Abstract
Diffuse Large B-cell Lymphoma (DLBCL) represents a clinically heterogeneous malignancy in which apoptotic regulation is profoundly altered by both cell-intrinsic mutations and microenvironmental stimuli. Among the central regulators of this process is the NF-κB signalling pathway, which regulates anti-apoptotic BCL2-family proteins, including BCL2, BCLXL, and MCL1. Aberrant NF-κB activity has been strongly implicated in therapy resistance, particularly under the influence of tumour microenvironment (TME)-derived signals. However, due to the regulatory complexity of this pathway and the functional redundancy among BCL2-family members, therapeutic targeting remains challenging.In this thesis, NF-κB activity and survival rewiring in DLBCL were systematically investigated through the integration of molecular profiling, computational modelling, and experimental perturbation. Flow cytometry, immunofluorescence, and in vivo studies were employed to characterise baseline NF-κB and BCL2-family protein abundance across multiple DLBCL cell lines. These datasets were used to inform a mechanistic, Ordinary Differential Equation- (ODE)-based, NF-κB-BCL2 model, which incorporated cooperative functional regulation to enable cell line and microenvironment-specific simulations. Substantial heterogeneity in NF-κB activity and anti-apoptotic protein abundances and dependencies were identified, both between and within cell lines. In U2932, for instance, clonal sub-populations displayed differential MCL1 abundance, resulting in divergent responses to venetoclax and highlighting the functional consequences of intratumoral heterogeneity. To mimic microenvironmental signalling, co-culture with CD40L-expressing fibroblasts was used to induce NF-κB pathway activation. This led to the acquisition of drug resistance through distinct transcriptional programmes: RelB-driven BCLXL upregulation and Venetoclax resistance in RIVA and U2932, and cRel-driven MCL1 upregulation in SUDHL8. The latter was found to depend on high basal RelA activity, establishing a mechanism of canonical-non-canonical NF-κB crosstalk. Model simulations generated mediators of apoptosis combinatorial Scores (MAC Score) to quantify these shifts and identify re-sensitisation strategies.
Co-inhibition of BCL2-family proteins was shown to reverse resistance in vitro, guided by model predictions. In parallel, upstream inhibition of NF-κB signalling was evaluated as a broader strategy to disrupt survival rewiring. BTK inhibition in SUDHL8 prevented RelA-driven p100 accumulation and suppressed cRel-mediated MCL1 induction. In contrast, inhibition of non-canonical signalling through Amgen16 reduced both p100 processing and IκBδ degradation, thereby limiting nuclear RelB and cRel activity. This intervention re-sensitised all three cell lines to BH3 mimetics, as demonstrated through Annexin-V staining, western blotting, and flow cytometric assessment of BCL2-family protein levels.
Together, these findings demonstrate that BH3 mimetic resistance in DLBCL is not fixed but dynamically regulated by upstream NF-κB activity. By combining quantitative profiling with computational modelling and functional perturbation, this thesis establishes a mechanistic framework for identifying context-specific vulnerabilities in NF-κB-driven lymphomas. Moreover, it provides proof-of-concept that survival rewiring can be selectively disrupted either through co-targeting of BCL2-family proteins or through upstream inhibition of canonical and non-canonical NF-κB signalling, offering a rationale for precision-guided BH3 mimetic strategies in stratified DLBCL.
Thesis embargoed. Available: 22/01/31
| Date of Award | Jan 2026 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Simon Mitchell (Supervisor), Chris Pepper (Supervisor) & Andrea Pepper (Supervisor) |