Biology subjects

Wheeler, L.

Publications and source records attributed to Wheeler, L..

7 recordsLinked to original sources

bioRxiv2026

Spatiotemporal Proteome Remodeling Directs Human Hematopoietic Stem Cells via a Metabolic-Cytoskeletal Axis

Hematopoietic stem cells (HSCs) sustain the lifelong production of all blood and immune cells and are central to transplantation and gene therapies. However, the clinical use of these approaches remains limited due to the functional attrition of HSCs during ex vivo adaptation and expansion in culture. As these transitions are executed at the protein and structural level, the primary drivers of cellular adaptation have remained elusive in transcriptional studies. Here, we overcome the technical resolution limits of rare primary cells by integrating a scalable, ultra-low input proteomics pipeline with 3D super-resolution imaging to map the proteomic and spatial organization of primary human HSCs during ex vivo expansion. Our analysis reveals a previously unrecognized, metabolic-cytoskeletal axis that coordinates organelle reorganization and structural polarity. Perturbation of this axis preserves structural polarity while uncoupling it from biomass expansion, demonstrating that growth and functional deterioration are separable processes in ex vivo HSC culture. These findings establish a framework for understanding ex vivo HSC adaptation and expansion and inform the rational development of next-generation cellular therapies.

cell biology↗

bioRxiv2026

CD180 identifies chemoresistant stem-like blasts and reveals a KMT2A-driven vulnerability in acute myeloid leukaemia

Relapse and chemoresistance remain major challenges in paediatric acute myeloid leukaemia (PAML), particularly in KMT2A-rearranged (KMT2A-r) subtypes where conventional markers such as CD34 are often absent, complicating measurable residual disease (MRD) detection. Leukaemia stem/regenerating cells (LSC/LRC) drive disease initiation, progression, and relapse, sharing stemness and chemoresistance properties that make them critical therapeutic targets. Using high-dimensional spectral flow cytometry, we identified CD180, a Toll-like receptor-like surface protein, as highly expressed on blasts and stem-like populations in KMT2A-r AML, while near absent on normal haematopoietic stem cells (HSCs). PAML KMT2A-r exhibits an unconventional immunophenotype dominated by CD34-CD180 populations. Integrated single-cell transcriptomics and functional profiling revealed CD180high clusters enriched for quiescence, oxidative phosphorylation, and KMT2A/LSC stemness signatures. CD180 cells demonstrated robust leukaemia-initiating capacity in xenograft models and persisted through therapy, re-emerging at relapse with phenotypic plasticity. Epigenomic analysis showed CD180 is a direct transcriptional target of the KMT2A::MLLT3 fusion complex, regulated by intragenic enhancers and downregulated by menin and BET inhibitors. Longitudinal single-cell analysis confirmed persistence and clonal evolution of CD180 populations during treatment and relapse, underscoring their mechanistic role in chemoresistance and disease progression. In summary, CD180 marks dynamic, relapse-driving populations in KMT2A-r PAML, persists through therapy, and importantly is near absent on normal HSCs, offering a selective therapeutic window. These findings position CD180 as a clinically actionable biomarker for MRD detection and a compelling therapeutic target for eradicating chemoresistant, stem-like cells in paediatric AML. Main PointsO_LICD180 marks chemoresistant, relapse-driving stem-like blasts in KMT2A-r paediatric AML, overcoming CD34-based MRD limitations. C_LIO_LIAbsent on normal HSCs, CD180 is a KMT2A::MLLT3 target and actionable for MRD, relapse prediction, and CD180-directed therapies. C_LI NoveltyThis study introduces CD180 as a novel biomarker and therapeutic target in AML, particularly KMT2A-rearranged subtypes where conventional markers are often absent. Unlike MRD strategies focused on bulk blasts, CD180 marks chemoresistant, stem-like populations driving relapse, critical reservoirs poorly defined in paediatric AML. This work fills a major gap in prognostic assessment and therapy by enabling precise detection of relapse-driving cells and offering a selective therapeutic window.

cancer biology↗

bioRxiv2025

Constitutive Androstane Receptor induces Ribonucleotide Reductase-M2 expression and maintains hepatocyte ploidy in mice

