Our studies using primary glioma cultures from PTEN-deficient and PTEN-intact PDGF-B driven mouse GBMs demonstrate that PTEN status influences mTOR's sensitivity to Akt inhibition with perifosine. When PTEN is intact, mTOR appears to be AKT independent. Our data agrees with recent studies in EGFR driven gliomas models that show mTOR can be regulated by the PKC pathway independent of Akt phosphorylation 
. Gliomas are one of the many tumor types where selectively inhibiting key signaling pathways appears to be a moving target. Recent studies have shown that in response to therapy, gliomas undergo critical changes in their molecular circuitry, such as loss of key tumor suppressor proteins, the selection for kinase-resistant mutants, and the deregulation of feedback loops 
. Our model supports the view that the presence or absence of a single regulatory component, such as PTEN, is enough to shift the signaling network, even in very similar tumors. However, tumors treated with a combination of perifosine and CCI-779 had dramatically decreased the levels of pAkt and pS6RP independent of PTEN status.
Synergistic effects between rapamycin and LY294002, an PI3K inhibitor that acts upstream of Akt, are commonly observed in vitro 
, but confirming those results in clinically relevant in vivo
models has proved to be more difficult. Since perifosine was able to penetrate the BBB in previous mouse glioma studies 
, we were able to follow up on the effective synergy we observed in our in vitro
culture studies and investigate the effectiveness of this combination in PTEN-intact and PTEN-deficient PDGF-B-driven Ink4a-ARF-deficient gliomas in vivo
. Five days of in vivo
treatment with perifosine resulted in variable but not statistically significant decreases of pAkt levels. Similarly, perifosine alone caused a variable and not statistically significant increase cell death. Since in cell culture, perifosine decreased pAkt levels dramatically and in a dose-dependant manner (data not shown), a possible explanation of the lack of statistically significance in vivo
could be as a result of low drug concentrations in the tumor due to the BBB. To further investigate this possibility, we treated tumor-bearing mice with a single dose of 120 mg/kg perifosine, and immunoblot analysis showed a statistically significant decrease in pAkt levels at 3 and 6 hours (data not shown). This further supports the hypothesis that, in our model, perifosine levels are limited by the BBB, and that we might be able to overcome this complication with an increased dose of perifosine. However, these elevated doses of perifosine were not well tolerated by the animals.
It is also worth addressing that, unlike the results observed in culture, Akt inhibition caused by perifosine alone did not have a drastically different effect when comparing the PTEN +/+ and PTEN −/− tumors. One possible explanation for this discrepancy is that the degree of Akt inhibition in vivo was much less dramatic than that observed in culture. When performing experiments in culture, we have precise control over both the concentration of the drug and the length of exposure to the drug. These parameters cannot be fully reproduced in vivo. While the exact cause for the discrepancy between these findings is a potential area for future study, it was encouraging that significant Akt inhibition was achieved when combined with CCI-779, further supporting the rational for using the combination of these inhibitors.
In contrast to perifosine, CCI-779 was much more consistent as a single agent, suggesting the BBB was not a complicating factor for this compound. This correlates with human clinical data, were CCI-779 has been shown to effectively penetrate the BBB of patients with recurrent glioblastoma 
. Treating animals for 5 days with 40 mg/kg decreased pS6RP levels in tumors independent from PTEN status, and the decrease in pS6RP corresponded with a decreased cellular proliferation rate. Of note, this effect was more dramatic in PTEN intact tumors, as demonstrated by both PCNA and Ki67 staining. While gliomas treated with CCI-779 in vivo
did show decreased pS6RP, they did not show increased pAkt levels. Interestingly, our results did not completely reproduce data observed by Cloughsey et al 
where fourteen recurrent PTEN null GBMs were biopsied both before and after treatment with rapamycin, in which half of the patients had no change in Akt activity and half had a slight increase in Akt activity. Although the human GBMs were selected based on PTEN status, they were not selected based on their molecular subgroup status. Recent studies have clearly shown at least three distinct subtypes of GBM, and these subtypes have very different molecular and signaling profiles
. By contrast, our study specifically focuses on PDGF-driven gliomas in the hope to better understand how the human PDGF-subgroup will respond to therapy. However, we do not believe we can compare our results in modeling PDGF-driven glioma to all subtypes of glioma, and thus we cannot compare our results directly to the previous human clinical trials.
When the two drugs were combined in vivo for 5 days, both pAkt and pS6RP levels were significantly decreased independent of PTEN status. This coincided with a striking decrease in cellular proliferation and a marked increase in cell death, although the relative decrease in proliferation and increase in cell death was greatest in PTEN null tumors. Therefore, we utilized PTEN-deficient tumors and MR-imaging to monitor the dynamics of the tumor response in real time by monitoring in vivo changes in cellularity and apparent diffusion value (ADC) changes and imaging characteristics. MRI allowed for tumor staging and thus gliomas could be entered into the study at a similar size, providing an opportunity to follow changes in tumor volumes over time for each of the four animal cohorts. In , the quantified diffusion values are provided as ADC values and were overlayed on the corresponding anatomical image in order to visualize the spatial variations of the changes within the tumor mass. If tumor cell killing occurs during treatment intervention, ADC values will increase in those regions of the tumor affected. What specifically shows is that the ADC values progressively increased from controls to Perifosine to CCI to combination therapy, as visualized by the increased intensity represented by the red regions in the image. As the ADC values are a reflection of the rate of cellular removal, these results indicate a progressive loss of cell viability over these 4 cohorts of animals.
It is interesting to note that tumor volume continued to gain during the treatment period, despite the marked increase in diffusion values. Since the gliomas had a rapid proliferation rate, it may be possible that the rate of cell removal, as evidenced by the increased ADC values (red regions in the color images), never quite overcame the rate of cellular proliferation, even in the combination treatment group. In the current study, changes in ADC values appear to correlate well with the reduction of tumor growth rates and serve as an indication of the efficacy of the treatment. Although overall tumor volume did not shrink, ADC values proved to be an exquisitely sensitive measurement for comparing different treatments, and this imaging biomarker can be easily implemented in clinical translation of promising therapies. We would anticipate that future treatments capable of producing tumor mass shrinkage would be reflected as an even greater response in tumor ADC values, and that this strategy would serve as a foundation on which to non-invasively monitor novel treatment protocols. Thus, DW-MRI may provide an opportunity to assess early treatment response in patients treated with agents targeting the PI3K/Akt/mTOR pathways.
In conclusion, Our preclinical data strongly supports the notion that the best clinical potential for inhibiting the PI3K/Akt/mTOR will be reached when multiple components of the pathway are inhibited and that DW-MRI may be used as an early imaging response biomarker to provide quantitative and spatial information related to tumor cell death during treatment administration. Furthermore, our studies represent the murine portion of a co-clinical trial where the human part of the trial is currently being run at Memorial Sloan Kettering Cancer Center [NCT01051557]. The trial is recruiting adults with recurrent gliomas to be treated with CCI-779 and perifosine as salvage therapy. Therefore, further insight as to how these two inhibitors work together, specifically in a PDGF-driven glioma model, is of significant importance to understanding the potential impact on the human disease.