TAK-901 – VPS34 inhibitor

Preclinical FLT-PET and FDG-PET imaging of tumor response to the multi-targeted Aurora B kinase inhibitor, TAK-901
Carleen Cullinane a,g,⁎, Kelly L. Waldeck a, David Binns c, Ekaterina Bogatyreva a, Daniel P. Bradley d,
Ron de Jong e, Grant A. McArthur a,b,f,g,1, Rodney J. Hicks a,c,f,g,1
a Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia b Division of Cancer Medicine, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia c Centre for Cancer Imaging, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia
d Millennium Pharmaceuticals, Cambridge, MA
e Takeda California, San Diego, CA
f Department of Medicine, St Vincent’s Hospital, The University of Melbourne, Parkville, Victoria, Australia
g Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia

a r t i c l e i n f o

Article history:
Received 7 October 2013
Received in revised form 1 November 2013 Accepted 11 November 2013

Keywords: FLT-PET FDG-PET
Aurora B kinase TAK-901

a b s t r a c t

Introduction: The Aurora kinases play a key role in mitosis and have recently been identified as attractive targets for therapeutic intervention in cancer. The aim of this study was therefore to investigate the utility of 3′-[18F]ﬂuoro-3′-deoxythymidine (FLT) and 2-deoxy-2-[18F]ﬂuoro-D-glucose (FDG) for assessment of tumor response to the multi-targeted Aurora B kinase inhibitor, TAK-901.
Methods: Balb/c nude mice bearing HCT116 colorectal xenografts were treated with up to 30 mg/kg TAK 901 or vehicle intravenously twice daily for two days on a weekly cycle. Tumor growth was monitored by calliper measurements and PET imaging was performed at baseline, day 4, 8, 11 and 15. Tumors were harvested at time points corresponding to days of PET imaging for analysis of ex vivo markers of cell proliferation and metabolism together with markers of Aurora B kinase inhibition including phospho-histone H3 (pHH3) and senescence associated β-galactosidase.
Results: Tumor growth was inhibited by 60% on day 12 of 30 mg/kg TAK-901 therapy. FLT uptake was significantly reduced by day 4 of treatment and this corresponded with reduction in bromodeoxyuridine and pHH3 staining by immunohistochemistry. All biomarkers rebounded towards baseline levels by the commencement of the next treatment cycle, consistent with release of Aurora B kinase suppression. TAK-901 therapy had no impact on glucose metabolism as assessed by FDG uptake and GLUT1 staining by immunohistochemistry.
Conclusions: FLT-PET, but not FDG-PET, is a robust non-invasive imaging biomarker of early HCT116 tumor response to the on-target effects of the multi-targeted Aurora B kinase inhibitor, TAK-901.
Advances in knowledge and implications for patient care: This is the first report to demonstrate the impact of the multi-targeted Aurora B kinase inhibitor, TAK-901 on tumor FLT uptake. The findings provide a strong rationale for the evaluation of FLT-PET as an early biomarker of tumor response in the early phase clinical development of this compound.

1. Introduction

Inhibition of mitosis has proven to be a highly successful strategy for therapeutic intervention in cancer. The widely used tubulin inhibitors including the vinca alkaloids and taxanes exert their therapeutic effects by destabilizing and stabilizing microtubules, respectively while the more recently developed epothilones function as microtubule stabi-

⁎ Corresponding author at: Division of Cancer Research, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria, Australia. Tel.: +61 3 9656 1275;
fax: +61 3 9656 1411.
E-mail address: [email protected] (C. Cullinane).
1 Contributed equally to the manuscript.

