (ECFCs) induced expression of multiple EndoMT marker genes. We hypothesized that CD45 may contribute to atherosclerosis; however, detailed molecular mechanisms underlying how CD45 may contribute to EndoMT and the impact of therapeutic manipulation of CD45 expression in atherosclerosis are unknown. MethodsWe generated a tamoxifen-inducible EC-specific CD45-deficient mouse strain (EC-iCD45KO) on an ApoE-deficient (WT/ApoE-/-) background and fed them a Western diet (WD) to produce atherosclerosis. We enriched mouse aortic ECs with anti-CD31 beads to perform single-cell RNA sequencing. Cellular, biochemical and molecular approaches were used to investigate the effect of endothelial CD45-specific deletion on EndoMT and lesion development in an ApoE-/- mouse model of atherosclerosis. ResultsEC-iCD45KO mice showed reductions in lesion development, plaque macrophage infiltration, and expression of cell adhesion molecules when compared to WT/ApoE-/- controls. Single-cell RNA sequencing revealed that loss of endothelial CD45 decreases EndoMT marker expression and TGF-{beta} signaling in atherosclerotic mice, which is associated with reduction of lesions. Mechanistically, CD45 loss increases Fibroblast Growth Factor Receptor 2 (FGFR2) expression in mouse aortic ECs and Kruppel-like Factor 2 (KLF2) expression in the aortic root. Endothelial CD45-deficiency also inhibits EndoMT and TGF{beta} signaling in atherosclerosis. ConclusionsOur findings demonstrate that genetic depletion of endothelial CD45 protects against EndoMT-driven atherosclerosis by promoting FGFR2 and KLF2 expression while inhibiting Transforming Growth Factor beta (TGF{beta}) signaling and EndoMT. Consequently, targeting endothelial CD45 may represent a novel therapeutic strategy to reduce EndoMT in atherosclerosis.

cell biology↗

bioRxiv2024

Targeting Liver Epsins Ameliorates Dyslipidemia in Atherosclerosis

BackgroundThe low-density lipoprotein receptor (LDLR) in the liver plays a crucial role in clearing low-density lipoprotein cholesterol (LDL-C) from the bloodstream. This process takes place mainly in the liver. Under atherogenic conditions, Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9), secreted by the liver, binds to LDLR on hepatocytes, preventing its recycling and enhancing its lysosomal degradation. This process reduces LDL-C clearance, promoting hypercholesterolemia. Epsins, a family of ubiquitin-binding endocytic adaptors, are key regulators of atherogenesis in lesional cells, including endothelial cells and macrophages. Given epsins canonical role in regulating endocytosis of cell surface receptors, we aimed to determine whether and how liver epsins contribute to PCSK9-mediated LDLR endocytosis and degradation, thereby impairing LDL-C clearance and accelerating atherosclerosis. MethodsLiver-specific epsin knockout (Liver-DKO) atherosclerotic models were generated in ApoE-/- and PCSK9-AAV8-induced atheroprone mice fed on a Western diet. We utilized single-cell RNA sequencing, along with molecular, cellular, and biochemical analyses, to investigate the physiological role of liver epsins in PCSK9-mediated LDLR degradation. Additionally, we explored the therapeutic potential of nanoparticle-encapsulated siRNAs targeting epsins 1 and 2 in ApoE-/- mice with established atherosclerosis. ResultsWestern diet (WD)-induced atherosclerosis was significantly attenuated in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls, as well as in PCSK9-AAV8-induced Liver-DKO mice compared with PCSK9-AAV8-induced wild-type (WT) mice accompanied by reductions in blood cholesterol and triglyceride levels. Mechanistically, single-cell RNA sequencing of hepatocytes and aortas isolated from atherosclerotic ApoE-/- and ApoE-/-/Liver-DKO mice revealed epsin-deficient Ldlrhi hepatocytes with diminished lipogenic potential. Notably, pathway analysis of hepatocytes showed increased LDL particle clearance and enhanced LDLR-cholesterol interactions under WD treatment in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls, correlating with decreased plasma LDL-C levels. Furthermore, pathway analysis of the aortas showed attenuated inflammation and endothelial activation, coupled with reduced lipid uptake, and enhanced cholesterol efflux under WD treatment in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls. Moreover, the absence of liver epsins led to an upregulation of LDLR protein expression in hepatocytes. We further demonstrated that epsins bind LDLR via the ubiquitin-interacting motif (UIM), enabling PCSK9-mediated LDLR degradation. Depleting epsins abolished this degradation, thereby preventing atheroma progression. Lastly, targeting liver epsins with nanoparticle-encapsulated epsins siRNAs effectively ameliorates dyslipidemia and inhibits atherosclerosis progression. These results are consistent with findings showing an increased epsin1 and epsin2 expression in atherosclerotic cardiovascular disease patients. ConclusionsLiver epsins drive atherogenesis by promoting PCSK9-mediated LDLR degradation, thereby elevating circulating LDL-C levels and heightening lesional inflammation. As such, targeting epsins in the liver represents a promising therapeutic strategy to mitigate atherosclerosis by preserving LDLR and enhancing LDL-C clearance in the liver.

cell biology↗

bioRxiv2024

Chemoresistance of TP53 mutant AML requires the mevalonate byproduct, GGPP, for regulation of ROS and induction of a mitochondria stress response

Acute myeloid leukemia (AML) with mutations in the tumor suppressor gene, TP53 (TP53mut AML), is fatal with a median survival of only 6 months. RNA sequencing on purified AML patient samples show TP53mut AML has higher expression of mevalonate pathway genes. We retrospectively identified a survival benefit in TP53mut AML patients who received chemotherapy concurrently with a statin, which inhibits the mevalonate pathway. Mechanistically, TP53mut AML resistance to standard AML chemotherapy, cytarabine (AraC), correlates with increased mevalonate pathway activity and a mitochondria stress response with increased mitochondria mass and oxidative phosphorylation. Pretreatment with a statin reverses these effects and chemosensitizes TP53mut AML cell lines and primary samples in vitro and in vivo. Mitochondria-dependent chemoresistance requires the geranylgeranyl pyrophosphate (GGPP) branch of the mevalonate pathway and novel GGPP-dependent synthesis of glutathione to manage AraC-induced reactive oxygen species (ROS). Overall, we show that the mevalonate pathway is a novel therapeutic target in TP53mut AML. SignificanceChemotherapy-persisting TP53mut AML cells induce a mitochondria stress response that requires mevalonate byproduct, GGPP, through its novel role in glutathione synthesis and regulation of mitochondria metabolism. We provide insight into prior failures of the statin family of mevalonate pathway inhibitors in AML. We identify clinical settings and strategies to successfully target the mevalonate pathway, particularly to address the unmet need of TP53mut AML.

cancer biology↗