Biology subjects

Leary, P.

Publications and source records attributed to Leary, P..

14 recordsLinked to original sources

bioRxiv2026

Acquired epithelial Wnt7b secretion establishes autocrine WNT dependency in BRAF mutant colorectal cancer

Colorectal cancer (CRC) subtypes differ fundamentally in their reliance on WNT signaling, yet the sources and regulation of WNT ligands as well as the subtype-specific requirements for ligand-driven activation remain poorly defined. Here, we generated genetically-engineered organoid models of Apc- and Braf-mutant CRC to systematically dissect ligand dependency across distinct genetic backgrounds and CRC subtypes. Through genetic perturbation and pharmacological inhibition of WNT secretion, we find that BRAF-driven organoids critically depend on autocrine WNT ligand production for survival and proliferation. In contrast, APC-mutant organoids remain viable but undergo discernible transcriptional changes upon ligand withdrawal. We identify Wnt7b as a non-redundant epithelial WNT ligand required to sustain beta-catenin-dependent signaling specifically in BRAF-mutant CRC. Chromatin accessibility and transcriptional profiling reveal that Wnt7b activation occurs early during transformation and coincides with cancer-specific opening of regulatory elements at the Wnt7b locus. Integrative regulatory analyses nominate the transcription factor RFX7 as a candidate upstream regulator of epithelial Wnt7b expression. Consistent with these findings, analysis of human CRC specimens demonstrates WNT7B expression in a subset of BRAF-mutant tumors and reveals an association with poor patient survival. Together, these results redefine ligand dependency in colorectal cancer and uncover autocrine WNT7B signaling as a mechanistically defined vulnerability in aggressive BRAF-mutant disease.

molecular biology↗

bioRxiv2026

High regeneration-associated stress defines a distinct HCC subgroup with therapeutically exploitable vulnerabilities

Background: To date, no precision oncology approach has been established for HCC. Despite the diverse underlying causes, HCC development exhibits a uniform pathophysiology characterised by chronic hyper-proliferation, resulting from hepatocyte apoptosis and compensatory liver regeneration. This chronic hyper-proliferative pressure, termed regeneration stress, drives genomic instability during HCC onset, yet its therapeutic potential remains poorly explored. This study aimed to identify targetable vulnerabilities tied to regeneration stress and establish clinically applicable markers for treatment stratification. Methods: Weighted gene co-expression network analysis (WGCNA) was applied on external bulk RNA-seq datasets to define a LIVer REgeneration Stress Signature (LIVRESS). The signature was functionally validated using HCC patient-derived organoids (HCC-Org), and vulnerabilities were mapped using mid-throughput drug screening, single-molecule and single-cell assays, and multi-omic integration. Results: High LIVRESS scores, characterised by enrichment in replication, mitotic and DNA damage repair pathways, identified a subset of HCC patients with aggressive disease and poorer survival across aetiologies. HCC-Org with high LIVRESS scores displayed exquisite sensitivity to multiple inhibitors of the checkpoint kinase ATR. Although HCC-Org models exhibited a baseline reduction in replication fork speed, sensitivity to ATR inhibitor (ATRi) was decoupled from replication fork dynamics and rather linked to intrinsic mitotic instability. ATR inhibition triggers mitotic failure and apoptosis in LIVRESSHigh HCC-Org. This killing effect was significantly potentiated by combining ATRi with PARPi or WEE1i. Multi-omic integration identified KPNA2 as a surrogate biomarker of ATRi sensitivity. Conclusion: Our findings demonstrate that a subset of HCC-Org, characterised by high liver regeneration-associated stress, is vulnerable to ATRi-based therapies. By focusing on a comprehensive regenerative stress model, we establish a framework to stratify HCC patients and implement biomarker-driven, ATR-based therapies for HCC patients with advanced disease. Impact and implications: Regeneration stress is a key factor that drives genomic instability in HCC, providing a basis for the LIVRESS to identify patients dependent on ATR-mediated checkpoints. These findings reveal a conceptual shift for researchers and trialists: ATRi efficacy is decoupled from replication fork dynamics and instead leverages mitotic fragility. Practically, the LIVRESS and its IHC surrogate marker (KPNA2) offer a scalable roadmap for physicians to improve patient stratification in ATRi-based precision oncology trials. While requiring prospective validation, these results pave the way toward biomarker-driven therapies for advanced HCC.

cancer biology↗

bioRxiv2026

Transcriptional profiling of extraocular motor neurons reveals sim1a as a candidate strabismus-related gene