lisers [1–3]. Recent advances in the understanding of the molecular events in mitosis have identified a range of key mitotic proteins with potential for therapeutic targeting, including the Aurora kinases, CENP-E, kinesin spindle protein and polo-like kinase 1 [2–4].
The Aurora kinases are a family of three (Aurora A, -B, -C) serine- threonine kinases that play a key role in the regulation of mitosis. Aurora A and, to a lesser extent, Aurora B are overexpressed or amplified in a wide range of human cancers (reviewed in [5]). While Aurora A is crucial for centrosome maturation, Aurora B is the catalytic subunit of a chromosomal passenger complex essential for accurate chromosome segregation and cytokinesis [6,7]. Since Aurora B acts at multiple points in the mitotic process, its inhibition leads to progression of the cell through mitosis with incorrectly attached

chromosomes and the failure of cytokinesis, which results ultimately in polyploidy and cell death [8].
The Aurora kinases are therefore attractive targets for therapeutic intervention. A range of ATP competitive small molecule Aurora inhibitors has been developed and evaluated in preclinical and clinical studies. These include pan-inhibitors (e.g. PHA-739358, VX-680) and compounds selective for Aurora A (e.g. MLN8054, MLN8237) or Aurora B (e.g. AZD1152) [5,9–11]. TAK-901 is a potent Aurora B inhibitor with activity against a broad range of kinases in vitro, but in the cellular context, TAK-901 displays much greater selectivity for Aurora B compared to other kinases [12]. Consistent with its Aurora B inhibitory activity, TAK-901 induces histone H3 phosphorylation and polyploidy both in vitro and in vivo and exerts antitumor activity against a range of solid tumor and leukemia models in vivo [12].
The development of a robust predictive biomarker of early tumor response to therapy has the potential to accelerate the drug develop- ment process and guide the clinical application of a multi-targeted Aurora B inhibitor such as TAK-901. Molecular imaging techniques such as positron emission tomography (PET) provide a powerful means by which to non-invasively and repeatedly investigate on-target drug induced changes in biological functions within a tumor. Indeed, PET imaging using [18F]-ﬂuorodeoxyglucose (FDG) has demonstrated clinical utility as an early marker of tumor response to molecular targeted agents including imatinib [13,14] and vemerafinib [14]. 3′-[18F] ﬂuoro-3′-deoxythymidine (FLT) PET imaging has shown promise in preclinical studies for monitoring treatment response to molecularly targeted therapies including crizotinib, cetuximab [15,16] and erlotinib
[16] although this was not shown to be superior to FDG clinically [17]. In a preclinical study by Chan et al [18], treatment of mice bearing HCT116 colorectal xenografts with CCT129202, an inhibitor of both Aurora A and B kinase, led to a reduction in tumor FLT uptake. This effect was associated with CCT129202 dependent reduction in expression of thymidine kinase 1 (TK1), the key enzyme responsible for the cellular retention of FLT. These findings provide a strong rationale to investigate the impact of Aurora B inhibition on tumor uptake of FLT. The aim of our study was therefore to investigate the utility of FLT-PET imaging to assess tumor response to Aurora B kinase
inhibition using the multi-targeted inhibitor, TAK-901.

2. Materials and methods

TAK-901 was synthesised by Millennium. Captisol was obtained from Cydex Pharmaceuticals, KS. FLT was prepared in house at Peter MacCallum Cancer Centre as described previously [19] and FDG was purchased from Cyclotek, Victoria, Australia.

2.1. Animal studies

All animal experiments were performed with approval from the Peter MacCallum Cancer Centre Animal Experimentation Ethics Com- mittee and performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 7th Edition. 3 × 106 HCT116 cells in 50% Matrigel were implanted subcutaneously on the ﬂank of 6–8 week old female Balb/c nude mice (Animal Resources Centre, WA, Australia). Once tumors reached a volume of 150 mm3, animals were randomised into three treatment groups of 8 animals and this was defined as day 1. TAK-901 (10 or 30 mg/kg) was given intravenously at 5 mL/kg in 12% Captisol in 25 mM sodium citrate, pH 3.0 twice daily over two days each week for two weeks.

