Biology subjects

Wu, T.

Publications and source records attributed to Wu, T..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Human trunk embryoids with patterned anterior-posterior and dorsal-ventral body axes: utility for understanding human development and disease

Human embryoid models enable mechanistic studies of development and disease. We generated trunk embryoids from human pluripotent stem cells that recapitulate posterior trunk formation at Carnegie stage (CS) 8-10, with patterned anterior-posterior (A-P) and dorsal-ventral (D-V) axes. These self-organizing structures comprise a ventral notochord, dorsal neural tube, floor plate and bilateral somites. Genetic and chemical perturbations of SHH signaling confirmed the notochords central role in D-V patterning. Moreover, VANGL1/2 loss-of-function mutations recapitulated mouse phenotypes, including axial truncation and somite segmentation failure. This model enables detailed study of key developmental events that underlie posterior trunk formation and provides a promising platform for human disease modeling.

developmental biology↗

bioRxiv2025

Dysregulated lactate metabolism synergizes with ALS genetic risk factors to accelerate motor decline

Neurons rely on glial lactate shuttling for metabolic support, which declines with aging and in neurodegenerative disease. Full disruption of lactate shuttling in peripheral nerves causes progressive axon degeneration, but we were interested to understand how partial disruption, a scenario more relevant to aging and disease, contributes to neurodegeneration risk. Pyruvate and lactate are interconverted by lactate dehydrogenases (LDHA and LDHB) in both lactate producing and consuming cells. We therefore began by investigating Ldhb knockout mice (loss of LDHA, the dominant LDH in liver and muscle, caused embryonic lethality), and discovered that they develop progressive neuromuscular junction atrophy and functional decline without axon degeneration. Because even Ldhb+/- heterozygosity significantly affects motor behavior, we also wondered about a potential link to congenital disease and pursued this by identifying rare loss-of-function LDHB variants among ALS patients. Next, to better understand how LDHB loss leads to motor decline, we selectively deleted it in defined cell types. SC-specific deletion caused robust motor defects, whereas motor neuron-specific deletion has little effect. Reasoning that neuronal LDHB deficiency could model age-associated decline in lactate metabolism, we asked whether it would interact with ALS genetic risk. Indeed, motor-neuron LDHB deficiency synergizes with relatively mild ALS risk variants-- TDP43Q331Kand Sod1D83G knock-in alleles--to produce early motor neuropathy, indicating that LDHB loss enhances disease risk. These findings establish lactate metabolism as a modifier of motor system vulnerability and highlight it as a therapeutic target in peripheral as well as central neurodegeneration.

neuroscience↗

bioRxiv2025

Phage toxin variants are linked to protection specificity in a defensive symbiont

Insects often depend on symbiotic bacteria for protection, yet the mechanisms by which these microbes target specific natural enemies remain poorly understood. In aphids, different strains of the facultative symbiont Hamiltonella defensa provide highly specific protection against particular species of parasitoid wasps. To uncover the genetic basis of this specificity, we analyzed 26 Hamiltonella genomes and their toxin-encoding APSE bacteriophages with distinct protective phenotypes. Our analyses revealed that Hamiltonella strains share a conserved core genome but differ significantly in accessory gene content, reflecting their distinct evolutionary origins. Strikingly, we show that variation in toxin types is the key distinguishing feature of APSE phages in Hamiltonella strains that protect against different parasitoid species. These toxin repertoires include several novel candidates, such as variants with MAC/perforin domains and leucine-rich repeat (LRR) proteins previously unreported in insect defensive symbionts. We also reveal cases of multiple co-integrated APSE phages carrying different toxins within a single genomic locus. These findings implicate phage-borne toxins as primary determinants of enemy-specific defense and point to phage-driven toxin diversification as a major force shaping the functional evolution of this symbiosis. This work highlights how mobile genetic elements influence the ecological roles and diversification of protective symbionts.