Strabismus, or misalignment of the eyes, is a heritable disorder frequently associated with vision loss and decreased quality of life. Incomitant strabismus, where the degree of misalignment differs based on gaze angle, can arise from mutations in genes that regulate the development of extraocular motor neurons. To date, few such genes have been identified. The extraocular motor system is highly conserved across vertebrates, suggesting a comparative transcriptomic discovery approach would be fruitful. Using bulk and single-cell sequencing in a small accessible vertebrate, the larval zebrafish, we identified genes expressed in subpopulations of extraocular motor neurons in cranial nuclei nIII/nIV. We next assessed extraocular motor neuron number and vestibulo-ocular reflex performance after CRISPR/Cas9-mediated mutagenesis of three genes with suggestive expression patterns: sim1a, nav2a, one-cut1, and one known to disrupt nIII/nIV motor neuron specification: phox2a. Loss of sim1a impaired the vestibulo-ocular reflex without change to nIII/nIV motor neuron number. Our data suggest that constitutive disruptions to sim1a can impair nIII/nIV-dependent eye movements. More broadly, our work illuminates considerable transcriptomic diversity among extraocular motor neuron subpopulations, and establishes a pipeline to identify genes relevant to ocular motor disease etiology.

neuroscience↗

bioRxiv2025

The nucleus accumbens shell regulates hedonic feeding via a rostral hotspot

The medial nucleus accumbens shell (medNAcSh) is increasingly recognized as a key node in the regulation of hedonic feeding, exerting strong control over reward consumption through its projections to downstream structures. Recent studies showed that one of the primary cellular mediators of these effects are dopamine 1 receptor positive striatal projection neurons (D1-SPNs). Specifically, population D1-SPN activity gets inhibited during reward consumption and such inhibition is necessary and sufficient to authorize reward consumption, independent of metabolic need. Anatomically, the medNAcSh spans a 1 to 1.5 mm distance along the rostro-caudal axis in mice and previous studies have reported functional gradients along this axis in behavioral control. For instance, classical pharmacological studies have suggested that rostral rather than caudal medNAcSh regulates appetitive behavior. However, the cellular, circuit, and molecular mechanisms underlying this topographical gradient remain unknown. Here we hypothesized that D1-SPNs contribute to this gradient by regulating hedonic feeding via a specific hotspot in the rostral medNAcSh. Using population level calcium monitoring with fiber photometry in mice, we show that rostral medNAcSh D1-SPNs demonstrate inhibitory responses during reward consumption, while caudal D1-SPNs do not. Consistently, optogenetic stimulation of rostral medNAcSh D1-SPNs inhibits consumption, while stimulation of caudal D1-SPNs had minimal effects, confirming the existence of a functional rostro-caudal gradient. Importantly, we observed no differences between rostral and caudal D1-SPNs in their behavioral or activity responses to aversive stimuli, suggesting that the D1-SPN gradient is specific to appetitive contexts. To investigate potential molecular correlates of this functional gradient, we leveraged open-source anatomy datasets and performed fluorescent in situ hybridization, identifying Stard5 and Peg10 as markers enriched in the rostral and caudal medNAcSh, respectively. Finally, we developed a novel Stard5-Flp driver mouse line, which allowed us to target the rostral hotspot selectively. We demonstrated that Stard5+ neurons recapitulate activity patterns of rostral medNAcSh D1-SPNs. Together, these findings establish a spatially confined rostral subregion of the medNAcSh as a critical regulator of reward consumption and introduce Stard5 as a molecular tool for its manipulation - offering new opportunities for intervention in dysregulated eating beyond classical homeostatic circuits.

