Assessment of the In vivo Antitumor Effects of ENMD-2076, a Novel Multitargeted Kinase Inhibitor, against Primary and Cell Line–Derived Human Colorectal Cancer Xenograft Models

Purpose: This in vivo study was designed to investigate the efficacy of ENMD-2076, a small-molecule kinase inhibitor with activity against the Aurora kinases A and B, and several other tyrosine kinases linked to cancer, including vascular endothelial growth factor receptor 2, cKit, and fibroblast growth factor receptor 1, against murine xenograft models of human colorectal cancer (CRC).

Experimental Design: HT-29 CRC cell line xenografts were treated with either vehicle or ENMD-2076 (100 or 200 mg/kg) orally daily for 28 days. Tumor growth inhibition, dynamic contrast-enhanced magnetic resonance imaging, and ¹⁸FDG-positron emission tomography were conducted to assess the antiproliferative, antiangiogenic, and antimetabolic responses, respectively. Effects on proliferation were also analyzed by immunohistochemical methods. Additionally, three patient-derived xenografts from primary and metastatic sites were treated with ENMD-2076 (100 mg/kg) and assessed for tumor growth inhibition.

Results: In the HT-29 xenograft model, ENMD-2076 induced initial tumor growth inhibition followed by regression. Treatment was associated with significant tumor blanching, indicating a loss of vascularity and substantial reductions in tumor vascular permeability and perfusion as measured by dynamic contrast-enhanced magnetic resonance imaging. Positron emission tomography scanning showed significant decreases in ¹⁸FDG uptake at days 3 and 21 of treatment, which was associated with a marked reduction in proliferation as assessed by Ki-67. All three of the patient-derived xenografts tested were sensitive to treatment with ENMD 2076 as measured by tumor growth inhibition.

Conclusions: ENMD-2076 showed robust antitumor activity against cell line and patient-derived xenograft models of CRC that is detectable by functional imaging, supporting clinical investigation of this agent in CRC. Clin Cancer Res; 16(11); 2989–98. ©2010 AACR.

This article is featured in Highlights of This Issue, p. 2919

Colorectal cancer (CRC) is the third most prevalent cancer type in men and women in the United States, accounting for 10% of estimated new cases, and is the third leading cause of cancer-related deaths in men and women (1). Current management of CRC includes systemic treatment combinations of 5-fluorouracil, oxaliplatin, or irinotecan chemotherapy with biologically targeted drugs such as inhibitors of the epidermal growth factor receptor or vascular endothelial growth factor (VEGF) pathways (2). Although multiple large clinical trials are under way to evaluate the most efficacious means of combining and sequencing these agents, advanced CRC remains incurable with an estimated 50, 000 deaths per year in the United States (1). Additionally, cytotoxic agents in particular have potentially serious side effects such as neutropenia and diarrhea with irinotecan, or debilitating peripheral neuropathy with oxaliplatin (3). Therefore, new agents and approaches to treatment are needed.

The Aurora kinases are a family of highly conserved serine-threonine kinases that have integral functions in mitotic cell division (4, 5). Mammalian cells express three isoforms of Aurora kinases: Aurora A (AurA), Aurora B (AurB), and Aurora C (AurC). Although most cells express AurA and AurB, AurC seems to be restricted primarily to the testes, in which it plays a role in spermatogenesis (6). During mitosis, AurA regulates centrosome formation, duplication, spindle assembly, and microtubule-kinetochore interactions, as well as chromosome condensation, alignment, and segregation (7–10). AurB is critical for accurate chromosomal segregation, cytokinesis, correct microtubule-kinetichore attachments, and regulation of the mitotic checkpoint (11–13). Given these essential roles in the proper execution of various mitotic events and the maintenance of genomic integrity, it is not surprising that the dysregulation of AurA and AurB has been linked to genomic instability, tumorigenesis, tumor progression, and poor prognosis in a wide variety of cancer types (4, 5, 14, 15). AurA is located on chromosome 20q13.2, a region commonly amplified in several malignancies, and overexpression of AurA has been reported in CRC, as well as in, ovarian, pancreatic, breast, bladder, and skin cancers (16–22). Although AurB is not typically amplified at the chromosomal level, increased mRNA and protein expression has been reported in tumors including CRC (23) and exogenous overexpression of AurB in Chinese hamster ovary cells results in chromosome segregation defects and increased invasiveness in vivo (24). Taken together, these observations indicate that aberrant Aurora kinase expression results in cellular genomic instability and tumorigenesis in a wide variety of malignancies, making them attractive targets for anticancer therapy (4, 14, 25, 26).

