[18 F]FDG-PET imaging is an early non-invasive pharmacodynamic biomarker for a first-in-class dual MEK/Raf inhibitor, RO5126766 (CH5126766), in preclinical xenograft models
© Tegnebratt et al.; licensee Springer. 2013
Received: 3 July 2013
Accepted: 8 September 2013
Published: 16 September 2013
Positron emission tomography (PET) with [2-18 F]-2-fluoro-2-deoxy-D-glucose ([18 F]FDG-PET) was acquired at multiple time-points a) to monitor the early response to RO5126766 (CH5126766) in xenograft models b) to evaluate non-invasive small animal [18 F]FDG-PET imaging as a biomarker for MEK inhibitors for translation into dose-finding studies in cancer patients and c) to explore the underlying mechanism related to FDG uptake in tumors treated with RO5126766.
[18 F]FDG uptake was studied in HCT116 (K-ras), COLO205 (B-raf) mutants and COLO320DM (wild type) xenografts from day 0 to 3 of RO5126766 treatment using a microPET Focus 120 and complemented with in vitro incubations, ex-vivo phosphor imaging and immunohistochemical (IHC) analyses.
In the HCT116 (K-ras) and COLO205 (B-raf) mutant xenografts, significant decreases in [18 F]FDG uptake were detected in vivo on day 1 with 0.3 mg/kg and ex vivo on day 3 with 0.1 mg/kg RO5126766. [18 F]FDG changes correlated with decreases in tumor cells proliferation (Ki-67) and with changes in expression levels of GLUT1. No effects were observed in drug resistant COLO320DM cells. The cellular fractionation and Western blotting analyses suggested that the change of [18 F]FDG uptake associated with RO5126766 is due to translocation of GLUT1 from membrane to cytosol, similar to the results reported in the literature with EGFR tyrosine kinase inhibitors, which also target the MAPK pathway.
RO5126766 inhibition resulted in a rapid time - and dose - dependent decline in [18 F]FDG uptake in both mutant xenografts. These results strongly resemble the clinical observations obtained with MEK/Raf inhibitors support the use of preclinical [18 F]FDG-PET as a translational tool for decision support in preclinical and early clinical development of MEK inhibitors.
The Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated (ERK) cascade transmits signals from the cell surface receptors to the nucleus and regulates cell cycle progression, cell proliferation, survival, differentiation and transformation. The genetic mutations in many of the components in this pathway have been found to be associated with cancers. The Ras/Raf/MEK/ERK pathway has a well-defined role in cancer biology and has become an important target in the development of cancer therapeutics [1–3]. Many drugs targeting the ligand-activated receptor tyrosine kinases and their downstream effectors such as Ras, Raf and MEK are currently being tested in clinical trials [4–7].
A major drawback in the clinical testing of the new drugs is the lack of pharmacodynamic biomarkers at early stage clinical trials. Non-invasive imaging techniques have demonstrated a potential for accelerating the drug development process by assessing therapeutic response and early identification of responders [8–10]. Positron emission tomography (PET) imaging with the fluorine-18 labeled glucose analog 2-fluoro-2-deoxy-D-glucose ([18 F]FDG-PET) is increasingly being included as a new functional endpoint in phase I to III clinical trials in oncology, in addition to conventional endpoints such as toxicity and decreases in tumor size. Many of these clinical studies have reported that [18 F]FDG-PET imaging can be successfully used for monitoring the efficacy of a range of targeted therapies [11–13].
MEK has a critical position in the Ras/Raf/MEK/ERK pathway with few direct upstream activators (e.g. Raf) and few downstream targets (e.g., ERK). The successful development of MEK inhibitors and their evaluations in various clinical trials is well summarized in recent reviews [14, 15]. [18 F]FDG-PET imaging has been included as a therapeutic read-out in several of these studies [16–18]. PET/CT imaging was also used as a primary therapeutic endpoint for sorafenib, inhibitor of Raf kinase activity .
