Biology subjects

Tan, L.

Publications and source records attributed to Tan, L..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Arabidopsis GALACTURONOSYLTRANSFERASE (GAUT) 1 synthesizes a homogalacturonan tightly bound to the cell wall and required for cell expansion

Arabidopsis GALACTURONOSYLTRANSFERASE1 (GAUT1) synthesizes homogalacturonan (HG), the most abundant pectin in growing plant cells. GAUT1 has the greatest in vitro enzyme activity of the six confirmed Arabidopsis HG biosynthetic GAUTs, but its biological activity remains elusive. Here we show that Arabidopsis GAUT1 homozygous mutants have a severe dwarfed seedling phenotype, survive several weeks as 2 to 3 mm seedlings, and have severely reduced shoot and root growth and hypocotyl epidermal, cortex and endodermal cell size. gaut1-1 pollen tubes are shorter than WT with increased bursting. Complementation of homozygous gaut1-1 with GAUT1 coding sequence driven by the GAUT1 promoter restored WT-like growth. The extreme dwarf phenotype of homozygous gaut1-1 seedlings precluded their use for detailed cell wall analysis, thus suspensions cultures were produced from callus generated from mutant and WT seedlings. Homozygous gaut1-1 suspension cells were smaller than WT with [~]30% reduced wall GalA content compared to WT. Sequential extraction of the walls with increasingly harsh solvents and sugar composition analysis revealed reduced GalA content in only the 4M KOH post-chlorite fraction, indicating that GAUT1-synthesized HG was held tightly in the wall by direct or indirect hydrogen bonding and/or oxidation-sensitive linkages. Treatment of wall fractions with endopolygalacturonase to hydrolyze HG and gel electrophoretic separation of hydrolysates exposed an HG-associated doublet band markedly downregulated in the homozygous gaut1-1 4M KOH post-chlorite fraction and to a lesser extent in 4M KOH and sodium chlorite fractions. NMR analysis identified the band as rhamnogalacturonan (RG)-II. Super resolution microscopy using anti-HG antibodies showed that, compared to WT, the homozygous gaut1-1 hypocotyl epidermal and callus cells had reduced content and length of HG nanofilaments, HG fibers associated with cell expansion in Arabidopsis. The results demonstrate that GAUT1-synthesized HG resides in a tightly-cell-wall-bound, RG-II-containing polymer required for HG nanofilament formation and seedling cell expansion.

plant biology↗

bioRxiv2025

Versatile high-speed volumetric imaging from microscopic to macroscopic scale by self-adaptive oblique plane microscopy

There is an increasing need for large-scale high-speed volumetric recording in complex multi-cellular model systems to define dynamic processes. Oblique plane microscopy (OPM) provides a solution that features oblique illumination, rapid optical scanning, and remote focusing to achieve real-time 4D microscopy. OPM implements light sheet imaging via a single primary objective lens, making the entire space below the objective accessible for large specimens, such as living mouse brain. Yet it is challenging to adopt OPM beyond a microscopic scale (i.e. size < 1mm), limiting its broad applications. Here we present a self-adaptive OPM that leverages Abbes sine condition to unlock its flexibility across a range of field-of-views (FOVs) (up to 8 mm2) and resolutions (down to 2.2 {micro}m3). This versatility enables brain-wide single neuron volumetric calcium imaging in behaving larval zebrafish (1x0.4 mm2 FOV at 5 Hz) and capillary blood cell tracking in living mouse brain (>3x3 mm2 FOV) with a sweeping 0.32 mm wide volume section at 100 Hz. In optically cleared mouse brain, the flexibility allows a screen-and-zoom capability by sequentially imaging the whole brain at low-and-high magnifications to locate and resolve subcellular structures such as dendritic tress and spines. By offering a switchable imaging resolution, volume, and speed, the self-adaptive OPM achieves a versatile platform for studying a wide range of multi-cellular model system, whether in vivo or fixed and optically cleared.

