Biology subjects

Wu, W.

Publications and source records attributed to Wu, W..

At least 91 records · Page 5Linked to original sources

bioRxiv2025

Creatine synthesis is a tumor suppressor pathway hypostatic to one-carbon metabolism

Methylene tetrahydrofolate reductase 2 (MTHFD2), the rate-limiting enzyme of mitochondrial one-carbon metabolism, is one of the most highly expressed metabolic enzymes across diverse cancers and lymphoproliferative disorders. However, its exact roles in oncogenic metabolism remain poorly defined. We show that MTHFD2 is a key regulator of mitochondrial energetics in Epstein-Barr virus-transformed B lymphoblastoid cell lines (LCLs), an in vitro model of post-transplant lymphoproliferative disorder (PTLD). We also delineate a role for MTHFD2 in fueling de novo creatine synthesis; MTHFD2 mediates the production of glycine, a necessary substrate for creatine synthesis, through serine catabolism. Aminomethyltransferase (AMT) suppression short-circuits the glycine cleavage system (GCS) to augment LCL mitochondrial glycine levels. Creatine synthesis is hypostatic to mitochondrial one-carbon metabolism; inhibition of creatine synthesis improves LCL fitness only when MTHFD2 is lost. Our findings emplace MTHFD2 at the nexus of amino acid and energy metabolism pathways in LCLs, with potential clinical ramifications for PTLD. Highlights* Complete activation of creatine synthesis in an in vitro cellular model of PTLD * Creatine synthesis is a major sink for mitochondrial 1C-derived glycine * Reverse GCS activity due to AMT deficiency in lymphoblastoid cells * Epistasis between mitochondrial 1C metabolism and creatine synthesis eTOC BlurbLeung et al. demonstrate that MTHFD2 is crucial for creatine synthesis in lymphoproliferative disorders. MTHFD2 supports forward 1C flux through SHMT and drives reverse GCS activity to augment mitochondrial glycine, a substrate for creatine synthesis. Tumor-suppressive effects of creatine synthesis are unmasked with MTHFD2 loss, exhibiting metabolic epistasis.

molecular biology↗

bioRxiv2025

Actionable biological programs to enhance EGFR-targeted therapy response unveiled by single-cell lineage tracing in clinically relevant lung cancer models

Developing high-resolution approaches to capture both tumor architectural clonality and transcriptional state(s) in individual cells within heterogeneous tumor cell populations could shed light on the evolution of pre-existing and newly emergent tumor subclones and their phenotypes, elucidating their trajectories in response to selective pressures such as drug treatment. Reports to date have focused primarily on analyzing the drug-induced evolution of lung cancer cells in in vitro preclinical models with limited complexity and a relative lack of characterization of actionable biological programs to induce durable responses. Here, we challenged this paradigm and employed a lineage tracing single-cell RNAseq method to track the evolution of primary non-small cell lung cancer (NSCLC) patient-derived organoids (PDOs) and tumor xenografts in response to the standard-of-care EGFR inhibitor osimertinib, with a focus on understanding drug persistence and resistance. Our single-cell lineage tracing-RNAseq system revealed the presence of a discrete set of lineages with distinct transcriptional phenotypes over the course of the treatment. We identified two lineage populations that became predominant during drug treatment and resisted therapy in the PDOs and tumor xenografts. These lineages were present before treatment and harbored Hedgehog pathway and FOXD1 transcriptional programs, respectively. These specific transcriptomic lineages were otherwise undetectable by lower-resolution profiling. Functional studies confirmed the protective role that the baseline expression of the Hedgehog pathway and FOXD1 programs in the lineage tumor cell sub-populations exerts upon targeted therapy. The potential clinical relevance of these regulatory programs was validated by cross-analysis of single-cell transcriptomic data obtained from human NSCLC specimens. Overall, our approach identified pre-existing seeds of resistance before therapy and convergent, adaptive mechanisms supporting tumor residual disease and resistant states. This study highlights the utility of high-resolution tracing of tumor clonal heterogeneity with matched single-cell profiling to reveal occult cell states and molecular mechanisms of therapy resistance and develop counteracting strategies.