evolutionary biology↗

bioRxiv2025

SIMAIS: A swarm intelligence inspired biosensor for rapid protein detection

Swarm intelligence, the collective behaviour of animals such as birds, fish, and ants, arises from simple local interactions and enables survival, efficient foraging, and decision-making without central control. Inspired by this principle, we aimed to harness swarm intelligence for biosensing applications. Conventional assays often struggle with limited sensitivity, long processing times, and the need for specialised instruments, restricting their use in decentralised healthcare. Here we developed a swarm intelligence of microbead-inspired, artificial intelligence (AI)-assisted, and smartphone-based (SIMAIS) biosensing platform that can transform invisible molecular recognition into visible, macroscale patterns. Millions of antibody-coated magnetic microbeads (MBs) organise into magnetically induced structures upon binding protein biomarkers, with the resulting patterns correlating directly to biomarker concentration. Using interferon-gamma (IFN-{gamma}) as a model biomarker, we demonstrate that this approach delivers results within 10 minutes and achieves a tenfold increase in sensitivity compared to conventional lateral flow assays. Robust performance in human serum and urine confirms clinical applicability. By mimicking natural swarm behaviour, this platform introduces a new biosensing paradigm that bridges biology and engineering. Its versatility, speed, and ease of use highlight its potential for decentralised diagnostics, early disease detection, and broader applications in point-of-care healthcare.

biophysics↗

bioRxiv2025

MYRF is Essential for Epicardial Development

Myelin Regulatory Factor (MYRF) is a transcription factor previously known for myelination of neurons in the central nervous system. Accumulating evidence has recently implicated MYRF mutations in the pathogenesis of cardiac urogenital syndrome (CUGS), in which patients exhibit a range of cardiac abnormalities, including atrial septal defect (ASD), ventricular septal defect (VSD), and hypoplastic left heart syndrome (HLHS). However, the mechanisms by which MYRF deficiency leads to cardiac anomalies remain poorly defined. Moreover, in part due to the lack of cell-type specific loss-of-function models, it is unclear which cardiac cell types contribute to the defects observed in MYRF-related CUGS. To address these questions, we examined the expression pattern of MYRF in the heart and found that it is most highly expressed in epicardial cells (EPCs) among all cardiac cell types. Importantly, Myrf global knockout (KO) mice displayed impeded epicardial development, with markedly reduced epicardial coverage of the myocardial surface. Furthermore, specifically deleting MYRF in EPCs results in severely degenerated epicardium, dramatically reduced epicardial derived cells (EPDCs), and thin myocardial wall. Conversely, ablating MYRF in cardiomyocytes (CMs) did not lead to any overt cardiac phenotypes, indicating that MYRF is dispensable for the developing CMs. Taken together, our findings suggest that compromised function of MYRF in EPCs may contribute to the pathogenesis of MYRF-related CUGS. To our knowledge, this is the first study to link impaired epicardial development to this disease.

developmental biology↗

bioRxiv2025

A Modular Fusion Neural Network Approach to Efficiently Predict Multi-Metal Binding Sites in Protein Sequences

Accurate identification of metal-binding residues is essential for the study of metalloproteins, like zinc finger proteins, haemoglobin, and DNA polymerase. Due to the high cost and time demands of experimental methods, computational prediction has been applied. However, computational complexity and inextensibility of rigid frameworks impeded the application. This work presents a two-stage, sequence-based deep learning framework, which can predict zinc, iron, and magnesium binding amino acids of proteins. In the first stage, tokenized sequences are processed by independent one-dimensional convolutional neural networks (CNNs) to generate single-residue probability maps. In the second stage, a lightweight fusion network integrates these maps to model inter-metal dependencies and refine predictions. The framework employs an imbalance-aware loss function and the ensemble evaluation to improve robustness. Structural agnostic and modular design enable efficient training and inference, making it suitable for the annotation of large-scale proteome. CCS CONCEPTS* Applied computing; * Life and medical sciences; * Bioinformatics

bioinformatics↗

bioRxiv2025

Direct and indirect regulation of fetal globin transcript by RNA-binding protein IGF2BP1