bioengineering↗

bioRxiv2025

De Novo Hepatic Pyrimidine Synthesis Regulates Systemic Energy Homeostasis

Fasting blood uridine is increased in obesity and type 2 diabetes (T2D), but the significance of hepatic uridine biosynthesis to the etiology of both remains elusive. We found that de novo pyrimidine synthesis in the liver is reduced by fasting and diet-induced obesity, while suppression of hepatic pyrimidine synthesis promotes obesity and insulin resistance. The metabolic sequalae of hepatic pyrimidine synthesis suppression, however, is not associated with altered plasma uridine concentration. Instead, it is associated with an increased hepatic glucose production and a decreased hepatic insulin clearance, two key functions of hepatocytes in regulating systemic energy homeostasis. We found that enhanced gluconeogenesis is the primary reason for increased hepatic glucose production. Moreover, uridine, which was maintained stable in the circulation by adipose tissue and the liver, preferentially shut down pyrimidine synthesis in hepatocytes but not adipocytes at blood concentrations that occur with fasting. Remarkably, uridine, at fasting levels, increases gluconeogenesis further in hepatocytes when de novo pyrimidine synthesis is suppressed, indicating a synergistical action of uridine and its biosynthesis pathway in promoting hepatic glucose production, a mechanism highly relevant to the pathophysiology of insulin resistance in obesity. Theologically, maintenance of blood uridine within the narrow range protects mammals from high-rate spontaneous tumorigenesis. Since obesity promotes an increase in blood uridine from adipocytes, suppressing uridine synthesis in hepatocytes becomes a critical response to lower spontaneous tumorigenesis. Pyrimidine synthesis suppression in hepatocytes, however, promotes gluconeogenesis and ultimately triggers obesity and T2D. These findings suggest a new paradigm for the etiology of metabolic deterioration in diet-induced obesity, in which perturbations in uridine promotes obesity and T2D.

pathology↗

bioRxiv2025

Sex-dependent cortico-amygdala circuits controlling emotion recognition

In social species, the ability to recognize others emotional states is essential for appropriate social interactions, yet it often declines with age and is impaired in various neurodevelopmental and neurodegenerative disorders. While emotion recognition has been characterized in both humans and rodents, the underlying neural circuits and how they vary by sex and age remain poorly understood. Here, we used a negative Emotional state Discrimination Task (EDT) in TRAP2 transgenic mice to map brain regions engaged during negative emotion recognition in young and aged animals. Young male and female mice successfully discriminated emotionally altered conspecifics, recruiting the basolateral amygdala (BLA) and medial orbitofrontal cortex (MO) in a sex-specific manner. Fiber photometry revealed distinct activation dynamics in these regions, and chemogenetic inhibition of bidirectional BLA-MO projections abolished emotion recognition in male but not female mice. Notably, young human participants also showed sex-specific recruitment of BLA and OFC during negative facial emotion recognition. Moreover, aging selectively impaired emotion recognition in male mice, coinciding with reduced BLA activity. Remarkably, chemogenetic activation of BLA in aged male mice rescued this deficit. Together, these findings identify a sex-dependent BLA-MO circuit as a conserved neural substrate for emotion recognition and demonstrate that age-related impairments can be reversed through targeted circuit-level intervention.

neuroscience↗

bioRxiv2025

An Immuno-Linguistic Transformer for Multi-Scale Modeling of T-Cell Spatiotemporal Dynamics

Understanding the spatiotemporal dynamics of T-cell clones is a critical challenge in immunology and immunotherapy, with direct implications for cancer treatment and vaccine design. While Large Language Models (LLMs) have demonstrated immense power in decoding complex sequential data, their application to the "language of immunity" remains nascent. Existing computational models often struggle to capture the hierarchical, multi-scale nature of immune responses and fail to model biologically plausible system perturbations. To bridge this gap, we propose the Immuno-Linguistic Spatiotemporal Transformer (ILST), a self-supervised framework inspired by LLM architectures. Our framework introduces two key innovations: a Biologically-Informed Perturbation (BIP) module that simulates systemic events (e.g., infection or therapy) by respecting the functional importance of key T-cell clones, and a Hierarchical Tissue-Scale Fusion (HTF) module that uses attention to dynamically weigh and combine representations from cellular, tissue, and systemic levels. We validate our model on several public graph datasets, which serve as effective proxies for complex biological networks with varying degrees of heterogeneity. ILST achieves consistently strong results in predicting node states. Notably, it significantly enhances performance on heterogeneous (disassortative) graphs, demonstrating its potential for robustly modeling T-cell dynamics in complex microenvironments like tumors.