The nuclear receptor Constitutive Androstane Receptor (CAR/NR1i3) is known for regulating various liver functions, including detoxification, nutrient metabolism, and hepatocyte proliferation. While CAR activation has been previously linked to higher ploidy, the underlying mechanisms are not fully known. Here, we uncover a basal role for CAR in maintaining hepatocyte ploidy, such that CAR deletion increases the number of diploid (2c) hepatocytes with a concomitant reduction in tetraploid (4c) hepatocytes. We demonstrate that CAR controls the de novo dNTP synthesis by directly transactivating the Ribonucleotide Reductase-M2 (RRM2) gene, which encodes the rate-limiting catalytic subunit of the enzyme, ribonucleotide reductase. Further, we find that the ligand-dependent CAR activation is sufficient to induce several genes involved in the de novo dNTP synthesis pathways, resulting in higher hepatic dATP and dTTP levels within the liver. Importantly, overexpressing RRM2 levels in CAR knockouts led to increased DNA synthesis and tetraploid (4c) hepatocytes compared to the control mice. Importantly, we demonstrate that CAR-mediated DNA synthesis in liver cells is dependent on catalytically active RRM2 function. Taken together, these findings reveal that the CAR-mediated RRM2 regulation contributes towards DNA synthesis and thereby maintains hepatocyte ploidy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/651109v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1750110org.highwire.dtl.DTLVardef@98f579org.highwire.dtl.DTLVardef@1c0e87org.highwire.dtl.DTLVardef@1fdfda5_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Interleukin-1 is overexpressed in injured muscles following spinal cord injury and promotes neurogenic heterotopic ossification

Neurogenic heterotopic ossifications (NHOs) form in periarticular muscles following severe spinal cord (SCI) and traumatic brain injuries. The pathogenesis of NHO is poorly understood with no effective preventive treatment. The only curative treatment remains surgical resection of pathological NHOs. In a mouse model of SCI-induced NHO that involves a transection of the spinal cord combined with a muscle injury, a differential gene expression analysis revealed that genes involved in inflammation such as interleukin-1{beta} (IL-1{beta}) were overexpressed in muscles developing NHO. Using mice knocked-out for the gene encoding IL-1 receptor (IL1R1) and neutralizing antibodies for IL-1 and IL-1{beta}, we show that IL-1 signaling contributes to NHO development following SCI in mice. Interestingly, other proteins involved in inflammation that were also overexpressed in muscles developing NHO, such as colony-stimulating factor-1, tumor necrosis factor or C-C chemokine ligand-2 did not promote NHO development. Finally using NHO biopsies from SCI and TBI patients, we show that IL-1{beta} is expressed by CD68+ macrophages. IL-1 and IL-1{beta} produced by activated human monocytes promote calcium mineralization of fibro-adipogenic progenitors isolated from muscles surrounding NHOs. Altogether these data suggest that interleukin-1 promotes NHO development in both humans and mice.

physiology↗

bioRxiv2020

Germline ERBB3 mutation in familial non-small cell lung carcinoma: expanding the role of the ErbB family in oncogenesis.

BackgroundLung cancer is the commonest cause of cancer deaths worldwide. Although strongly associated with smoking, predisposition to lung cancer is also heritable with multiple common risk variants identified. Rarely, dominantly inherited non-small-cell lung cancer (NSCLC) has been reported due to somatic mutations in EGFR/ErbB1 and ERBB2. MethodsGermline exome sequencing was performed in a multi-generation family with autosomal dominant NSCLC, including an affected child. Tumour samples were also sequenced. Full-length wild-type (wtErbB3) and mutant ERBB3 (mutErbB3) constructs were transfected into HeLa cells. Protein expression, stability, and sub-cellular localisation were assessed; and cellular proliferation, pAkt/Akt, and pERK levels were determined. ResultsA novel germline variant in ERBB3 (c.1946T>G: p.Iso649Arg), coding for receptor tyrosineprotein kinase erbB-3 (ErbB3), was identified, with appropriate segregation. There was no loss-of-heterozygosity in tumour samples. Both wtErbB3 and mutErbB3 were stably expressed. MutErbB3-transfected cells demonstrated an increased ratio of the 80kD form (which enhances proliferation) compared to the full-length (180kD) form. MutErbB3 and wtErbB3 had similar punctate cytoplasmic localisation pre- and post-EGF stimulation; however, EGFR levels decreased faster post-stimulation in mutErbB3-transfected cells, suggesting more rapid processing of the mutErbB3/EGFR heterodimer. Cellular proliferation was increased in mutErbB3-transfected cells compared to wtErbB3 transfection. MutErbB3-transfected cells also showed decreased pAkt/tAkt ratios and increased pERK/tERK 30 minutes post-stimulation compared to wtErbB3 transfection, demonstrating altered signalling pathway activation by mutErbB3. Cumulatively, these results support this mutation as tumorogenic. ConclusionsThis is the first reported family with a germline ERBB3 mutation causing heritable NSCLC, furthering understanding of the ErbB family pathway in oncogenesis.

