Date Published: July 13, 2017
Publisher: Public Library of Science
Author(s): Nada M. S. Al-Saffar, Alice Agliano, Lynley V. Marshall, L. Elizabeth Jackson, Geetha Balarajah, Jasmin Sidhu, Paul A. Clarke, Chris Jones, Paul Workman, Andrew D. J. Pearson, Martin O. Leach, Daniel Monleon.
Recent experimental data showed that the PI3K pathway contributes to resistance to temozolomide (TMZ) in paediatric glioblastoma and that this effect is reversed by combination treatment of TMZ with a PI3K inhibitor. Our aim is to assess whether this combination results in metabolic changes that are detectable by nuclear magnetic resonance (NMR) spectroscopy, potentially providing metabolic biomarkers for PI3K inhibition and TMZ combination treatment. Using two genetically distinct paediatric glioblastoma cell lines, SF188 and KNS42, in vitro1H-NMR analysis following treatment with the dual pan-Class I PI3K/mTOR inhibitor PI-103 resulted in a decrease in lactate and phosphocholine (PC) levels (P<0.02) relative to control. In contrast, treatment with TMZ caused an increase in glycerolphosphocholine (GPC) levels (P≤0.05). Combination of PI-103 with TMZ showed metabolic effects of both agents including a decrease in the levels of lactate and PC (P<0.02) while an increase in GPC (P<0.05). We also report a decrease in the protein expression levels of HK2, LDHA and CHKA providing likely mechanisms for the depletion of lactate and PC, respectively. Our results show that our in vitro NMR-detected changes in lactate and choline metabolites may have potential as non-invasive biomarkers for monitoring response to combination of PI3K/mTOR inhibitors with TMZ during clinical trials in children with glioblastoma, subject to further in vivo validation.
Glioblastomas (grade IV astrocytomas) are very aggressive tumours and are one of the leading causes of brain tumour-related deaths in children . Although these tumours are morphologically similar to malignant gliomas that arise in adults, increasing evidence indicates that the molecular pathways activated in brain neoplasms in children substantially differ from those in adults [2–6]. Despite these molecular differences, both adult and childhood malignant gliomas are generally treated similarly post-operatively, with the Stupp regimen of concomitant radiotherapy and temozolomide (TMZ) followed by adjuvant TMZ . However, the published paediatric literature suggests that TMZ may be less effective in high-grade astrocytomas in children compared to adults [8–10]. Recent studies have investigated the differential mechanisms of resistance to TMZ in a series of paediatric cell lines [11, 12]. Results from one study  showed that, as in adults, in the majority of paediatric glioblastoma cell lines TMZ resistance was linked to a lack of promoter methylation of the gene encoding the repair protein DNA methyltransferase MGMT (O6-methylguanine-DNA-methyl-transferase). However, in glioblastoma cells not expressing MGMT, resistance to TMZ was shown to be associated with a PI3K–mediated HOX/stem cell gene signature, and this resistance was reversed by inhibition of the PI3K signalling pathway using the dual pan-class I PI3K/mTOR inhibitor PI-103 [13–15]. Thus, combination of TMZ with PI3K inhibition may provide one therapeutic strategy for those children who tumours either have innate resistance to, or which acquire resistance to, TMZ treatment.
It is generally well established that combinations of anti-cancer treatments offering complementary mechanisms and synergistic pharmacodynamic interactions can be more effective in anti-cancer therapy than single-target approaches [31, 32]. The PI3K-AKT-mTOR pathway is activated in both adult and paediatric glioblastoma, and contributes to resistance to the standard-of-care chemotherapeutic agent TMZ [12, 33, 34]. Increasing evidence suggested clinical benefits are obtained by combining TMZ with inhibitors of the PI3K-AKT-mTOR pathway for the treatment of adult and paediatric glioblastoma ([12, 35] and references therein). Identification of non-invasive biomarkers of target inhibition and potentially of tumour response to this novel combination treatment would be of value in the clinical development of combination treatments, in particular for the treatment of childhood brain tumours, where repeated biopsy post-administration of a new treatment is typically too invasive and therefore not routinely carried out. We investigated whether a combination of the dual pan-Class I PI3K/mTOR inhibitor PI-103 [13–15] with TMZ in paediatric glioblastoma cell lines would show changes in their metabolic profiles that could be detected with NMR. Furthermore, we aimed to identify mechanisms underlying the detected metabolic changes.