Wedge, S. R. | bioRxiv Science

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↗

bioRxiv2023

Synthetic STING agonists elicit powerful vaccine adjuvancy providing robust central memory and anti-tumour effects

Drugs that target the innate immune sensor STING are known to be effective in modulating the immune infiltrate of the tumour microenvironment. STING agonists have potential to enhance responses to checkpoint inhibitor therapy, however, their ability to influence and shape adaptive immune responses is poorly understood. Here, we investigated the impact of a range of synthetic STING agonists on antigen specific CD8+ T-cell responses to soluble antigen using the murine OT-1 adoptive transfer model with Ovalbumin as the antigen to monitor T cell responses. Our data demonstrate that synthetic STING agonists are able to stimulate antigen specific T-cell expansion in response to challenge in mice. This effect required expression of STING, an intact myeloid compartment and Type-I IFN and TNF signalling. Expanded T-cells post treatment differed from those induced by the established immune adjuvant, anti-CD40 antibody through lower induction of the immune checkpoint receptor PD-1. Furthermore, our data revealed a marked increase in the induction and persistence of CD8+ central memory cells after STING agonist and antigen challenge. Finally, we demonstrate that following rechallenge, STING agonism produced larger secondary responses that could be translated into enhanced tumour protection and survival. Therefore, synthetic STING agonists are capable of acting as potent immune adjuvants and can induce robust memory formation leading to better recall and tumour control. Critically, these benefits along with the lower expression of PD-1, have implications for their use as adjuvants for multiple immunotherapy and vaccine applications.

immunology↗