Abstract
BACKGROUND
Patients with glioblastoma are confronted with a high likelihood of recurrence and poor prognosis despite an aggressive treatment-regime involving surgery followed by radiation therapy (RT) and temozolomide (TMZ). RT and TMZ cause extensive DNA damage and replication stress, thus activating tumour cell death pathways. Upregulation of the DNA repair mechanisms significantly reduces effective treatment response and contributes to poor patient outcomes. We investigated the effect of inhibiting ATM- and Rad3-Related protein (ATR), a crucial sensor of replication stress and initiator of cell cycle arrest in tumour cells, using the potent and selective ATR inhibitor, gartisertib.
MATERIAL AND METHODS
Twelve patient-derived glioblastoma cell lines were grown as monolayer cultures in serum-free media and treated with TMZ, RT and/or gartisertib. Cell viability of treated cells was assessed after a 7-day incubation using the MTT method, while cell confluence, apoptosis and cell death were examined using the Incucyte S3 Live-Cell Analysis System (Sartorius, Germany). Gene expression of glioblastoma cell lines treated with TMZ+RT and/or gartisertib was assessed 4-days post-treatment.
RESULTS
As a single agent, gartisertib potently reduced glioblastoma cell viability, while 8-fold less potent in human astrocyte cells. Glioblastoma cell lines with mutated DNA damage response-related genes were more sensitive to gartisertib treatment. Live-cell imaging of glioblastoma cells treated with gartisertib (1 µM) plus TMZ (35µM) and RT (2Gy) showed a significant increase in cell death and apoptosis compared to gartisertib or TMZ+RT alone. ATR inhibition by gartisertib strongly synergised with TMZ, while moderately synergising with RT in reducing glioblastoma cell growth. Gartisertib, alone and in combination with TMZ+RT, increased gene expression in pro-inflammatory cytokines, antigen presentation, and pattern recognition pathways while decreasing genes involved in hypoxia and epithelial-mesenchymal transition pathways.
CONCLUSION
These data suggest the potential for ATR inhibition as an effective chemo- and radiosensitiser in glioblastoma tumours. Whether the change in gene expression induced by ATR inhibition reduces hypoxia within or provokes an immunogenic response directed at glioblastoma tumours, requires further investigation.