Analysis of the kinomic and immune cancer synapse in GBM
Item statusRestricted Access
Embargo end date08/12/2022
Al Shboul, Sofian
A deeper understanding of the signalling networks operating in vivo is needed to develop more physiologically relevant ex-vivo models and support the discovery of new therapeutics in human cancer. An example is human brain cancer where new therapeutics using essentially cancer cell models has failed to deliver therapeutic that work well in vivo. The main objective of this PhD project is to analyse the kinome of GBM tissues versus their derived cancer stem cell lines, then to use this data to identify any missing features of the cell models. This will be linked to the immune landscape of GBM tissues and CDKN2A deletion. Such approach will allow us to develop the most genetically physiologically resembling models, therefore, improving our cancer drug discovery understanding of tumour molecular biology. In this PhD project, I undertook a functional proteomics (kinomics) approach using patient-derived tissue to identify bioactive kinases that are highly expressed in GBM tissue lysates but not in matched patient derived cancer stem cell models. The kinomics screen used a cell-free biochemical assay that measures ATP-dependent peptide-array phosphorylation. The data indicate that patient-derived GBM tumour tissue, rather than tumour-derived GBM cell lines, is a superior source of total kinase activity. Kinomics profiling of 30 GBM tumour and 6 non-tumour brain tissues (adjacent to tumour) revealed that the latter showed kinomic malignant signature and that GBM intratumoural heterogeneity can be observed at the kinome levels. The kinome data indicated that Bruton tyrosine kinase (BTK)-associated activity was identified as a dominant kinase activity in GBM tissue lysates but not in matched patient derived cell lines. Immunoblotting analysis of 16 GBM, two human neural stem cells and a panel of brain tissues included 32 GBM tumours and 6 non-tumour tissues showed that BTK is widely expressed in GBM tissues but not detectable in all examined cell models. Immunohistochemical staining of a 127 patient glioma tumour tissue microarray demonstrated that BTK protein was expressed in at least two distinct cell types within GBM tissue; tumour cells expressing SOX2 and immune cells expressing CD163. However, a larger proportion of BTK-positive cells were SOX2-negative and CD163-negative suggesting a third cell population exists with respect to BTK expression. Since BTK is associated with various immune cells including macrophages, the expression of main immune cells in glioma TMA was examined. I found that CD163 and CD68 macrophages are the most dominant immune cells within GBM TME and the corelate with tumour grade. Additionally, few CD3, CD8 and FOXp3 cells were observed among all grades with no significant change between grades. Using corresponding clinical data, increased BTK protein expression in GBM tissue was correlated with longer patient survival. In stark contrast to reduced survival associated with M2 CD163 TAMs expression. Dual-immunofluorescence microscopy analysis indicated that BTK and SOX2 are co-expressed in approximately 12% of cells within GBM tissue, but the majority of SOX2- positive cells are BTK-negative. These latter data suggest that patient derived cancer stem cell lines, which are BTK-negative, represent the dominant cancer stem cell population within GBM tissue. The data exemplifies the importance of assessing whole tumour tissue when developing and assessing the clinical utility of therapeutic agents such as protein kinases. The emerging use of BTK inhibitors in GBM clinical trials would benefit from understanding the cell-specific expression of BTK within GBM tissue to modify patient stratification and create more effective GBM targeted therapeutics.