Despite these applications of [18 F]FDG-PET as an efficacy biomarker for MEK inhibitors in humans, very few preclinical studies have been reported. Early studies demonstrated instead the utility of the thymidine analog 3’-deoxy-3’-18 F-fluorothymidine (FLT) for therapeutic monitoring of the MEK inhibitor PD0325901 in a V600E B-raf mutant SK-MEL-28 melanoma model [8, 20]. [18 F]FDG-PET was used to evaluate inhibitors of PI3K/AKT/mTOR and epidermal growth factor receptor (EGFR) pathways either alone or in combination with a MEK inhibitor. For instance, PET/CT together with magnetic resonance imaging demonstrated the synergistic effects of NVP-BEZ235, a dual PI3K/mTOR inhibitor, and ARRY-142886 (AZD6244/selumetinib), an allosteric MEK inhibitor, on K-ras mutated tumor in a genetically engineered mouse model of lung adenocarcinoma .
The value of using [18 F]FDG-PET as an early surrogate marker has been demonstrated in several preclinical models. Studies in xenografts sensitive to gefitinib, an EGFR tyrosine kinase inhibitor (EGFR-TKI), revealed up to a 55% decrease in [18 F]FDG uptake within 48 hours after start of treatment . [18 F]FDG-PET could also be a surrogate marker for the efficacy of erlotinib, another EGFR-TKI, in preclinical human head and neck carcinoma models  and of the c-KIT inhibitor, imatinib, in models with activating c-KIT mutations in gastrointestinal stromal tumors (GISTs). Preclinical PET imaging revealed that [18 F]FDG uptake in tumors sensitive to the drug was significantly reduced as early as 4 hours after imatinib treatment while no response was observed in resistant tumors .
The main goal of our study was to explore whether the effects of MEK/Raf inhibitors in humans revealed with [18 F]FDG-PET could be replicated in animals and whether [18 F]FDG-PET can therefore be used in preclinical models as an endpoint for early detection of therapeutic activity and dose-finding studies for this class of inhibitors. For this purpose, we have used RO5126766, a first-in-class orally active and highly selective dual protein kinase inhibitor, specific for Raf and MEK. RO5126766 is a novel chemical class allosteric inhibitor of MEK activity and prevents MEK from phosphorylation by Raf through stable Raf-MEK complex formation. RO5126766 inhibits ERK signalling more effectively that a standard MEK inhibitors. It suggests a new therapeutic approach for ras tumors by blocking feedback activation of ERK signalling . RO5126766 has shown potent in vivo anti-tumor efficacy in diverse human tumor xenografts models and has recently been evaluated in a phase I dose-escalation study in humans in which [18 F]FDG-PET was included as one of the biomarker assessments [18, 25]. Our results show that in vivo [18 F]FDG-PET imaging of preclinical tumor models can be used to successfully monitor therapeutic response to MEK inhibition.
Cell culture and reagents
The human colon cancer cell lines HCT116, COLO205 and COLO320DM were purchased from the American Type Culture Collection (ATCC). All cells were maintained in the designated media and indicated concentrations of heat-inactivated fetal bovine serum (Gibco) and L-glutamine (Sigma) according to the ATCC recommendations. Cells were grown at 37°C in an atmosphere of 5% CO2. RO5126766 (CH5126766) was synthesized in Chugai Pharmaceuticals Co., Ltd. For in vitro and in vivo studies, the drug was dissolved in DMSO (Wako Chemicals GmbH) to yield a 2.5 mg/mL stock solution concentration and stored at -20°C. The solutions of RO5126766 used for in vitro and in vivo experiments were freshly prepared on each experimental day. The vehicle and RO5126766 stock solutions were diluted 1:20 with the diluent (10.5% aqueous solution of 2-hydroxypropyl-β-cyclodextrin (Celdex HP- β-CD, HPCD, Sigma)) on each dosing day.
[18 F]FDG uptake in vitro
[18 F]FDG uptake was determined in untreated HCT116, COLO205 and COLO320DM cells as well as treated with vehicle only as a control or RO5126766 at indicated concentrations. 1×105 cells/well were seeded in 6-well plates (Costar®) together with appropriate doses of RO5126766 for indicated times. Cell culture medium was changed to glucose-free and 0.37 MBq of [18 F]FDG was added to each well and incubated for 1 hour in 5% CO2 atmosphere at 37°C. The cells were washed three times with ice cold PBS and radioactivity was measured using a 1480 Automatic gamma counter Wizard3 (Perkin Elmer). [18 F]FDG incorporation was determined and expressed relative to total protein concentration. Protein content was determined using the Thermo Scientific Pierce BCA Protein Assay Kit (Waltham, USA).