Despite extensive investigation, the molecular control of developmental hemoglobin expression remains incompletely elucidated. Hemoglobin switching is controlled by transcription factors, miRNAs, and RNA-binding proteins (RBPs) that enforce gene regulatory changes through development. Here we examine the role of the heterochronically silenced N-6 methyladenosine (m6A) RNA-binding protein IGF2BP1 that was previously described to regulate HBG1/2 indirectly by suppressing BCL11A expression through an unknown mechanism. We find that IGF2BP1 binds and activates HIC2, itself a BCL11A repressor. Furthermore, we identify that IGF2BP1 plays a BCL11A-independent role by direct binding to HBG1/2 to promote its translation. Stop codon-proximal m6A-modified coding sequences within HBG2 transcripts are necessary and sufficient for direct positive regulation mediated by IGF2BP1. This work deepens the mechanistic understanding of hemoglobin switching and suggests a physical relationship between heterochronic RBPs and globin transcripts.

genetics↗

bioRxiv2025

Transcriptional Readthrough at Atf4 Locus Suppresses Rps19bp1 and Impairs Heart Development

BACKGROUNDActivating Transcription Factor 4 (ATF4) functions as a transcriptional regulator in various cell types and tissues under both physiological and pathological conditions. While previous studies have linked ATF4 activation with promoting cardiomyocyte (CM) death in dilated cardiomyopathy (DCM), atrial fibrillation, and heart failure, its role in developing CMs remains unexplored. METHODSWe generated multiple distinct CM-specific (Atf4cKO(e2/3/pA) and Atf4cKO(e2)) and global Atf4 knockout (Atf47del/7del and Atf41ins/1ins) mouse models targeting different Atf4 regions, as well as cardiomyocyte-specific deletion of Rps19bp1 to study cardiac phenotypes. Detailed morphological and molecular analyses were performed. RESULTSAtf4cKO(e2/3/pA) (targeting exon 2-3 including the polyadenylation signal (polyA)) mice exhibited severe cardiac defects and died before E17.5, likely due to ectopic activation of p53 signaling pathway resulting from Rps19bp1 downregulation, a potent suppressor of p53. Further investigation revealed that deleting the polyA signal of Atf4 in Atf4cKO(e2/3/pA) mice led to transcriptional readthrough, resulting in the formation of an Atf4-Cacna1i fusion transcript and Rps19bp1 downregulation. To avoid readthrough while abolishing ATF4 function, we introduced small indels into exon 3 of Atf4 in mice (Atf47del/7del and Atf41ins/1ins), which showed normal Rps19bp1 expression and cardiac morphology. Importantly, CM-specific deletion of Rps19bp1 recapitulated the cardiac defects and transcriptional change seen in Atf4cKO(e2/3/pA) mice. CONCLUSIONSWe found that the downregulation of Rps19bp1, not loss of ATF4 function, underlying the cardiac phenotypes in Atf4cKO(e2/3/pA) mice. The reduced expression of Rps19bp1 in Atf4cKO(e2/3/pA) mice is likely due to the unintentional deletion of Atf4 polyA signal and subsequent transcriptional readthrough, underscoring the essential role of RPS19BP1, not ATF4, in cardiac development. Consistent Rps19bp1 downregulation has been observed in other tissue-specific Atf4 knockout models utilizing the Atf4fl(e2/3/pA) allele, suggesting that previously reported Atf4 KO phenotypes may result from Atf4 transcriptional readthrough effects. These findings reveal a locus-dependent transcriptional interference mechanism and emphasize the importance of avoiding confounding cis effects in genetically engineered models. TRANSLATIONAL PERSPECTIVEOur findings clarify ATF4s role in heart development by showing that cardiac defects in cardiomyocyte-specific ATF4 knockout mice--using a widely employed floxed ATF4 line--result from unintended downregulation of RPS19BP1 caused by transcriptional readthrough. This shifts the focus from ATF4 to RPS19BP1, a key regulator of p53 activity, as a potential driver of cardiac developmental abnormalities. Clinically, these insights caution against misinterpretation of genetic knockout models and highlight RPS19BP1 as a promising target for congenital heart disease and related cardiac dysfunctions, with potential implications for future therapies.