ENMD-2076 (EntreMed, Inc.) is a novel, orally bioavailable multitargeted antiangiogenic and Aurora kinase inhibitor. Its targets include VEGF receptor 2 (VEGFR2/KDR), fibroblast growth factor receptor 1 (FGFR1), src, c-Kit, Flt-3 as well as AurA and AurB, with much greater potency against AurA (Table 1). In this respect, ENMD-2076 represents a new class of targeted cancer therapy that exerts its effects through multiple mechanisms of action, including the inhibition of growth factor signaling pathways, inhibition of angiogenesis, and antimitotic activity. ENMD-2076 has shown preclinical activity against in vitro models across a variety of cancer types including CRC-derived cell lines with IC₅₀s in the nanomolar range. In vivo studies of 28-day continuous oral dosing with ENMD-2076 showed an acceptable and manageable toxicology profile suggesting a potential use for this drug in a variety of solid and hematologic human cancers (EntreMed Clinical Investigator's Brochure).

Recent studies have shown that dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) and 18-fluoro-2-deoxy-d-glucose positron emission tomography (¹⁸FDG-PET) may provide functional imaging end points for responders earlier in the course of targeted treatment therapy before alterations in tumor size can be detected (27, 28). Therefore, the overall goal of the current study was to rigorously assess the antitumor and imaging-based pharmacodynamic effects of ENMD-2076 against the well-defined human CRC xenograft model, HT-29, with the intent of preclinically validating feasible biomarkers in preparation for clinical studies. In addition, we sought to determine whether the antitumor efficacy observed in the HT-29 cell line xenograft model could be recapitulated in three patient-derived, KRAS mutant, CRC xenografts.

ENMD-2076 (2-(phenylvinyl-4-[4-methylpiperazin-1-yl)]-6-(5-methy-2H-pyrazol-3-yl-amino)-pyrimidine L(+) tartrate salt) was provided by EntreMed, Inc. For in vivo studies, this compound was prepared as a suspension in sterile-filtered water by brief vortexing followed by sonication for 10 minutes.

The human colon cancer cell line HT-29 (Kras wild-type, p53 mutant) was obtained from the American Type Culture Collection and was cultured according to American Type Culture Collection recommendations. Cells were maintained in DMEM (Cellgro Mediatech) supplemented with 10% fetal bovine serum (Invitrogen), 1% nonessential amino acids (Cellgro Mediatech) at 37°C under an atmosphere of 95% O₂, 5% CO₂. The cells were routinely screened for the presence of Mycoplasma (MycoAlert, Cambrex Bio Science). Before use in xenograft studies, all cells tested Mycoplasma free. The HT-29 cell line was tested and authenticated by the University of Colorado Cancer Center DNA Sequencing and Analysis Core. DNA from HT-29 cells was analyzed using the Profiler Plus kit (Applied Biosystems). The cell line DNA profile data was compared with the American Type Culture Collection data to ensure authenticity. Verification of the HT-29 cell line was last done in September 2009.