Cellular fractionation and Western blotting
Plasma membrane fractionation was performed using a membrane protein extraction kit from BioVision. According to the manufacturer's recommendations, 5×108 HCT116 cells were used for protein extraction. The purity of the plasma membrane protein fraction was assessed by Western blot analysis of the plasma membrane marker (mouse monoclonal antibodies to Na+, K+-ATPase from Abcam). For Western blot analysis, 15 μg of plasma membrane-associated or of the cytosol proteins were separated on 4-12% poly-acrylamide gels (Invitrogen) and Western blotting was performed. The rabbit polyclonal antibodies to glucose transporters GLUT1 and GLUT3 were from Abram (Cambridge, UK). (Secondary HRP-linked anti-mouse and anti-rabbit IgG were from Cell Signaling (In Vitro Sweden AB, Stockholm, Sweden). Bands were visualized with Western blotting Luminol Reagent (Santa Cruz). The images were captured using a LAS-1000 from Fujifilm or exposed to X-ray film (Fujifilm, Tokyo).
Xenograft tumor models
Female athymic nude (nu/nu) mice, age 5–6 weeks (18-22 g) were purchased from Scanbur AB (Sollentuna, Sweden). Animal care, handling and health monitoring were carried out in accordance with the Guidelines for Accommodation and Care of Laboratory Animals. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care committee.
For the tumor xenografts, 5×106/mouse human cancer colon carcinoma cells were inoculated subcutaneously in the right flank of Balb-nu/nu mice. Once tumors were established (150–200 mm3), mice were randomized into groups with similar mean tumor volumes at the start of the study. Tumor volume and body weight were measured two times per week. Tumor volumes were determined with digital caliper using the formula (tumor length × width 2 )/2. Tumor growth inhibition (TGI) was calculated using the following formula: TGI = [1 − (T − T 0 )/(C − C 0 )] × 100, where T and T 0 are the mean tumor volumes on a specific experimental day and on the first day of treatment, respectively, for the experimental groups and likewise, where C and C 0 are the mean tumor volumes for the control group. The daily administration of RO5126766 was performed orally at doses 0.1, 0.3 and 1.0 mg/kg. The doses were selected based on the results of preliminary studies. The maximal tolerated dose (MTD) was defined as the maximum dose associated with <20% weight loss and no toxic deaths. The MTD in the three xenograft models was 1.5 mg/kg for RO5126766.
MicroPET imaging was performed by standard protocols as described previously . Mice were fasted for 6–8 hours prior to start of imaging session . [18 F]FDG (obtained as an aliquot from daily clinical productions at Karolinska University Hospital, 7–8 MBq per mouse, maximum volume of 200 μl) was administered to awake, warmed (37°C) mice by a bolus injection via the tail vein. Forty to sixty minutes after the tracer injection, the mice were anaesthetized with isoflurane, controlled by an E-Z anaesthesia vaporizer (5% initially and then 1.5% to maintain anaesthesia, blended with 7:3 air/O2 and delivered through a Microflex non-rebreather mask from Euthanex Corporation, Palmer, PA). The mice were placed on a heating pad (37°C) on the camera bed, with most of the body in the field-of-view (7.68 cm). Emission data were collected for 20 minutes in list mode with MicroPET Focus 120 scanner (CTI Concorde Microsystems). Data were processed using MicroPET Manager (CTI Concorde Microsystems). PET data were acquired in fully three-dimensional (3-D) mode and images were reconstructed by standard 2-D filtered back projection using a ramp filter. The matrix size of the reconstructed images was 128 × 128 × 95 with a spatial resolution of 1.3 mm. Data were corrected for randoms, dead time and decay. Standard Uptake Values (SUV) were calculated for 3D regions of interest (ROI), using Inveon Research Workplace software (Siemens Medical Solutions). Tumor ROIs drawn on the images employed a 75% threshold of the maximum intensity voxel. The ROI counts were normalized to the injected dose and body weight and converted to SUV. The drug effect on tumor metabolism was estimated as%SUVmax change day 1, 2, or 3 compared to day 0 (baseline).