bioinformatics↗

bioRxiv2025

Establishing a continuum of cell types in the visual cortex

The mammalian cerebral cortex is composed of neurons whose properties vary in a continuous fashion rather than falling into discrete cell types. In the mouse visual cortex, excitatory neurons in layer 2 and 3 (L2/3) form such a continuum along cortical depth, patterned by the graded expression of hundreds of genes. Here we sought to understand how this continuum develops and contributes to cortical wiring. Using single-nucleus multiomics (RNA- and ATAC-Seq) and spatial transcriptomics, we show that the L2/3 continuum is established in two phases. During the first postnatal week, a genetically hardwired program establishes a primitive continuum of cell identities spanning the depth of L2/3. The second program, promoted by visual experience, is later superimposed upon the preexisting continuum. This second phase is driven by activity-regulated transcription factors that drive the L2/3 depth-dependent expression of genes linked to synaptic function and plasticity. We show that neurons at different positions along the L2/3 continuum project preferentially to distinct higher visual areas and that visual deprivation disrupts targeting to some higher visual areas while sparing others. Thus, cortical continua emerge through a stepwise process in which genetic programs and sensory experience specify neuronal identity and sculpt intracortical wiring specificity.

neuroscience↗

bioRxiv2025

Memorability of Images Positively Influences Associative Memory Retention

Enhancing memory function is essential for daily living and cognitive health, particularly amid population aging and cognitive decline. However, current methods for memory enhancement often require specialized interventions or effortful practice. Here, we present evidence that the intrinsic memorability of images can serve as a simple, scalable, and involuntary strategy for improving associative memory. We studied its effects on associative memory in adults of distinct ages, memory retention in cognitively impaired older adults, and vocabulary learning in foreign language learners. Our results show that highly memorable images significantly enhance the recall of associated words, especially when the cue image and target word are semantically unrelated. Notably, these effects persist for at least one week and are robust across age groups and cognitive status. In a foreign language vocabulary task, pairing words with memorable images led to either improved recall accuracy or reduced learning time. These findings highlight that, in addition to being an intrinsic memory-enhancing property, image memorability also serves as a general facilitator of associative memory, via a method that is easy to adopt, requires minimal conscious effort, and can benefit the general public, particularly learners and cognitive impairment patients in educational and clinical settings.

neuroscience↗

bioRxiv2025

Temporal Interference Stimulation Enhances Neural Regeneration

Neural regeneration therapies aim to treat neurodegeneration by promoting the proliferation and maturation of exogenous or endogenous neural progenitor cells (NPCs). However, their efficacy has been limited. Deep brain stimulation (DBS) via implanted electrodes has been shown to promote neurogenesis. However, its invasiveness precludes deployment in research and widespread clinical use. Temporal interference (TI) has emerged as a strategy for non-invasive, high-precision DBS using multiple kHz-range electric fields, with a frequency difference within the range of neural activity. Here, we validate the potential of TI stimulation for neural regeneration augmentation. We demonstrate that TI stimulation with a theta-band frequency difference enhances the maturation of embryonic neural progenitor cells in vitro. We then demonstrate that theta-band TI stimulation targeting the hippocampus enhances endogenous hippocampal neurogenesis in an in vivo mouse model of Alzheimers disease. By uncovering frequency-specific control of stem cell fate, we propose a clinically relevant regeneration strategy which avoids pharmacological or genetic manipulation. Our results demonstrate focal, non-invasive augmentation of deep-brain neural regeneration. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/670811v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@984165org.highwire.dtl.DTLVardef@1ed8bbdorg.highwire.dtl.DTLVardef@715057org.highwire.dtl.DTLVardef@151882f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

scSTAR2: a multiomics integration algorithm to reveal disease specific cellular signatures by bridging single-cell resolution features and clinical metadata

Single-cell techniques, pivotal in characterizing intricate cell and spatial structures within tissues, face challenges in contextualizing clinical phenotypes. Currently, most investigations of the clinical phenotype related cell/spatial heterogeneities relied on the phenotype information from single-cell data. However, a wealth of underutilized clinical metadata exists within conventional bulk sequencing data. Current methods for correlating clinical information with individual cells or spatial regions remain limited and lack robustness. Here we present scSTAR2, a novel algorithm that transcends existing limitations by reconstructively integrating multiomics data to identify cells associated with clinical phenotypes. Unlike existing methods, scSTAR2 reconstructs single-cell data guided by specific phenotypes, significantly reducing interference from irrelevant noise. By employing scSTAR2 to integrate scRNA-seq, scATAC-seq, and spatial transcriptomics and bulk RNA-seq, we not only confirmed a new heat-shock Treg subtype in tumors but also more sensitively identified TLS (tertiary lymphoid structure) areas than traditional methods. In conclusion, scSTAR2 has proven to significantly enhance single-cell data interpretation across diverse clinical scenarios.