cancer biology↗

bioRxiv2025

Deep-learning-enabled multi-omics analyses for prediction of future metastasis in cancer

Metastasis remains the leading cause of cancer-related mortality, yet predicting future metastasis is a major clinical challenge due to the lack of validated biomarkers and effective assessment methods. Here, we present EmitGCL, a deep-learning framework that accurately predicts future metastasis and its corresponding biomarkers. Based on a comprehensive benchmarking comparison, EmitGCL outperformed other computational tools across six cancer types from seven cohorts of patients with superior sensitivity and specificity. It captured occult metastatic cells in a patient with a lymph node-negative breast cancer, who was declared to have no evidence of disease by conventional imaging methods but was later confirmed to have a metastatic disease. Notably, EmitGCL identified HSP90AA1 and HSP90AB1 as predictable biomarkers for future breast cancer metastasis, which was validated across five independent cohorts of patients (n=420). Furthermore, we demonstrated YY1 transcription factor as a key driver of breast cancer metastasis which was validated through in-silico and CRISPR-based migration assays, suggesting that YY1 is a potential therapeutic target for deterring metastasis.

bioinformatics↗

bioRxiv2025

G Protein Coupled Estrogen Receptor Signaling Maintains β Cell Identity in Female Mice

Type 2 diabetes (T2D) arises in the context of obesity and overnutrition; however, additional demographic features including age and biological sex contribute to T2D risk. Estradiol (E2) is thought to play a protective metabolic role that may govern sex differences in the development of T2D. The mechanisms by which E2 exerts these effects and the impact of reduced E2 signaling in {beta} cells during menopause remain incompletely understood. We analyzed publicly available whole islet transcriptome datasets from female and male cadaveric donors and showed significant age-related modulation of gene expression, including changes in pathways related to {beta} cell function, in islets from female donors. Importantly, these patterns were not observed in islets from male donors. To test the in vivo relationship between E2 signaling and {beta} cell function, 10-week- old female C57BL6/J mice underwent an ovariectomy (OVX) or sham (CTR) surgery, followed by 4 weeks of high-fat diet (HFD) treatment. HFD-OVX mice exhibited obesity-induced glucose intolerance, increased cell mass, and reduced expression of {beta} cell identity markers. Furthermore, ex vivo treatment of islets with the G protein coupled estrogen receptor (GPER)- specific agonist G-1 restored {beta} cell identity gene expression. Together, these data identify a novel connection between GPER signaling and {beta} cell identity and suggest that menopausal loss of E2 signaling through GPER may be linked with loss of {beta} cell identity.

physiology↗

bioRxiv2025

Targeting USP2 induces degradation of PML-RARα with or without drug-resistant mutations in acute promyelocytic leukemia

Despite the high efficacy of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) in treating acute promyelocytic leukemia (APL), approximately 10-20% of patients develop drug resistance due to mutations in PML-RAR and other factors. Here, it is demonstrated that inhibiting USP2 with ML364 or silencing USP2 reduces PML-RAR protein levels in both ATRA-sensitive and -resistant APL cells, an effect reversed by proteasome inhibition. Conversely, USP2 overexpression enhances PML-RAR stability. Mechanistically, USP2 interacts with and deubiquitinates PML-RAR, including drug-resistant mutants. Consistent with PML-RAR degradation, ML364 treatment significantly induces apoptosis in APL cell lines and primary leukemia cells. In conclusion, this study identifies USP2 as a novel deubiquitinating enzyme for PML-RAR and highlights USP2 inhibition as a potential therapeutic strategy for APL with PML-RAR mutations.

cell biology↗

bioRxiv2025

Anti-CSF-1R therapy with combined immuno- chemotherapy coordinate an adaptive immune response to eliminate macrophage enriched Triple Negative Breast Cancers

