Simultaneous targeting of glycolysis and oxidative phosphorylation as a therapeutic strategy to treat diffuse large B-cell lymphoma.

BACKGROUND: We evaluated the therapeutic potential of combining the monocarboxylate transporter 1 (MCT1) inhibitor AZD3965 with the mitochondrial respiratory Complex I inhibitor IACS-010759, for the treatment of diffuse large B-cell lymphoma (DLBCL), a potential clinically actionable strategy to target tumour metabolism. METHODS: AZD3965 and IACS-010759 sensitivity were determined in DLBCL cell lines and tumour xenograft models. Lactate concentrations, oxygen consumption rate and metabolomics were examined as mechanistic endpoints. In vivo plasma concentrations of IACS-010759 in mice were determined by LC-MS to select a dose that reflected clinically attainable concentrations. RESULTS: In vitro, the combination of AZD3965 and IACS-010759 is synergistic and induces DLBCL cell death, whereas monotherapy treatments induce a cytostatic response. Significant anti-tumour activity was evident in Toledo and Farage models when the two inhibitors were administered concurrently despite limited or no effect on the growth of DLBCL xenografts as monotherapies. CONCLUSIONS: This is the first study to examine a combination of two distinct approaches to targeting tumour metabolism in DLBCL xenografts. Whilst nanomolar concentrations of either AZD3965 or IACS-010759 monotherapy demonstrate anti-proliferative activity against DLBCL cell lines in vitro, appreciable clinical activity in DLBCL patients may only be realised through their combined use.

Highly Potent Clickable Probe for Cellular Imaging of MDM2 and Assessing Dynamic Responses to MDM2-p53 Inhibition.

MDM2 is a key negative regulator of the p53 tumor suppressor. Direct binding of MDM2 to p53 represses the protein's transcriptional activity and induces its polyubiquitination, targeting it for degradation by the proteasome. Consequently, small molecule inhibitors that antagonize MDM2-p53 binding, such as RG7388, have progressed into clinical development aiming to reactivate p53 function in TP53 wild-type tumors. Here, we describe the design, synthesis, and biological evaluation of a trans-cyclooctene tagged derivative of RG7388, RG7388-TCO, which showed high cellular potency and specificity for MDM2. The in-cell reaction of RG7388-TCO with a tetrazine-tagged BODIPY dye enabled fluorescence imaging of endogenous MDM2 in SJSA-1 and T778 tumor cells. RG7388-TCO was also used to pull down MDM2 by reaction with tetrazine-tagged agarose beads in SJSA-1 lysates. The data presented show that RG733-TCO enables precise imaging of MDM2 in cells and can permit a relative assessment of target engagement and MDM2-p53 antagonism in vitro.

Identification of a novel ligand for the ATAD2 bromodomain with selectivity over BRD4 through a fragment growing approach.

ATAD2 is an ATPase that is overexpressed in a variety of cancers and associated with a poor patient prognosis. This protein has been suggested to function as a cofactor for a range of transcription factors, including the proto-oncogene MYC and the androgen receptor. ATAD2 comprises an ATPase domain, implicated in chromatin remodelling, and a bromodomain which allows it to interact with acetylated histone tails. Dissection of the functional roles of these two domains would benefit from the availability of selective, cell-permeable pharmacological probes. An in silico evaluation of the 3D structures of various bromodomains suggested that developing small molecule ligands for the bromodomain of ATAD2 is likely to be challenging, although recent reports have shown that ATAD2 bromodomain ligands can be identified. We report a structure-guided fragment-based approach to identify lead compounds for ATAD2 bromodomain inhibitor development. Our findings indicate that the ATAD2 bromodomain can accommodate fragment hits (Mr < 200) that yield productive structure-activity relationships, and structure-guided design enabled the introduction of selectivity over BRD4.

Inhibition of monocarboxyate transporter 1 by AZD3965 as a novel therapeutic approach for diffuse large B-cell lymphoma and Burkitt lymphoma.

