Biology subjects

Doody, G.

Publications and source records attributed to Doody, G..

3 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↗

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↗