genomics↗

bioRxiv2019

A rare SMAD9 mutation identifies the BMP signalling pathway as a potential osteoanabolic target

To identify targets for novel anabolic medicines for osteoporosis, we recruited a large cohort with unexplained high bone mass (HBM). Exome sequencing identified a rare (minor allele frequency 0.0014) missense mutation in SMAD9 (c.65T>C, p.Leu22Pro) segregating with HBM in an autosomal dominant family. The same mutation was identified in another two unrelated individuals with HBM. In-silico protein modelling predicts the mutation severely disrupts the MH1 DNA-binding domain of SMAD9. Affected individuals have bone mineral density [BMD] Z-Scores +3 to +5, with increased volumetric cortical and trabecular BMD, increased cortical thickness, and low/normal bone turnover. Fractures and nerve compressions are not seen. Both genome-wide, and gene-based association testing of heel estimated-BMD in >362,924 UK-Biobank British subjects showed strong associations with SMAD9 (PGWAS=6x10-16; PGENE =8x10-17). Smad9 is highly expressed in murine osteocytes and zebrafish bone tissue. Our findings support SMAD9 as a novel HBM gene, and a potential novel osteoanabolic target.

genetics↗

bioRxiv2018

Longitudinal expression profiling of CD4+ and CD8+ cells in patients with active to quiescent Giant Cell Arteritis

BackgroundGiant cell arteritis (GCA) is the most common form of vasculitis affecting elderly people. It is one of the few true ophthalmic emergencies. GCA is a heterogenous disease, symptoms and signs are variable thereby making it challenging to diagnose and often delaying diagnosis. A temporal artery biopsy is the gold standard to test for GCA, and there are currently no specific biochemical markers to categorize or aid diagnosis of the disease. We aimed to identify a less invasive method to confirm the diagnosis of GCA, as well as to ascertain clinically relevant predictive biomarkers by studying the transcriptome of purified peripheral CD4+ and CD8+ T lymphocytes in patients with GCA.\n\nMethods and FindingsWe recruited 16 patients with histological evidence of GCA at the Royal Victorian Eye and Ear Hospital (RVEEH), Melbourne, Australia, and aimed to collect blood samples at six time points: acute phase, 2-3 weeks, 6-8 weeks, 3 months, 6 months and 12 months after clinical diagnosis. CD4+ and CD8+ T-cells were positively selected at each time point through magnetic-assisted cell sorting (MACS). RNA was extracted from all 195 collected samples for subsequent RNA sequencing. The expression profiles of patients were compared to those of 16 age-matched controls. Over the 12-month study period, polynomial modelling analyses identified 179 and 4 statistically significant transcripts with altered expression profiles (FDR < 0.05) between cases and controls in CD4+ and CD8+ populations, respectively. In CD8+ cells, we identified two transcripts that remained differentially expressed after 12 months, namely SGTB, associated with neuronal apoptosis, and FCGR3A, which has been found in association with Takayasu arteritis (TA), another large vessel vasculitis. We detected genes that correlate with both symptoms and biochemical markers used in the acute setting for predicting long-term prognosis. 15 genes were shared across 3 phenotypes in CD4 and 16 across CD8 cells. In CD8, IL32 was common to 5 phenotypes: a history of Polymyalgia Rheumatica, both visual disturbance and raised neutrophils at the time of presentation, bilateral blindness and death within 12 months. Altered IL32 gene expression could provide risk evaluation of GCA diagnosis at the time of presentation and give an indication of prognosis, which may influence management.\n\nConclusionsThis is the first longitudinal gene expression study undertaken to identify robust transcriptomic biomarkers of GCA. Our results show cell type-specific transcript expression profiles, novel gene-phenotype associations, and uncover important biological pathways for this disease. These data significantly enhance the current knowledge of relevant biomarkers, their association with clinical prognostic markers, as well as potential candidates for detecting disease activity in whole blood samples. In the acute phase, the gene-phenotype relationships we have identified could provide insight to potential disease severity and as such guide us in initiating appropriate patient management.

genomics↗