bioinformatics↗

bioRxiv2025

The ASNS inhibitor ASX-173 potentiates L-asparaginase anticancer activity

Cancer cells reprogram metabolic pathways to meet increased energy and biosynthetic demands. Among those pathways, elevated asparagine metabolism regulated by asparagine synthetase (ASNS) has been linked to tumor progression in various cancers, driving cell proliferation, chemoresistance, and metastasis. ASNS inhibition represents a promising therapeutic strategy, but inhibitors have shown limited efficacy due to poor specificity and cell permeability. Through phenotypic screening, we identified ASX-173, a cell-permeable small molecule that inhibits ASNS at nanomolar concentrations. Biochemical and cellular assays confirm the specificity of ASX-173 activity and demonstrate its potentiation of the anti-cancer activity of L-asparaginase (ASNase), a key component of childhood acute lymphoblastic leukemia therapy. Mechanistically, the combination treatment disrupted nucleotide synthesis and induced cell cycle arrest and apoptosis. In a mouse model of acute myeloid leukemia, the combination significantly delayed the growth of OCI-AML2 xenografts. Analysis of data from The Cancer Genome Atlas (TCGA) revealed that ASNS mRNA expression is associated with poor survival in some cancer types and that ASNS protein levels are elevated in multiple solid tumors compared with the levels in normal tissues, suggesting possible broad utility of ASNS inhibition across the landscape of cancer. Together, these findings establish ASX-173 as a promising ASNS inhibitor and, for the first time, demonstrate a viable strategy to target ASNS therapeutically--an approach that has long remained elusive.

cancer biology↗

bioRxiv2025

Downregulation of LATS1/2 Drives Endothelial Senescence-Associated Stemness (SAS) and Atherothrombotic Lesion Formation

