Biophysical and Functional Characterisation of IRF4 DNA Binding Domain Mutations Associated with Lymphoid Malignancies

Abstract

Interferon Regulatory Factor 4 (IRF4) is a transcription factor that is uniquely restricted in its expression to lymphocytes. IRF4 gene regulation is documented to play a critical role in many lymphocyte subsets, including in the development and terminal differentiation of B and T cells. Mutations in this transcription factor have been associated with a slew of haematological malignancies including B cell-related chronic lymphocytic leukemia and adult T cell leukemia. Interestingly, a large proportion of disease-causing mutations appear to localise in the DNA binding domain (DBD) of IRF4. This suggests the observed disease outcomes may stem from altered interactions of IRF4 with regulatory DNA sequences. This thesis used a two-prong approach, combining immunophenotyping mouse models and biophysical characterisations to investigate the impacts of three select IRF4 DBD mutations (K59R T95R and L116R). For these mutations, CRISPR generated mouse models were generated and immunophenotyping of major lymphoid organs (spleen, lymph nodes and peritoneal cavity) was conducted. With the primary focus on naive B cells populations, we observed unique effects for each mutation with regards to B1 cell and marginal zone populations. To further understand these in vivo effects, biophysical characterisation of the mutants was conducted. Interestingly, differential scanning fluorimetry and surface plasmon resonance studies found all mutations were impacted similarly compared to wild type. Specifically, mutant DBD's were observed to be less stable in apo forms but increased affinity for known IRF4 DNA binding motifs. Using X-ray crystallography, we were able to investigate the structural basis of these interactions. In the process, we crystallised IRF4WT and IRF4K59R DBDs with a canonical IRF DNA motif which both found to form homodimers. This allowed us to draw comparisons and investigate the structural basis for the observed increase in DNA binding affinities in the mutants. Comparing the interactions of the IRF4WT DBD with that of IRF4K59R, it is clear that not only do these mutations result in localised structural alterations involving the mutated residue, but they also cause global conformational changes to the interacting DNA-protein complex. From these findings, we concluded that, on a molecular level, each IRF4 mutation acts in a gain of function manner. As such, we attempted to design a peptide that would disrupt the increased DNA affinity. IRF4 is known to be critical for the differentiation of plasma cells and the survival of multiple myeloma cells. In order to test the efficacy of this peptide, in vitro trials on plasma cell differentiation and multiple myeloma cell lines were conducted. Although the peptide effectively blocked plasma cell differentiation, it was less successful at inhibiting multiple myeloma cell survival. From these studies, it was clear that although our peptide has potential as a lead compound, more work is needed to improve its efficacy and bioavailability. To conclude, IRF4 is a transcription factor that is involved in many critical immunological pathways however, the wide presentation of IRF4 mutations is still poorly understood. This thesis, primarily focused on naive B cell immunology, highlights the complex nature in which select IRF4 mutations that act very similarly at a molecular level have vastly differing in vivo presentations. Show more