Inhibition of monocarboxylate transporter 1 has been proposed as a therapeutic approach to perturb lactate shuttling in tumor cells that lack monocarboxylate transporter 4. We examined the monocarboxylate transporter 1 inhibitor AZD3965, currently in phase I clinical studies, as a potential therapy for diffuse large B-cell lymphoma and Burkitt lymphoma. Whilst extensive monocarboxylate transporter 1 protein was found in 120 diffuse large B-cell lymphoma and 10 Burkitt lymphoma patients' tumors, monocarboxylate transporter 4 protein expression was undetectable in 73% of the diffuse large B-cell lymphoma samples and undetectable or negligible in each Burkitt lymphoma sample. AZD3965 treatment led to a rapid accumulation of intracellular lactate in a panel of lymphoma cell lines with low monocarboxylate transporter 4 protein expression and potently inhibited their proliferation. Metabolic changes induced by AZD3965 in lymphoma cells were consistent with a feedback inhibition of glycolysis. A profound cytostatic response was also observed in vivo: daily oral AZD3965 treatment for 24 days inhibited CA46 Burkitt lymphoma growth by 99%. Continuous exposure of CA46 cells to AZD3965 for 7 weeks in vitro resulted in a greater dependency upon oxidative phosphorylation. Combining AZD3965 with an inhibitor of mitochondrial complex I (central to oxidative phosphorylation) induced significant lymphoma cell death in vitro and reduced CA46 disease burden in vivo These data support clinical examination of AZD3965 in Burkitt lymphoma and diffuse large B-cell lymphoma patients with low tumor monocarboxylate transporter 4 expression and highlight the potential of combination strategies to optimally target the metabolic phenotype of tumors.

Mechanisms that influence tumour response to VEGF-pathway inhibitors.

There has been significant investment in developing novel therapies to target solid tumour vasculature. Different technical approaches have been utilized with the aim of inhibiting tumour angiogenesis or compromising the function or stability of pre-existing tumour blood vessels. The vascular endothelial growth factor (VEGF) signalling axis remains the most widely studied, with biological and small-molecule therapeutics now registered for clinical use. However, despite these successes, the activity of these agents is not as widespread as was first postulated. The present review discusses the clinical successes of the VEGF inhibitors, the factors that may limit their utility, and the potential opportunities to maximize benefit from treatment with these agents in the future.

Modulation of ERK5 Activity as a Therapeutic Anti-Cancer Strategy.

The extracellular signal-regulated kinase 5 (ERK5) signaling pathway is one of four conventional mitogen-activated protein (MAP) kinase pathways. Genetic perturbation of ERK5 has suggested that modulation of ERK5 activity may have therapeutic potential in cancer chemotherapy. This Miniperspective examines the evidence for ERK5 as a drug target in cancer, the structure of ERK5, and the evolution of structurally distinct chemotypes of ERK5 kinase domain inhibitors. The emerging complexities of ERK5 pharmacology are discussed, including the confounding phenomenon of paradoxical ERK5 activation by small-molecule ERK5 inhibitors. The impact of the recent development and biological evaluation of potent and selective bifunctional degraders of ERK5 and future opportunities in ERK modulation are also explored.

Fragment expansion with NUDELs - poised DNA-encoded libraries.

Optimisation of the affinity of lead compounds is a critical challenge in the identification of drug candidates and chemical probes and is a process that takes many years. Fragment-based drug discovery has become established as one of the methods of choice for drug discovery starting with small, low affinity compounds. Due to their low affinity, the evolution of fragments to desirable levels of affinity is often a key challenge. The accepted best method for increasing the potency of fragments is by iterative fragment growing, which can be very time consuming and complex. Here, we introduce a paradigm for fragment hit optimisation using poised DNA-encoded chemical libraries (DELs). The synthesis of a poised DEL, a partially constructed library that retains a reactive handle, allows the coupling of any active fragment for a specific target protein, allowing rapid discovery of potent ligands. This is illustrated for bromodomain-containing protein 4 (BRD4), in which a weakly binding fragment was coupled to a 42-member poised DEL via Suzuki-Miyaura cross coupling resulting in the identification of an inhibitor with 51 nM affinity in a single step, representing an increase in potency of several orders of magnitude from an original fragment. The potency of the compound was shown to arise from the synergistic combination of substructures, which would have been very difficult to discover by any other method and was rationalised by X-ray crystallography. The compound showed attractive lead-like properties suitable for further optimisation and demonstrated BRD4-dependent cellular pharmacology. This work demonstrates the power of poised DELs to rapidly optimise fragments, representing an attractive generic approach to drug discovery.

Targeting Cytotoxic Agents through EGFR-Mediated Covalent Binding and Release.

A major drawback of cytotoxic chemotherapy is the lack of selectivity toward noncancerous cells. The targeted delivery of cytotoxic drugs to tumor cells is a longstanding goal in cancer research. We proposed that covalent inhibitors could be adapted to deliver cytotoxic agents, conjugated to the ß-position of the Michael acceptor, via an addition-elimination mechanism promoted by covalent binding. Studies on model systems showed that conjugated 5-fluorouracil (5FU) could be released upon thiol addition in relevant time scales. A series of covalent epidermal growth factor receptor (EGFR) inhibitors were synthesized as their 5FU derivatives. Achieving the desired release of 5FU was demonstrated to depend on the electronics and geometry of the compounds. Mass spectrometry and NMR studies demonstrated an anilinoquinazoline acrylate ester conjugate bound to EGFR with the release of 5FU. This work establishes that acrylates can be used to release conjugated molecules upon covalent binding to proteins and could be used to develop targeted therapeutics.

