Biology subjects

Thomas, J. R.

Publications and source records attributed to Thomas, J. R..

13 recordsLinked to original sources

bioRxiv2026

A JAK-STAT-activated myeloid hub at ulceration sites underlies resistance to anti-TNF therapy in Crohn's disease

Despite major advances in immunotherapies, durable disease control in immune-mediated inflammatory diseases (IMIDs) is frequently limited by resistance driven by maladaptive immune programs, resulting in progressive tissue damage. Myeloid cells are central effectors across IMIDs, including inflammatory bowel disease (IBD), yet how discrete myeloid states govern disease progression and therapeutic response remains incompletely defined. Here, using integrative single-cell transcriptomic and molecular analyses of inflamed human ileum in Crohns disease (CD), we identify combinatorial transcriptional programs that revealed previously unrecognized axes of monocyte-macrophage (mo-mac) heterogeneity. These myeloid states and programs were conserved and validated across multiple independent cohorts. We delineate a pathogenic mo-mac subset, interferon-associated inflammatory monocytes (IFIM), shaped by convergent NF-{kappa}B and IFN{gamma} signaling and enriched in advanced disease. Baseline abundance of IFIM programs predicted resistance to anti-TNF therapy and severe postoperative recurrence, with IFIM-derived ligands sustaining epithelial injury. IFIM exhibited robust JAK-STAT activation, indicating susceptibility to JAK inhibition with clinically approved agents, including upadacitinib. Exemplified by IFIM-driven inflammation, these findings identify pathogenic myeloid state remodeling as a molecular basis of anti-TNF resistance and provide a framework for mechanism-guided therapeutic strategies across chronic inflammatory diseases.

immunology↗

bioRxiv2025

Human chronic inflammation is orchestrated by spatially restricted inflammation-activated Dendritic cells

Dendritic cells (DCs) form coordinated networks that orchestrate inflammatory responses across tissues. Upon activation, conventional dendritic cells (DCs) undergo changes in their transcriptome, phenotype, and function, depending on the tissue microenvironment. The role of DC activation during human chronic inflammation has yet to be explored. To address this, we aimed to investigate activated DCs across a range of chronically inflamed human tissues, with a focus on cervical lymph nodes (LNs). We performed scRNAseq and flow cytometry on healthy, acutely, chronically inflamed, and cancer-associated human cervical (LNs) and identified a novel inflammation-associated DC (iaDC) cell state, found only within chronically inflamed LNs. iaDCs were defined by a unique cytokine expression profile (CXCL9, CXCL10, IL1B) and could be phenotypically distinguished from other activated DCs by their elevated expression of CD1C, CD206, CD319, and CD274. Functionally, we found that IFN{gamma} signaling induces DC2s to enter the iaDC cell state and that this process can be abrogated by inhibition of JAK-STAT signaling. Using spatial transcriptomics, we observed LN iaDC residing within a specific chronic inflammatory niche, which was enriched for inflammatory monocytes, NK cells, and effector memory CD4+ and CD8+ T cells. Extrapolating our findings to other chronic inflammation-associated diseases, we similarly observed the emergence of iaDCs within the intestines of treatment-resistant Crohns Disease patients, synovial membranes and lymph nodes of rheumatoid arthritis patients and lungs of sarcoidosis patients. Similarly to the lymph node, iaDCs were found to reside in conserved chronic inflammation-associated spatial niches within the small intestine of affected individuals and displayed equivalent transcriptional characteristics and surrounding cellular neighborhoods. Collectively, these data highlight a previously unexplored role of iaDCs in human chronic inflammatory diseases and propose a conserved spatially restricted iaDC-populated chronic inflammatory niche associated with resistance to therapy. These findings highlight novel avenues to shape inflammatory trajectories by targeting iaDCs and their spatial niches, for example, by inhibiting JAK-STAT signaling.

immunology↗

bioRxiv2024

Photoaffinity enabled transcriptome-wide identification of splice modulating small molecule-RNA binding events in native cells