Patients diagnosed with metastatic triple negative breast cancer (mTNBC) have limited treatment options, are more prone to develop resistance and are associated with high mortality. A cold tumor immune microenvironment (TIME) characterized by low T cells and high tumor associated macrophages (TAMs) in mTNBC is associated with the failure of standard-of-care chemotherapy and immune checkpoint blockade (ICB) treatment. We demonstrate that the combination of immunomodulatory metronomic Cyclophosphamide (CTX) coupled with anti-CSF-1R antibody targeted therapy (SNDX-ms6352) and anti-PD-1 (ICB), was highly effective against aggressive metastatic syngeneic Trp53 null TNBC genetically engineered mouse models (GEMMs) that present with high macrophage infiltration. Mechanistically, CSF-1R inhibition along with CTX disrupted the M-CSF/CSF-1R axis which upregulated IL-17, IL-15 and type II interferon resulting in elevated B- and T cell infiltration. Addition of an anti-PD-1 maintenance dose helped overcome de novo PD-L1 intra-tumoral heterogeneity (ITH) associated recurrence in lung and liver mTNBC.

cancer biology↗

bioRxiv2025

Novel HIV-1 fusion peptide immunogens using glycan-engineered alphavirus-like particles

Immunofocusing on conserved, subdominant epitopes is critical for vaccines against highly diverse viruses such as HIV-1, influenza, and SARS-CoV-2. The eight-residue N-terminus of the HIV-1 fusion peptide (FP) is one such example of a promising yet small target. We developed new FP immunogens using three alphavirus-like particles (VLPs) and introduced additional glycans to mask shared carrier-specific epitopes. In two independent guinea pig studies, sequential immunization with heterologous carriers enhanced FP-directed antibody titers, which were further improved with glycan engineering. Separately, using diverse FP variants sharing the same N-terminal six amino acids increased neutralizing antibody titers. When combined, these two strategies led to higher FP-directed titers and, after Env trimer boosting, induced FP-directed neutralizing antibodies against multi-clade wild-type HIV-1 in nearly all animals. These findings established the importance of minimizing recurrent off-target epitopes across immunizations and support the engineered VLPs as a promising platform for peptide immunization. HighlightsO_LINovel HIV-1 fusion peptide immunogens using glycan-engineered alphavirus-like particles C_LIO_LIImproved FP-directed response by minimizing recurrent carrier-specific epitopes across immunizations C_LIO_LIImproved neutralizing response by sequential immunization with diverse FP variants C_LIO_LIFP-directed antibodies neutralizing multi-clade wildtype viruses in nearly all animals C_LI

immunology↗

bioRxiv2025

PASTA: Versatile Tyramine-oligonucleotide Amplification for Multi-modal Spatial Biology

Spatial proteomics techniques have revolutionized our understanding of tissue architecture, but are frequently limited by detection sensitivity, bioconjugation limitations, multiplexing capacity, and multi-modal integration. Here we present Protein and nucleic Acid Serial Tyramine Amplification (PASTA), a novel signal amplification approach that significantly enhances detection sensitivity while maintaining compatibility with diverse spatial profiling methodologies. PASTA utilizes horseradish peroxidase (HRP) recruitment pathways to generate tyramine radicals that deposit oligonucleotides, enabling adaptable signal amplification across multiple biomarkers at high-plex via cyclical imaging using complementary fluorophore-labeled oligonucleotides. We demonstrate that PASTA achieves up to 100-fold signal enhancement for markers with minimal background in blank controls. The method is compatible with in situ hybridization for DNA/RNA detection, proximity ligation assays for protein-protein interactions, sequential antibody staining protocols, or any modular combination thereof. PASTA enables antibody rescue of markers with suboptimal signal-to-noise ratios and is versatile in its applications to unconjugated antibodies, and multi-round probe-based RNA detection systems beyond current capabilities. This technique addresses key limitations in spatial-omics by enhancing sensitivity for challenging targets while maintaining compatibility with established multiplexing strategies, providing a versatile, cost-efficient, and valuable tool for comprehensive spatial tissue analysis in both research and clinical applications.

molecular biology↗

bioRxiv2025

Miniaturized and accessible functional ultrasound imaging system for freely moving mice