A Phase I Dose-escalation Study of AZD3965, an Oral Monocarboxylate Transporter 1 Inhibitor, in Patients with Advanced Cancer.

PURPOSE: Inhibition of monocarboxylate transporter (MCT) 1-mediated lactate transport may have cytostatic and/or cytotoxic effects on tumor cells. We report results from the dose-escalation part of a first-in-human trial of AZD3965, a first-in-class MCT1 inhibitor, in advanced cancer. PATIENTS AND METHODS: This multicentre, phase I, dose-escalation and dose-expansion trial enrolled patients with advanced solid tumors or lymphoma and no standard therapy options. Exclusion criteria included history of retinal and/or cardiac disease, due to MCT1 expression in the eye and heart. Patients received daily oral AZD3965 according to a 3+3 then rolling six design. Primary objectives were to assess safety and determine the MTD and/or recommended phase II dose (RP2D). Secondary objectives for dose escalation included measurement of pharmacokinetic and pharmacodynamic activity. Exploratory biomarkers included tumor expression of MCT1 and MCT4, functional imaging of biological impact, and metabolomics. RESULTS: During dose escalation, 40 patients received AZD3965 at 5-30 mg once daily or 10 or 15 mg twice daily. Treatment-emergent adverse events were primarily grade 1 and/or 2, most commonly electroretinogram changes (retinopathy), fatigue, anorexia, and constipation. Seven patients receiving ≥20 mg daily experienced dose-limiting toxicities (DLT): grade 3 cardiac troponin rise (n = 1), asymptomatic ocular DLTs (n = 5), and grade 3 acidosis (n = 1). Plasma pharmacokinetics demonstrated attainment of target concentrations; pharmacodynamic measurements indicated on-target activity. CONCLUSIONS: AZD3965 is tolerated at doses that produce target engagement. DLTs were on-target and primarily dose-dependent, asymptomatic, reversible ocular changes. An RP2D of 10 mg twice daily was established for use in dose expansion in cancers that generally express high MCT1/low MCT4).

Expression of stromal genes associated with the angiogenic response are not differentiated between human tumour xenografts with divergent vascular morphologies.

Human tumour xenografts have commonly been used to explore the mechanisms of tumour angiogenesis and the interaction of tumour cells with their microenvironment, as well as predict potential utility of anti-angiogenic inhibitors across different tumour types. To investigate how well human tumour xenografts can be used to differentiate the effects of stromal targeting agents we performed a comparative assessment of the murine angiogenic response across a panel of pre-clinical tumour xenografts. By analysing a panel of 22 tumour xenografts with a range of vascular morphologies, micro-vessel densities and levels of fibroblast and inflammatory infiltrate, we have examined the relationship between angiogenic stroma and human tumour models. These models were studied using a combination of immunohistochemistry and species specific mRNA profiling to differentiate the tumour and stromal transcript mRNA profiles. Principal Component Analysis (PCA) and regression analysis was used to investigate the transcriptional relationships between the individual models and the correlation with the stromal architecture. We found the human tumour cell expressed factors to be independent of the murine host responses such as microvessel density, and fibroblast or macrophage cellular infiltrate. Moreover mRNA profiling of the mouse stroma suggested that the host response to the different tumours was relatively uniform despite differences in stromal structures within the tumour. Supporting this, models with different stromal compositions responded similarly to cediranib, a small molecule inhibitor of VEGF signalling. The data indicate that although the angiogenic response to the tumour results in reproducible stromal architectures, these responses are not differentiated at the level of gene expression.

Parallel Optimization of Potency and Pharmacokinetics Leading to the Discovery of a Pyrrole Carboxamide ERK5 Kinase Domain Inhibitor.

The nonclassical extracellular signal-related kinase 5 (ERK5) mitogen-activated protein kinase pathway has been implicated in increased cellular proliferation, migration, survival, and angiogenesis; hence, ERK5 inhibition may be an attractive approach for cancer treatment. However, the development of selective ERK5 inhibitors has been challenging. Previously, we described the development of a pyrrole carboxamide high-throughput screening hit into a selective, submicromolar inhibitor of ERK5 kinase activity. Improvement in the ERK5 potency was necessary for the identification of a tool ERK5 inhibitor for target validation studies. Herein, we describe the optimization of this series to identify nanomolar pyrrole carboxamide inhibitors of ERK5 incorporating a basic center, which suffered from poor oral bioavailability. Parallel optimization of potency and in vitro pharmacokinetic parameters led to the identification of a nonbasic pyrazole analogue with an optimal balance of ERK5 inhibition and oral exposure.

