Overcoming hypoxia mediated resistance to radiotherapy-PARP inhibitor combinations in glioblastoma.

Background: Glioblastoma (GBM) is a cancer of unmet need; average survival is approximately 12 months. The only efficacious treatments are DNA damaging agents: radiotherapy and temozolomide. Overexpression of DNA damage response proteins in GBM correlates with radioresistance and poor prognosis. GBM are also hypoxic, which exacerbates radioresistance and worsens outcomes.

The Vens and Chalmers laboratories have led preclinical and early clinical development of poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) as radiosensitizers.

PARPi increase radiosensitivity in GBM models and clinical studies have shown both olaparib and veliparib to be well-tolerated in combination with radical radiotherapy in GBM patients. To date, however, there is no evidence that PARPi improve efficacy.

Addition of veliparib to radiotherapy and adjuvant temozolomide did not improve survival in MGMT unmethylated GBM, and randomised data for olaparib are awaited.

One potential barrier is hypoxia, which is highly prevalent in GBM and associates with radioresistance and poor outcomes.

Giaccia recently showed that moderate hypoxia (2% oxygen) promotes resistance to PARPi, with tirapazamine restoring sensitivity.

Hypoxia mediated PARPi resistance was attributed to reduced induction of reactive oxygen species (ROS) and diminished DNA damage.

Since the radiosensitising effects of PARPi involve elevated ROS and DNA damage we hypothesised that moderate hypoxia would similarly reduce the radiosensitising activity of PARPi in GBM.

Higgins has shown that mitochondrial targeting agents like atovaquone can combat hypoxia and increase radiosensitivity by inhibiting oxygen consumption. Atovaquone is clinically approved and exhibits excellent brain penetration.

Aim: We will evaluate the ability of atovaquone to reduce hypoxia and overcome resistance to the radiosensitizing effects of PARPi in hypoxic, clinically relevant models of GBM.

Methods: In vitro studies will utilise 2- and 3-dimensional cultures of patient-derived GBM cell lines (G7, E2, R10, Ox5); in vivo work will use our clinically relevant orthotopic G7 xenograft model.

Mechanistic studies will measure effects of atovaquone and olaparib on oxygen consumption rate (Seahorse), hypoxia, ROS and DNA damage (immunofluorescence); radiosensitizing effects will be measured by clonogenic survival and spheroid growth delay.

In vivo, hypoxia will be measured by in vivo imaging (oxygen-enhanced and glucose CESL-MRI) and validated by immunohistochemistry.

Efficacy studies will evalute all combinations of atovaquone, olaparib and radiotherapy in the G7 model, using established SARRP protocols for micro-irradiation and symptom-free survival as endpoint.

How results will be used: Mechanistic and efficacy data will inform development of an early phase clinical trial in patients with newly diagnosed, hypoxic GBM.