Female athymic nude (nu/nu) mice, ages 4 to 6 weeks, were purchased from the National Cancer Institute. Mice were housed at the University of Colorado Center for Comparative Medicine, a facility accredited by the American Association for Accreditation of Laboratory Animal Care. Animals were allowed to acclimatize for 1 week and then were caged in groups of five. Animals were exposed to a 12-hour/12-hour light/dark cycle and autoclaved food and water was supplied ad libitum. The research protocol for this study was approved by the University of Colorado at Denver Institutional Animal Care and Use review board. For xenograft production, HT-29 human colon cancer cells were grown in 75-cm² culture flasks in DMEM supplemented with 10% fetal bovine serum until they reached ∼60% confluency and were in the log phase growth. Cells were harvested with trypsin/EDTA, pelleted by centrifugation, and resuspended in a solution consisting of 50% serum-free DMEM/50% Matrigel (BD Biosciences), v/v. Approximately 2.5 × 10⁶ cells in a volume of 100 μL were injected s.c. unto the left and right flanks of each mouse using a 1-cc syringe with a 23-g needle. Resulting xenograft tumors were measured daily until tumor volumes of 100 to 150 mm³ were reached. Mice were then randomized into three groups (n = 12 mice per group), (a) vehicle control (sterile water), (b) ENMD-2076 (100 mg/kg), and (c) ENMD-2076 (200 mg/kg). ENMD-2076 and vehicle were dosed daily by oral gavage at volumes that varied between 90 and 120 μL, based on body weight. Mice were monitored daily for signs of toxicity and were weighed twice weekly. Tumor size was evaluated twice per week by caliper measurements using the following formula: tumor volume = (length × width²)×0.52. Tumor volume and body weight data were collected using the Study Director software package (Studylog Systems). At the end of a 28-day cycle, mice were euthanized by isoflurane anesthesia and tumor samples were collected for gross anatomic and immunohistochemical analyses.

The human CRC explant xenografts were generated according to previously published methods (29) Briefly, surgical specimens from patients undergoing either removal of a primary CRC or metastatic tumor at the University of Colorado Hospital were reimplanted s.c. into five mice for each patient. CU-CRC-027 was obtained from a primary tumor, whereas CU-CRC-001 and CU-CRC-012 originated from peritoneal and pelvic metastatic sites, respectively. The human primary tumor explants were assayed for KRAS mutational status by the University of Colorado Cancer Center Pathology Core with the DxS Scorpion method (DxS) using the manufacturer's instructions. Briefly, template DNA was analyzed for a set of seven known KRAS point mutations using the Therascreen KRAS Mutation Detection kit (DxS Ltd.). Reactions and analysis were done on a Lightcycler 480 real-time PCR instrument (LC480) that was calibrated using a dye calibration kit provided by the manufacturer. Cycle cross point values were calculated using the LC480 Fit-point software suite and the control cross point was subtracted from the cross point of each mutation-specific primer set (30). CU-CRC-001, CU-CRC-012, and CU-CRC-027 all contained KRAS mutations. Tumor samples were then passaged into subsequent generations of mice for drug studies. Briefly, tumors were allowed to grow to a size of 1,000 to 1,500 mm³ (F1) at which point they were harvested, divided, and transplanted to an additional five mice (F2) to maintain the tumor bank. After a subsequent growth passage, tumors were excised, transplanted onto both flanks of nude mice, and expanded into cohorts of ≥25 mice for treatment. All experiments were conducted on F3 to F5 generations. Tumors from this cohort were allowed to grow until reaching approximately 150 to 300 mm³, at which time they were equally distributed by size into the two treatment groups: control and ENMD-2076 treated. Because of the variability in take rates of the human patient explant material, enough mice were designated into each group based on the number of overall tumors (n = 12 tumors per group). Mice were treated for 28 days with either vehicle control (sterile water) or ENMD-2076 (100 mg/kg) once daily by oral gavage. Monitoring of mice and measurements of tumors was conducted as described above. The relative tumor growth index was calculated by taking the tumor volume of control or ENMD-2076–treated mice at study end as a percentage of the tumor volume at day 1 of treatment.

All of the xenograft studies were conducted in accordance with the NIH guidelines for the care and use of laboratory animals, were conducted in a facility accredited by the American Association for Accreditation of Laboratory Animal Care, and received approval from the University of Colorado Animal Care and Use Committee before initiation. Obtaining tissue from CRC patients at the time of removal of a primary tumor or metastectomy was conducted under a Colorado Multi-Institutional Review Board–approved protocol.