genetics↗

bioRxiv2025

An Objective Detection Method for Tinnitus using Memristors

Approximately 10-20% of the world population experiences tinnitus, which is increased with age and comorbid with frustration, annoyance, anxiety, depression, cognitive dysfunction, insomnia and stress. The underlying mechanisms of tinnitus remain largely unknown and there are no quantifiable biomarkers, making this prevalent disorder a global burden. There are no FDA-approved therapeutics and the current assessment mainly relies on self-reports (e.g., questionnaires), which underscore the need for diagnostic tools for tinnitus. Memristors, nanoscale electronic components mimicking synaptic plasticity, can monitor and control neuronal activity, hold transformative potential for detecting and characterizing tinnitus objectively. Here, we harvested the blood from human and mice with tinnitus, and engineered memristors via magnetron sputtering and made the functional layer with serum. Its core mechanism lies in regulating the reversible formation and rupture of conductive filaments which is formed by utilizing abnormally elevated glutamate concentrations in serum assisted Ag+ ions. This serum-based memristor achieves unprecedented discrimination between tinnitus and non-tinnitus individuals. Furthermore, this platform provides mechanistic insights into tinnitus pathogenesis, with potential implications in other neurological disorders.

pathology↗

bioRxiv2025

Actinium-225 dendrimer-radioconjugates combined with low-dose standard-of-care chemotherapy: site-independent treatment of triple negative breast cancer metastases

PURPOSEMetastatic triple negative breast cancer (mTNBC) is incurable largely due to the development of drug resistance, the lack of selective cell targeting, and/or limitations in tumor drug delivery that vary depending on the different (metastatic) tumor locations. METHODSThe potential of a single type of systemic, targeted alpha-particle therapy (TAT) was investigated for addressing the above challenges of TNBC tumors implanted at different anatomic sites in mice. Actinium-225 dendrimer-radioconjugates alone, and/or after pretreatment with low-dose standard-of-care cisplatin, were assessed in vitro and on immune-competent 4T1-Balb/c mouse models with tumors implanted intracranially, orthotopically or subcutaneously. RESULTSIn vitro, TATs efficacy was enhanced by cisplatin. In vivo, treatment was initiated well after tumors had grown (Vto = 39 {+/-} 14 mm3 in the intracranial model, and Vto=100mm3 in the orthotopic and subcutaneous models). Across all tumor implantation sites, a unified correlation was observed between animal mean survival and the dendrimer-delivered tumor absorbed doses, which were selectively increased by low-dose cisplatin pretreatment. Importantly, in all animal models, the mean survival following systemic treatment with both modalities was significantly longer vs. each modality alone and/or vs. no treatment, at injected doses that did not cause long-term (10-month) toxicities in tumor-free mice. CONCLUSIONSystemically-injected dendrimer-delivered TAT, combined with low-dose cisplatin pretreatment, can safely extend survival independent of mTNBC tumors anatomic site, potentially presenting a single type of therapy to simultaneously treat multi-site mTNBC.

bioengineering↗

bioRxiv2025

BACH2 dosage establishes the hierarchy of stemness and finetunes antitumor immunity in CAR T cells