Ex-vivo phosphor imaging
Immediately after the last MicroPET scan, the animals were sacrificed and their tumors were rapidly frozen. Tumor slices (20 μm thickness) were obtained using a cryomicrotome (CM 3050S, Leica Microsystems, Wetzlar, Germany) and were placed on Superfrost Plus microscope slides (Menzel-Glaser, Germany). The sections were placed in a BAS exposure cassette with a 2325 imaging plate (Fujifilm Corporation, Japan) and exposed for at least 1 hour. The quantitative autoradiography data (photo-stimulated luminescence, PSL, unit/mm2) were normalized to the injected dose and body weight.
Tumor slices were obtained as described above. Frozen tumor slices sections were kept at −80°C until needed. For IHC, frozen sections (20 μm) were air dried and fixed for 10 minutes with ice-cold acetone. The slides were incubated in 2.5% normal horse blocking serum in PBS before primary antibodies, rabbit polyclonal to Ki-67 (Abcam), were added followed by incubation for 1 hour. As the secondary antibody, ImmPRESS™ reagent, anti-rabbit Ig, peroxidase (Vector) was used. The staining’s were visualized by using an ImmPact™ DAB substrate kit (Vector). The counter stain was performed in methyl green for 2 minutes at room temperature. The images were analyzed with an Olympus UC30 digital color camera (Olympus Color Management Technology) and Cell Imaging software for Life Sciences microscopy.
Statistical significance was examined by Student t test. P-values less than 0.05 were considered a statistically significant difference.
Effects of RO5126766 on cellular determinants of the uptake and retention of [18 F]FDG
In order to identify the cellular components determining the uptake and retention of [18 F]FDG in these cell lines, the expression levels of glucose transporters (GLUTs) and hexokinases were analyzed by Western blotting. GLUT1 was detected in all cell lines. GLUT3 was expressed in COLO320DM and HCT116 but not in COLO205 cells. Hexokinase II was expressed in all cell lines (Figure 1d). To further examine possible mechanisms behind the RO5126766-induced changes in [18 F]FDG uptake, we used the HCT116 cell line because of its higher basal glucose utilization. We detected significant decreases in the expression of the cellular transmembrane protein GLUT1 in the plasma membrane fraction after 24 hours of treatment with 1.3 μM of RO5126766, compared to the vehicle treated cells. In parallel, an increase of GLUT1 in the cytosol fraction was observed during treatment (Figure 1e). We did not detect significant changes in hexokinase II activity during the treatment of HCT116 cells with 1.3 μM of RO5126766 for 24 hours (Figure 1f).
Anti-tumor activities of RO5126766 and FDG-PET imaging results in human colon carcinoma xenografts in balb nu/nu mice
[18 F]FDG uptake was measured in tumors of mice treated with RO5126766 at 0.1, 0.3 or 1.0 mg/kg versus vehicle from day 0 (baseline) to day 3. PET imaging revealed no significant effect on [18 F]FDG uptake in COLO320DM tumors during the treatment (Figure 2d). In contrast, RO5126766 treatment HCT116 (K-ras) tumors demonstrated significant decrease in metabolic activity on day 3, compared to day 0 (p < 0.05 at 0.3- and 1.0 mg/kg doses and vehicle (p < 0.01). No significant effect was observed in the 0.1 mg/kg dosing group (Figure 2e). Despite the low basal [18 F]FDG-uptake in COLO205 tumor xenografts, we could detect inhibition by RO5126766 at both 0.3 mg/kg (p < 0.05) and 1.0 mg/kg (p < 0.05) doses. Representative coronal PET images in Figure 2g-i demonstrate the FDG uptake observed in COLO320DM (wt) tumors, HCT116 (K-ras) and COLO205 (B-raf), respectively, at days 0 and 3 of treatment with vehicle versus RO5126766 (only 1.0 mg/kg dose shown).