neuroscience↗

bioRxiv2025

Deep visual multi-omics profiling reveals mechanisms that underly cancer cell differentiation and aggressiveness in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) exhibits significant intra-tumoral heterogeneity (ITH) at both morphological and genetic levels, complicating treatment and contributing to disease progression. Among these, ccRCCs with focal rhabdoid differentiation stand out as highly aggressive tumors distinguished by cells with unique morphological features. However, the correlation between distinct morphological phenotypes, specific molecular alterations, and their influence on tumor behavior remains poorly understood. In this study, we integrated advanced AI-based image analysis with single-cell isolation and multi-omics profiling to dissect the link between clinically relevant morphological and molecular features of ccRCC cells. Using a novel digital pathology workflow, we quantified low-grade, high-grade, and rhabdoid morphologies in ccRCC diagnostic images with unprecedented precision. Subsequently, isolation of two sets of 1,000 morphologically distinct cells for detailed mRNA and protein expression analyses, revealed significant increasing dysregulation associating with higher histopathological grades. Rhabdoid ccRCC cells (grade 4) demonstrated unique molecular profiles, including upregulated FOXM1-driven proliferation, disrupted cell-matrix interactions, and enhanced immune evasion pathways. Despite high T-cell infiltration in rhabdoid areas, we identified a rhabdoid-specific immunosuppressive network driven by cytokines, IFN-beta, and integrin signaling, likely contributing to T-cell exhaustion. Rhabdoid ccRCC cells develop a distinct immunosuppressive signaling network, involving PD-L1 and novel immunomodulatory factors such as CD38 and ITGB2. These findings provide a basis for novel therapeutic strategies targeting these pathways in combination with immunotherapy to improve outcomes for patients with aggressive rhabdoid ccRCC. Key PointsO_LIccRCC is characterized by well-established morphological heterogeneity but the correlation with the underlying molecular aberrations remained elusive. C_LIO_LIBy integrating AI-based image analysis with single cell isolation and deep multi-omics profiling, we dissect the molecular intricacies of ccRCC, from targeted collection of 1,000 morphologically distinct cells. C_LIO_LIOur results demonstrate significant dysregulation of gene and protein expression correlating with higher histopathological grades in ccRCC. C_LIO_LIAggressive ccRCC cells with rhabdoid differentiation (grade 4) display distinct molecular profiles, as they upregulate FOXM1-mediated proliferation, ECM remodeling and the immune evasion responses, suggesting new therapeutic avenues enhancing ICI efficacy in these patients. C_LI

cancer biology↗

bioRxiv2025

Extrachromosomal circular DNA promotes inflammation and hepatocellular carcinoma development

Two decades after the initial report on increased micronuclei in human chronic liver disease (CLD) and hepatocellular carcinoma (HCC), their role in HCC development is still poorly understood. Here we show that micronuclei in hepatocytes trigger hepatic immune response and promote HCC development via an increased level of extrachromosomal circular DNA (eccDNA). Livers from a CLD model (Mcl1{Delta}hep mice) show increased micronuclei in parallel to eccDNA. Circular sequencing confirms higher eccDNA levels in micronuclei compared to primary nuclei. We developed nuclei-segregated DNA fiber (NuSeF) assay to show that micronuclei are more susceptible to replication stress, showing increased replication fork slowing. By comparing different murine liver disease models, high eccDNA is correlated with increased tumor incidence. EccDNA is a strong immunostimulant and promotes a crosstalk between hepatocytes and immune cells through the cGAS-STING pathway. Deletion of Sting1 in Mcl1{Delta}hep mice reduces immune cell chemotaxis, as well as tumor incidence. Our findings suggest that eccDNA from micronuclei mediates inflammation-driven liver carcinogenesis in CLD.

cancer biology↗

bioRxiv2024

prolfquapp - A User-Friendly Command-Line Tool Simplifying Differential Expression Analysis in Quantitative Proteomics

AbstractMass spectrometry is a cornerstone of quantitative proteomics, enabling relative protein quantification and differential expression analysis (DEA) of proteins. As experiments grow in complexity, involving more samples, groups, and identified proteins, traditional interactive data analysis methods become impractical. The prolfquapp addresses this challenge by providing a command-line interface that simplifies DEA, making it accessible to non-programmers and seamlessly integrating it into workflow management systems. Prolfquapp streamlines data processing and result visualization by generating dynamic HTML reports that facilitate the exploration of differential expression results. These reports allow for investigating complex experiments, such as those involving repeated measurements and multiple explanatory variables. Additionally, prolfquapp supports various output formats, including XLSX files, SummarizedExperiment objects and rank files, for further interactive analysis using spreadsheet software, the exploreDE Shiny application, or gene set enrichment analysis software. By leveraging advanced statistical models from the prolfqua R package, prolfquapp offers a user-friendly, integrated solution for large-scale quantitative proteomics studies, combining efficient data processing with insightful, publication-ready outputs. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/617391v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@10930a2org.highwire.dtl.DTLVardef@57f5dborg.highwire.dtl.DTLVardef@ce0d73org.highwire.dtl.DTLVardef@1d531da_HPS_FORMAT_FIGEXP M_FIG C_FIG This visual table of contents illustrates the workflow and key features of the prolfquapp tool for differential expression analysis in proteomics. On the left are the inputs, like the CSV for annotation and quantification results, YAML for parameters, and FASTA files for protein information. In the center are the prolfquapp and prolfqua R packages and supporting tools like crosstalk and knitr, representing the core processing components. On the right side, the figure highlights the various outputs generated by prolfquapp O_LIXLSX files containing protein abundances, group summaries, and differential expression results. C_LIO_LIHTML reports with text, graphs, interactive volcano plots, and dynamic tables for data exploration. C_LIO_LIPDF documents with detailed protein boxplots and peptide-level matrix plots. C_LIO_LIIntegration with exploreDE for interactive data visualization. C_LI This diagram concisely summarizes the flow from data input to the creation of analysis-ready outputs, offering a clear overview of the prolfquapp toolset.

