Differential activation of pro‐survival pathways in response to CD40LG/IL4 stimulation in chronic lymphocytic leukaemia cells (2018)

Chronic lymphocytic leukaemia (CLL) is characterized by the progressive accumulation of mature monoclonal B lymphocytes, which are dependent on the microenvironment of proliferation centres for survival (Burger et al, 2009). The pro-survival signals activated in these tissues include the ERK, AKT, STAT3 and NF-κB pathways (Chen et al, 2016). The secretion of the T cell chemokines CCL3/4/17/22 by CLL cells is also important for proliferation (Burger, 2010). This microenvironment can be mimicked by a combination of CD40LG (also termed CD154) and interleukin-4 (IL4), which improves viability and enhances the expression of pro-survival signals and increases resistance to chemotherapeutic drugs and targeted agents (Vogler et al, 2009). However, the time course and magnitude of the activation of the pathways induced have not been fully investigated. We found that CD40LG/IL4 stimulation, which improved the viability of primary CLL cells (Figs 1A and S1A), activated STAT3 within the first 12 h, reaching its highest values within 24 h before returning to basal levels by 72 h (Fig 1B). The NF-κB pathways were also quickly induced through increased expression of RelB (RELB) and p65 (RELA), suggesting both non-canonical and canonical signalling activation. The expression of NF-κB (RelB) reached maximal levels within the first 12 h, followed by a slight decrease, while the expression of NF-κB (p65) was lower and gradually increased. In contrast to the quick activation on NF-κB and STAT3 signals, the ERK (MAPK1) and AKT (AKT1) pathways were not significantly enhanced in the first 24 h, but markedly increased 48 h after stimulation. Our data suggests the importance of the early activation of NF-κB signalling for CLL cell survival. Consistent with this hypothesis, the expression of NF-κB targets and anti-apoptotic proteins BCL2, Bcl-xL (BCL2L1) and MCL1 also increased within 12 h (Fig 1C). To further understand this phenomenon, we measured the expression of APRIL (a proliferation-inducing ligand, also termed TNFSF13) and BAFF (B cell-activating factor of the TNF family, also termed TNFSF13B), as well as their receptors BAFFR (TNFRSF13C), TACI (transmembrane activator or the calcium modulator and cyclophilin ligand-interactor; TNFRSF13B) and BCMA (B-cell mutation antigen; now termed TNFRSF17), all incorporated in NF-κB pathways (Kern et al, 2004). In unstimulated CLL cells, basal levels of APRIL were low compared to BAFF, and BCMA levels were lower than those of the other two receptors, BAFFR and TACI (Figure S1B). Expression after CD40LG/IL4 stimulation showed that APRIL progressively increased in a subset of patients, while the induction of BAFF was more variable and only transient (Figs 1D and S2 ). TACI was also increased in the first 12 hs in around half the cases, followed by a decrease to basal levels or below in all patients. BCMA progressively increased in most patients, while BAFFR was suppressed. We also found that the basal expression of CCL3/4/17/22 was low in unstimulated CLL cells (Figure S1B). CD40LG/IL4 stimulation decreased the expression of CCL3, which in some patients eventually recovered and then increased, while CCL4 was mostly suppressed (Figs 1D and S2 ). CCL17 and CCL22 were greatly increased at early time points in most patients and then levels were reduced, although they remained higher than that in non-stimulated cells. These results together confirm that the microenvironment in proliferation centres activates a diverse array of pathways. We show here that they follow diverse temporal patterns, suggesting that they contribute differently to CLL survival. While AKT and ERK are important survival stimuli for CLL cells (Chen et al, 2017), they are not immediately activated. Instead, when cells enter the proliferation centres they may initially rely on NF-κB, which is quickly induced at high levels. STAT3 may be helping in this initial stage but it may later be dispensable. Some cells showed a strong upregulation of NF-κB related genes, especially TNFSF13 (encoding APRIL) and TNFRSF17 (encoding BCMA), while the effect was not so evident in others. Chemokines were differentially regulated, with CCL17/22 peaking early and remaining induced and CCL3/4 generally maintaining low levels. Our data suggests that the response of CLL cells to the microenvironment in proliferation centres is biphasic, with an initial input from STAT3 and NF-κB pathways and a maintenance of the signal dependent on NF-κB, ERK and AKT, with CCL17/22. Of note, the range of responses were varied, which provides a rationale for defining factors that could predict the expression of the proteins involved. As we did not observe any pattern within the available clinical information for these samples (Table S1), a larger cohort and more parameters need to be investigated. We propose that identification of patients with high activation of NF-κB pathways could provide a rational approach at designing better precision therapies (Dyer et al, 2013). Work supported by Kay Kendall Leukaemia Fund (#KKL982/999), M.C. Andreu Memorial Fund, Leicester Haematology Research Fund and Leicester Experimental Cancer Medicine Centre (C325/A15575). YC and SM designed the experiments, analysed the data and prepared the manuscript, with MJSD and SJ. YC and CP performed the experiments. All authors reviewed the manuscript. The authors declare no conflict of interest. Table S1. Information for the primary CLL samples used. Table S2.qPCR primers used. Figure S1.(A) Same as Fig 1A, using cells from two other patients (#7 and 8, see Table S1). Figure S2.(A) Individual expression levels of the qPCR data shown in Fig 1D. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.