2.2. PET imaging and analysis

Baseline PET scans were performed on day 1 prior to drug treatment. For FLT imaging, mice were injected intravenously with
14.8 MBq FLT and 90 min later anaesthetised using isoﬂurane in 50% oxygen in air. Mice were placed on the bed of a Philips Mosaic PET

scanner and imaged over 10 min. Once all animals had recovered from anaesthetic they were dosed intravenously with vehicle or drug as appropriate. FLT-PET imaging was subsequently performed on days 4, 9, 11 and 15. For FDG-PET imaging mice were fasted for a minimum of three hours before being anaesthetised and intravenous adminis- tered with 14.8 MBq FDG. Anaesthesia was maintained for a further 20 min to minimise skeletal muscle uptake of the tracer. At 90 min post tracer injection the mice were again anesthetised and scanned as described above. FDG-PET imaging was performed on days 1, 4, 8, 11 and 15. PET scans were corrected for decay and random emissions before the images were reconstructed using a 3D RAMLA algorithm. The results were expressed as the maximum Standardised Uptake Value (SUVmax).

2.3. Biomarker studies

Mice were injected with 100 mg/kg bromodeoxyuridine (BrdU) one hour prior to sacrifice. Sections from formalin fixed, paraffin embedded tumors were stained with anti-BrdU antibody (Becton Dickinson, #347580) using standard protocols. Image analysis was then performed using MetaMorph software and the percentage of BrdU positive cells per field was determined in three fields from each tumor. Analysis of samples for phospho-histone H3 (pHH3) and GLUT-1 staining was performed similarly using anti-pHH3 antibody (Upstate, # 06-570) and anti-GLUT-1 antibody (Dako, #A3536).
For β-galactosidase staining, sections were cut from OCT frozen tumors on the day of the assay, fixed in 2% paraformaldehyde/0.2% glutaraldehyde and stained overnight with X-gal according to the manufacturer’s instructions. The sections were counterstained with Nuclear Fast Red and analysed using Metamorph software. Each image was analysed for total pixel area stained blue (therefore positive for β-gal), and for total pixel area stained (total of positive blue area and pink counterstained area). The percent β-gal positive area was then determined as the percent of the total area that was positive for β-galactosidase staining.

2.4. Statistical analysis

Data were analysed using SigmaStat for one-way analysis of variance and Dunnett’s post hoc test or Student’s unpaired t-test as appropriate.

3. Results

3.1. TAK-901 reduces FLT uptake into HCT116 tumors

The HCT116 colorectal cell line was selected for the study on the basis of the previously described activity of the compound in this model [12]. Nude mice bearing HCT116 tumors were randomised into treatment groups before being imaged by FLT-PET. The mice were then treated with vehicle, 10 or 30 mg/kg TAK-901 BID for two days each week and PET scans performed on four subsequent days over two weeks as summarised in Fig. 1a. PET scans were performed at similar times over two treatment cycles, with one scan at the commencement of a treatment cycle and another other, mid-cycle. The effects of TAK-901 on tumor growth are summarised in Fig. 1b. TAK-901 therapy resulted in dose dependent antitumor effects with percent tumor growth inhibition (%TGI) of 21% (P = N.S.) and 60% (P b 0.01) at 10 and 30 mg/kg TAK-901, respectively on day 12. The drug was well tolerated with no significant weight loss observed in any of the treatment groups (Fig. S1).
HCT116 tumors demonstrated robust FLT uptake at baseline which remained reasonably constant in the vehicle treated group over the time course of the experiment (Fig. 1c). In contrast, following treatment with 30 mg/kg TAK-901, tumor FLT uptake declined on days 4 and 11 but partially recovered on days 9 and 15. Tumor FLT

