AbstractB-cell Receptor (BCR) signalling plays a critical role in the progression of Chronic Lymphocytic Leukaemia (CLL) and BCR-targeted kinase inhibitors, such as ibrutinib and idelalisib, have recently revolutionised its treatment. Despite these advances, CLL remains an incurable malignancy and the identification of novel molecular targets is paramount. This thesis investigates Toll-Like Receptor 9 (TLR9), a pattern recognition receptor of the innate immune system, which becomes activated in response to unmethylated CpG DNA. Importantly, our team have shown significantly increased levels of unmethylated CpG DNA motifs within the plasma of CLL patients relative to healthy individuals. We hypothesise that this arises from CLL cell apoptosis and the subsequent exposure of unmethylated mitochondrial self-DNA could be fuelling CLL progression through an auto-stimulatory feedback loop.
CLL cells traffic from the peripheral blood to the secondary lymphoid tissues where they become activated and proliferate, and CLL cell migration is therefore vital for survival and progression. Data presented in this thesis demonstrates that activation of primary CLL cells through TLR9 (using the agonist ODN 2006) induces a more activated and migratory phenotype including the upregulation of cell surface CD69, CD49d and CD38. Furthermore, the expression of intracellular TLR9 is higher in migrated vs non-migrated CLL cells. Interestingly, despite these uniform phenotypic changes, ODN 2006 stimulated an increase in CLL cell migration in only a subset of ‘Responder’ patient samples. These migratory responses to ODN 2006 were inhibited by the use of a TLR9 antagonist (ODN INH-18), showing these responses to be TLR9-dependent.
CLL patients can be divided into two main prognostic subgroups according to the mutational status of the BCR immunoglobulin gene (IGHV) and previous studies have reported patients with unmutated IGHV genes (U-CLL) to have a poorer clinical outcome due to a heightened propensity for BCR signalling relative to patients with mutated IGHV genes (M-CLL). ODN 2006 stimulated a significant increase in CLL cell migration in the M-CLL but not U-CLL subgroup. Many U-CLL patient samples were non-responsive, and a subset even showed a decrease in CLL cell migration following TLR9 agonism. Preliminary data suggests TLR9 ‘Responder’ and ‘Non/Reverse Responder’ samples to have different NF-κB activation signatures in response to ODN 2006.
In view of these results, the potential rationale for the therapeutic inhibition of both the BCR and TLR9 was investigated. In Responder samples, the simultaneous inhibition of BCR and TLR9 signalling (with ODN INH-18 and ibrutinib) showed a
synergistic effect on the reduction of ODN 2006-induced CLL cell migration and NFκB activation. Additionally, ibrutinib exposure appeared to increase the sensitivity of U-CLL Non/Reverse Responder samples to stimulation with ODN 2006. This indicates that TLR9 signalling could mediate ibrutinib resistance in both M-CLL and U-CLL patients. Taken together, these data highlight a role for TLR9 in CLL cell activation and migration and suggest that dual targeting of the BCR and TLR9 may be a promising therapeutic strategy for CLL patients.
|Date of Award||Jul 2021|
|Supervisor||Andrea Pepper (Supervisor) & Chris Pepper (Supervisor)|