Self-renewing stem-like T cells promote the efficacy of cancer immunotherapy and are a heterogeneous population with sub-lineages demonstrating different degrees of stemness. At the apex of this hierarchy is long-term (LT) stem-like T cells with the highest capacity of persistence, repopulation and response to immune checkpoint inhibitors (ICI). However, the pathway that establishes the hierarchy of stemness in chimeric antigen receptor (CAR) T cells and its role in antitumor efficacy of CAR T cells are unclear. Here, we demonstrate that LT stem-like differentiation and antitumor immunity of CAR T cells are dose-dependently regulated by BACH2. Pre-infusion LT stem-like CAR T cells showed epigenetic activation of BACH2 and superior antitumor response in mice and humans. After clearing tumor in vivo, LT stem-like T cells emerged as a CAR subset that transcriptionally and epigenetically upregulated BACH2 and downregulated TOX. Loss-of-function experiments revealed an essential role of BACH2 in the antitumor response of CAR T cells and the transcriptional program of LT stem-like CAR T cells. In exhaustion-prone GD2 CAR T cells with high tonic signaling, we showed that quantitative control of BACH2 protein level by a small molecule drug finetuned the degree of stemness and exhaustion in CAR T cells. Chemically inducing temporal BACH2 activation during manufacture of GD2 CAR T cells imprinted greater antitumor immunity against solid tumor after infusion. Together, we show that BACH2 dosage establishes the hierarchy of stem-like CAR T cells and can be temporally and tunably controlled in CAR T cells to optimize differentiation and antitumor immunity.

cancer biology↗

bioRxiv2025

Single-cell clonal lineage tracing identifies the transcriptional program controlling the cell fate decisions by neoantigen-specific CD8+ T cells

Neoantigen-specific T cells specifically recognize tumor cells and are critical for cancer immunotherapies. However, the transcriptional program controlling the cell fate decisions by neoantigen-specific T cells is incompletely understood. Here, using joint single-cell transcriptome and TCR profiling, we mapped the clonal expansion and differentiation of neoantigen-specific CD8+ T cells in the tumor and draining lymph node in mouse prostate cancer. Compared to other antitumor CD8+ T cells and bystanders, neoantigen-specific CD8+ tumor-infiltrating lymphocytes (TILs) upregulated gene signatures of T cell activation and exhaustion. In the tumor draining lymph node, we identified TCF1+TOX- TSCM, TCF1+TOX+ TPEX, and TCF1-TOX+ effector-like TEX subsets among neoantigen-specific CD8+ T cells. Clonal tracing analysis of neoantigen-specific CD8+ T cells revealed greater clonal expansion in divergent clones and less expansion in clones biased towards TEX, TPEX, or TSCM. The TPEX subset had greater clonal diversity and likely represented the root of neoantigen-specific CD8+ T cell differentiation, whereas highly clonally expanded effector-like TEX cells were positioned at the branch point where neoantigen-specific clones exited the lymph node and differentiated into TEX TILs. Notably, TSCM differentiation of neoantigen-specific CD8+ clones in the lymph node negatively correlated with exhaustion and clonal expansion of the same clones in the tumor. In addition, the gene signature of neoantigen-specific clones biased toward tumor infiltration relative to lymph node residence predicted a poorer response to immune checkpoint inhibitor. Together, we identified the transcriptional program that controls the cell fate choices by neoantigen-specific CD8+ T cells and correlates with clinical outcomes in cancer patients.

cancer biology↗

bioRxiv2025

Spatial Dynamics in the Yeast Galactose Switch Revealed Through RDME-ODE Hybrid 4D Simulations

