Poster Presentation NSW State Cancer Conference 2023

Inhibition of ATR synergistically enhances cell death in combination with temozolomide, radiation and lomustine in glioblastoma cells (#314)

Mathew Lozinski 1 2 3 , Nikola Bowden 1 2 , Moira Graves 1 2 3 , Michael Fay 1 2 3 4 , Bryan Day 5 , Brett Stringer 6 , Paul Tooney 2 3 7
  1. School of Medicine and Public Health, University of Newcastle, Callaghan, NSW, Australia
  2. Drug Repurposing and Medicines Research Program, Hunter Medical Research Institute, New Lambton, NSW, Australia
  3. Mark Hughes Foundation Centre for Brain Cancer Research, University of Newcastle, Callaghan, NSW, Australia
  4. GenesisCare, Gateshead, NSW, Australia
  5. QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia
  6. College of Medicine and Public Health, Adelaide, SA, Australia
  7. School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW, Australia

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. In the recurrent setting,  lomustine (CCNU) is given as the standard of care, however there is limited clinical evidence of its survival advantage for the majority of glioblastoma patients. Upregulation of DNA repair mechanisms significantly reduces effective treatment response and contributes to poor patient outcomes. We investigated the effect of inhibiting ataxia telangiectasia and rad3-related protein (ATR), a crucial sensor of replication stress and initiator of cell cycle arrest in tumour cells, using the ATR inhibitors BAY1895344 (elimusertib) and AZD6738 (ceralasertib).

Methods: In silico blood-brain barrier (BBB) prediction models (DeePred-BBB[1], AdmetSAR[2], CNS MPO score[3] and Brain Penetration Predictor[4]) were used to identify the potential BBB penetrance of BAY1895344 and AZD6738. Known BBB penetrant chemotherapy drugs (TMZ, CCNU and carmustine) and the ATM inhibitor, AZD1390, were used as positive controls. Patient-derived glioblastoma cell lines (n=12) were grown as monolayer cultures in serum-free media and treated with TMZ, RT, CCNU and/or ATR inhibitor (elimusertib or ceralasertib). Endpoint images were taken after a 7-day incubation using the IncucyteⓇ SX5 Live-Cell Analysis System (Sartorius, Germany). Cell viability (%), cell death (%) and apoptosis (%) were quantified using the IncucyteⓇ Cell-by-Cell Analysis Software Module (Sartorius, Germany). Synergy analysis of ATR inhibitor combinations was performed using the synergyfinder module[5] in R.

Results: In silico BBB modelling revealed that BAY1895344 and AZD6738 had consistent and favourable BBB penetrance scores across the different models, especially for BAY1895344. As a single agent, BAY1895344 potently reduced glioblastoma cell viability in low nanomolar ranges, while AZD6738 was significantly less potent. Across the varying treatment doses, ATR inhibition using BAY1895344 synergised with TMZ, RT and CCNU in reducing glioblastoma cell viability. Notably, such combinations had significantly stronger synergy in enhancing cell death and apoptosis.

Conclusion: These data suggest the potential for ATR inhibition as an effective chemo- and radiosensitiser in glioblastoma tumours.

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