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Four patterns of CD180-mediated signalling in CLL cells have been identified: AKT-signallers (AKT-S); p38MAPK signallers (p38MAPK-S); nonsignallers (NS); and a minor subset of double AKT/p38MAPK signallers (DS). 24 out of the 60 CD180+ CLL samples, responded to CD180 ligation by a significant upregulation of AKT-P (AKT-S) (Figures 3.1A). It was reported that activation of AKT is essential for the BCR-mediated growth and survival of B lymphocytes (Woodland

et al., 2008) as well as the survival of CLL cells (Longo et al., 2008; Packham et al., 2010; Downward, 2014). CD180 ligation resulted in increased

phosphorylation of p38MAPK in 16 out of 36 remaining samples (p38MAPK-S) (Figures 3.1B) and only in a small cohort of 6 CLL samples showed activation of both AKT-P and p38MAPK-P(DS) upon stimulation with anti-CD180 mAb (Figures 3.1A and B). The use of specific inhibitors of AKT and p38MAPK signalling pathways to confirm that, activation of AKT and p38MAPK pathways is exclusive will be discussed in chapter 4. Activation of alternative pathways appears to be a feature of CLL cells, and not of normal B cells as they responded to CD180 ligation by increasing both AKT-P and p38MAPK-P (Figures 3.1A, B). CD180 ligation led to significant drop in p38MAPK-P basal levels in the AKT-S cells (Figures 3B) and AKT-P basal levels in p38MAPK-S cells (Figures 3A). Petlickovski et al., (2005) reported that BCR stimulation causes a decrease in signal intensity of p38MAPK, rather than an increase, but not linked to AKT activation. This data suggests the regulatory influence of one pathway on another. 14 CD180+ CLL samples did not respond to CD180 ligation by activating either of the two pathways (NS, Figures 3.1 A and B). It is possible to suggest that those NS CLL cells may use an alternative pathway or that they are totally refractive to ligation of CD180. Interestingly, those CD180 NS CLL samples remained unresponsive to the ligation of sIgM suggesting their anergic status (Packham et

al., 2014). The distribution of CD180-mediated signalling categories of CLL cells

121 Figure 3.9: Proportion of CLL samples exhibiting four different patterns of signalling. Treatment with anti-CD180 mAb of CD180+CLL samples or treatment with anti-IgM F(ab)2 of CD180+_sIgM+ _{CLL samples. AKT-S, AKT-signallers; p38MAPK-S,} p38MAPK signallers; DS, double AKT/p38MAPK signallers; NS, nonsignallers (Porakishvili et al., 2015)

Porakishvili et al., (2005) have demonstrated that more U-CLL samples (71%) are CD180+_sIgM+ _{than CD180}+_sIgMneg/low _{attributing U-CLL samples mostly to the}

AKT-S and NS signalling categories. However, in this study M-CLL samples were found to be evenly distributed between AKT-S, p38MAPK-S and NS categories. Interestingly, all six DS CLL samples belonged to M-CLL group, which is associated with increased anergy (Packham et al., 2014). There were no differences in surface expression of the CD180, sIgM, CD79b or CD38 between the four signalling groups (data not shown). However, p38MAPK-S cells expressed significantly higher ex vivo levels of sIgD compared to AKT-S cells: 83.5±15.1% versus 48.5±33.0%, p=0.0055, n=10. Although the relevance of this is currently unclear, it is possible to suggest that sIgD could be involved in the p38MAPK-pathway mediated through CD180 which our research group is currently investigating.

122 3.3.2 CD180-mediated AKT-signalling pathway in CLL involves activation of BTK and is pro-survival, while p38MAPK activation favours apoptosis

There are several studies showing that BTK plays an important role in BCR signalling pathway (Burger et al., 2010). Moreover, its role in CLL cell survival has also been reported (Ponader et al., 2012). As per this data, CD180 ligation in AKT-S CLL samples activated pro-survival BTK kinase while suppressing p38MAPK pathway. Activation of BTK led to the reduction in apoptosis in this category of CLL samples (Figure 3.2). However, recruitment of BTK in p38MAPK- S samples was not observed, resulting in the increased apoptosis (Figure 3.2). The role of p38MAPK-mediated signalling in CLL is so far unclear. Activation of p38MAPK has been associated with proliferation of various cells in response to CpG-ODN. Takeshita et al., (2004) reported that CpG DNA/TLR9-mediated cellular signalling involves in activation of p38MAPK in macrophages. CPG-DNA driven activation of p38MAPK in murine macrophage cell line, RAW264.7 has been reported by Ahmad-Nejad et al., (2002). Moreover, in mice dendritic cells, CpG DNA induced survival signals via activation of PI3K and p38MAPK (Park et

al., 2002). Peng (2005) showed the importance of activation of p38MAPK in TLR4

signalling in B cells for transcription of inflammatory genes and in TLR9 signalling for activation, proliferation and Ig secretion. CpG DNA augmented BCR-mediated signals for the activation of p38MAPK which lead to synergistic production of cytokines and induction of splenic mature B cells proliferation (Kyung-Ae et al., 2003). On the contrary, Ntoufa et al., (2012) demonstrated that p38MAPK activation led to tolerant status in CLL cells.

Moreover, it has been documented that p38MAPK is associated with regulation of cell survival in CLL (Grumont et al.,1998; Furman et al., 2000; Piatelli et al., 2004). p38MAPK has been shown to induce apoptosis in murine splenocytes and human lymphoma B-cell lines (Graves et al., 1998: Yan et al., 2008). Later Negro et al., (2012) demonstrated the significance of p38MAPK in apoptosis of CLL cells. Current data strongly confirm that CD180-mediated activation of p38MAPK is associated with apoptosis of CLL cells. This might suggest the potential use of p38MAPK-signalling as a new profiling tool for those patients who are unresponsive to BTK inhibitors.

To summarise, current data show that CD180 ligation activates BTK/AKT signalling pathway in AKT-S cells leading to their survival but not in p38MAPK-S or NS CLL samples (Figures 3.1, 3.2 and 3.3). Therefore, considering that CLL

123

cells receive various stimuli from their microenvironment in vivo, including via CD180, these findings suggest that BTK inhibitors might be limited to AKT-S CLL cells.

3.3.3 sIgM ligation favours pro-survival BTK/AKT signalling pathways in