Eukaryotic cells are spatially organized into functionally-distinct compartments. This three-dimensional(3D) organization generates intracellular heterogeneities that can modulate regulatory dynamics. Despite this knowledge of subcellular organization, most quantitative gene-regulation models still assume a well-mixed environment in which molecules can react regardless of their spatial positions. Here, we use the well-established galactose switch in budding yeast (Saccharomyces cerevisiae) to develop spatially-resolved models that integrate experimentally-derived intracellular architectures, including chromosome organization, the endoplasmic reticulum (ER) and spatially distinct ribosome populations. We implement a hybrid stochastic-deterministic framework in which gene expression is modeled using a reaction-diffusion master equation that enforces locality (i.e., reactions occur only when molecules are in physical proximity), while metabolic and transport processes are captured by ordinary differential equations. Guided by electron microscopy and biochemical constraints, we quantify how accounting for intracellular spatial organization alters regulatory predictions in the galactose switch. We show that chromosome geometry has little effect on Gal2p output, whereas ER-associated translation reduces Gal2p delivery to the plasma membrane; the largest decrease of Gal2p abundance occurs when translation of GAL2 mRNA is restricted to a population of ribosomes physically bound to the ER. Together, these results demonstrate that more realistic 3D cellular architectures and local reaction rules can qualitatively change regulatory predictions, motivating integration of intracellular organization in future whole-cell models. Author SummaryEukaryotic cells are highly organized spaces with distinct subcellular compartments and heterogeneous distributions of molecules which influence how cells function. Yet, most computational models of gene regulation assume a spatially homogeneous intracellular environment. To quantify how intracellular architecture influences regulatory predictions, we developed spatially resolved models of the galactose switch in budding yeast, a well-characterized gene regulatory system controlling the response to extracellular galactose. We compared conventional well-stirred simulations with models that explicitly incorporate experimentally informed intracellular organization, including chromosome positioning, endoplasmic reticulum geometry, and functionally distinct ribosome populations. Incorporating these spatial features substantially altered the dynamics of predicted gene activity, protein production, and intracellular sugar levels. Our results demonstrate that the three-dimensional organization of eukaryotic cells can significantly change regulatory outcomes of computational models, underscoring the need for integrating realistic spatial architectures in future models of eukaryotic gene regulation.

systems biology↗

bioRxiv2025

Single Cell Proteomics in the Developing Human Brain

Proteins are the functional effectors of virtually all biological processes, and accurately measuring their abundance and dynamics is essential for understanding development and disease. Although mRNA levels have historically been used as proxies for protein expression, growing evidence, especially from studies of the human cerebral cortex, has revealed widespread discordance between transcript and protein abundance. To directly address this limitation, we developed a rigorously optimized workflow that combines single-cell mass spectrometry with precise sample preparation to resolve, for the first time, quantitative proteomes of individual cells from the developing human brain. Our platform achieved deep proteomic coverage ([~]800 proteins per cell) even in immature prenatal human neurons (5-10 m diameter, [~]100 pg of protein per cell), capturing major brain cell types and enabling proteome-wide characterization at single-cell resolution. This approach revealed extensive transcriptome-proteome discordance across cell types, with particularly strong discrepancies in genes associated with neurodevelopmental disorders, a finding validated through orthogonal experiments. Proteins exhibited markedly higher cell-type specificity than their mRNA counterparts, underscoring the importance of proteomic-level analysis for resolving cellular identity and function. By reconstructing developmental trajectories from radial glia to excitatory neurons at the proteomic level, we identified dynamic stage-specific protein co-expression modules and pinpointed the intermediate progenitor-to-neuron transition as a molecularly vulnerable phase linked to autism. Altogether, by enabling single cell proteomics, this study establishes a foundational resource and technological advance for developmental neuroscience. It demonstrates that single-cell proteomics can capture critical developmental events and disease mechanisms that are undetectable at the transcript level. As this technology continues to improve in sensitivity and scalability, single-cell proteomics will become an indispensable tool for uncovering the molecular logic of brain development and for illuminating pathophysiological processes underlying neurodevelopmental disorders.

neuroscience↗

bioRxiv2025

Complement C3aR deletion does not attenuate neurodegeneration in a tauopathy model or alter acute inflammation-induced gene expression changes in the brain

Aberrant activation of the classical complement pathway in the brain is implicated in contributing to synapse loss and neurodegeneration in various neurodegenerative conditions. Given that C3aR is a druggable target in the complement pathway, we evaluated the potential of C3aR KO to rescue neurodegeneration in a tauopathy model and neuroinflammatory responses in an acute endotoxemia model. We found that C3aR KO did not rescue tau pathology, microglia activation markers, neurodegeneration, or behavioral abnormalities in tauopathy model mice. While we found that endotoxemia resulted in numerous transcriptional changes including distinct alterations in subpopulations of microglia, astrocytes and oligodendrocytes, C3aR KO did not impact these alterations. Together, our results suggest that the beneficial effects of blocking the complement classical pathway in neurodegeneration models is likely independent of C3aR activation and raise questions about the rationale for therapeutically targeting C3aR for neurodegenerative disease.