Functional ultrasound (fUS) imaging provides brain-wide activity maps with high spatiotemporal resolution and deep penetration, positioning it as a key technology for future non-invasive brain-computer interfaces (BCIs). Realizing this potential, particularly for chronic BCI applications requiring long-term monitoring in naturalistic settings, critically depends on significant system miniaturization to overcome the cost and complexity limitations of current platforms. Addressing this challenge, we present mini-fUS, a miniaturized, cost-effective fUS platform engineered for accessibility without compromising core performance for demanding neuroscience research. The system features a compact transmit-receiver, low-noise power supply, and high-speed data transfer, achieving pulse repetition frequencies up to 5 kHz with negligible jitter. Real-time GPU-accelerated beamforming and singular value decomposition (SVD) enable whole-brain activity mapping, demonstrated here in freely moving mice at up to 3.57 Hz with [~]100 {micro}m spatial resolution and 15 mm penetration depth. Validated through recordings of brain activity during sensory stimulation, anesthesia, and behavior, this design defines a practical hardware-software framework for fUS. By significantly improving accessibility and enabling robust monitoring in mobile subjects, this work advances the development of fUS for both fundamental research and future BCI technologies, while clarifying essential fUS operational principles.

neuroscience↗

bioRxiv2025

Integration of the hammerhead ribozyme into structured RNAs to measure ligand-binding events for riboswitch candidates and aptamers

Some structured RNAs, such as riboswitches and aptamers, can bind their cognate ligands and have been used in biosensors and gene expression control elements. However, current methods for detecting ligand binding to structured RNAs are either severely limited or inconvenient. In this study, we design a multi-base pair bridge to integrate a hammerhead ribozyme into structured RNAs to detect ligand binding events. The experimental results demonstrate that the length of the bridge has a significant effect on the cleavage of the ribozyme; optimal cleavage can be achieved with three to six base pairs in the bridge. The dissociation constant (KD) values obtained through this method are in agreement with those determined by in-line probing techniques, and 1 pmol of allosteric ribozyme RNA is sufficient for measurement. We apply this method to evaluate the binding affinity of the riboswitch candidate Motif_9307. Our findings indicate that this motif has no binding affinity for S-adenosylmethionine or several other tested ligands, which is consistent with the results of the in-line probing experiments. Notably, our method reveals an increase in cleavage activity when yeast extract is added as a mixture of ligands, suggesting that the ligand of Motif_9307 is present in the extract. In conclusion, we develop an alternative approach for measuring ligand binding events associated with riboswitch candidates and aptamers.

molecular biology↗

bioRxiv2025

Connecting the Dots: A Computational Framework linking Molecular Regulation, Synaptic Plasticity, and Brain Disorders

Neuroplasticity supports learning, development, and adaptive behavior, but also underlies maladaptive circuit remodeling in neuropsychiatric disorders. Although genetic disruptions of synaptic signaling are widely implicated in these conditions, the causal chain from molecular perturbation to systems-level dysfunction remains unclear. Here we introduce a multiscale computational framework--a parameter-free Boolean regulatory model of glutamatergic signaling--that captures the dynamic control of synaptic plasticity. The model generates emergent attractor states corresponding to long-term potentiation and depression and reveals how these states arise from underlying Hebbian principles. In silico gene-knockout simulations show that specific molecular perturbations destabilize these attractors, impairing synaptic plasticity and producing circuit-level alterations characteristic of diverse brain disorders. Remarkably, the model recapitulates the opposing developmental trajectories of autism spectrum disorder and schizophrenia, offering a mechanistic account of their divergent cortical phenotypes. This computationally tractable and generalizable framework quantitatively links genetic variation to synaptic instability and clinical severity, aligning with experimental and clinical observations. By moving beyond correlative associations, it establishes a causal scaffold for tracing how molecular disruptions propagate through synaptic regulatory networks to impair brain function, informing integrative diagnostics and mechanism-based therapeutic strategies.

neuroscience↗

bioRxiv2025

An unrecognized and crucial role of chloroplast division in leaf variegation in Arabidopsis thaliana