Splice modulating small molecules have been developed to promote the U1 snRNP to engage with pre-mRNAs with strong and altered sequence preference. Transcriptomic profiling of bulk RNA from compound treated cells enables detection of RNAs impacted; however, it is difficult to delineate whether transcriptional changes are a consequence of direct compound treatment or trans-acting effects. To identify RNA targets that bind directly with splice modulating compounds, we deployed a photoaffinity labeling (PAL)-based Chem-CLIP approach. Through this workflow, we identify the telomerase lncRNA (TERC) as a previously unknown target of this class of clinically relevant small molecules. Using cellular {Delta} SHAPE-MaP, we orthogonally validate and further define the compound binding site as likely to be the conserved CR4/5 domain. Additionally, a thorough analysis of the PAL-based Chem-CLIP data reveals that considering competed RNAs, irrespective of magnitude of enrichment, adds a rich dimension of hit calling. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/623654v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1ef467eorg.highwire.dtl.DTLVardef@1fd201dorg.highwire.dtl.DTLVardef@1478f83org.highwire.dtl.DTLVardef@11ef59a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Identification of NanoLuciferase Substrates Transported by Human ABCB1 and ABCG2 and their Zebrafish Homologs at the Blood-Brain Barrier

ATP-binding cassette (ABC) transporters expressed at the blood-brain barrier (BBB) impede delivery of therapeutic agents to the brain, including agents to treat neurodegenerative diseases and primary and metastatic brain cancers. Two transporters, P-glycoprotein (P-gp, ABCB1) and ABCG2, are highly expressed at the BBB and are responsible for the efflux of numerous clinically useful chemotherapeutic agents, including irinotecan, paclitaxel, and doxorubicin. Based on a previous mouse model, we have generated transgenic zebrafish in which expression of NanoLuciferase (NanoLuc) is controlled by the promoter of glial fibrillary acidic protein, leading to expression in zebrafish glia. To identify agents that disrupt the BBB, including inhibitors of ABCB1 and ABCG2, we identified NanoLuc substrates that are also transported by P-gp, ABCG2, and their zebrafish homologs. These substrates will elevate the amount of bioluminescent light produced in the transgenic zebrafish with BBB disruption. We transfected HEK293 cells with NanoLuc and either human ABCB1, ABCG2, or their zebrafish homologs Abcb4 or Abcg2a, respectively, and expressed at the zebrafish BBB. We evaluated the luminescence of ten NanoLuc substrates, then screened the eight brightest to determine which are most efficiently effluxed by the ABC transporters. We identified one substrate efficiently pumped out by ABCB1, two by Abcb4, six by ABCG2, and four by Abcg2a. These data will aid in the development of a transgenic zebrafish model of the BBB to identify novel BBB disruptors and should prove useful in the development of other animal models that use NanoLuc as a reporter. Significance StatementThe ATP-Binding Cassette (ABC) transporters ABCB1 and ABCG2 at the blood-brain barrier (BBB) hinder pharmacological treatment of brain-related diseases. Consequently, there is a need for tools to identify BBB disruptors. We conducted a screen of ten NanoLuciferase substrates, identifying the brightest and those that were transported by human and zebrafish ABC transporters at the BBB. This work supports and complements our development of a transgenic zebrafish model, in which NanoLuciferase is expressed within glial cells, enabling detection of BBB disruption.

pharmacology and toxicology↗

bioRxiv2023

Abcg2a is the functional homolog of human ABCG2 expressed at the zebrafish blood-brain barrier

BackgroundA principal protective component of the mammalian blood-brain barrier (BBB) is the high expression of the multidrug efflux transporters P-glycoprotein (P-gp, encoded by ABCB1) and ABCG2 (encoded by ABCG2) on the lumenal surface of endothelial cells. The zebrafish P-gp homolog Abcb4 is expressed at the BBB and phenocopies human P-gp. Comparatively little is known about the four zebrafish homologs of the human ABCG2 gene: abcg2a, abcg2b, abcg2c, and abcg2d. Here we report the functional characterization and brain tissue distribution of zebrafish ABCG2 homologs. MethodsTo determine substrates of the transporters, we stably expressed each in HEK-293 cells and performed cytotoxicity and fluorescent efflux assays with known ABCG2 substrates. To assess the expression of transporter homologs, we used a combination of RNAscope in situ hybridization probes and immunohistochemistry to stain paraffin-embedded sections of adult and larval zebrafish. ResultsWe found Abcg2a had the greatest substrate overlap with ABCG2, and Abcg2d appeared to be the least functionally similar. We identified abcg2a as the only homolog expressed at the adult and larval zebrafish BBB, based on its localization to claudin-5 positive brain vasculature. ConclusionsThese results demonstrate the conserved function of zebrafish Abcg2a and suggest that zebrafish may be an appropriate model organism for the studying the role of ABCG2 at the BBB.