Fig. 1. TAK-901 inhibits HCT116 tumor growth and biomarkers of cell proliferation in vivo. Nude mice bearing HCT116 tumors were treated with vehicle or TAK-901 and subjected to serial FLT-PET imaging. (a) Schema for TAK-901 treatment and PET imaging. (b) Tumor volume was determined using calliper measurements and is expressed as mean tumor volume ± SEM, n = 8. Graphs are shown until the first animal from the group was removed upon reaching the maximum ethically allowed tumor size. Statistical analysis was performed on day 12 (*P b 0.05). (c) Serial FLT-PET maximum intensity projection (upper) and transaxial (lower) images are shown of a representative mouse each from the vehicle and 30 mg/kg TAK-901 groups. Mice were injected intravenously with 14.8 MBq FLT and imaged 90 min later under isoﬂurane anaesthesia for 10 min. The arrows and arrow heads indicate the position of the tumor and bladder, respectively. (d) FLT SUV tumor max for each group was determined and is expressed as the mean ± SEM. (*P b 0.05; **P b 0.001). Tumors were harvested from mice treated with vehicle or 30 mg/kg TAK-901 on days corresponding to PET scanning and analysed using IHC for bromodeoxyuridine (BrdU) staining.
(e) The images from sections stained for BrdU were quantified as described in the Materials and methods and the results expressed as the mean percentage of cells staining positive for BrdU (±SEM; n = 4 animals/group). (*P b 0.05 vs baseline).

uptake was quantified and the results summarised in Fig. 1d show that uptake, as measured by SUVmax, in the vehicle treated mice remained stable over time. In contrast, 30 mg/kg TAK-901 signifi- cantly reduced FLT uptake at all time points tested (P b 0.001). While FLT uptake was reduced to 46% (P b 0.001) on day 4 of cycle 1, on day 9 (following the second dose in the second treatment cycle), tumor FLT uptake had recovered to 26% of the baseline level. FLT uptake again declined to 51% of the baseline level on day 4 of the second treatment cycle (day 11) before once more recovering to 25% immediately prior to the commencement of the third treatment cycle. In the 10 mg/kg TAK-901 treatment group, the time course of reduction and recovery of tumor FLT uptake was similar, but less pronounced than that in the high drug dose group (Fig. 1d). These results are consistent with robust inhibition of cell proliferation early after TAK-901 treatment (days 4 and 11) and that is overcome upon commencement of the following treatment cycle.
To further validate the antiproliferative effects of TAK-901 suggested by the FLT imaging study, a second experiment was performed in which HCT116 tumors were harvested from mice at

time points corresponding to the PET imaging experiment. The tumors were then assayed for incorporation of BrdU by immunohistochem- istry and the results are summarised in Fig. 1e. Cell proliferation, as measured by the percentage of BrdU positive cells, remained relatively consistent in the vehicle treated tumors over the time course of the experiment. In contrast, the percentage of BrdU positive cells was reduced in the TAK901 treated mice on day 4 of each cycle (days 4 and 11) (P b 0.05) and had partially recovered prior to the commencement of dosing for cycles 2 and 3 (days 8 and 15). These results are highly consistent with the FLT-PET imaging findings (Fig. 1d) that suggest TAK-901 exerts immediate antiproliferative effects in HCT116 tumors that are overcome between treatment cycles.
Interestingly, increased FLT uptake into bone marrow was apparent in mice treated with TAK-901 on days 4 and 11 (Fig. 1c). Quantitation of FLT uptake into the proximal humerus region showed no change in uptake in vehicle treated mice over the course of the experiment but significantly (P b 0.05) elevated FLT uptake in the 30 mg/kg TAK-901 treated animals on days 4 and 11 (Fig. S2). These findings correlate inversely with tumor FLT uptake and BrdU

incorporation and suggest normal tissue response to TAK-901 therapy is markedly different from that of HCT116 tumors.

3.2. TAK-901 has no impact on FDG uptake into HCT116 tumors

The effect of TAK-901 therapy on glucose metabolism was investigated using FDG-PET imaging in the HCT116 xenograft model. Tumor bearing mice were treated with vehicle or 30 mg/kg TAK-901 and imaged by FDG-PET at similar time points as the previous study. As observed in the FLT experiment, TAK-901 significantly inhibited HCT116 tumor growth (TGI = 60% on day 16; P b 0.001; Fig. S3). The FDG-PET imaging results demonstrate only modest baseline FDG uptake (mean SUV = ~ 1) in HCT116 tumors and which remained relatively constant in the vehicle treated group over the 15 day time course of the experiment (Fig. 2a). In contrast to its effects on FLT uptake, 30 mg/kg TAK-901 treatment was not

associated with any change in HCT116 tumor FDG uptake (Fig. 2b), suggesting that HCT116 tumor response to the TAK-901 treatment regimen does not impact on glucose metabolism. Correlative biomarker studies were performed to independently validate the FDG-PET findings. Expression of the major glucose transporter, GLUT-
1 was therefore examined at time points corresponding to PET imaging. While HCT116 tumors stained strongly for GLUT-1 expres- sion, the extent of positively stained tumor cells remained constant in both groups over the 15 day time course, consistent with the FDG-PET imaging results (Fig. 2c).