For noninvasive measurements of tumor vascularity, selected animals from HT-29 xenograft vehicle control and ENMD-2076 (100 mg/kg) groups were subjected to DCE-MRI (n = 4–8 tumors per group). DCE-MRI was done at baseline (before start of treatment), on day 7 of treatment, and at the end of the treatment cycle (day 21) as previously described (31). Briefly, animals were anesthetized with ketamine/xylazine (80/10 mg/kg) and a catheter preloaded with gadolinium (Gd; Magnevist, Berlex Schering AG) was placed in the lateral tail vein. Animals were placed into a 4.7 Tesla Bruker PharmaScan MRI (Bruker Medical) equipped with a 31-mm-diameter Bruker volume receiver/transmitter coil. A series of fast T1-weighted modified driven equilibrium Fourier transforms pulses were applied for total acquisition time of 15 minutes. The scan parameters were as follows: field of view = 4.0 cm; slice thickness = 1.5 mm; echo time/repetition time = 9.3/116.6 ms; number of slices = 4; number of averages = 1; matrix size = 128 × 256; number of evolutions = 60; resolution time = 15 seconds. After 1 minute of image acquisition (precontrast), 0.2 mmol/kg Magnevist was injected through the tail vein catheter and T1-weighted Gd-enhanced MRI scans were continuously taken for another 14 minutes. Images were analyzed with the Bruker ParaVision software version 3.0.2 by hand drawing regions of interest with the “track” command from each set of slices.

The area under the Gd uptake (T1 signal intensity) versus time curve (AUC) and the initial AUC (IAUC, taken over the first 60 s post-Gd injection) were calculated using the trapezoidal rule in Microsoft Excel 2003. Data were also fitted to a two-compartment model (32) described in equation 1, with SAAMII version 1.2 (University of Washington) to estimate K^trans and K_ep

¹⁸FDG-PET scans were done at baseline (baseline − before start of treatment) and on days 3 and 18 of treatment. Selected mice from the HT-29 xenograft vehicle control and ENMD-2076 (100 mg/kg) groups were fasted for 4 to 8 hours and ∼250 μCi of ¹⁸FDG, obtained through PetNet Solutions (Knoxville TN) at the University of Colorado Hospital, was administered to conscious animals by tail vein injection. Animals were maintained unanesthetized in cages for ∼1 hour to allow for ¹⁸FDG uptake in tumors. Under 2.5% isoflurane anesthesia, mice were placed on a heated pad (m2m Imaging) and a 10-minute emission was acquired using a Siemens Inveon micro-PET scanner (Siemens Medical). Images were reconstructed and analyzed with AsiProVM (Concorde Microsystems). Regions of interest were drawn with the trace command around the tumors on axial slices and the total activity of all tumor slices was summed. Total activity was divided by the time-corrected dose delivered [time corrected dose = dose injected × exp (−0.006317*t), in which t is the time between the injection and scan time] and is presented as the percentage of the respective tumor's baseline scan.

At study end, animals were euthanized and HT-29 xenograft tumors were resected and weighed. Tumors were then sectioned and portions were fixed in neutral buffered formalin for 24 hours followed by immersion in 70% ethanol. Tumors were then processed and embedded in paraffin and cut in 3-μm sections and mounted on glass slides for immunohistochemistry as previously described (33). Briefly, the procedure consisted of antigen retrieval using the DAKO target retrieval solution followed by a 10-minute peroxide block in water. Primary antibody for Ki-67 (1:100, DAKO) was incubated for 1 hour followed by a 30-minute secondary antibody incubation using EnVision anti-rabbit peroxidase 3,3′-diaminobenzidine–conjugated antibody (DAKO). Finally, incubation with substrate solution was carried out for 7 minutes using 3,3′-diaminobenzidine plus 20 μL chromagen (DAKO) followed by counterstaining for 1 minutes with hematoxylin.