cell biology↗

bioRxiv2022

Autism-related behavioral phenotypes of three Camk2a mutant mouse lines with reduced CaMKIIalpha expression and/or activity

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a key modulator of excitatory synaptic transmission, gene expression, learning and memory. Mutations in the CAMK2A gene, which encodes CaMKII and is highly expressed in multiple regions in the forebrain, have been recently linked to neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability (ID). Our lab generated and characterized a knock-in (KI) mutant mouse with a glutamate-183 to valine (E183V) CaMKII mutation detected in several children diagnosed with ASD or ID. The E183V mutation reduces CaMKII activity and expression levels but the contributions of these two changes to the ASD-related behavioral phenotypes of these mice are unclear. Therefore, we performed side-by-side comparisons of the behavioral phenotypes of CaMKII E183V-KI mice with two other mutant mouse lines with either a complete loss of CaMKII expression (CaMKII Null mice) or reduced kinase activity (due to a threonine-286 to alanine mutation that abrogates autophosphorylation at this site) with no significant change in expression levels (CaMKII T286A-KI mice). In all three lines, homozygous mutant mice displayed increased stereotypic jumping behavior and hyperactivity, without alterations in anxiety or social interactions. Interestingly, homozygous mutant mice in all three lines also displayed a substantial reduction in tactile sensitivity using the Von Frey filament test. Together, these data suggest that reductions of either CaMKII expression or activity in mice disrupted normal motor and sensory functions.

neuroscience↗

bioRxiv2022

Human HLFneg placental erythro-myeloid progenitors give rise to HLA Class IIneg Hofbauer cells

The earliest macrophages are generated during embryonic development from erythro-myeloid progenitors (EMPs) via primitive haematopoiesis. This process is still poorly understood in humans but is generally thought to be spatially restricted to the yolk sac. Human fetal placental macrophages, Hofbauer cells (HBC), arise during the primitive haematopoietic wave, yet are unlikely to be yolk sac derived as they appear prior to placental vascularisation. Here we identify a population of placental erythro-myeloid progenitors (PEMPs) in the early human placenta that give rise to HBC. PEMP are fetal CD34+CD43+ progenitors found exclusively at early gestational timepoints. Transcriptomic analyses reveal that PEMP have a unique transcriptome with some conserved features of primitive yolk sac EMPs, including the lack of HLF expression. Using in vitro single-cell culture experiments we show that PEMP generate HBC-like cells which lack HLA-DR expression, a conserved feature of all fetal primitive macrophages in humans. These findings indicate that HBC are derived locally from PEMP and demonstrate that human primitive haematopoiesis is not restricted to the yolk sac, occurring also in the placenta.

immunology↗

bioRxiv2021

Chemoproteomics Enabled Discovery of Selective Probes for NuA4 Factor BRD8

Bromodomain-containing proteins frequently reside in multisubunit chromatin complexes with tissue or cell state-specific compositions. Recent studies have revealed tumor-specific dependencies on the BAF complex bromodomain subunit BRD9 that are a result of recurrent mutations afflicting the structure and composition of associated complex members. To enable the study of ligand engaged complex assemblies, we established a chemoproteomics approach using a functionalized derivative of the BRD9 ligand BI-9564 as an affinity matrix. Unexpectedly, in addition to known interactions with BRD9 and associated BAF complex proteins, we identify a previously unreported interaction with members of the NuA4 complex through the bromodomain-containing subunit BRD8. We apply this finding, alongside homology model guided design, to develop chemical biology approaches for the study of BRD8 inhibition, and to arrive at first-in-class selective and cellularly active probes for BRD8. These tools will empower further pharmacological studies of BRD9 and BRD8 within respective BAF and NuA4 complexes.

biochemistry↗

bioRxiv2021

Is Palmer's elm leaf goldenrod real? The Angiosperms353 kit provides within-species signal in Solidago ulmifolia s.l.