BackgroundAtherothrombosis, the main event leading to acute coronary syndrome (ACS), is strongly linked to disturbed blood flow (d-flow) regions. Although the involvement of the Hippo pathway and its kinases Large Tumor Suppressor Kinase 1and 2 (LATS 1 and 2) in mechanical stress responses is known, the mechanisms by which d-flow simultaneously induces senescence, proliferation, and atherothrombosis remain unclear. MethodsThe role of endothelial cells (EC)-specific LATS1/2 was examined using EC specific knock-out (EKO) mice in a partial left carotid ligation (PLCL) model. Plaque spatial multi-omics analysis was performed by integrating imaging mass cytometry, sequential immunofluorescence (COMET), and spatial metabolomics at the single-cell level in human and mouse atherosclerotic plaques. ResultsIn tamoxifen-inducible Lats1homo(-/-) /Lats2 homo(-/-) EC-specific knockout (EKO) mice, deletion of LATS1/2 induced by tamoxifen led to fatal outcomes, characterized by severe systemic edema and markedly increased vascular permeability. In contrast, Lats1het(+/-)/Lats2 homo(-/-)-EKO mice survived and developed atherothrombotic plaques exhibiting neovascularization even without further additional dietary or genetic intervention. Spatial proteomics analysis revealed that LATS1/2 depletion in ECs triggered a senescence-associated stemness (SAS) phenotype, primarily driven by CD38 upregulation. Complementary spatial metabolomics profiling demonstrated a significant increase in sulfite and taurine within LATS1/2-deficient plaques, indicating lowered sulfite oxidase (SUOX) activity. Mechanistically, CD38 upregulation was found to suppress SUOX expression, induce the reverse mode of mitochondrial complex V, and increase succinate dehydrogenase (SDH) activity along with ATP consumption. Paradoxically, despite ATP depletion, this metabolic disturbance enhanced glutamate metabolism and the tricarboxylic acid (TCA) cycle, sustaining EC proliferation under energetically stressed conditions. The combined effect of LATS1/2 deletion and CD38 activation established a unique EC phenotype defined by increased SAS, leading to proliferation, senescence, and eventual cell death. These pathological processes culminated in the formation of atherothrombotic plaques, which were attenuated by inhibition of CD38. Notably, a similar phenotype--marked by metabolically active ECs--was observed in human atherothrombotic plaques, suggesting translational relevance. ConclusionLoss of LATS1/2 in ECs induces SAS state that promotes excessive EC proliferation, senescent cell accumulation, and the development of structurally fragile, leaky neo vessels--hallmarks of atherothrombotic lesions. CD38-mediated SUOX deficiency further amplifies this pathological process by inducing mitochondrial dysfunction, depleting ATP, and triggering compensatory upregulation of glutamate and TCA cycle metabolism. These findings identify a novel LATS1/2-CD38-SUOX axis in ECs that orchestrates SAS-driven atherothrombosis. Targeting CD38 may represent a promising therapeutic strategy to mitigate vascular dysfunction and plaque instability in high-risk ACS patients. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/660635v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@149d313org.highwire.dtl.DTLVardef@1c0988borg.highwire.dtl.DTLVardef@15efec9org.highwire.dtl.DTLVardef@1a96e03_HPS_FORMAT_FIGEXP M_FIG C_FIG Under normal physiological conditions, LATS1/2 and Lamin A work together to suppress CD38 expression. Lamin A binds directly to the CD38 promoter to repress transcription, and LATS1/2 interact with Lamin A to reinforce this suppression. This collaboration helps maintain low CD38 activity and preserves cellular NAD levels. However, under disturbed flow (d-flow), both LATS1/2 and Lamin A are downregulated. The loss of this dual repression leads to increased CD38 NADase expression. Elevated CD38 accelerates NAD consumption, causing NAD depletion--a hallmark of cellular senescence. Reduced NAD disrupts key metabolic and stress-response pathways, contributing to the onset of the senescent state. At the same time, CD38 suppresses sulfite oxidase (SUOX), leading to sulfite accumulation and mitochondrial redox imbalance. This shift activates the reverse mode of mitochondrial Complex V, which decreases ATP production and increases mitochondrial ROS, intensifying metabolic and oxidative stress in endothelial cells (ECs). In response, ECs compensate by upregulating succinate dehydrogenase (SDH), enhancing TCA cycle activity and glutamate metabolism. This metabolic adaptation provides the biosynthetic building blocks needed for cell growth and proliferation. As a result, ECs adopt a paradoxical phenotype: they show classical features of stress-induced senescence (such as NAD depletion, oxidative stress, and cell cycle arrest signals), while simultaneously undergoing metabolic activation and proliferation, also mediated by YAP. This defines a non-canonical endothelial program known as senescence-associated stemness (SAS), characterized by the formation of abnormal, proliferative, yet fragile neovessels. These dysfunctional vessels contribute to atherothrombosis, setting this process apart from the more stable lesions typical of conventional atherosclerosis.

molecular biology↗

bioRxiv2025

A conserved glycan motif overcomes antigenic variation inducing broadly reactive antibodies against the zoonotic pathogen Streptococcus suis

Streptococcus suis is a largely neglected but emerging bacterial zoonotic pathogen of global concern for animal welfare, antibiotic resistance development and human health. No effective vaccines are currently available. Here, we identified and characterized the function and structure of two cell wall polysaccharide variants in pathogenic S. suis strains using genetic deletion and (heterologous) complementation, lectin staining, glycan composition analysis and specialized NMR spectroscopy. Both glycan variants were anionic polymers that differed in the presence of glucose in the side-chain as a result of allelic variation in a glycosyltransferase gene. Deletion of this variable glycosyltransferase revealed an identical glycan core and affected S. suis morphology and lysozyme resistance. Immunization of pigs with this core domain induced antibodies recognizing a wide range of antigenically-diverse pathogenic S. suis strains. This study provides new insights for developing next-generation glycoconjugate vaccines, whereby a single-glycan target could protect against the emerging zoonotic pathogen S. suis.

microbiology↗

bioRxiv2025

CDK12 condensation in nuclear speckles confers sensitivity to cyclin K molecular glue degraders

