This study was designed to investigate the potential for targeting the NF-kB inducing kinase, NIK, in two common B-cell malignancies, chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). Using a selective NIK inhibitor, CW15337, we were able to demonstrate that cell lines and primary tumor cells were sensitive to the effects of NIK inhibition, whilst normal lymphocytes were significantly more resistant to its cytotoxic effects. Sensitivity to CW15337 was associated with the nuclear expression of the NF-kB subunit, p52. Importantly, tumor samples from a subset of poor prognosis CLL patients, with mutations in a gene called BIRC3, showed elevated p52 expression and were particularly sensitive to NIK inhibition. Furthermore, the combination of CW15337 and ABT-199 (venetoclax) reversed the drug resistance observed when treating tumor cells with ABT-199 alone. Our study shows the potential for targeting NIK in both CLL and MM.

Chronic lymphocytic leukemia (CLL) remains incurable despite B-cell receptor-targeted inhibitors revolutionizing treatment. This suggests that other signaling molecules are involved in disease escape mechanisms and resistance. Toll-like receptor 9 (TLR9) is a promising candidate that is activated by unmethylated cytosine guanine dinucleotide-DNA. Here, we show that plasma from patients with CLL contains significantly more unmethylated DNA than plasma from healthy control subjects (P < .0001) and that cell-free DNA levels correlate with the prognostic markers CD38, β2-microglobulin, and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (hazard ratio, 4.0; P = .003). We also show that TLR9 expression was associated with in vitro CLL cell migration (P < .001), and intracellular endosomal TLR9 strongly correlated with aberrant surface expression (sTLR9; r = 0.9). In addition, lymph node-derived CLL cells exhibited increased sTLR9 (P = .016), and RNA-sequencing of paired sTLR9hi and sTLR9lo CLL cells revealed differential transcription of genes involved in TLR signaling, adhesion, motility, and inflammation in sTLR9hi cells. Mechanistically, a TLR9 agonist, ODN2006, promoted CLL cell migration (P < .001) that was mediated by p65 NF-κB and STAT3 transcription factor activation. Importantly, autologous plasma induced the same effects, which were reversed by a TLR9 antagonist. Furthermore, high TLR9 expression promoted engraftment and rapid disease progression in a NOD/Shi-scid/IL-2Rγnull mouse xenograft model. Finally, we showed that dual targeting of TLR9 and Bruton’s tyrosine kinase (BTK) was strongly synergistic (median combination index, 0.2 at half maximal effective dose), which highlights the distinct role for TLR9 signaling in CLL and the potential for combined targeting of TLR9 and BTK as a more effective treatment strategy in this incurable disease.

Combined analyses of the pairs of biomarkers A IGHV mutation status and CD49d, B IGHV mutation status and telomere length and C telomere length and CD49d as predictors of progression-free survival (PFS) in CLL patients. D Shows the overlaid Kaplan–Meier curves for the ARCTIC/ADMIRE cohort, which demonstrate that patients with mutated IGHV genes and short telomeres have a similar, inferior PFS to the unmutated IGHV subset. Furthermore, patients with mutated IGHV genes, long telomeres and low CD49d expression have a significantly longer PFS than patients with mutated IGHV genes, long telomeres and high CD49d expression. E An additional cohort, derived from the FC-treated arm of the UK CLL4 trial, confirmed the findings from the ARCTIC/ADMIRE cohort. F Shows a schematic diagram of the propose a risk-adapted approach to treatment selection, which would contra-indicate frontline chemoimmunotherapy for ~83% of patients.