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.