3.3. TAK-901 induces changes in downstream biomarkers of Aurora B kinase inhibition

As histone H3 is a direct substrate of Aurora B kinase we investigated its phosphorylation status following TAK-901 treatment

Fig. 2. TAK-901 therapy does not modulate glucose metabolism in HCT116 tumors. HCT116 tumor bearing mice treated with vehicle or 30 mg/kg TAK-901 were imaged using FDG- PET over 15 days. (a) Representative serial FDG-PET images of vehicle and TAK-901 treated mice. Mice were injected intravenously with 14.8 MBq FDG and imaged 90 min later under isoﬂurane anaesthesia for 10 min. (b) FDG SUV tumor max was determined at each time point and is expressed as the mean ± SEM (n = 8/group). (c) Tumors harvested from mice treated with vehicle or 30 mg/kg TAK-901 on days corresponding to PET scanning were analysed for GLUT1 expression by IHC. Images are shown at ×20 magnification.

Fig. 3. Effects of TAK-901 on cellular markers of Aurora B kinase inhibition. (a) Tumors were harvested from mice at the conclusion of the experiment shown in Fig. 1 at 4 hours following treatment with vehicle or TAK-901 on day 16 (second dose of third treatment cycle). Tumor sections were then analysed by IHC for pHH3 expression and the results expressed as the mean percent positively stained cells ±SEM (n = 8/group). (b) Tumors were harvested from mice treated with vehicle or 30 mg/kg TAK-901 on days corresponding to PET scanning and analysed using IHC for pHH3 expression. The results are expressed as the mean percent positively stained cells ± SEM (n = 4/group). (c) Representative images of tumor sections from vehicle and TAK-901 treated animals stained for β-galactosidase expression on day 8 of treatment. (d) The mean percentage area of viable tissue staining positive for β-galactosidase was determined at each time-point (n = 4/group; mean ± SEM).

in vivo. Histone H3 phosphorylation (pHH3) in HCT116 tumors was performed at an early time point (4 hours) following TAK-901 treatment on day 16 of therapy (second dose of third treatment cycle). As seen in Fig. 3a, there was a dose dependent decrease in pHH3 at this early time point after dosing with 56% inhibition at 30 mg/kg (P = 0.003) compared to vehicle control levels. Analysis of pHH3 staining at time points corresponding to PET imaging revealed a 42% reduction (P b 0.05) on day 4 of TAK-901 treatment (ie 24 hours following the final dose in the first treatment cycle) compared to baseline levels that then rebounded by day 8 (Fig. 3b). The time course of changes in tumor pHH3, although not as pronounced, mirrored the changes in FLT uptake at the same time points. Together, these results suggest that TAK-901 levels were sufficient to inhibit Aurora B early after treatment with 30 mg/kg TAK-901 and are consistent with the time course of TAK-901 exposure in tumor tissue and associated pHH3 suppression pharmacodynamic response [12].
To further explore the differential effects of TAK-901 therapy on glucose metabolism and proliferation identified in the PET imaging studies, we investigated the drug effects on key pathways associated with tumor response to therapy. Tumor samples were analysed for cleavage of caspase 3 as a marker of apoptosis. However, no evidence of caspase 3 cleavage was seen at any of the imaging time points (data not shown). As inhibition of the Aurora kinases has also been associated with induction of accelerated senescence [20,21], we also evaluated tumors for biochemical and morphological markers of senescence. Tumor sections from vehicle and 30 mg/kg TAK-901 treated mice were stained for senescence associated β-galactosidase. The representative images in Fig. 3c show increased β-galactosidase staining in the drug treated compared to the control tumors on day

