Biology subjects

O'Brien, J. J.

Publications and source records attributed to O'Brien, J. J..

6 recordsLinked to original sources

bioRxiv2026

DCN1 inhibitor induces fetal hemoglobin through self-limited regulation of CUL3 neddylation

Few genetic loci are as well-characterized as the globin gene locus, and the substitution of healthy {gamma}-globin (HbF) for missing or mutated {beta}-globin (HbB) is an established therapeutic strategy for {beta}-hemoglobinopathies including sickle cell disease (SCD) and {beta}-thalassemia. Although substantial progress has been made in understanding HbF derepression and globin switching, many current therapeutic strategies involving small molecules increase HbF through broad epigenetic perturbations or cytotoxic stress, raising concerns about dose-limiting cytopenias and off-target effects. By coupling single-cell transcriptomics, genetic perturbations, and functional genomics, we identified an unknown role of neddylation in the regulation of fetal hemoglobin (HbF). Partial impairment of neddylation of cullin ubiquitin ligase 3 (CUL3) through defective in cullin neddylation 1 (DCN1) inhibition leads to highly selective chromatin changes, histone demethylation, and globin locus binding of known activators of HbF transcription. Further, DCN1 inhibition drives globin switching and HbF increases in vitro and in vivo with minimal off-target transcriptional effects and no evidence of cytotoxicity or stress erythropoiesis. To therapeutically target this axis, we report the discovery and characterization of CLY-124, a first-in-class, covalent DCN1 inhibitor with favorable pharmacology properties. In a humanized mouse model, CLY-124 showed a dose-dependent increase in HbF as monotherapy and in synergy with hydroxyurea (HU), a current standard of care. Collectively, these findings highlight the power of single-cell transcriptomics to elucidate undiscovered biologic insights with therapeutic potential, and the promise of DCN-1 inhibitors like CLY-124 to address {beta}-hemoglobinopathies. With an appropriate nonclinical safety profile, a first-in human study of safety, pharmacokinetics and HbF assessments in healthy volunteers and participants with SCD is ongoing for CLY-124. One-sentence SummaryDCN1 is a promising target for {beta}-hemoglobinopathies

molecular biology↗

bioRxiv2024

Automated preparation of plasma lipids, metabolites, and proteins for LC/MS-based analysis of a high-fat diet in mice

Blood plasma is one of the most commonly analyzed and easily accessible biological samples. Here, we describe an automated liquid-liquid extraction (LLE) platform that generates accurate, precise, and reproducible samples for metabolomic, lipidomic, and proteomic analyses from a single aliquot of plasma while minimizing hands-on time and avoiding contamination from plasticware. We applied mass spectrometry to examine the metabolome, lipidome, and proteome of 90 plasma samples to determine the effects of age, time of day, and a high-fat diet in mice. From 25 L of mouse plasma, we identified 907 lipid species from 16 different lipid classes and subclasses, 233 polar metabolites, and 344 proteins. We found that the high-fat diet induced only mild changes in the polar metabolome, upregulated Apolipoproteins, and induced substantial shifts in the lipidome, including a significant increase in arachidonic acid (AA) and a decrease in eicosapentaenoic acid (EPA) content across all lipid classes.

physiology↗

bioRxiv2019

Coupling Terrestrial Laser Scanning with 3D Fuel Biomass Sampling for Advancing Wildland Fuels Characterization

The spatial pattern of surface fuelbeds in fire-dependent ecosystems are rarely captured using long-standing fuel sampling methods. New techniques, both field sampling and remote sensing, that capture vegetation fuel type, biomass, and volume at super fine-scales (cm to dm) in three-dimensions (3D) are critical to advancing forest fuel and wildland fire science. This is particularly true for computational fluid dynamics fire behavior models that operate in 3D and have implications for wildland fire operations and fire effects research. This study describes the coupling of new 3D field sampling data with terrestrial laser scanning (TLS) data to infer fine-scale fuel mass in 3D. We found that there are strong relationships between fine-scale mass and TLS occupied volume, porosity, and surface area, which were used to develop fine-scale prediction equations using TLS across vegetative fuel types, namely grasses and shrubs. The application of this novel 3D sampling technique to high resolution TLS data in this study represents a major advancement in understanding fire-vegetation feedbacks in highly managed fire-dependent ecosystems.

ecology↗

bioRxiv2019

Simulating groundcover community assembly in a frequently burned ecosystem using a simple neutral model

Fire is a global process that drives patterns of biodiversity. In frequently burned fire-dependent ecosystems, surface fire regimes allow for the coexistence of high plant diversity at fine-scales even where soils are uniform. The mechanisms on how fire impacts groundcover community dynamics are however, poorly understood. Because fire can act as a stochastic agent of mortality, we hypothesized that a neutral mechanism might be responsible for maintaining plant diversity. We used the demographic parameters of the Unified Neutral Theory of Biodiversity (UNTB) as a foundation to model groundcover species richness, using a southeastern U.S. pine woodland as an example. We followed the fate of over 7,000 individuals of 123 plant species for four years and two prescribed burns in frequently burned Pinus palustris sites in NW FL, USA. Using these empirical data and UNTB-based assumptions, we developed two parsimonious autonomous agent models, which were distinct by spatially explicit and implicit local recruitment processes. Using a parameter sensitivity test, we examined how empirical estimates, input species frequency distributions, and community size affected output species richness. We found that dispersal limitation was the most influential parameter, followed by mortality and birth, and that these parameters varied based on scale of the frequency distributions. Overall, these nominal parameters were useful for simulating fine-scale groundcover communities, although further empirical analysis of richness patterns, particularly related to fine-scale burn severity is needed. This modeling framework can be utilized to examine our premise that localized groundcover assemblages are neutral communities at high fire frequencies, as well as examine the extent to which niche-based dynamics determine community dynamics when fire frequency is altered and spatial fire intensity patterns differ.

ecology↗

bioRxiv2019

Systematic identification of cancer cell vulnerabilities to natural killer cell-mediated immune surveillance

Only a subset of cancer patients respond to T-cell checkpoint inhibitors, highlighting the need for alternative immunotherapeutics. We performed CRISPR-Cas9 screens in a leukemia cell line to identify perturbations that enhance natural killer effector functions. Our screens defined critical components of the tumor-immune synapse and highlighted the importance of cancer cell interferon-{gamma} signaling in modulating NK activity. Surprisingly, disrupting the ubiquitin ligase substrate adaptor DCAF15 strongly sensitized cancer cells to NK-mediated clearance. DCAF15 disruption induced an inflamed state in leukemic cells, including increased expression of lymphocyte costimulatory molecules. Proteomic and biochemical analysis revealed that cohesin complex members were endogenous client substrates of DCAF15. Genetic disruption of DCAF15 was phenocopied by treatment with indisulam, an anticancer drug that functions through DCAF15 engagement. In AML patients, reduced DCAF15 expression was associated with improved survival. These findings suggest that DCAF15 inhibition may have useful immunomodulatory properties in the treatment of myeloid neoplasms.

cancer biology↗

bioRxiv2018

eIF2B activator prevents neurological defects caused by a chronic Integrated Stress Response

The Integrated Stress Response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a novel eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complex in vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of other disease contexts.

neuroscience↗