Biology subjects

Tooze, R.

Publications and source records attributed to Tooze, R..

6 recordsLinked to original sources

bioRxiv2025

Structural, biophysical and biological analysis and characterisation of IRF4 DNA-binding domain mutations associated with multiple myeloma

IRF4, a transcription factor in the interferon regulatory factor family, is a key regulator in immune cell differentiation indicated to have an essential role in the development of lymphoid malignancies. Genome-wide association studies previously identified a set of overlapping mutations within the IRF4 DNA-binding domain in T-cell lymphoma and multiple myeloma, several of which appeared to be associated with better prognosis. Mapping these mutations to the known crystal structure of the IRF4:PU.1:DNA ternary complex and a new structure of the IRF4 DNA-binding domain in the apo state suggested they might interfere with DNA-binding, directly or via destabilisation of domain structure. We characterised these cancer-associated IRF4 mutants experimentally using the recombinant IRF4 DNA-binding domain (DBD) in vitro and examined the clinically relevant mutant K123R in cellulo. Using fluorescence polarisation, surface plasmon resonance, differential scanning fluorimetry and molecular dynamics, we find that mutation may give rise to significant differences in DNA-binding kinetics and thermal stability without compromising the affinity of IRF4 DNA-binding. The K123R IRF4 mutant showed increased transcriptional activity via a luciferase reporter assay and increased nuclear partitioning, which may be preferentially selected for in multiple myeloma. We discuss our observations in relation to the improved prognosis conferred by this mutation.

biophysics↗

bioRxiv2024

Enforced MYC expression selectively redirects transcriptional programs during human plasma cell differentiation

MYC provides a rheostat linking cell growth and division during plasma cell (PC) differentiation. Precise control of MYC is central to the network controlling differentiation. Deregulation of MYC drives transformation in aggressive B-cell neoplasms and is often accompanied by apoptotic protection conferred by BCL2. We assess how MYC and BCL2 deregulation impacts on the ability of human B-cells to complete PC differentiation. Under permissive conditions for PC differentiation we find such deregulation does not transform cells. While driving loss of normal PC surface phenotype, MYC deregulation has little impact on components of regulatory circuitry controlling B-cell identity. This contrasts with profound impact on initiation of secretory output and secretory reprogramming, coupled to dampening of XBP1 and immunoglobulin gene enhancement and a shift toward distinct metabolic programs. The establishment of this aberrant state depends on MYC homology boxes (MB0 and MBII). Dependence on MBII is profound and resolves to residue W135.

cell biology↗

bioRxiv2020

Co-activation of NF-κB and MYC renders cancer cells addicted to IL6 for survival and phenotypic stability

NF-{kappa}B and MYC are found co-deregulated in human B and plasma-cell cancers. In physiology, NF-{kappa}B is necessary for terminal B-to-plasma cell differentiation, whereas MYC repression is required. It is thus unclear if NF-{kappa}B/MYC co-deregulation is developmentally compatible in carcinogenesis and/or impacts cancer cell differentiation state, possibly uncovering unique sensitivities. Using a mouse system to trace cell lineage and oncogene activation we found that NF-{kappa}B/MYC co-deregulation originated cancers with a plasmablast-like phenotype, alike human plasmablastic-lymphoma and was linked to t(8;14)[MYC-IGH] multiple myeloma. Notably, in contrast to NF-{kappa}B or MYC activation alone, co-deregulation rendered cells addicted to IL6 for survival and phenotypic stability. We propose that conflicting oncogene-driven differentiation pressures can be accommodated at a cost in poorly-differentiated cancers. SignificanceOur studies improve the understanding of cancer pathogenesis by demonstrating that co-deregulation of NF-{kappa}B and MYC synergize in forming a cancer with a poorly-differentiated state. The cancers in the mouse system share features with human Plasmablastic lymphoma that has a dismal prognosis and no standard of care, and with t(8;14)[MYC-IGH] Multiple myeloma, which is in overall resistant to standard therapy. Notably, we found that NF-{kappa}B and MYC co-deregulation uniquely render cells sensitive to IL6 deprivation, providing a road-map for patient selection. Because of the similarity of the cancers arising in the compound mutant mouse model with that of human Plasmablastic lymphoma and t(8;14)[MYC-IGH] Multiple myeloma, this model could serve in preclinical testing to investigate novel therapies for these hard-to-treat diseases. HighlightsO_LINF-{kappa}B and MYC co-activation originates (pre)plasmablast-like cancer C_LIO_LINF-{kappa}B/MYC+ renders cancer cells addicted to IL6 for survival and phenotypic stability C_LIO_LINF-{kappa}B/MYC+ cancers are alike a fraction of human plasmablastic lymphoma C_LIO_LIt(8;14)[MYC-IGH] multiple myeloma is linked to a NF-{kappa}B/MYC co-activation signature C_LI

cancer biology↗

bioRxiv2019

A dichotomy in association of core transcription factors and gene regulation during the activated B-cell to plasmablast transition.