neuroscience↗

bioRxiv2025

Bringing the Genetically Minimal Cell to Life on a Computer in 4D

We present a whole-cell spatial and kinetic model for the 100 minute cell cycle of the genetically minimal bacterium, JCVI-syn3A. This is the first simulation of a complete cell cycle in 4D including all genetic information processes, metabolic networks, growth, and cell division. Integrating hybrid computational methods, dynamics of the morphological transformations were achieved. Growth is driven by synthesis of lipids and membrane proteins and constrained by new fluorescence imaging data. Chromosome replication and segregation is controlled by essential SMC and topoisomerase proteins in Brownian dynamics simulations with replication rates responding to dNTP pools from metabolism. The model captures the origin to terminus ratio measured in our DNA sequencing and recovers other experimental measurements like doubling time, mRNA half-lives, protein distributions, and ribosome counts. Because of stochasticity, each replicate cell is unique. Not only do we predict average behavior for partitioning to daughter cells, we predict the heterogeneity among them.

biophysics↗

bioRxiv2025

A single MHCII neoepitope mRNA vaccine elicits CD4 T- and B- cell responses promoting endogenous CD8 anti-tumor immunity

Recent progress in therapeutic cancer vaccines has shown promising clinical activity, especially when targeting MHC class I (MHCI) neoantigen-specific CD8+ T cell responses in post-surgical patients. To explore the role of CD4+ T cells in vaccine-dependent tumor rejection, we constructed an mRNA lipoplex vaccine encoding a single MHCII-restricted neoantigen. The vaccine elicited Tfh and Th1 cell responses while decreasing Tregs, leading to rejection of established tumors in mice. IL-21 and IFN-{gamma}, crucial for Tfh and Th1 function respectively, contributed to anti-tumor activity. B cells and neoantigen-specific antibodies were also shown to participate in vaccine efficacy. Additionally, conventional type 1 dendritic cells (cDC1s) were essential for eliciting vaccine-induced CD4+ T cells, and both cDC1s and CD4+ T cells were required to enhance endogenous CD8+ responses, which were crucial for tumor control. Our results suggest that immunizing against MHCII neoantigens alone is sufficient to orchestrate a potent and cooperative immune response against cancer.

immunology↗

bioRxiv2025

LIN28A-Dependent Kinome and Phosphoproteome Reprogramming Promotes Imatinib Resistance

Chronic myeloid leukemia (CML) resistance to BCR-ABL tyrosine kinase inhibitors (TKIs) can arise from ABL kinase domain mutations, BCR-ABL fusion gene amplification, or kinase-independent mechanisms. To investigate imatinib-resistance, we performed quantitative mass spectrometry comparing the proteome and phosphoproteome of K562 cells (a standard CML model) and ImR cells, an imatinib-resistant K562 derivative that also exhibits cross-resistance to second- and third-generation BCR-ABL TKIs. In addition to revealing global proteome and phosphoproteome changes associated with drug resistance, we identified LIN28A--a multi-functional RNA-binding protein--as a critical mediator of imatinib resistance. LIN28A was significantly overexpressed and hyperphosphorylated in ImR cells. Depleting LIN28A via shRNA restored imatinib sensitivity, while its ectopic expression in parental K562 cells induced imatinib resistance. Mechanistically, LIN28A coordinates an extensive kinase-substrate network regulating proliferation, survival, and metabolism to drive resistance. Notably, pharmacological inhibition of LIN28A-dependent kinases (PKC, AKT, SGK1, and RPS6K) suppressed ImR proliferation. Midostaurin, a clinical PKC/FLT3 inhibitor used in FLT3-ITD--positive AML, potently re-sensitized ImR cells to imatinib. Our findings suggest that targeting LIN28A and its downstream effectors, particularly PKC, could overcome resistance to imatinib and next-generation BCR-ABL inhibitors.

cancer biology↗