bioinformatics↗

bioRxiv2024

Tau load in select brainstem neurons predicts the severity and nature of balance deficits in the absence of cell death

Patients with tauopathies present with profoundly different clinical symptoms1, even within the same disorder2. A central hypothesis in the field, well-supported by biomarker studies3,4 and post-mortem pathology5-7, is that clinical heterogeneity reflects differential degeneration of vulnerable neuronal populations responsible for specific neurological functions. Recent work has revealed mechanisms underlying susceptibility of particular cell types8-10, but relating tau load to disrupted behavior -- es- pecially before cell death -- requires a targeted circuit-level approach. Here we studied two distinct balance behaviors in larval zebrafish11 expressing a human 0N/4R-tau allele12 in select populations of evolutionarily-conserved and well-characterized brainstem vestibular circuits13,14. We observed that human tau load predicted the severity of circuit-specific deficits in posture and navigation in the ab- sence of cell death. Targeting expression to either mid- or hindbrain balance neurons recapitulated these particular deficits in posture and navigation. By parametrically linking tau load in specific neu- rons to early behavioral deficits, our work moves beyond cell type to close the gap between pathological and neurological conceptions of tauopathy.

neuroscience↗

bioRxiv2024

Development of the Motor Periphery is the Rate-Limiting Step in the Ontogeny of the Vestibulo-ocular Reflex

Vertebrates stabilize gaze using a neural circuit that transforms sensed instability into compensatory counter-rotation of the eyes. Sensory feedback tunes this vestibulo-ocular reflex throughout life. Gaze stabilization matures progressively, either due to similar tuning, or to a slowly developing circuit component. Here we studied the functional development of vestibulo-ocular reflex circuit components in the larval zebrafish, with and without sensation. Blind fish stabilize gaze normally, and neural responses to body tilts mature before behavior. Instead, synapses between motor neurons and the eye muscles mature with a timecourse similar to behavioral maturation. Larvae without vestibular sensory experience, but whose neuromuscular junction was mature, had a strong vestibulo-ocular reflex. Development of the neuromuscular junction, and not sensory experience, determines the rate of maturation of an ancient behavior.

neuroscience↗

bioRxiv2024

Motor neurons are dispensable for the assembly of a sensorimotor circuit for gaze stabilization

Sensorimotor reflex circuits engage distinct neuronal subtypes, defined by precise connectivity, to transform sensation into compensatory behavior. Whether and how motor neuron populations specify the subtype fate and/or sensory connectivity of their pre-motor partners remains controversial. Here, we discovered that motor neurons are dispensable for proper connectivity in the vestibular reflex circuit that stabilizes gaze. We first measured activity following vestibular sensation in premotor projection neurons after constitutive loss of their extraocular motor neuron partners. We observed normal responses and topography indicative of unchanged functional connectivity between sensory neurons and projection neurons. Next, we show that projection neurons remain anatomically and molecularly poised to connect appropriately with their down-stream partners. Lastly, we show that the transcriptional signatures that typify projection neurons develop independently of motor partners. Our findings comprehensively overturn a long-standing model: that connectivity in the circuit for gaze stabilization is retrogradely determined by motor partner-derived signals. By defining the contribution of motor neurons to specification of an archetypal sensorimotor circuit, our work speaks to comparable processes in the spinal cord and advances our understanding of principles of neural development.

neuroscience↗

bioRxiv2023

Choline supplementation in early life improves and low levels of choline can impair outcomes in a mouse model of Alzheimer's disease.