The activated B-cell (ABC) to plasmablast transition is the cusp of antibody secreting cell (ASC) differentiation but is incompletely defined. We apply expression time-courses, parsimonious gene correlation network analysis, and ChIP-seq to explore this in human cells. The transition initiates with input signal loss leading within hours from cell growth dominant programs to enhanced proliferation, accompanied from 24h by ER-stress response, secretory optimization and upregulation of ASC features. Clustering of genomic occupancy for ASC transcription factors (TFs) IRF4, BLIMP1 and XBP1 with CTCF and histone marks defines distinct patterns for each factor in plasmablasts. Integrating TF-associated clusters and modular gene expression identifies a dichotomy: XBP1 and IRF4 significantly link to gene modules induced in plasmablasts, but not to modules of repressed genes, while BLIMP1 links to modules of ABC genes repressed in plasmablasts but is not significantly associated with modules induced in plasmablasts. Pharmacological inhibition of the G9A (EHMT2) histone-methytransferase, a BLIMP1 co-factor that catalyzes repressive H3K9me2 marks, leaves functional ASC differentiation intact but de-represses ABC-state genes. Thus, in human plasmablasts IRF4 and XBP1 emerge as the dominant association with ASC gene expression, while BLIMP1 links to repressed modules with particular focus in repression of the B-cell activation state.

immunology↗

bioRxiv2019

B cell tetherin: a flow-cytometric cell-specific assay for response to Type-I interferon predicts clinical features and flares in SLE

ObjectiveType I interferon (IFN-I) responses are broadly associated with autoimmune disease including SLE. Given the cardinal role of autoantibodies in SLE, we investigated whether a B lineage cell-specific IFN assay might correlate with SLE activity. MethodsB cells and PBMCs were stimulated with IFN-I and IFN-II. Gene expression was scrutinised for pathway-related membrane protein expression. A flow-cytometric assay for tetherin (CD317), an IFN-induced protein ubiquitously expressed on leucocytes, was validated in vitro then clinically against SLE diagnosis, plasmablast expansion, and BILAG-2004 score in a discovery cohort (156 SLE; 30 RA; 22 healthy controls). A second longitudinal validation cohort of 80 patients was also evaluated for SLE flare prediction. ResultsIn vitro, a close cell-specific and dose-responsive relationship between IFN-I responsive genes and cell surface tetherin in all immune subsets existed. Tetherin expression was selectively responsive to the IFN-I compared to IFN-II and -III. In the discovery cohort memory B-cell tetherin was best associated with diagnosis (SLE/HC: effect size=0.11, p=0.003;SLE/RA: effect size=0.17, p<0.001); plasmablast numbers in rituximab-treated patients (Rho=0.38, p=0.047) and BILAG-2004. Association were equivalent or stronger than interferon score or monocyte tetherin. The validation cohort confirmed this relationship with memory B-cell tetherin predictive of future clinical flares (Hazard Ratio=2.29 (1.01-4.64), p=0.022). ConclusionMemory B cell surface tetherin, a reflection of cell-specific IFN response in a convenient flow cytometric assay, was associated with SLE diagnosis, disease activity and predicted flares better than other cell subsets or whole blood assays in independent validation cohorts.

immunology↗

bioRxiv2018

Growth-factor like gene regulation is separable from survival and maturation in antibody secreting cells

Recurrent mutational activation of the MAP kinase pathway in plasma cell myeloma implicates growth factor-like signaling responses in the biology of antibody secreting cells (ASCs). Physiological ASCs survive in niche microenvironments, but how niche signals are propagated and integrated is poorly understood. Here we dissect such a response in human ASCs using an in vitro model. Applying time course expression data and parsimonious gene correlation networking analysis (PGCNA), we map expression changes that occur during the maturation of proliferating plasmablast to quiescent plasma cell under survival conditions including the potential niche signal TGFB3. This analysis demonstrates a convergent pattern of differentiation, linking UPR/ER stress to secretory optimization, co-ordinated with cell cycle exit. TGFB3 supports ASC survival while having a limited effect on gene expression including up-regulation of CXCR4. This is associated with a significant shift in response to SDF1 in ASCs with amplified ERK1/2 activation, growth factor-like immediate early gene regulation and EGR1 protein expression. Similarly, ASCs responding to survival conditions initially induce partially overlapping sets of immediate early genes, without sustaining the response. Thus, in human ASCs growth factor-like gene regulation is transiently imposed by niche signals but is not sustained during subsequent survival and maturation.

immunology↗