Analysis of the kinomic and immune cancer synapse in GBM
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Date
08/12/2021Author
Al Shboul, Sofian
Metadata
Abstract
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.