AbstractPediatric diffuse intrinsic pontine glioma (DIPG), an incurable, aggressive childhood malignancy, arises in a region- and age-specific nature. The underlying pathophysiology suggests dysregulation of postnatal neurodevelopmental processes causing aborted cell differentiation. The cell of origin is as yet unclear, but recent data suggests an oligodendrocytic lineage. This is supported by the over-expression of transcription factors such as Olig1 and Olig2 in 80% of DIPG cases. Results from in-depth bioinformatics and principal component analysis (PCA) of genes involved in brain development and DIPG led us to explore GPR17. GPR17, an orphan G protein-coupled receptor has been identified in a number of physiological and pathological processes, such as oligodendrocyte differentiation, spinal cord injury and brain injury. Bioinformatic analyses of publicly available mRNA datasets (GSE26576), indicated a significant up-regulation of GPR17 in DIPG patients compared to normal brainstem and normal brain . PCA analysis shows GPR17 clusters closely to Olig1 and Olig2. GPR17 expression was confirmed in cell lines (VUMC-DIPG-A, VUMC-DIPG-08 and SU-DIPG-IV) at the mRNA level using RT-qPCR and the protein level using Western blot, IHC and Flow Cytometry.
To understand the role of GPR17 in our DIPG cells, we used HAMI3379, an experimental drug identified as an antagonist for GPR17 has been shown to work via blocking GPR17 and promote oligodendrocyte development. Treatment with HAMI3379 causes a reduction in GPR17 expression, mRNA and protein, resulting in a change in cell morphology, an increase in O4 and nuclear localisation of CREB. This resulted in a reduction in sphere size, cell motility, cell velocity and self-renewal capacity. When DIPG cells had pre-treatment with HAMI3379 in combination with known cytotoxic agents, there was a decrease in cell viability compare to cytotoxic alone.
In conclusion I have provided data to support my hypotheses that GPR17 is over expressed by DIPG cell lines and by altering expression levels using a small molecule agonist (MDL 299,51) and antagonist (HAMI3379). The result included altered cell genotype and phenotype. The inactivation or blocking of GPR17 caused the DIPG cells to be more sensitive to potential therapies.
|Date of Award
|Helen Fillmore (Supervisor)