1023 - Targeting NF-κB-inducing kinase (NIK) in chronic lymphocytic leukaemia

Background
Aberrant NF-κB signalling appears to play a key role in the pathogenesis of CLL, including several recurrent genetic mutations in NF-κB-activating genes[1-3]. Constitutive NF-κB activity is associated with a more aggressive disease[1,4] and is implicated in the development of resistance to both ibrutinib and venetoclax[5]. Inhibiting NF-κB signalling is therefore a potential therapeutic approach. However, given the key role of canonical NF-κB signalling on multiple cellular processes, direct pharmacological targeting of NF-κB (e.g. through IKKβ inhibitors) has so far failed due to associated toxicities.

We investigated the potential for targeting NF-κB inducing kinase (NIK), the central kinase regulating non-canonical NF-κB signalling, in CLL. We evaluated three NIK inhibitors, CW15337, Amgen16 and BO22, in the MEC-1 cell line and in primary CLL cells. Given that NIK expression levels are very low under physiological conditions, and constitutive activation is usually only present in pathological contexts[6], NIK may represent a tumour-selective therapeutic target. We hypothesised that NIK inhibition may represent a promising strategy in the treatment of CLL by targeting CLL cells in the lymphoid tissue environment where they are particularly reliant on non-canonical NF-κB signalling.

Results
All three NIK inhibitors caused a dose-dependent G1 arrest in the cell cycle in the MEC-1 cell line. Mean proportion of MEC-1 cells in G1 with no drug = 48%, whilst at the highest drug concentration = 69%, 68% and 66% for CW15337, Amgen16 and BO22, respectively. All three agents were cytotoxic in both in the MEC-1 cell line (LC50 = 1.63µM, 30µM and 19.3µM for CW15337, Amgen16 and BO22, respectively) and in primary samples (LC50 = 2.05µM, 15.23µM and 14.4µM for CW15337, Amgen16 and BO22, respectively). CW15337 was the most potent drug in both MEC-1 and primary samples. Nuclear expression of the non-canonical NF-κB subunit, p52, was correlated with sensitivity to CW15337 (p = 0.01; r2 = 0.39).
When co-cultured with CD40L-expressing fibroblasts, as a model of the lymphoid niche, NIK inhibitors were able to overcome this cytoprotective environment and cause cell death. This was seen with both MEC-1 and primary samples. Furthermore, all three agents significantly inhibited MEC-1 cell migration against a chemokine gradient (p < 0.001) in a dose-dependent manner. Co-culture on CD40L-expressing cells induced both canonical and non-canonical subunit expression in nuclear extracts, which promoted in vitro resistance against fludarabine and ABT-199 (venetoclax) but not CW15337. Furthermore, the combination of CW15337 with fludarabine or ABT-199 showed cytotoxic synergy. Mechanistically, CW15337 caused the selective inhibition of non-canonical NF-κB subunits and the transcriptional repression of BCL2L1, BCL2A1 and MCL1 anti-apoptotic gene transcription. Taken together, these data suggest that the NIK inhibitor, CW15337, exerts its effects via suppression of the non-canonical NF-κB signalling pathway, and reverses venetoclax resistance in the context of CD40L stimulation through repression of BCL2 family proteins.

In conclusion, NIK inhibitors can overcome the cytoprotective environment conferred by co-culture, suggesting that they could be effective at targeting disease in lymphoid tissues, where CLL cells are more reliant on NF-κB signalling. The inhibition of MEC-1 migration suggests that NIK inhibitors could block re-entry of CLL cells to the lymph node microenvironment. Furthermore, they may be effective in targeting residual disease in the lymph node by suppressing the non-canonical NF-κB signalling pathway, which resensitises CLL cells to venetoclax.

References:
1. Cuní S., Pérez-Aciego P., Pérez-Chacón G.et al. A sustained activation of PI3K/NF-κB pathway is critical for the survival of chronic lymphocytic leukemia B cells. Leukemia. 2004;18:1391–1400.
2. Baliakas P., Hadzidimitriou A., Sutton L.A.et al. Recurrent mutations refine prognosis in chronic lymphocytic leukemia. Leukemia. 2015;29:329–336.
3. Mansouri L., Sutton L.A., Ljungström V.et al. Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia. J. Exp. Med. 2015;212:833–843.
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5. Jayappa KD, Portell CA, Gordon VL, Capaldo BJ, Bekiranov S, Axelrod MJ, et al. Microenvironmental agonists generate de novo phenotypic resistance to combined ibrutinib plus venetoclax in CLL and MCL. Blood Adv. 2017;1(14):933–46.
6. Thu YM, Richmond A. NF-κB inducing kinase: a key regulator in the immune system and in cancer. Cytokine Growth Factor Rev. 2010;21(4):213–26.