Transcriptional machinery often operates in the configuration of biomolecular condensates, presenting emerging opportunities to develop therapies targeting transcriptionally addicted malignancies. In this study, we discovered that cyclin-dependent kinase 12 (CDK12), a key regulator of transcription elongation, formed dynamic liquid-like droplets within nuclear speckles, a process enhanced by high CDK12 expression and CDK12 inhibitor treatment. Recognizing this unique property, we rationally designed ZSQ253, a modular cyclin K molecular glue degrader that coupled binding moieties for both CDK12 and DDB1. By promoting phase separation-driven assembly and subsequent proteasomal degradation of the CDK12-cyclin K complex, ZSQ253 interfered with oncogenic transcription and demonstrated potent antitumor capability. Further structure-guided optimization yielded HQY1428, a derivative with a multi-site gluing effect and improved drug activity. Given the frequent genomic co-amplification of CDK12 and ERBB2 across diverse human cancers, we showed that combining HQY1428 with HER2 inhibitors provided synergistic therapeutic benefits. Collectively, our findings establish CDK12 condensation within nuclear speckles as an exploitable vulnerability, and introduce a novel mechanism of action underlying cyclin K molecular glue degraders.

cancer biology↗

bioRxiv2025

Amyloid-β precursor protein promotes tumor growth by establishing an immune-exclusive tumor microenvironment

During initiation and progression, cancerous tissue hijacks a series of elaborate tissue homeostatic mechanisms to avoid immune surveillance, including neuro-immune interactions. Here, we show that amyloid-{beta} precursor protein (APP) and its {beta}-cleavage product amyloid-{beta}1-42 (A{beta}1-42), well-known in the pathogenesis of Alzheimers disease (AD), are expressed in multiple cancer tissues. However, the oncogenic activity of APP is due to its E1 domain, instead of A{beta}1-42. Mechanistically, APP restricts immune cells influx into tumor microenvironment (TME) and impairs CD8+ T cell and NK cell-based immunity, by dampening type I interferon (IFN) response in TME. We also provide proof-of-concept that vaccination targeting APP is effective for cancer prevention. Our current study reveals a previously unrecognized role of APP in cancer immune surveillance, and provides a new strategy for cancer prevention and treatment by targeting APP.

cancer biology↗

bioRxiv2025

A perturbation proteomics-based foundation model for virtual cell construction

Building a virtual cell requires comprehensive understanding of protein network dynamics of a cell which necessitates large-scale perturbation proteome data and intelligent computational models learned from the proteome data corpus. Here, we generate a large-scale dataset of over 38 million perturbed protein measurements in breast cancer cell lines and develop a neural ordinary differential equation-based foundation model, namely ProteinTalks. During pretraining, ProteinTalks gains a fundamental understanding of cellular protein network dynamics. Our model encodes protein networks and exhibits consistently improved predictive accuracy across various downstream tasks, highlighting its generalization capabilities and adaptability. In cancer cells, ProteinTalks robustly predicts drug efficacy and synergy, identifies novel drug combinations, and, through its interpretability, uncovers resistance-associated proteins. When applied to more complex system, patient-derived tumor xenografts, ProteinTalks predicts potential responses to drugs. Its integration with clinical patient data enhances the prognosis prediction of breast cancer patients. Collectively, we present a foundational model based on proteome dynamics, offering potential for various downstream applications, including drug discovery, and providing a basis for developing virtual cells.

systems biology↗

bioRxiv2025

Neoadjuvant anti-4-1BB confers protection against spontaneous metastasis through low-affinity intratumor CD8+ T cells in triple-negative breast cancer

Neoadjuvant immunotherapy seeks to harness the primary tumor as a source of relevant tumor antigens to enhance systemic anti-tumor immunity through improved immunological surveillance. Despite having revolutionized the treatment of patients with high-risk early-stage triple-negative breast cancer (TNBC), a significant portion of patients remain unresponsive and succumb to metastatic recurrence post-treatment. Here, we found that optimally scheduled neoadjuvant administration of anti-4-1BB monotherapy was able to counteract metastases and prolong survival following surgical resection. Phenotypic and transcriptional profiling revealed enhanced 4-1BB expression on tumor-infiltrating intermediate (Tint), relative to progenitor (Tprog) and terminally exhausted (Tterm) T cells. Furthermore, Tint was enriched in low-affinity T cells. Treatment with anti-4-1BB drove clonal expansion of Tint, with reduced expression of tissue-retention marker CD103 in Tprog. This was accompanied by increased TCR clonotype sharing between paired tumors and pre-metastatic lungs. Further interrogation of sorted intratumor T cells confirmed enhanced T cell egress into circulation following anti-4-1BB treatment. In addition, gene signature extracted from anti-4-1BB treated Tint was consistently associated with improved clinical outcomes in BRCA patients. Combinatorial neoadjuvant anti-4-1BB and ablation of tumor-derived CXCL16 resulted in enhanced therapeutic effect. These findings illustrate the intratumor changes underpinning the efficacy of neoadjuvant anti-4-1BB, highlighting the reciprocity between local tissue-retention and distant immunologic fortification, suggesting treatment can reverse the siphoning of intratumor T cells to primary tumor, enabling redistribution to distant tissues and subsequent protection against metastases.