The Student's t test was used to determine statistical significance between two groups when data were normally distributed. Analysis was done with Prism version 4.03 (GraphPad Software, Inc.). Values of P < 0.05 were considered statistically significant.

To determine the effects of ENMD-2076 on human HT-29 CRC xenograft tumor growth kinetics, athymic tumor-bearing nude mice were randomized into three groups, vehicle, ENMD-2076 (100 mg/kg), and ENMD-2076 (200 mg/kg), and treated daily by oral gavage as described in Materials and Methods. Figure 1 depicts the HT-29 tumor growth curves of mice treated with vehicle or ENMD-2076 at doses of either 100 or 200 mg/kg over a period of 28 days. Xenograft tumor volumes in mice treated with either dose of ENMD-2076 remained static for ∼17 days of treatment compared with vehicle controls and then subsequently showed modest levels of tumor regression, with a slightly higher degree of regression at the 200 mg/kg dose. The doses and schedule used in this study (100 and 200 mg/kg, daily) are well within the tolerability limits for ENMD-2076 in mice and no animals exhibited obvious signs of toxicity as indicated by outward morbidity or weight loss. Similar xenograft experiments in mice using the colorectal cell line HCT-116 have been done at EntreMed using 200 mg/kg twice daily doses of ENMD-2076 (400 mg/kg daily) in which eventual weight loss and mortality was observed in study animals.⁴

An extensive toxicokinetic study has been done in rats and dogs with ENMD-2076 in its preclinical phase of development and these data will be reported in subsequent publications. At study end, the mice were euthanized and the xenograft tumors were resected for gross pathologic examination. As depicted in Fig. 2A, the HT-29 tumors from vehicle control–treated mice showed a high degree of vascularity, whereas tumors from mice treated with 200 mg/kg ENMD-2076 displayed blanching with an apparent loss of tumor vasculature. Additionally, tumor samples were fixed and subjected to immunohistochemical analysis for Ki-67, a marker of proliferating cells. As depicted in Fig. 2B, there was a marked decrease in the number of proliferating cells in tumors from mice treated with ENMD-2076 (200 mg/kg) compared with vehicle-treated controls.

To assess the effects of ENMD-2076 on HT-29 xenograft tumor vascularity, selected animals from the vehicle- and ENMD-2076–treated (100 mg/kg) groups underwent DCE-MRI scans at baseline, before treatment, and at day 7 and day 21 after the initiation of treatment. Figure 3 depicts representative DCE-MRI images acquired at baseline, and day 7 and day 21 posttreatment with ENMD-2076 or vehicle. The left image from each series represents precontrast tumor slice; the right image was taken at 2.5 minutes post-Gd injection. As early as day 7, there was a marked decrease in the size of the tumors as well as Gd uptake in the ENMD-2076 group compared with controls. By day 21 posttreatment, tumors from mice treated with ENMD-2076 continue to show low Gd uptake, reflecting a decrease in vascular perfusion and permeability. Figure 4A to D shows model-independent IAUC (A) and AUC (B), and model-dependent K^trans (C) and K_ep (D) kinetic parameters of Gd uptake in subcutaneous HT-29 tumor xenografts from mice treated with vehicle or ENMD-2076 (100 mg/kg). The area under the Gd curve (AUC) of the tumor was significantly lower versus control in the ENMD-2076 treatment group on day 7 (P < 0.05), indicating decreased vascular perfusion and permeability (Fig. 4B). K^trans and K_ep data were calculated using the two-compartment Tofts model (32). K^trans, the volume transfer constant of contrast agent between blood plasma and the tumor extravascular extracellular space, was decreased by ∼80% compared with vehicle controls 1 and 3 week posttreatment with ENMD-2076, indicating a decrease tumor vascular permeability. Similarly, K_ep, the reflux coefficient, which assesses the transfer of contrast agent from the tumor back to the blood, was inhibited ∼70% after ENMD-2076 treatment.

