To assess the effect of inhibition of AKT using GSK2141795 on the phosphorylation of the downstream target PRAS40 in vivo, mice bearing SKOV3 xenografts were treated with 30 mg/kg GSK2141795, and after 5 hours animals were sacrificed, tumours harvested and the levels of total and pPRAS40 were determined in each sample by ELISA.
In addition mice bearing xenograft tumours were treated with combination of 1.5 mg/kg cisplatin and 30 mg/kg GSK2141795 or only cisplatin. When two drugs were combined, animals were treated with GSK2141795 two hour prior to the receiving of the cisplatin. Tissues were harvested 3 hours after the dosing with cisplatin and total protein was extracted from each tumour. Level of pPRAS40 in each sample was determined using ELISA.
Levels of phosphorylated PRAS40 were significantly reduced in SKOV3 xenografts following treatment with either GSK2141795 as a single agent (p<0.01) or in combination with cisplatin (p<0.05; Figure 4.12). This effect was not observed when cisplatin was used as a single agent in the xenografts, and therefore suggests that FDG-PET is a useful PD biomarker for AKT inhibition even in the context of combination cisplatin treatment.
138 vehicle cd dp GSK2 1417 95 cddp + GS K214 1795 0.0 0.5 1.0 1.5
**
#*
P R AS 4 0 (p T 2 4 6 )/ to ta l P RA S 4 0 %Figure 4-12 Concentration-dependent effect of AKT inhibition by GSK2141795 on phospho-PRAS40 (Thr246) level in SKOV3 xenografts
GSK2141795 (30 mg/Kg) abolished phosphorylation of PRAS40 at Thr246, both as single agent and in combination with cisplatin (cddp), in SKOV3 tumour xenografts, indicating the effective inhibition of AKT by GSK2141795 (mean of each treatment group is normalised to the mean of the vehicle group. n= 5 tumours). Data are presented as a phospho-PRAS40 / total PRAS40 decrease relative to untreated samples. Data are shown as the mean ± SEM for n = 5 animals (tumours) . *p<0.05 and **p<0.01, where the symbols * and # represent significant differences when compared to vehicle and cisplatin data, respectively.
139
4.3 Discussion
Findings described in chapter 3 suggested the potential ability of GSK2141795 to be used in combination with cisplatin to restore sensitivity to platinum in ovarian cancer patients. However, combining anti-cancer agents in the clinic is not always easy; doses of drugs used in combination often have to be reduced compared to when they are given as mono- therapies. Yet it is not always clear that the reduced dose will have the same effects on the target pathway, i.e. what is the minimal level of pathway inhibition required to maintain efficacy in a given situation. Ideally, quantification of pathway inhibition would be done with serial biopsies of a patient’s tumour before and after drug treatment, in order to evaluate levels of phosphorylated AKT substrates. However, clinical implementation of this approach is challenging due to the reluctance of many patients to undergo repeat biopsies, lack of biopsiable tumours in all patients and tumour heterogeneity amongst other reasons. Therefore having a way to measure pathway inhibition in a non-invasive fashion would be very helpful to this process.
Positron emission tomography (PET) with [18F]fluorodeoxy glucose (FDG-PET) evaluates cancer cells glycolysis as a surrogate for tumour response to chemotherapy. Two independent studies in 2002 and 2003 showed that early changes in FDG-PET signal can be used to predict imatinib response in gastrointestinal stromal tumours (Van den Abbeele and Badawi 2002, Stroobants, Goeminne et al. 2003). These findings led to much interest in using FDG-PET imaging as a predictive marker of response in development of novel cancer drugs.
Mechanistically, AKT activation causes increased transcription and plasma membrane localisation of Glucose Transporter 1 (GLUT1), a protein which facilitates transport of
140
glucose across the plasma membrane of mammalian cells. Membrane bound GLUT1 is an important mediator of FDG uptake in most of cancer cells (Brown, Goodman et al. 2002, Plas and Thompson 2005). Immunohistochemical studies have revealed decreased GLU1 staining in both plasma membrane and cytosol following PI3K/AKT/mTOR pathway inhibition in pancreatic tumour cells indicating that AKT inhibition inactivated GLUT1 transcription (Ma, Jacene et al. 2009). In addition, immunohistochemical studies on pancreatic tumours from patients treated with the mTOR inhibitor, temsirolimus, indicated a higher cytosolic to plasma membrane fraction of GLUT1, suggesting that PI3K/AKT/mTOR pathway inhibition disrupts the translocation of GLUT1 to plasma membrane (Ma, Jacene et al. 2009). AKT also regulates the translocation of Glut4 to the plasma membrane. Given the role of AKT in the regulation of glucose metabolism, [18F]FDG-PET could serve as a powerful tool in assessing the effectiveness of AKT inhibitors in tumour cells.
The preclinical FDG-PET studies described in this chapter provide the justification for using FDG-PET to evaluate the effects of GSK2141795 on tumour glucose metabolism in the clinic, as well as the confidence that inhibition of glucose metabolism correlates strongly with AKT inhibition. Identical FDG-PET studies were performed using SKOV3 cells as (1) monolayers, (2) MTS and (3) in vivo xenografts. This allowed for comparison of the data obtained with the three models in order to determine the inter-predictability of each model and the relationship between changes in phospho-PRAS40 and FDG uptake in various models. Data described in this chapter shows that although phospho-PRAS40 levels were decreased by >50% in all 3 model systems, only MTS and in vivo xenografts showed >50% decrease in FDG uptake, suggesting that MTS may be a better in vitro representation of in vivo physiology than monolayer cultures. The decreases in FDG uptake and phospho- PRAS40/total PRAS40 were strongly positively correlated. Given that PRAS40 phosphorylation is a validated PD biomarker of AKT inhibition by GSK2141795, this finding
141
suggests that FDG uptake can also be used as a PD biomarker for GSK2141795 mediated target inhibition.
Although the decrease in FDG uptake by GSK2141795 was observed in both 2D and 3D in vitro models used in this study, the effectiveness of the compound was shown to be greater in the 3D model and more closely resembled the effects observed in vivo. This observation suggests that 3D models may be more suitable, efficient and predictive models than 2D models in determining dose and schedule optimisation prior to in vivo experiments. This may be important in minimising the cost of the studies and the use of animals in translational approaches.
The MTS studies also demonstrated a concentration-dependent decrease in FDG imaging signal following GSK2141795 administration, which was reproduced in xenograft studies. These data, coupled with the PK data obtained from xenograft studies, predict that concentrations ≥1 μM GSK2141795 would be required to determine the maximal decrease in phospho-PRAS40 and FDG uptake.
In summary, these results demonstrate that the pan-AKT inhibitor, GSK2141795, inhibits AKT signalling in platinum-resistant ovarian cancer cells lines in vitro and in vivo, and that [18F]FDG uptake could be used as a non-invasive pharmacodynamic marker for guiding dose selection in ovarian cancer patients in future studies. These results found the rational for a phase I clinical trial of GSK2141795, an open label study to investigate the pharmacokinetics and pharmacodynamics of repeat escalating doses of the oral AKT inhibitor GSK2141795 by 18F FDG PET analysis in subjects with ovarian cancer (www.clinicaltrials.gov).
142