8 of treatment. The images were quantified for β-galactosidase staining and the results were summarised in Fig. 3d. In the TAK-901 treated tumors, a non-significant increase in cells staining positive for β-galactosidase compared to baseline was observed from day 8 onwards. In contrast, no change was observed over the time course in the vehicle treated tumors. Tumor cells were also examined for morphological changes associated with senescence. Enlarged, multi- nucleated cells with increased DNA content were also apparent in TAK-901 treated, but not vehicle treated tumors, consistent with Aurora B inhibition and senescence phenotype (Fig. S4). Western blots were also performed for changes in the cyclin dependent kinase inhibitor, p21 as an additional marker of senescence. The results shown in Fig. S4 reveal increased staining of p21 in the drug treated groups on all days as compared to baseline. Together, the β- galactosidase positive staining, morphological changes and up- regulation of p21 suggest that TAK-901 induces senescence in HCT116 tumors in vivo.

4. Discussion

In this study we performed preclinical studies to investigate the potential utility of FLT-PET imaging as a non-invasive biomarker of early tumor response to the on-target effects of TAK-901, a novel multi- targeted Aurora B inhibitor that has recently entered Phase I clinical trials. Treatment of HCT116 tumor bearing mice with 30 mg/kg TAK- 901 resulted in substantial inhibition of tumor growth (60% on day 12, Fig. 1) and this was indeed associated with early changes in FLT uptake. A significant reduction in tumor FLT uptake was observed within 2 days of completion of the first TAK-901 treatment cycle (day 4, Fig. 1d).

These findings are consistent with the reported effects of a range of Aurora inhibitors on tumor FLT uptake in the HCT116 tumor model including the Aurora B inhibitor, AZD1152 [22], the Aurora A inhibitor, alisertib [23] and the Aurora A and B inhibitor, CCT129202 [18].
In our study, the reduction in FLT uptake at day 4 was not sustained as demonstrated by the rebound in FLT uptake towards baseline levels observed upon the commencement of the subsequent cycle of TAK-901 treatment. These imaging findings were validated in tumors ex vivo using BrdU staining as a marker of cell proliferation and pHH3 as a marker of Aurora kinase B inhibition. The apparent resumption of cell proliferation between treatment cycles may reﬂect both the ongoing proliferation of cells that escaped Aurora B inhibition and the slow cell cycle progression of endoreduplicated cells generated following their inappropriate exit from mitosis upon Aurora B inhibition.
Our findings also suggest that the dose and schedule of TAK-901 used in the current study did not achieve sustained and potent Aurora B kinase inhibition that may be required for maximal antitumor activity. As with other molecularly targeted therapies, maximal therapeutic benefit of TAK-901 will be achieved using a dose and schedule that leads to sustained pathway inhibition, which in our study is measured by inhibition of histone H3 phosphorylation and indirectly by FLT uptake. As the drug dose and schedule used in the current study were not associated with any toxicity (Fig. S1), a further increase in dose, or more particularly, more frequent dosing should be explored to achieve more prolonged Aurora B inhibition and pronounced antitumor effects. Furthermore, our findings also demonstrate the potential use of FLT- PET imaging as a highly sensitive non-invasive pharmacodynamic marker for optimisation of the dose and schedule of TAK-901 to maximise target inhibition in human clinical trials.
FLT imaging of TAK-901 treated mice also revealed that while tumor FLT uptake declined on days 4 and 11 of TAK-901 treatment, FLT uptake into the bone marrow was consistently increased above baseline at these time points (Fig. 1c and S2). FLT uptake into both tissues then rebounded towards baseline levels prior to commence- ment of a subsequent treatment cycle. Previous studies by Chan et al
[18] showed Aurora kinase inhibition using CCT129202 leads to induction of the cdk inhibitor, p21 which in turn leads to the inhibition of the expression of E2F regulated genes including TK1. While this is consistent with our FLT findings in tumor cells, one possible explanation for the enhanced bone marrow FLT uptake in the absence of TK1 is via effects of the TAK-901 therapy on expression of the nucleoside transporters responsible for FLT uptake into cells. TAK- 901 therapy may delay bone marrow cells in a cell cycle phase where the expression of the equilibrative nucleoside transporter, ENT1 is at basal levels [24,25]. As bone marrow cells express high levels of concentrative nucleoside transporters (CNT) as compared to tumor cells, the net effect of Aurora B inhibition in these cells may be enhanced FLT accumulation due to reduced ENT1 mediated FLT efﬂux [26,27]. However, further investigation of the differential effects of TAK-901 on bone marrow and tumor cells is clearly warranted.
In contrast to the robust reduction in FLT uptake upon HCT116 tumor response to TAK-901, there was no change in glucose metabolism as assessed by FDG uptake or GLUT-1 expression. These imaging findings are consistent with those observed by Moroz et al.
[22] with Aurora B inhibitor AZD1152 in both HCT116 and SW620 xenografts. Furthermore, treatment of MDA-MB-435 tumor bearing mice with the novel paclitaxel formulation, Abraxane resulted in an increase in FDG uptake, despite induction of tumor regression [28]. These unexpected findings are not limited to compounds targeting mitosis since HCT116 tumor response to the topoisomerase I inhibitor, irinotecan is also associated with an increase in FDG uptake but a reduction in FLT [29].
The basis of the maintenance of FDG uptake in tumors responding to this broad range of therapies remains to be elucidated. It has been speculated that the response may reﬂect FDG uptake by inﬂammatory cell infiltrate in tumors responding to therapy, increased DNA repair