Phosphor imaging of [18 F]FDG uptake and immunohistochemistry in excised tissue sections
Histopathology in RO5126766 treated mice
In this study we have demonstrated the feasibility of using [18 F]FDG-PET imaging as an early surrogate endpoint for monitoring biological and anti-tumor activity of MEK/Raf inhibitors given for the treatment of human cancers. We first used in vitro experiments to investigate whether RO5126766 effects on sensitive tumor cells would be accompanied by changes in the uptake of labeled glucose. We found that [18 F]FDG uptake was reduced in a dose (0.3-1.3 μM) - and time (0-48 h) - dependent manner in HCT116 and COLO205 tumor cells carrying K-ras and B-raf mutations, respectively, whereas RO5126766 did not affect [18 F]FDG uptake in COLO320DM cells, which has no mutation in these two genes and no apparent levels of phospho-MEK and phospho-ERK in the cells. Both mutant tumor cell lines demonstrated metabolic sensitivity to the drug, confirming their feasibility for [18 F]FDG-PET imaging of RO5126766 efficacy. However, in vitro results showed variations in basal [18 F]FDG-uptake among three cell lines, with the lowest levels observed in COLO205.
The transport of glucose through the cell membrane via glucose transporter proteins and its subsequent intracellular phosphorylation by hexokinases are key steps required for its cellular accumulation . The expression levels of glucose transporters and hexokinases are changed in many cancers [29, 30]. Chung et al.  suggested that increased numbers of glucose transporters at the plasma membrane of cancer cells may be a cause of increased [18 F]FDG uptake, at least in colon cancers. Yun et al.  reported that GLUT1 expression levels were consistently upregulated and that glucose uptake was enhanced in K-ras and B-raf mutated cells compared to wild type cells. Drug-induced changes in [18 F]FDG uptake together with the expression levels of GLUTs and hexokinases in tumor cells may therefore serve as good predictors for how well [18 F]FDG-PET can be used for monitoring response in vivo in xenografts from a particular cell line. We observed that GLUT1 expression levels decreased in the plasma membrane and increased in the cytosol fractions of HCT116 cells treated with RO5126766. These results are indicative of a RO5126766-induced translocation of GLUT1 from the plasma membrane to the cytosol, which could be a possible mechanism behind the observed reductions in [18 F]FDG uptake in the drug-treated cells. Similar translocation effects on glucose transporters have been reported for the EGFR inhibitors, gefitinib  and erlotinib .
This study shows that in RO5126766-sensitive cells MEK and Raf inhibition results in a rapid decrease in [18 F]FDG uptake. In contrast, in COLO320DM resistant cells (no detectable pMEK and pERK levels, suggesting no activation of RAF and MEK), RO5126766 did not affect the glucose uptake. These results support the applicability of FDG-PET as a pharmacodynamic biomarker for MEK/Raf inhibitors.
In vivo imaging revealed significant reductions in [18 F]FDG uptake as early as after 1 day of treatment with 0.3 mg/kg of RO5126766 in both HCT116 and COLO205 xenografts (37% and 43% , respectively). The FDG change paralleled but preceded the drug-induced reductions in xenograft sizes. In HCT116 tumors the [18 F]FDG uptake was increasingly reduced over time (for example, at the dose of 0.3 mg/kg, for 17% at day 1, 19% at day 2 and 35% on day 3) and exposure-dependent, showing a decrease from baseline on day 3 (range from 97 to 52% at the doses range 0.1-1.0 mg/kg) compared to an increase in vehicle treated group (126%). These observations are consistent with reports elsewhere of early decreases in [18 F]FDG uptake for mTOR inhibition in experimental lymphoma model , and for combined PI3K/mTOR (PF-04691502) and MEK (PD-0325901) inhibitors in a K-ras G12D ; Pten mutated mouse model of ovarian cancer .
Partial volume effects associated with imaging tissues close in size to the 1–2 mm resolutions of small animal PET and the heterogeneous nature of tumors including varying amounts of necrosis and non-tumor tissue can affect in vivo quantifications . In order to account for these limitations and further validate the results obtained, we used PET imaging combined with ex vivo phosphor imaging to evaluate minimal effective doses and times for RO5126766 efficacy in the tumor xenografts. With an order of magnitude higher resolution, ex vivo phosphor imaging can serve as a useful single time point complement to the longitudinal in vivo information obtained from small animal PET imaging . Using ex vivo phosphor imaging, reductions in [18 F]FDG uptake could also be detected even for the lowest administered dose (0.1 mg/kg) on day 3 of treatment in HCT116 tumors. We also observed high [18 F]FDG uptake in necrotic-free tumor fractions of vehicle treated mice, compared to low uptake in tumors of the drug treated animals. Thus ex vivo tissue sampling was helpful in defining the dose- and time- dependency while using microPET to examine MEK inhibition at selected doses over time.