Leaf variegation provides a valuable model for elucidating the molecular mechanisms that underlie chloroplast biogenesis, including both chloroplast development and division. While there has been notable advancement in comprehending the role of the Arabidopsis Yellow Variegated2 (VAR2), which encodes the AtFtsH2 subunit of the thylakoid FtsH metalloprotease complex, the exact mechanism governing leaf variegation formation remains elusive. In this study, time-course microscopy analyses of chloroplast number per cell from the first leaf pair revealed that most var2 cells lack chloroplasts. Interestingly, chloroplast biogenesis in the green sector of var2 leaves is significantly delayed but prolonged compared to the wild type (WT), leading to increased heterogeneity in chloroplast number and size among cells. These results suggest that chloroplast biogenesis in the green sector can partially compensate for the defect in the white sector. Additionally, approximately 15% of cells in var2 white sectors are devoid of plastids, and chloroplast number in guard cells is significantly lower in var2 than in WT, indicating that VAR2 mutations impair chloroplast division. Consistently, the var2 phenotype is exacerbated by knocking out key plastid division genes, including Paralog of Accumulation and Replication of Chloroplasts6 (PARC6) and Plastid Division1 (PDV1), but is fully rescued by overexpressing PDV1 or PDV2. Furthermore, VAR2 mutations inhibit etioplast development, while accelerating chloroplast biogenesis by knocking out Constitutively Photomorphogenic1 (COP1) rescues the var2 phenotype. Together, our findings reveal a novel role for VAR2 in plastid division and its essential function in chloroplast biogenesis, providing new insights into the mechanism underlying variegated leaf formation.

plant biology↗

bioRxiv2025

Helminth infection favors reprogramming and proliferation of lung neutrophils

Neutrophils are a granulocytic population of myeloid cells that have critical effector functions during infectious disease but are generally thought to be short-lived and nonproliferative with markedly limited activation states. In these studies, we directly compared lung neutrophil activation following infection with different groups of pathogens including bacteria, fungi, and helminths. Our results demonstrate considerable heterogeneity depending on the type of infectious agent. In contrast to bacterial and fungal infection, after helminth infection neutrophils expressed markers associated with characteristic type 2 responses and unexpectedly upregulated genes associated with cell cycling and protein synthesis. Further studies showed reduced neutrophil cell death following helminth infection and increased proliferation, which was dependent on IL-4R signaling. This distinct subset of proliferating neutrophils expanded following helminth infection and was released from the endothelial niche to colocalize with invading parasites in the airways. These studies demonstrate a novel long-lived cycling phenotype for neutrophils following helminth infection.

immunology↗

bioRxiv2025

Tumor cell villages define the co-dependency of tumor and microenvironment in liver cancer

Spatial cellular context is crucial in shaping intratumor heterogeneity. However, understanding how each tumor establishes its unique spatial landscape and what factors drive the landscape for tumor fitness remains significantly challenging. Here, we analyzed over 2 million cells from 50 tumor biospecimens using spatial single-cell imaging and single-cell RNA sequencing. We developed a deep learning-based strategy to spatially map tumor cell states and the architecture surrounding them, which we referred to as Spatial Dynamics Network (SDN). We found that different tumor cell states may be organized into distinct clusters, or villages, each supported by unique SDNs. Notably, tumor cell villages exhibited village-specific molecular co-dependencies between tumor cells and their microenvironment and were associated with patient outcomes. Perturbation of molecular co-dependencies via random spatial shuffling of the microenvironment resulted in destabilization of the corresponding villages. This study provides new insights into understanding tumor spatial landscape and its impact on tumor aggressiveness.

cancer biology↗

bioRxiv2025

TSHR-targeting nucleic acid aptamer treats Graves' ophthalmopathy via novel allosteric inhibition

Abnormal thyrotropin receptor (TSHR) signaling drives a spectrum of diseases affecting hundreds of millions globally, including Graves disease (GD), Graves ophthalmopathy (GO), hyperthyroidism, and thyroid cancers. Therefore, TSHR is considered an attractive therapeutic target with immense drug discovery potential. Currently, there are no clinically available TSHR inhibitors. To address this unmet medical need, we developed YC3, a functional nucleic acid aptamer targeting TSHR, via an innovative approach combining protein-targeting cell-SELEX and functional selection. YC3 exhibited nanomolar affinity with potent TSHR inhibition. Its therapeutic potential was comprehensively evaluated both in vitro and in vivo for GO. In GO patient-derived orbital fibroblasts (OFs), YC3 reversed thyroid-stimulating antibodies (TSAbs)-induced cell activation, suppressing inflammatory cytokines and extracellular matrix (ECM) secretion. In a GO mouse model, YC3 treatment markedly attenuated orbital inflammation, ECM deposition, and fibrosis, ameliorating the pathological remodeling of orbital tissue. Mechanistically, YC3 bound to a previously unidentified allosteric site within the leucine-rich repeat domain of TSHR, thereby inhibiting receptor activation. This study identifies YC3 as a promising TSHR-targeted therapy and unveils a new druggable site for inhibitor design. It also provides the first preclinical evidence supporting pharmacological TSHR inhibition for GO treatment, advancing therapeutic development for TSHR-related diseases.