immunology↗

bioRxiv2024

Ultra-Fast Multi-Organ Proteomics Unveils Tissue-Specific Mechanisms of Drug Efficacy and Toxicity

Rapid and comprehensive analysis of complex proteomes across large sample sets is vital for unlocking the potential of systems biology. We present UFP-MS, an ultra-fast mass spectrometry (MS) proteomics method that integrates narrow-window data-independent acquisition (nDIA) with short-gradient micro-flow chromatography, enabling profiling of >240 samples per day. This optimized MS approach identifies 6,201 and 7,466 human proteins with 1- and 2-min gradients, respectively. Our streamlined sample preparation workflow features high-throughput homogenization, adaptive focused acoustics (AFA)-assisted proteolysis, and Evotip-accelerated desalting, allowing for the processing of up to 96 tissue samples in 5 h. As a practical application, we analyzed 507 samples from 13 mouse tissues treated with the enzyme-drug L-asparaginase (ASNase) or its glutaminase-free Q59L mutant, generating a quantitative profile of 11,472 proteins following drug treatment. The MS results confirmed the impact of ASNase on amino acid metabolism in solid tissues. Further analysis revealed broad suppression of anticoagulants and cholesterol metabolism and uncovered numerous tissue-specific dysregulated pathways. In summary, the UFP-MS method greatly accelerates the generation of biological insights and clinically actionable hypotheses into tissue-specific vulnerabilities targeted by ASNase.

systems biology↗

bioRxiv2024

Therapeutic modulation of ROCK overcomes metabolic adaptation of cancer cells to OXPHOS inhibition and drives synergistic anti-tumor activity

Genomic studies have identified frequent mutations in subunits of the SWI/SNF chromatin remodeling complex including SMARCA4 and ARID1A in non-small cell lung cancer. Previously, we and others have identified that SMARCA4-mutant lung cancers are highly dependent on oxidative phosphorylation (OXPHOS). Despite initial excitements, therapeutics targeting metabolic pathways such as OXPHOS have largely been disappointing due to rapid adaptation of cancer cells to inhibition of single metabolic enzymes or pathways, suggesting novel combination strategies to overcome adaptive responses are urgently needed. Here, we performed a functional genomics screen using CRISPR-Cas9 library targeting genes with available FDA approved therapeutics and identified ROCK1/2 as a top hit that sensitizes cancer cells to OXPHOS inhibition. We validate these results by orthogonal genetic and pharmacologic approaches by demonstrating that KD025 (Belumosudil), an FDA approved ROCK inhibitor, has highly synergistic anti-cancer activity in vitro and in vivo in combination with OXPHOS inhibition. Mechanistically, we showed that this combination induced a rapid, profound energetic stress and cell cycle arrest that was in part due to ROCK inhibition-mediated suppression of the adaptive increase in glycolysis normally seen by OXPHOS inhibition. Furthermore, we applied global phosphoproteomics and kinase-motif enrichment analysis to uncover a dynamic regulatory kinome upon combination of OXPHOS and ROCK inhibition. Importantly, we found converging phosphorylation-dependent regulatory cross-talk by AMPK and ROCK kinases on key RHO GTPase signaling/ROCK-dependent substrates such as PPP1R12A, NUMA1 and PKMYT1 that are known regulators of cell cycle progression. Taken together, our study identified ROCK kinases as critical mediators of metabolic adaptation of cancer cells to OXPHOS inhibition and provides a strong rationale for pursuing ROCK inhibitors as novel combination partners to OXPHOS inhibitors in cancer treatment.

cancer biology↗