[18 F]FDG uptake reductions in drug-treated tumors correlated with decreased number of proliferating cells in RO5126766 treated tumors measured with Ki67. This is in agreement with recently published studies suggesting that cellular proliferation and metabolism are tightly linked processes that share common regulatory pathways in tumour cells . It has been shown [38, 39] that some oncoproteins (such as Ras, c-Myc, Akt) participate in the control of cancer cell metabolism. Therefore, in addition to the metabolic studies with [18 F]FDG, we also investigated the proliferation status of the tumor cell lines during treatment with RO5126766. In this study, we observed significant decreases in the number of proliferating cells in HCT116 tumors upon exposure to the drug, which supports also the use of proliferation PET tracers such as [18 F]FLT  for evaluating the anti-proliferative activity of RO5126766.
Both EGFR-TKIs, for which [18 F]FDG-PET as already demonstrated utility in monitoring efficacy , and MEK/Raf inhibitors are targeting the MAPK pathway. EGFR inhibitors block the initiation of the pathway at the upstream receptor site while MEK/Raf inhibitors block pathway signaling at one of the effector sites downstream from the receptor. In our study, we observed similarities between the effect obtained with the MEK/Raf inhibitor and the one reported in the literature with EGFR inhibitor. In both cases drug treatment was associated with a reduction of FDG-PET uptake and in both cases this was accompanied by a translocation of GLUT1 from the plasma membrane to the cytosol. The similarity observed between MEK/Raf and EGFR inhibitors provides further evidence that cellular glycolytic metabolism as measured by the uptake and retention of [18 F]FDG provides an effective downstream pharmacodynamic read-out for therapeutic strategies targeting inhibition of signaling components of the MAPK pathway.
These preclinical studies were performed in parallel with phase 1 dose escalation clinical studies of the dual inhibitor, RO5126766, in patients with locally advanced and/or metastatic solid tumors without specific genotype. The reduction in FDG uptake observed in the current pre-clinical study mimics the results observed clinically. In both studies, the decrease in FDG uptake was dose dependent with similar overall reduction in FDG uptake (approximately 35% on day 3 in drug-sensitive xenograft models (HCT116, COLO205) compared to 28% on day 15 in patients with melanoma ).
Our preclinical PET imaging studies support the use of [18 F]FDG-PET imaging as an early pharmacodynamic biomarker in preclinical studies of MEK and Raf inhibitors, with strong decreases in SUVmax observed as early as 24 hours post treatment. The decrease in [18 F]FDG uptake was dose-dependent and increased with treatment exposure, therefore strongly paralleling and supporting the observations obtained with this class of compounds in patients [18, 25]. The effect in [18 F]FDG uptake in vitro was more rapid in B-raf mutant cell line COLO205, reflecting the increased sensitivity of B-raf mutated tumors to MEK inhibition. Data obtained by cellular fractionation and Western blotting suggest that the change of [18 F]FDG uptake associated with MEK inhibition might be due to translocation of GLUT1 from membrane to cytosol. A future study, using preclinical dynamic [18 F]FDG-PET imaging and kinetic parameters analysis in response to RO5126766 treatment and its correlation with our in vitro findings would be very interesting.
The authors thank Dr.Yasushi Tomii, preclinical leader of the RO5126766 project in Chugai Pharmaceutical Co., Ltd for providing the background information for all in vivo studies of this drug.
This project was performed in the framework of the Roche Postdoc Fellowship Program and was mentored and financially supported by Hoffman-La-Roche. Additional financial support from the Karolinska Institutet, Swedish Research Council, the Swedish Governmental Agency for Innovation Systems and the Swedish Foundation for Strategic Research is gratefully acknowledged. The authors thank the production unit of the Neuroradiology Department at the Karolinska University Hospital for the delivery of the radiotracers and the staff of the Department of Comparative Medicine for skilled assistance and advice in the animal handling.
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