To assess the effects of ENMD-2076 on glucose uptake, ¹⁸FDG-PET was performed on mice bearing HT-29 human CRC xenografts at baseline, before the start of treatment, and at day 3 and 21 posttreatment with vehicle or ENMD-2076 (100 mg/kg). As depicted in Fig. 5A, differences in ¹⁸FDG tumor uptake between vehicle and ENMD-2076 were visible as early as day 3, suggesting that ENMD-2076 rapidly alters tumor glucose metabolism. At day 21 of treatment with ENMD-2076, tumors were difficult to detect or completely undetectable by ¹⁸FDG-PET, indicating that the effects of ENMD-2076 on tumor glucose metabolic activity are persistent. Figure 5B depicts the quantification of the ¹⁸F radioactivity PET images in mice treated with vehicle or ENMD-2076 at baseline and day 3 or day 21 after treatment (n = 6 per group) as described in Materials and Methods. Again, decreases in ¹⁸FDG uptake in tumors can be measured at day 3 posttreatment and uptake is significantly decreased at day 21 when compared with baseline or as a percentage of day 3 activity.

Lastly, we sought to determine whether the efficacy of ENMD-2076 observed against the HT-29 xenograft model would be recapitulated against patient-derived CRC explants from primary and metastatic sites, grown as xenografts in athymic nude mice. As depicted in Fig. 6, ENMD-2076 induced tumor growth inhibition against all patient-derived xenografts by day 30, whereas two of the three (derived from a peritoneal metastasis and primary tumor) tumors exhibited more dramatic effects throughout the treatment period. These results indicate that more recently derived human CRC xenograft models behave similarly to the historically benchmarked HT-29 model and, interestingly, against Kras mutant tumors.

ENMD-2076 is a novel, orally bioavailable small-molecule inhibitor of AurA kinase as well as several other tyrosine kinases involved in cancer including flt-3, VEGFR2, VEGFR3, fibroblast growth factor receptor 1, and Src. As such, ENMD-2076 is a member of the class of so-called “multitargeted” kinase inhibitors that can potentially affect tumor growth and progression at multiple levels, including the blockade of mitotic, angiogenic, and growth factor signaling pathways. The intent of this study was to use a well-defined xenograft model of human CRC, HT-29, along with preclinical functional imaging to clearly define the antitumor efficacy and pharmacodynamic effects of ENMD-2076. Additionally, patient-derived human CRC explants were treated with ENMD-2076 to determine whether they responded similarly to the HT-29 model, with the intent of using the data generated in these studies to design future clinical studies of ENMD-2076 in CRC.

Our studies showed that ENMD-2076 exhibits robust antitumor activity against in vivo models of human CRC. We observed tumor stasis and regression against the HT-29 CRC nude mouse xenograft model as well as strong tumor growth inhibition in three patient-derived CRC explant models. Interestingly, and as anticipated from the profile of kinase inhibition of ENMD-2076, we observed both antiproliferative and antiangiogenic effects of the agent against the HT-29 xenograft model as seen by tumor growth kinetics and gross examination, DCE-MRI, and immunohistochemistry. We will be the first to acknowledge that it is difficult to ascertain the contribution of the AurA versus angiogenic kinase inhibition to the antitumor activity observed with ENMD-2076. Studies of other dual AurA/AurB, or selective AurA or AurB inhibitors have shown similar tumor growth inhibition profiles against HT-29 or similar CRC xenograft models, although actual tumor regression is less common and greater weight loss has been observed with some agents (34–37). Likewise, preclinical studies of antiangiogenic agents such as bevacizumab, DC101 (murine VEGFR2 antibody), or sunitinib in CRC xenografts have reported significant tumor growth inhibition with regressions observed with sunitinib, emphasizing the exquisite dependence of subcutaneous xenografts on angiogenesis (38–41). Importantly, in addition to the well-characterized HT-29 CRC model, we observed efficacy in three xenografts derived directly (no in vitro passage) from primary or metastatic tumors from CRC patients, reflecting a more contemporary and genetically heterogeneous group.

