Characterization of a potent inhibitor of class 1 phosphatidylinositide-3’-kinases (PI3K) in human glioma

A243 Gliomas are primary tumours of the brain. The current treatment options for these tumours following surgery are limited to radiotherapy and/or treatment with DNA damaging agents that result in poor overall prognosis and survival rates. Gliomas frequently exhibit deregulation of PI3K signaling with loss of PTEN, mutations of PIK3CA or activation of EGFR. Therefore, inhibition of deregulated PI3K signaling is a potential therapeutic strategy, and there has been considerable interest in the use of EGFR-inhibitors or analogues of rapamycin for the treatment of these tumours. Here we have characterised the activity of PI103, an inhibitor of class I PI3K signaling (Raynaud et al. (2007) Cancer Res. 67:5840), in a panel of human glioma cell lines. The GI 50 for the panel ranged from 130-528nM. The molecular response to PI103 treatment occurred within 15 minutes and exhibited some similarities to treatment with rapamycin, for example decreased phosphorylation of thr 389 -p70S6K, but also clear differences, such as decreased phosphorylation of thr 308 -Akt, consistent with inhibition of PI3K. PI103 also exhibited activity in vivo against human U87MG glioma xenografts, with a T/C of 45.8% following 14 days of 70mg/kg ip PI103. There was also molecular evidence of modulation of the pathway following in vivo treatment. Treatment with PI103 resulted in a cell cycle arrest, with a robust G1 block and slowing of progression through G2/M. Gene expression profiling detected decreased expression of regulators of G1 and G2/M phases of cell cycle. Immunoblotting confirmed these changes, for example cyclin D1 or Cdc6, which occurred within 6 hours of treatment and preceded the induction of p27 kip1 and decreased phosphorylation of Rb after 12 hours exposure. Treatment with PI103 did not result in apoptosis, but induced the appearance of heavily vacuolated cells that exhibited increased cleavage of LC-3, a marker of autophagy. Consistent with these observations, PI103-treated cells retained the ability to proliferate when recultured in fresh media without PI103. Given the cytostatic response of glioma cells to PI103 treatment, we investigated the effects of combining PI103 with clinically relevant cytotoxic agents used for the treatment of glioma. PI103 was combined with vincristine (anti-microtubule agent), BCNU (bifunctional DNA alkylating agent) or temozolomide (DNA methylating agent approved for treatment of glioma). Median effect analysis demonstrated either additivity or synergy when PI103 was combined with vincristine or BCNU. Significantly, a high degree of synergy was detected when PI103 was combined with temozolomide. PI103 represents a valuable tool for exploring the biological functions of the PI3K family and is a lead for further optimisation of this novel class of molecular therapeutics. There is a clear unmet medical need for new therapies for glioma. Our results demonstrate the ability of PI103 to inhibit the proliferation of glioma cells in vitro and in vivo and significantly to potentiate the effects of commonly used cytotoxic agents. This suggests that similar combinations of this class of PI3K inhibitor with current cytotoxics may have potential for use in the clinic for the treatment of glioma.