The genus Solidago represents a taxonomically challenging group due to its sheer number of species, putative hybridization, polyploidy, and shallow genetic divergence among species. Here we use a dataset obtained exclusively from herbarium specimens to evaluate the status of Solidago ulmifolia var. palmeri, a morphologically subtle taxon potentially confined to Alabama, Arkansas, Mississippi, and Missouri. A multivariate analysis of both discrete and continuous morphological data revealed no clear distinction between S. ulmifolia var. palmeri and Solidago ulmifolia var. ulmifolia. Solidago ulmifolia var. palmeris status was also assessed with a phylogenomic and SNP clustering analysis of data generated with the "Angiosperms353" probe kit. Neither analysis supported Solidago ulmifolia var. palmeri as a distinct taxon, and we suggest that this name should be discarded. The status of Solidago delicatula (formerly known as Solidago ulmifolia var. microphylla) was also assessed. Both morphological and phylogenic analyses supported the species status of S. delicatula and we suggest maintaining this species at its current rank. These results highlight the utility of the Angiosperms353 probe kit, both with herbarium tissue and at lower taxonomic levels. Indeed, this is the first study to utilize this kit to identify genetic groups within a species.

evolutionary biology↗

bioRxiv2018

Covalent Ligand Screening Uncovers a RNF4 E3 Ligase Recruiter for Targeted Protein Degradation Applications

Targeted protein degradation has arisen as a powerful strategy for drug discovery allowing the targeting of undruggable proteins for proteasomal degradation. This approach most often employs heterobifunctional degraders consisting of a protein-targeting ligand linked to an E3 ligase recruiter to ubiquitinate and mark proteins of interest for proteasomal degradation. One challenge with this approach, however, is that only few E3 ligase recruiters currently exist for targeted protein degradation applications, despite the hundreds of known E3 ligases in the human genome. Here, we utilized activity-based protein profiling (ABPP)-based covalent ligand screening approaches to identify cysteine-reactive small-molecules that react with the E3 ubiquitin ligase RNF4 and provide chemical starting points for the design of RNF4-based degraders. The hit covalent ligand from this screen reacted with either of two zinc-coordinating cysteines in the RING domain, C132 and C135, with no effect on RNF4 activity. We further optimized the potency of this hit and incorporated this potential RNF4 recruiter into a bifunctional degrader linked to JQ1, an inhibitor of the BET family of bromodomain proteins. We demonstrate that the resulting compound CCW 28-3 is capable of degrading BRD4 in a proteasome- and RNF4-dependent manner. In this study, we have shown the feasibility of using chemoproteomics-enabled covalent ligand screening platforms to expand the scope of E3 ligase recruiters that can be exploited for targeted protein degradation applications.

biochemistry↗

bioRxiv2018

Harnessing the Anti-Cancer Natural Product Nimbolide for Targeted Protein Degradation

Nimbolide, a terpenoid natural product derived from the Neem tree, impairs cancer pathogenicity across many types of human cancers; however, the direct targets and mechanisms by which nimbolide exerts its effects are poorly understood. Here, we used activity-based protein profiling (ABPP) chemoproteomic platforms to discover that nimbolide reacts with a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. Nimbolide impairs breast cancer cell proliferation by disrupting RNF114 substrate recognition, leading to inhibition of ubiquitination and degradation of the tumor-suppressor p21, resulting in its rapid stabilization. We further demonstrate that nimbolide can be harnessed to recruit RNF114 as an E3 ligase in targeted protein degradation applications and show that synthetically simpler scaffolds are also capable of accessing this unique reactive site. Our study highlights the utility of ABPP platforms in uncovering unique druggable modalities accessed by natural products for cancer therapy and drug discovery applications.

cancer biology↗

bioRxiv2018

Eps8 is a convergence point integrating EGFR and integrin trafficking and crosstalk