Increasingly, DCE-MRI is used preclinically and in the early phases of clinical development to assess the efficacy of antiangiogenic and vascular-disrupting compounds (31, 42, 43). DCE-MRI is sensitive to differences in vascular perfusion and permeability, and is based on repetitive, rapid T1-weighted MRI scans before and following a Gd-containing i.v. injection (27). Here, we used DCE-MRI to evaluate the antiangiogenic potential of ENMD-2076 in HT29 xenograft models and to preclinically validate the use of DCE-MRI in early clinical trials. We found that as early as day 7, along with a decrease in the size of the tumors, the IAUC (reflecting vascular perfusion) as well as K^trans (reflecting vascular permeability) were drastically reduced in the ENMD-2076–treated animals compared with controls, consistent with an antiangiogenic mechanism. These results are similar to what others have observed with VEGF-targeted agents and are consistent with what we observed in a preclinical study of the HT-29 xenograft model treated with the dual epidermal growth factor receptor/VEGF inhibitor, vandetanib (31, 44, 45). Therefore, DCE-MRI represents a reasonable noninvasive pharmacodynamic biomarker of the antiangiogenic activity of ENMD-2076 that could be incorporated into early clinical trials.

As an additional measure of the antitumor effects of ENMD-2076, we also used ¹⁸FDG-PET in our HT-29 xenograft model. Because changes in tumor metabolism typically precede tumor size reductions, ¹⁸FDG-PET has the potential to detect early responses to chemotherapeutic and targeted agents (46). This has been shown clinically in gastrointestinal stromal tumor (GIST) in which ¹⁸FDG-PET correctly predicted tumor response to imatinib in 95% of patients as early as 1 month after the start of treatment (47). In our study, we showed the effects of ENMD-2076 on tumor metabolic activity as early day 3 posttreatment, indicating that FDG-PET may be useful clinically to predict efficacy at earlier time points, before changes in tumor size can be detected. Taken together, both DCE-MRI and ¹⁸FDG-PET are preclinically validated noninvasive functional imaging tools that should be applied in early clinical trials of ENMD-2076 to document biological effects and for potential use as early biomarkers of efficacy.

In this study, we used a direct human tumor explant model of CRC, and significantly, the tumors originated from primary and metastatic sites and were mutant for the KRAS gene. As CRC is now commonly segregated for treatment options into KRAS mutant and KRAS wild-type tumors, this is an important consideration for the development of ENMD-2076 in advanced CRC. In contrast to cetuximab, ENMD-2076, at least preliminarily, seems to be effective in KRAS mutant and wild-type tumors, therefore representing a potential option for the enlarging population of patients with KRAS/BRAF/phosphoinositide 3-kinase–mutated tumors. This apparent nonselectivity for tumors that harbor mutations downstream of growth factor receptors is consistent with what has been observed with antiangiogenic agents including bevacizumab (48, 49), whereas the effect of AurA/AurB inhibition in the setting of KRAS mutation deserves further investigation. Combinations of ENMD-2076 with irinotecan should be considered as a potential “chemopotentiation” strategy for patients with KRAS-mutated tumors.

In summary, we have shown that ENMD-2076, a novel multitargeted kinase inhibitor, is effective against preclinical models of CRC in vivo. ENMD-2076 is currently being investigated in phase I dose escalation studies in patients with advanced solid tumors and advanced hematologic malignancies. The drug is well tolerated with the expected mechanism-based toxicity of hypertension. Preliminary results show stabilization of disease and tumor marker reductions in a subset of patients with colorectal and ovarian cancer, associated with a decrease in plasma-soluble VEGFR2 (50). The results presented here, along with the early clinical trial results, suggest a pathway forward in CRC in which novel strategies are urgently needed.

J.J. Tentler: commercial research grant, EntreMed, Inc.; G.C. Fletcher and M.R. Bray, consultants, EntreMed, Inc.; S.G. Eckhardt, advisory board, EntreMed, Inc.

Grant Support: Grants CA106349 and CA148581 (S.G. Eckhardt), CA046934 (University of Colorado Cancer Center Support Grant), and research funding from EntreMed, Inc. (J.J. Tentler).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.