Maternal choline supplementation (MCS) improves cognition in Alzheimers disease (AD) models. However, effects of MCS on neuronal hyperexcitability in AD are unknown. We investigated effects of MCS in a well-established mouse model of AD with hyperexcitability, the Tg2576 mouse. The most common type of hyperexcitability in Tg2576 mice are generalized EEG spikes (interictal spikes; IIS). IIS also are common in other mouse models and occur in AD patients. Im mouse models, hyperexcitability is also reflected by elevated expression of the transcription factor {Delta}FosB in the granule cells (GCs) of the dentate gyrus (DG), which are the principal cell type. Therefore we studied {Delta}FosB expression in GCs. We also studied the the neuronal marker NeuN within hilar neurons of the DG because other studies have reduced NeuN protein expression is a sign of oxidative stress or other pathology. This is potentially important because hilar neurons regulate GC excitability. Tg2576 breeding pairs received a diet with a relatively low, intermediate or high concentration of choline. After weaning, all mice received the intermediate diet. In offspring of mice fed the high choline diet, IIS frequency declined, GC {Delta}FosB expression was reduced, and NeuN expression was restored. Using the novel object location task, spatial memory improved. In contrast, offspring exposed to the relatively low choline diet had several adverse effects, such as increased mortality. They had the weakest hilar NeuN immunoreactivity and greatest GC {Delta}FosB protein expression. However, their IIS frequency was low, which was surprising. The results provide new evidence that a diet high in choline in early life can improve outcomes in a mouse model of AD, and relatively low choline can have mixed effects. This is the first study showing that dietary choline can regulate hyperexcitability, hilar neurons, {Delta}FosB and spatial memory in an animal model of AD.

neuroscience↗

bioRxiv2022

Type I interferons govern Zika virus resistance of human macrophages and microglia

The role of the human type I interferon (IFN-I) system in restricting Zika virus (ZIKV) is uncertain. Here, genetic and pharmacological ablation of IFN-I signalling enhanced ZIKV replication and cytopathicity in macrophages and microglia, key cells in ZIKV transmission and pathogenesis. Thus, despite the extensive IFN-I countermeasures employed by ZIKV, IFN-I dictates the outcome of infection in macrophages. Therapeutic manipulation of the IFN-I system may bring clinical benefit in ZIKV.

cell biology↗

bioRxiv2022

Enhanced excitability of the hippocampal CA2 region and its contribution to seizure generation in a mouse model of temporal lobe epilepsy

The hippocampal CA2 region, an area important for social memory, has been suspected to play a role in temporal lobe epilepsy (TLE) because of its resistance to the degeneration observed in neighboring CA1 and CA3 regions in both human and rodent models of TLE. However, little is known about whether alterations in CA2 properties serve to promote seizure generation or propagation. Here we have used the pilocarpine-induced status epilepticus (PILO-SE) model of TLE to explore the role of CA2. Ex vivo electrophysiological recordings from acute hippocampal slices revealed a set of coordinated changes that enhance CA2 intrinsic excitability, reduce CA2 local inhibitory input, and increase CA2 excitatory output to its major CA1 synaptic target. Moreover, selective silencing of CA2 pyramidal cells using a chemogenetic approach caused a significant decrease in the frequency of spontaneous seizures. These findings provide the first evidence that CA2 actively contributes to TLE seizure activity and may thus be a promising therapeutic target.

neuroscience↗

bioRxiv2021

Regulation of CHK1 inhibitor resistance by a c-Rel and USP1 dependent pathway

We previously discovered that deletion of c-Rel in the E-Myc mouse model of lymphoma results in earlier onset of disease, a finding that contrasted with the expected function of this NF-{kappa}B subunit in B-cell malignancies. Here we report that c-rel -/- E{micro}-Myc cells have an unexpected and major defect in the CHK1 pathway, with almost undetectable levels of CHK1 and CLSPN protein leading to therapeutic resistance to the highly specific CHK1 inhibitor (CHK1i) CCT244747. Similar downregulation of CHK1 levels was also seen in CCT244747 resistant U20S osteosarcoma cells. Further investigation revealed that downregulation of the deubiquitinase USP1 is responsible, at least in part, for these effects. Importantly, we demonstrate that c-rel -/- E{micro}-Myc lymphoma cells survive though upregulation of compensatory PI3K/AKT pathway activity. Moreover, targeting this pathway with Pictilisib (GDC-0941) effectively killed c-rel -/- E{micro}-Myc in vivo, while having no effect on wild type E-Myc cells. This data reveals an NF-{kappa}B regulated pathway controlling CHK1 activity in cancer cells and identifies a potential mechanism for both acquiring and overcoming CHK1i resistance in cancer patients.

cancer biology↗