| dc.description.abstract | Chronic Lymphocytic Leukaemia (CLL) is characterized by the accumulation of CD5/19+ B cells in the blood, bone marrow and lymph nodes. The tumour microenvironment of the bone marrow and secondary lymphoid organs promotes cell proliferation, survival and protection from drug induced apoptosis. Selectins, integrins and chemokines mediate the trafficking of CLL cells to these protective niches. An important adhesion molecule in this process is L-selectin (CD62L) which is involved in initial tethering and rolling of CLL cells in the high endothelial venules (HEV), allowing migration between the blood and the secondary lymphoid organs. Within the tumour microenvironment, the B-cell receptor (BCR) signalling pathway is the most important pathway involved in microenvironmental crosstalk and CLL cell survival, and has recently been shown to interact with the Signal Transducer and Activator of Transcription 3 (STAT3) signalling pathway. STAT3 is a transcription factor that regulates a number of genes involved in cell survival, proliferation and migration. Elucidating how CLL cells traffic to, and their interactions within, the tumour microenvironment may provide new avenues for therapeutic intervention in CLL. In this thesis, the role of STAT3 in the trafficking and microenvironmental interactions of CLL cells, and its potential as a therapeutic target, was investigated.
The role of STAT3 in CLL pathogenesis is unclear; however, it is constitutively phosphorylated on serine residue 727 (serine pSTAT3) in CLL cells. Phosphorylation on tyrosine residue 705 (tyrosine pSTAT3) was seen in 6 out of 28 CLL samples isolated from the peripheral blood of patients, and correlated with poor prognostic indicators (unmutated immunoglobulin heavy chain variable (IGVH) genes and NOTCH1 mutated patient samples). STAT3 targeting agents, Cucurbitacin and S3I-201, had a divergent effect on CLL cell survival, being pro-apoptotic in some and pro-survival in others. Cucurbitacin induced apoptosis was concurrent with a downregulation in serine pSTAT3. The upstream JAK inhibitor Ruxolitinib had no effect on serine pSTAT3 and apoptosis of CLL cells.
We investigated the role of STAT3 in the regulation of a panel of adhesion molecules involved in the migration of lymphocytes. Pharmacologically targeting STAT3, using Cucurbitacin (p<0.0001, n=29) and S3I (p<0.0001, n=21), and siRNA mediated STAT3 knockdown, resulted in a significant decrease in the cell surface expression of CD62L in CLL cells. The effect of Cucurbitacin on CD62L downregulation was greater in patient samples positive for the expression of the poor prognostic marker CD38.
We investigated the adhesion of CLL cells to human umbilical vein endothelial cells (HUVEC) under fluid shear flow conditions to model the interactions of CLL cells with endothelial cells in the HEV. We found that that blocking CD62L, using and anti-CD62L
antibody, and treatment with Cucurbitacin resulted in a decrease in the adhesion of CLL cells to HUVECs under fluid shear flow (p<0.001, n=5). In addition to inhibition of CLL cell adhesion, pharmacological targeting of STAT3 using Cucurbitacin and S3I resulted in a significant decrease in CLL cell migration in response to the chemokine CXCL12 (p<0.001, n=8).
We investigated the effect of microenvironmental stimuli on the activation of STAT3 and the regulation of CD62L in CLL cells. Hs5 bone marrow stromal cell (BMSC) co-cultures resulted in an increase in tyrosine pSTAT3 and a significant increase in CD62L positive CLL cells (p<0.01, n=7). However; this did not overcome Cucurbitacin and S3I downregulation of CD62L.
The BCR is the most prominently activated pathway in CLL cells from the lymph node compartment. Stimulation of the BCR induced significantly increased tyrosine and serine phosphorylation of STAT3 in CLL cells with unmutated immunoglobulin (IGVH) genes compared to cells with mutated IGVH genes (p<0.05, n=26) . We show that BCR stimulation-mediated STAT3 phosphorylation is prolonged and suggest that it is a key target of the BCR signalling pathway. Stimulation of BCR also resulted in a significant increase in CD62L expression in patient samples, with increased upregulation seen in samples with UM-IGVH compared to M-IGVH (p< 0.05, n=23). Gene expression studies showed BCR stimulation also resulted in an upregulation of CD62L gene expression in CLL cells with unmutated IGVH genes. BCR stimulation-induced tyrosine pSTAT3 and upregulation of CD62L was abrogated by pre-treatment with the Janus kinase (JAK)/STAT3 inhibitor Ruxolitinib and the BCR inhibitor Ibrutinib (p<0.05, n=5).
In conclusion, this thesis shows a role for STAT3 in the pathogenesis of CLL. We have shown that STAT3 is an important regulator of the key adhesion molecule CD62L and is an important downstream mediator of the BCR signalling pathway. The down-regulation of CD62L expression, through BCR signalling pathway inhibition using Ibrutinib, and inhibition of STAT3 activation using Ruxolitinib, may have implications for the trafficking of CLL cells and present new potential therapeutic avenues for targeting CLL. | en |
