Despite the plethora of targeted inhibitors identified and studied preclinically in GBM, appreciable improvements in patient survival have not followed.2
One issue is the plasticity of GBM with its redundant signaling inputs22
and ability to bypass blockade of individual molecules by feedback loops.11,23
Paramount among these is the negative feedback loop by which inhibition of mTOR can trigger a negative feedback loop that results in PI3K/Akt activation. Therefore a combinatorial approach of PI3K inhibition, the common target for multiple RTKs, along with abrogation of mTOR to prevent feedback activation of Akt has been proposed and validated preclinically in multiple tumor types.24–29
XL765 is a novel, orally bioavailable PI3K/mTOR dual inhibitor that can be safely administered with no established maximally tolerated dose in phase I trials.21
In our study, we examined the effects of XL765 in a panel of genetically diverse GBM xenografts ± TMZ, the standard-of-care chemotherapeutic agent in postoperative GBM treatment.1
To our knowledge, this is the first study to evaluate a PI3K/mTOR inhibitor in GBM using multiple, distinct, and characterized xenografts. Given the genetic heterogeneity of GBM, this is an important consideration to identify susceptible genotypes.
Regardless of genetic background, XL765 demonstrated specific inhibition of the PI3K/mTOR pathway (Fig. ). All 5 xenografts showed highly robust inhibition of pAkt (biomarker for PI3K) and pS6 (biomarker for mTOR), with >50% inhibition of phosphorylation within the 2- to 4-µM range. GBM 8 and GBM 12 (both of which are EGFR wt [amplified], p16 null, and MGMT hypermethylated) were relatively resistant to p4EBP1 modulation even at high concentrations of XL765. Expression of unrelated phosphoproteins pp38 and pJNK were also queried and showed no change after exposure to XL765 (data not shown). Similarly, there was no effect on non-phosphoproteins in the PI3K pathway.
Activation of the PI3K pathway has been shown to promote survival-related genes,30
invasion of surrounding brain parenchyma,31
and recruitment of neural stem cells that are pro-invasion.32
Given this central role of PI3K in GBM, it is not surprising that XL765 resulted in in vitro cytotoxicity. Since TMZ is an important component of multimodality GBM treatment, we combined it with XL765 and showed statistically significant chemosensitization in 4 of 5 xenografts. To determine the mechanism of XL765-induced cytotoxicity, we performed several additional in vitro assays, including staining with Annexin V–fluorescein isothiocyanate to quantify apoptosis, propidium iodide staining to measure cell cycle effects, and clonogenic assays to evaluate reproductive cell death (data not shown). These experiments demonstrated equivocal results when XL765-treated cells were compared with controls. This was likely due to the fact that our xenografts were removed from animals and cultured in vitro for short periods of time and not used to establish primary cell cultures, which are more amenable to prolonged in vitro manipulation.
To evaluate the effects of XL765 ± TMZ in vivo, we injected mice intracranially with luciferase-expressing GBM 39 xenografts. We began treatments on day 21 after tumor injection, and our XL765 dosing schedule of 30 mg/kg was given twice per day (Monday–Friday, 6 h apart) for a period of 2 consecutive weeks without evidence of clinical toxicity or significant weight loss. A dosing schedule of 30 mg/kg twice per day was chosen over 100 mg/kg every other day due to preliminary evidence of increased toxicity with the latter dose (data not shown). XL765 showed benefits in growth reduction (~13 fold), survival, and in vivo reduction in pS6 expression by IHC compared with control, differences that were all statistically significant.
In GBMs that have MGMT
hypermethylation, patients have improved outcomes particularly when TMZ is used.33
Thus we were cognizant of the fact that our GBM 39 xenograft model (with MGMT
hypermethylation) would likely be sensitive to TMZ. Despite this, combination of XL765 with TMZ still demonstrated a greater than 10-fold decrease in average tumor bioluminescence compared to TMZ alone. As seen in Figure B, levels of bioluminescence in the combination group were essentially the same as background, indicating very robust tumor regression. While mice in the combination group did have a longer median survival than mice receiving TMZ alone, this did not quite reach the level of statistical significance (p
= 0.09). This is likely due to the MGMT
status of GBM 39, which already contributes to baseline TMZ sensitivity.
In our study we have shown that a combined PI3K/mTOR inhibitor can be successfully utilized against GBM xenografts with a diverse genetic background. We hypothesize that XL765 would be most effective in patients with activating mutations in the PI3K pathway, specifically when EGFR
is activated (through amplification of the wild-type form in GBMs 8, 12, and GS-2 or the vIII variant in GBMs 6 and 39) or PTEN
is homozygously deleted (GBM 8 and GS-2). This hypothesis is based on two lines of reasoning: first, mTOR inhibitors are particularly effective against tumors with increased PI3K activity39
; second, erlotinib, a small-molecule inhibitor of epithelial growth factor receptor (EGFR), is most valuable in tumors with permissive genetic backgrounds that include activating EGFR
The utility of surrogate biomarkers in predicting responses to EGFR inhibitors in prospective clinical trials of GBMs is controversial. In a randomized trial by the European Organisation for Research and Treatment of Cancer comparing progression-free survival (PFS) using TMZ versus erlotinib in recurrent GBM, the activity of the latter could not be predicted by activating mutations in EGFR
or expression of pAkt or PTEN.41
Similar conclusions were reached in 2 other multi-institutional single-arm prospective clinical trials that failed to identify biomarkers that predict response to erlotinib in recurrent GBMs.42,43
In studies in which erlotinib has been used up-front with chemoradiation as part of primary GBM treatment, attempts to identify biomarkers have also resulted in contradictory findings: while a single-arm prospective study by the North Central Cancer Treatment Group44
failed to find a relationship between biomarkers and tumor response, a similar study by UCSF45
found an improvement in overall survival in a subset of patients with MGMT
hypermethylation and intact PTEN
. Though it may be tempting to conclude that failure of biomarkers to predict erlotinib activity in some of these trials implies a lack of benefit of EGFR inhibition in GBMs, there are several non–mutually exclusive alternate hypotheses: (1
) use of a single EGFR inhibitor simply forces cancer cells to adapt by coactivating nontargeted tyrosine kinases,22
) there exists an excess toxicity of erlotinib,46
) erlotinib exists in tumors in subtherapeutic concentration,42
) insufficient patients were in surrogate biomarker groups to reach statistical significance.
The xenograft tested in our in vivo experiment had EGFR vIII
amplification and wild-type PTEN and was indeed relatively sensitive to XL765. We would predict that a xenograft with EGFR vIII
amplification and loss of PTEN would exhibit even greater sensitivity to XL765, and these experiments are ongoing. Furthermore, tumors without PI3K activation (granted, a minority of GBMs) may exhibit less in vivo sensitivity to XL765, and we are testing this hypothesis. Genetic background has bearing on a second issue—that of sensitization of tumors to TMZ. In vitro, inhibition of the PI3K pathway sensitized xenografts to TMZ in nearly all cases; and in vivo, XL765 showed marked sensitization to TMZ in a xenograft with MGMT
hypermethylated. Whether such dramatic sensitization to TMZ will be evident in GBMs without MGMT
hypermethylated is a topic for further studies. Lastly, combining erlotinib with XL765 is intriguing from mechanistic and efficacy viewpoints, particularly given recent clinical data using the former in GBM.34
Based in part on our preclinical data, a multi-institutional phase I trial studying the safety and tolerability of XL765 combined with TMZ is currently accruing.35