Darren Edwards, Neil D. Perkins, James M. Allan, Elaine Willmore, Susan J. Tudhope, Giacomo Berretta, Colin J. Suckling, Andrew Paul, Helen Marr, Aaron Gardner, Louise C. Young, Robin Plevin, Jill E. Hunter, Marie Boyd, Andrew Herridge, Jonathan P. Wallis, David Breen, and Simon P. Mackay
The stress-inducible transcription factor, NF-κB, plays an important in role in B-cell malignancies, regulating expression of a plethora of genes including those that drive proliferation, apoptosis and survival. Non-canonical ('alternative') NF-κB signalling, resulting in processing of the p100 subunit to p52 and dimerization with RelB, is less well-studied than the canonical pathway. Previous studies on NF-κB signalling, particularly in CLL, showed that the RelA (canonical) subunit is highly expressed and associated with poor outcome. In multiple myeloma and lymphoma, mutations in genes that alter NF-κB activity (NOTCH1, BIRC3, MYD88, TRAF2/3) can promote cell survival, by increasing activation of the CD40 receptor or NIK (NF-κB-inducing kinase), resulting in constitutive non-canonical NF-κB signalling. More recently, a comprehensive study showed that recurrent mutations in TRAF3, BIRC3 and NFKB2, all of which regulate non-canonical NF-κB signalling, also occur in CLL (Puente XS et al, Nature 2015). Taken together, these data suggest that non-canonical NF-κB signalling is important in CLL and that this pathway represents a novel therapeutic target. We hypothesised that receptor-activated proliferation in CLL cells is reliant on non-canonical NF-κB-regulated gene transcription, and that targeting this pathway would decrease CLL cell survival. Since the IKKα kinase phosphorylates p100, which in turn leads to active p52/RelB dimers in the nucleus, it is a key driver of the non-canonical signalling pathway. We used cell line models and patient-derived CLL cells to evaluate a series of selective, first-in-class IKKα inhibitors. Initially, we assessed 20 novel IKKα inhibitors in MEC-1 (CLL-like, BIRC2/3 and TP53 deleted) and Maver-1 (mantle cell lymphoma, TRAF3 deleted) cell lines, as these are representative of B-cell malignancies with constitutive activation of non-canonical NF-κB. Compounds S and U were found to be potent (cell-free IC50 values < 50nM) and selective (e.g. >50-fold over IKKβ). In growth inhibition studies, compound S inhibited cell proliferation with GI50 values of 4.3 μM (MEC-1) and 2.7 μM (Maver-1) whereas compound U gave GI50 values of 0.47 μM (MEC-1) and 0.5 μM (Maver-1). We then examined selected cases from our CLL cohort. Western blotting of nuclear extracts showed that expression of NF-κB subunits in patient-derived CLL cells was heterogeneous: RelA was constitutively expressed in most cases, and although expression of the non-canonical subunits, p52 and RelB, varied, high levels of p52 were associated with high RelB expression, indicating that non-canonical signalling is active in CLL. The panel of 20 IKKα inhibitors were also tested in viability and apoptosis assays in patient-derived CLL cells, revealing LC50 values ranging from 0.5 μM (Compound U) - 5μM (48 hr exposure). Decreasing cell survival correlated with increased caspase 3/7 activation. To model the tumour microenvironment, we used CD40L-expressing fibroblasts in co-culture with primary CLL cells to stimulate NF-κB signalling and CLL cell proliferation. CD40L stimulation led to increased levels of nuclear RelB and p52 levels after 2-8 hrs co-culture, concomitant with activation of IKKα, as demonstrated by rapid phosphorylation of p100 subunit. Compound S was evaluated in CD40L-stimulated CLL cells where it was shown to inhibit p100 processing and decrease levels of nuclear RelB and p52. Compound S significantly reduced CD40L-stimulated proliferation of CLL cells following 7 day treatment (1μM), in a similar fashion to the clinically-used PI3Kδ inhibitor, Idelalisib, and was also effective at reducing proliferation of TP53-mutated patient-derived CLL cells. Compound S had little or no effect on accumulation of nuclear RelA (p65), indicating selectivity of these inhibitors for IKKα over IKKβ and suggesting that targeting receptor-stimulated IKKα in B cells may avoid 'global' toxicity anticipated by use of IKKβ-targeting therapies. Future studies will examine the gene expression profile following IKKα inhibition in CLL, and also interrogate the underlying genotypes among patients that may drive NF-κB signaling. These data provide proof of concept that targeting non-canonical NF-κB signaling is a valid therapeutic strategy in CLL. Disclosures No relevant conflicts of interest to declare.