or apoptosis [29]. In our study there was no evidence of increased tumor infiltration by inﬂammatory cells (data not shown) or apoptosis in TAK-901 treated tumors. The tumors did however demonstrate a phenotype consistent with the induction of accelerated senescence, as observed with other Aurora kinase inhibitors [20,21]. As senescent cells undergo irreversible cell cycle arrest these cells are expected to demonstrate low FLT uptake but as they remain viable, maintain a requirement for glucose for their ongoing metabolism. While the senescent cell phenotype is consistent with the FLT and FDG imaging phenotypes observed in response to TAK-901 therapy (maintenance of metabolism but inhibition of proliferation), such cells make up only a very small proportion of the tumor (2–3%) and are hence unlikely to fully account for the FDG-PET results.

5. Conclusion

The FLT-PET imaging findings from our study are consistent with the antitumor effects of the multi-targeted Aurora B kinase inhibitor, TAK-901 being mediated through its inhibition of Aurora kinase B. Further support of the on-target activity of TAK-901 is demonstrated by the ex-vivo biomarker studies, in particular, the reduction in phosphorylation of the Aurora B kinase substrate, Histone H3 and the lack of effect of TAK-901 on tumor FDG uptake, an indirect marker of cell viability. The development of non-invasive biomarkers for early clinical development of targeted agents such as TAK-901 may facilitate decision making and accelerate their clinical progression. Given the low FLT signal to noise ratio observed in human tissue compared with that in rodents however, careful consideration of the tumor site chosen for the clinical evaluation of FLT in monitoring response to Aurora B inhibitors would be essential. The findings of this preclinical study demonstrate that FLT-PET but not FDG-PET is a valuable biomarker of early tumor response to the Aurora kinase B inhibitor, TAK-901 and supports its further evaluation for response assessment in the clinical development of this compound.

Conﬂict of interest

This work was supported by a grant received by Grant McArthur and Rodney Hicks from Millennium Pharmaceuticals. Grant McArthur is an uncompensated consultant for Millennium Pharmaceuticals. Daniel Bradley and Ron de Jong are employees of Millennium: The Takeda Oncology Company. The remaining authors have no compet- ing interests.
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.nucmedbio.2013.11.001.

Acknowledgments

We thank Susan Jackson and Alison Slater for technical support.

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