Authors
Iona Ashworth, Emma Kennedy, Lauren Stott, John Jones, Rosalynd Johnston, Simon P. Mackay, Silvia Deaglio, Tiziana Vaisitti, Fabio Simoes, Simon Mitchell, Andrea G.S. Pepper and Chris Pepper.
Background
Aberrant NF-κB signalling plays a critical role in chronic lymphocytic leukaemia (CLL), driven by both recurrent mutations in NF-κB-regulating genes (Cuní et al. 2004, Baliakas et al. 2015, Mansouri et al. 2015) the protective lymph node (LN) microenvironment (Herishanu et al. 2011). Elevated NF-κB activity is associated with disease aggressiveness (Cuní et al. 2004, Hewamana et al. 2008) and resistance to therapies such as ibrutinib and venetoclax (Jayappa et al. 2017). This resistance is partially mediated by the upregulation of anti-apoptotic Bcl-2 family members in the LN niche (Smit et al. 2006). We investigated the therapeutic potential of targeting NF-κB-inducing kinase (NIK), a central regulator of non-canonical NF-κB signalling. NIK expression is upregulated as CLL cells migrate into the LN, where they acquire therapy resistance (Jayappa et al. 2017). Since NIK is normally constitutively active only in pathological contexts (Thu et al. 2011), it presents a promising tumour-selective target. We hypothesised that NIK inhibition would preferentially affect LN-resident CLL cells reliant on non-canonical NF-κB signalling. We evaluated three selective NIK inhibitors (CW15337, Amgen16, and B022) in MEC-1 cells and primary CLL samples, cultured with CD40L-expressing fibroblasts to mimic the LN microenvironment. Non-transfected fibroblasts (NTLs) served as controls.
Results
All three NIK inhibitors induced dose-dependent reductions in nuclear p52 levels, confirming on-target inhibition of non-canonical NF-κB signalling. In parallel, they significantly impaired MEC-1 cell migration toward CXCL12 (p < 0.001) and induced a concentration-dependent G1 cell cycle arrest (p=0.001). Importantly, the compounds also reduced p52 activation and triggered dose-dependent cytotoxicity in both MEC-1 cells and 25 primary CLL samples cultured on CD40L-expressing fibroblasts. Despite CD40L-mediated enhancement of both canonical and non-canonical NF-κB signalling, NIK inhibition consistently overcame this cytoprotective effect. LC50 values were significantly lower in CD40L co-cultures compared to non-transfected fibroblasts (NTLs) for Amgen16 (p=0.01) and B022 (p=0.01), and comparable for CW15337 (p=0.9). Sensitivity to NIK inhibition was correlated with basal non-canonical NF-κB activity, as measured by nuclear p52 (p=0.006; r²=0.46 for B022). CD40L stimulation increased protein levels of the anti-apoptotic proteins Bcl-xL (p < 0.0001) and Mcl-1 (p=0.0003), but not Bcl-2 (p=0.8), and upregulated transcription of BCL2L1 and BCL2A1. Treatment with NIK inhibitors reversed these effects, leading to dose-dependent downregulation of Bcl-xL (p=0.001) and Mcl-1 (p < 0.001), with no significant effect on Bcl-2 expression (p=0.45). At the transcript level, NIK inhibition suppressed BCL2A1 and BCL2L1 expression in a dose-dependent manner, without affecting MCL1 or BCL2. Consistent with prior reports (Vogler et al. 2009, Haselager et al. 2020), CD40L co-culture induced in vitro resistance to venetoclax (ABT-199), which was effectively reversed by co-treatment with NIK inhibitors. Combination therapy yielded strong synergy, with mean synergy scores of 21.0, 23.9, and 32.3 for Amgen16, B022, and CW15337, respectively (p < 0.0001).
Conclusions
NIK inhibition effectively neutralises the cytoprotective effects of the LN microenvironment, likely through suppression of CD40L-induced anti-apoptotic signals and NF-κB pathway activation. NIK inhibitors target CLL cells in protective niches and inhibit their migration, suggesting utility in eliminating LN-resident disease and preventing niche re-entry. Furthermore, NIK inhibition resensitises CLL cells to venetoclax, supporting a rationale for combination strategies.
Future Work
Given that NF-κB signalling is a key driver of Richter’s transformation (Rohan et al. 2024) and that the lymph node microenvironment is a critical component of the disease biology, NIK represents a compelling therapeutic target. We now plan to evaluate the efficacy of NIK inhibition in this high-risk context using both in vitro and in vivo models.
Keywords : NF-κB-inducing kinase, tumour microenvironment, drug resistance
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