Abstract 5399: Inhibition of the PI3K/mTOR pathway potentiates irradiation effects in paediatric glioblastoma inducing metabolic alterations detected by MRS.
Authors: Agliano Alice , Balaragah Geetha , Ciobota Daniela M. , Raynaud Florence l. , Clarke Paul A. , Jones Chris , Workman Paul , Pearson Andrew D. , Leach Martin O. , Al-Saffar Nada M.S. 
Published: August 2014

Although radiotherapy is used to treat more than 50% of all cancer patients, in glioblastomas the percentage of success is limited due to the radioresistance of glioma cells. Reports have shown that part of this phenomenon has to be attributed to the deregulation of the PI3K/mTOR pathway (AC Begg, Nat Rev, 2011). Furthermore, p110α isoform specific PI3K inhibitors produced additive cytotoxic effects in combination with radiation therapy in in vitro and in vivo glioblastoma models (Chen JS et al. MCT. 2008). Based on this, we have decided to study if the treatment with the dual PI3K/mTOR inhibitor NVP-BEZ235 potentiates the effects of the irradiation and whether the combination would result in metabolic changes that can be evaluated by MRS and hence may be utilized as non-invasive biomarkers for the detection of response to combination therapy during early stage clinical trials in children with glioma.

For this purpose we treated the paediatric high-grade glioma SF188 cell line with a low dose of X-ray irradiation (5 Gy) and NVP-BEZ235 (2xGI50, 28nM) both as single treatments and in combination. Our results showed a higher decrease in the number of treated cells with the combination (P&lt;0.002) than with the single treatments alone (P&lt;0.01) compared to the control.

MRS spectra from NVP-BEZ235 treated cells showed a significant (P&lt;0.01) decrease in the levels of Lactate (Lac), phosphocholine (PC) and total choline (tCho). In contrast, irradiation resulted in an increase in PC, glycerolphosphocholine and tCho (P=0.001). The combination showed similar metabolic effects to NVP-BEZ235 single treatment, including a decrease in the levels of Lac and PC.

As expected, we observed inhibition of the PI3K/mTOR pathway. Protein expression levels of pAKT (Ser473) and pS6RP (Ser240/244) in NVP-BEZ235 and combination treated cells were decreased. Irradiation induced-apoptosis was detected by increased levels of cleaved PARP in cells treated with X-ray or combination but not with NVP-BEZ235 alone.

We also explored the causes of metabolic changes detected by MRS. Both in the NVP-BEZ235 and combination treatments, the decrease in PC levels was associated with a decrease in the expression of ChoKα, the enzyme responsible for choline phosphorylation into PC. Furthermore, a decrease in the levels of the glycolytic enzymes HKII and LDH-A was detected following NVP-BEZ235 treatment and the combination. These enzymes were not affected by X-ray alone.

We have shown that inhibition of the PI3K/mTOR pathway increases the cytotoxic activity of the irradiation in paediatric glioblastoma. MRS allows monitoring of changes occurring during the combination treatment, suggesting that PC and Lac may be used as non-invasive biomarkers to detect response to PI3K/mTOR inhibition in combination with radiotherapy.

Citation Format: Alice Agliano, Geetha Balaragah, Daniela M. Ciobota, Florence l. Raynaud, Paul A. Clarke, Chris Jones, Paul Workman, Andrew D. Pearson, Martin O. Leach, Nada M.S. Al-Saffar. Inhibition of the PI3K/mTOR pathway potentiates irradiation effects in paediatric glioblastoma inducing metabolic alterations detected by MRS. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5399. doi:10.1158/1538-7445.AM2013-5399