Molecular Characterisation of Interaction between Nurse-like cells and Chronic Lymphocytic Leukaemia cells

Ishaque Mohammad
Chronic Lymphocytic Leukaemia (CLL) is a proliferative B cell malignancy characterised by an accumulation of mature B cells in the peripheral blood, lymph nodes and bone marrow. The disease is heterogenous in both clinical presentation and response to treatment. Despite the well-established immunochemotherapy and recently introduced molecularly targeted therapies such as ibrutinib and venetoclax, CLL is still incurable. One possible cause of relapse in patients is the involvement of the tissue
more » ... icroenvironment which supports CLL-cell survival and confers drug resistance. Nurse like cells (NLCs) are a major component of the CLL microenvironment. However, the exact molecular mechanisms mediating interactions between CLL cells and NLCs are still not fully understood. The aim of this study was to investigate how NLCs and CLL cells influence each other at the level of gene expression and to uncover molecules and pathways that are responsible for prolonging survival and conferring drug resistance of CLL cells. I therefore independently characterised the development of NLCs using fresh peripheral blood samples from CLL patients and applied a co-culture system where primary CLL cells were cultured with NLCs. I showed that the development of NLCs varies considerably between the individual CLL samples. Consequently, I developed an NLC scoring system to reflect the variable nature of the NLC development. I also confirmed the pro-survival effect of NLCs on CLL cells. To address the issue of variation in developing NLCs, I developed a cell-line model using human THP-1 monocytic leukemic cells to mimic NLCs. The cell line model closely resembled the morphology and phenotype of NLCs. Like NLCs, it also provided pro-survival signals to CLL cells when in co-culture, as THP-1 cell-derived macrophages protected CLL cells from spontaneous and fludarabine-induced apoptosis. Finally, I prepared mRNA samples from the co-cultured CLL cells and primary NLCs, together with their respective cells cultured alone as controls, and generated the comprehensive, global gene expression datasets using next generation sequencing technology (RNA-seq). Through the application of contemporary bioinformatics analysis techniques, I identified 326 out of 19,595 expressed genes that are significantly differentially expressed in co-cultured CLL cells. Gene set enrichment analysis revealed that gene expression profile of CLL cells co-cultured with NLCs resembled that of the tissue resident CLL cells from the lymph nodes, thus validating the use of the co-culture system in mimicking ii the CLL microenvironment. Further analysis of differentially expressed genes led to identification of several signalling pathways that are critically involved in mediating the interaction between CLL cells and NLCs. These include some known pathways such as toll-like receptor and tumour necrosis factor, activation of which all lead to eventual activation of NF-ĸB, the master transcription factor of B cells. Furthermore, my analysis revealed other less well-known signalling pathways such as hypoxiainducible factor that are also involved in cross-talk between CLL cells and NLCs. The novel observations from my study has thus provided a rational basis for future studies to investigate the molecular mechanisms responsible for the functions of these signalling pathways and associated molecules, which may lead to identification of potential targets for therapeutic intervention to overcome microenvironment-mediated drug resistance in CLL.
doi:10.17638/03114800 fatcat:upddrgiuyjcbjktrjw45eesbpq