Crosstalk between adhesion and growth factor receptors plays a critical role in tissue morphogenesis and repair, and aberrations contribute substantially to neoplastic disease. However, the mechanisms by which adhesion and growth factor receptor signalling are integrated, spatially and temporally, are unclear.\n\nWe used adhesion complex enrichment coupled with quantitative proteomic analysis to identify rapid changes to adhesion complex composition and signalling following growth factor stimulation. Bioinformatic network and ontological analyses revealed a substantial decrease in the abundance of adhesion regulatory proteins and co-ordinators of endocytosis within 5 minutes of EGF stimulation. Together these data suggested a mechanism of EGF-induced receptor endocytosis and adhesion complex turnover.\n\nCombinatorial interrogation of the networks allowed a global and dynamic view of adhesion and growth factor receptor crosstalk to be assembled. By interrogating network topology we identified Eps8 as a putative node integrating 5{beta}1 integrin and EGFR functions. Importantly, EGF stimulation promoted internalisation of both 5{beta}1 and EGFR. However, perturbation of Eps8 increased constitutive internalisation of 5{beta}1 and EGFR; suggesting that Eps8 constrains 5{beta}1 and EGFR endocytosis in the absence of EGF stimulation. Consistent with this, Eps8 regulated Rab5 activity and was required for maintenance of adhesion complex organisation and for EGF-dependent adhesion complex disassembly. Thus, by co-ordinating 5{beta}1 and EGFR trafficking mechanisms, Eps8 is able to control adhesion receptor and growth factor receptor bioavailability and cellular contractility.\n\nWe propose that during tissue morphogenesis and repair, Eps8 functions to spatially and temporally constrain endocytosis, and engagement, of 5{beta}1 and EGFR in order to precisely co-ordinate adhesion disassembly, cytoskeletal dynamics and cell migration.

cell biology↗

bioRxiv2018

Integrin αVβ6-EGFR crosstalk regulates bidirectional force transmission and controls breast cancer invasion

The mechanical properties of the extracellular matrix within tumours control multiple cellular functions that drive cancer invasion and metastasis. However, the mechanisms controlling microenvironmental force sensation and transmission, and how these regulate transcriptional reprogramming and invasion, are unclear. Our aim was to understand how mechanical inputs are transmitted bidirectionally and translated into biochemical and transcriptional outputs to drive breast cancer progression. We reveal that adhesion receptor and growth factor receptor crosstalk regulates a bidirectional feedback mechanism co-ordinating force-dependent transcriptional regulation and invasion.\n\nIntegrin V{beta}6 drives invasion in a range of carcinomas and is a potential therapeutic target. V{beta}6 exhibits unique biophysical properties that promote force-generation and increase matrix rigidity. We employed an inter-disciplinary approach incorporating proteomics, biophysical techniques and multi-modal live-cell imaging to dissect the role of V{beta}6-EGFR crosstalk on transmission of mechanical signals bidirectionally between the extracellular matrix and nucleus.\n\nWe show that V{beta}6 expression correlates with poor prognosis in triple-negative breast cancer (TNBC) and drives invasion of TNBC cells. Moreover, our data show that a complex regulatory mechanism exists involving crosstalk between V{beta}6 integrin and EGFR that impacts matrix stiffness, force transmission to the nucleus, transcriptional reprogramming and microenvironment rigidity. V{beta}6 engagement triggers EGFR & MAPK signalling and V{beta}6-EGFR crosstalk regulates mutual receptor trafficking mechanisms. Consequently, EGF stimulation suppresses V{beta}6-mediated force-application on the matrix and nuclear shuttling of force-dependent transcriptional co-activators YAP/TAZ. Finally, we show that crosstalk between V{beta}6 & EGFR regulates TNBC invasion.\n\nWe propose a model whereby V{beta}6-EGFR crosstalk regulates matrix stiffening, but also the transmission of extracellular forces into the cell in order to co-ordinate transcriptional reprogramming and invasion. To exploit adhesion receptors and receptor tyrosine kinases therapeutically, it will be essential to understand the integration of their signalling functions and how crosstalk mechanisms influence invasion and the response of tumours to molecular therapeutics.

cancer biology↗