The cellular kinases inhibitory-κB kinase (IKK) α and Nuclear Factor-κB (NF-κB)-inducing kinase (NIK) are well recognised as key central regulators and drivers of the non-canonical NF-κB cascade and as such dictate the initiation and development of defined transcriptional responses associated with the liberation of p52-RelB and p52-p52 NF-κB dimer complexes. Whilst these kinases and downstream NF-κB complexes transduce pro-inflammatory and growth stimulating signals that contribute to major cellular processes, they also play a key role in the pathogenesis of a number of inflammatory-based conditions and diverse cancer types, which for the latter may be a result of background mutational status. IKKα and NIK, therefore, represent attractive targets for pharmacological intervention. Here, specifically in the cancer setting, we reflect on the potential pathophysiological role(s) of each of these kinases, their associated downstream signalling outcomes and the stimulatory and mutational mechanisms leading to their increased activation. We also consider the downstream coordination of transcriptional events and phenotypic outcomes illustrative of key cancer ‘Hallmarks’ that are now increasingly perceived to be due to the coordinated recruitment of both NF-κB-dependent as well as NF-κB–independent signalling. Furthermore, as these kinases regulate the transition from hormone-dependent to hormone-independent growth in defined tumour subsets, potential tumour reactivation and major cytokine and chemokine species that may have significant bearing upon tumour-stromal communication and tumour microenvironment it reiterates their potential to be drug targets. Therefore, with the emergence of small molecule kinase inhibitors targeting each of these kinases, we consider medicinal chemistry efforts to date and those evolving that may contribute to the development of viable pharmacological intervention strategies to target a variety of tumour types.

Cancer cells appear to depend heavily on antiapoptotic proteins for survival and so targeted inhibition of these proteins has therapeutic potential. One innovative strategy is to inhibit the cyclin‐dependent kinases (CDKs) responsible for the regulation of RNA polymerase II (RNAPII). In our study, we investigated the detailed cellular mechanism of a novel small‐molecule CDK inhibitor (CDKI‐71) in cancer cell lines, primary leukemia cells, normal B ‐ & T‐ cells, and embryonic lung fibroblasts and compared the cellular and molecular responses to the clinical CDK inhibitor, flavopiridol. Like flavopiridol, CDKI‐71 displayed potent cytotoxicity and caspase‐dependent apoptosis induction that were closely associated with the inhibition of RNAPII phosphorylation at serine‐2. This was caused by effective targeting of cyclinT–CDK9 and resulted in the downstream inhibition of Mcl‐1. No correlation between apoptosis and inhibition of cell‐cycle CDKs 1 and 2 was observed. CDKI‐71 showed a 10‐fold increase in potency in tumor cell lines when compared to MRC‐5 human fibroblast cells. Significantly, CDKI‐71 also demonstrated potent anti‐chronic lymphocytic leukemia activity with minimal toxicity in normal B‐ and T‐cells. In contrast, flavopiridol showed little selectivity between cancer and normal cells. Here, we provide the first cell‐based evidence that flavopiridol induces DNA double‐strand breaks: a fact which may explain why flavopiridol has such a narrow therapeutic window in preclinical and clinical settings. Taken together, our data provide a rationale for the development of selective CDK inhibitors as therapeutic agents and CDKI‐71 represents a promising lead in this context.

CD49d is a remarkable prognostic biomarker of chronic lymphocytic leukemia (CLL). The cutoff value for the extensively validated 30% of positive CLL cells is able to separate CLL patients into 2 subgroups with different prognoses, but it does not consider the pattern of CD49d expression. In the present study, we analyzed a cohort of 1630 CLL samples and identified the presence of ∼20% of CLL cases (n = 313) characterized by a bimodal expression of CD49d, that is, concomitant presence of a CD49d+ subpopulation and a CD49d− subpopulation. At variance with the highly stable CD49d expression observed in CLL patients with a homogeneous pattern of CD49d expression, CD49d bimodal CLL showed a higher level of variability in sequential samples, and an increase in the CD49d+ subpopulation over time after therapy. The CD49d+ subpopulation from CD49d bimodal CLL displayed higher levels of proliferation compared with the CD49d− cells; and was more highly represented in the bone marrow compared with peripheral blood (PB), and in PB CLL subsets expressing the CXCR4dim/CD5bright phenotype, known to be enriched in proliferative cells. From a clinical standpoint, CLL patients with CD49d bimodal expression, regardless of whether the CD49d+ subpopulation exceeded the 30% cutoff or not, experienced clinical behavior similar to CD49d+ CLL, both in chemoimmunotherapy (n = 1522) and in ibrutinib (n = 158) settings. Altogether, these results suggest that CD49d can drive disease progression in CLL, and that the pattern of CD49d expression should also be considered to improve the prognostic impact of this biomarker in CLL.

Several lines of evidence suggest that homing of tumor cells to lymphoid tissue contributes to disease progression in chronic lymphocytic leukemia (CLL). Here we demonstrate that lymph node (LN)-derived CLL cells possess a distinct phenotype and exhibit enhanced capacity for T-cell activation and superior immune synapse formation when compared to paired peripheral blood samples (PB). LN-derived CLL cells manifest a proliferative, CXCR4^dimCD5^bright, phenotype compared to those in the PB and higher expression of T-cell activation molecules including CD80, CD86 and HLA-DR. In addition, LN-CLL cells have higher expression of α4β1 (CD49d) which, as well as being a co-stimulatory molecule, is required for CLL cells to undergo transendothelial migration (TEM) and enter the proliferation centers of the LNs. Using an in-vitro system that models circulation and TEM, we showed that the small population of CLL cells that migrate are CXCR4^dimCD5^bright with higher CD49d, CD80, CD86 and HLA-DR compared to those that remain circulating; a phenotype strikingly similar to LN-derived CLL cells. Furthermore, sorted CD49d^hi CLL cells showed enhanced capacity to activate T-cells compared to CD49d^lo sub-populations from the same patient. Thus, although PB-CLL cells have reduced capacity to form immune synapses and activate CD4+ T-cells, this was not the case for LN-CLL cells or those with the propensity to undergo TEM. Taken together, our study suggests that CLL cell immunological function is not only modulated by microenvironmental interactions but is also a feature of a sub-population of PB-CLL cells that are primed for lymphoid tissue homing and interaction with T-cells.