biochemistry↗

bioRxiv2025

Conserved and Unique Features of Terminal Telomeric Sequences in ALT-Positive Cancer Cells

A significant portion of human cancers utilize a recombination-based pathway, Alternative Lengthening of Telomeres (ALT), to maintain telomere length. Targeting the ALT is of growing interest as a cancer therapy, yet a substantial knowledge gap remains regarding the basic features of telomeres in ALT-positive cells. To address this, we adopted END-seq, an unbiased sequencing-based approach, to define the identity and regulation of the terminal sequences of chromosomes in ALT cells. Our data reveal that the terminal portions of chromosomes in ALT cells contain canonical telomeric sequences with the same 5 terminus bias (-ATC) observed in non-ALT cells. Furthermore, as reported for non-ALT cells, POT1 is required to preserve the precise regulation of the 5 end in cells that maintain telomere length using the ALT pathway. Thus, the regulation of the terminal 5 of chromosomes occurs independently of the mechanism of telomere elongation. Additionally, we employed an S1 endonuclease-based sequencing method to determine the presence and origin of single-stranded regions within ALT telomeres. These data shed light on conserved and unique features of ALT telomeres.

molecular biology↗

bioRxiv2025

Cell surface proteomics of patient-derived malignant rhabdoid tumor organoids identifies ROBO1 as potential CAR T cell target for pediatric solid tumors.

Malignant rhabdoid tumors are highly aggressive pediatric malignancies with limited treatment options and poor outcomes. To expand therapeutic options, we employed mass spectrometry-based cell surface proteomics on malignant rhabdoid tumor and patient-matched normal kidney organoids to identify potential CAR T cell targets. Integrating these findings with transcriptomics and protein expression data, we revealed ROBO1 as a promising target, showing strong and uniform expression across malignant rhabdoid and other pediatric tumors. ROBO1-targeted CAR T cells displayed potent anti-tumor activity in vitro, effectively eliminating tumor cells in co-cultures with organoids from malignant rhabdoid tumors, rhabdomyosarcoma, and neuroblastoma. In vivo, ROBO1 CAR T cells infiltrated tumors, induced potent tumor regression and significantly increased survival in malignant rhabdoid tumor-bearing mice. These findings establish ROBO1 as a compelling therapeutic target for CAR T cell therapy and offer a promising approach to address the critical need for effective treatments in high-risk pediatric solid tumors. Statement of significanceThis study identifies ROBO1 as a promising CAR T cell target for pediatric solid tumors. Using patient-derived tumor organoids and tissues, we demonstrate strong ROBO1 expression across several tumor entities and robust anti-tumor efficacy of ROBO1-targeted CAR T cells in vitro and in vivo, underscoring its potential for clinical translation.

cancer biology↗

bioRxiv2025

Incongruent end structures of leading and lagging telomeres dictate the nature of end replication problem

The end replication problem refers to the incomplete replication of parental DNA at telomeres, a process whose molecular depiction is hampered by the complex nature of telomere ends. Here we recapitulate this process using a synthetic de novo telomere in yeast and delineate distinct molecular fates of telomere ends in vivo. We show that the lagging strand telomere carries a [~]10 nt 3 overhang, while the leading strand telomere has a Yku-protected blunt end, which is prevalent on native telomeres. In addition, RNase H2 but not RNase H1 is mainly responsible for the removal of the last RNA primer. Consistently, in the absence of RNase H2 activity, RNA primer is retained on the lagging strand telomere, attenuating telomere erosion and delaying senescence in telomerase-null cells. These findings highlight incongruent end structures on telomeres and clarify that the primary culprit behind end replication problem is the incompletely replicated lagging strand telomere.

molecular biology↗