DNAPK Inhibition Preferentially Compromises the Repair of Radiation-induced DNA Double-strand Breaks in Chronically Hypoxic Tumor Cells in Xenograft Models.

Radiation-induced DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) and nonhomologous end joining (NHEJ). Recently, it has been found that chronic tumor hypoxia compromises HR repair of DNA DSBs but activates the NHEJ protein DNAPK. We therefore hypothesized that inhibition of DNAPK can preferentially potentiate the sensitivity of chronically hypoxic cancer cells to radiation through contextual synthetic lethality in vivo In this study, we investigated the impact of DNAPK inhibition by a novel selective DNAPK inhibitor, NU5455, on the repair of radiation-induced DNA DSBs in chronically hypoxic and nonhypoxic cells across a range of xenograft models. We found that NU5455 inhibited DSB repair following radiation in both chronically hypoxic and nonhypoxic tumor cells. Most importantly, the inhibitory effect was more pronounced in chronically hypoxic tumor cells than in nonhypoxic tumor cells. This is the first in vivo study to indicate that DNAPK inhibition may preferentially sensitize chronically hypoxic tumor cells to radiotherapy, suggesting a broader therapeutic window for transient DNAPK inhibition combined with radiotherapy.

An Alkynylpyrimidine-Based Covalent Inhibitor That Targets a Unique Cysteine in NF-κB-Inducing Kinase.

NF-κB-inducing kinase (NIK) is a key enzyme in the noncanonical NF-κB pathway, of interest in the treatment of a variety of diseases including cancer. Validation of NIK as a drug target requires potent and selective inhibitors. The protein contains a cysteine residue at position 444 in the back pocket of the active site, unique within the kinome. Analysis of existing inhibitor scaffolds and early structure-activity relationships (SARs) led to the design of C444-targeting covalent inhibitors based on alkynyl heterocycle warheads. Mass spectrometry provided proof of the covalent mechanism, and the SAR was rationalized by computational modeling. Profiling of more potent analogues in tumor cell lines with constitutively activated NIK signaling induced a weak antiproliferative effect, suggesting that kinase inhibition may have limited impact on cancer cell growth. This study shows that alkynyl heterocycles are potential cysteine traps, which may be employed where common Michael acceptors, such as acrylamides, are not tolerated.

Structure-Based Design of Potent and Orally Active Isoindolinone Inhibitors of MDM2-p53 Protein-Protein Interaction.

Inhibition of murine double minute 2 (MDM2)-p53 protein-protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.

Placental growth factor neutralising antibodies give limited anti-angiogenic effects in an in vitro organotypic angiogenesis model.

Vascular Endothelial Growth Factor Receptor (VEGFR) mediated signalling drives angiogenesis. This is predominantly attributed to the activity of VEGFR-2 following binding of VEGF-A. Whether other members of the VEGFR and ligand families such as VEGFR-1 and its ligand Placental Growth Factor (PlGF) can also contribute to developmental and pathological angiogenesis is less clear. We explored the function of PlGF in VEGF-A dependent angiogenesis using an in vitro co-culture assay in which endothelial cells are cultured on a fibroblast feeder layer. In the presence of 2% FS MCDB media (containing limited growth factors) in vitro endothelial tube formation is driven by endogenous angiogenic stimuli which are produced by the fibroblast and endothelial cells. Under these conditions independent sequestration of either free VEGF-A or PlGF with polyclonal and monoclonal antibodies inhibited tube formation suggesting that both ligands are required to drive an angiogenic response. Endothelial tube formation could only be driven within this assay by the addition of exogenous VEGF-A, VEGF-E or VEGF-A/PlGF heterodimer, but not by PlGF alone, implying that activation of either VEGFR-2/VEGFR-1 heterodimers or VEGFR-2 homodimers were responsible for eliciting an angiogenic response directly, but not VEGFR-1 homodimers. In contrast to results obtained with an endogenous angiogenic drive, sequestration of PlGF did not affect endothelial tube formation when the assay was driven by 1 ng/ml exogenous VEGF-A. These data suggest that although neutralising PlGF can be shown to reduce endothelial tube formation in vitro, this effect is only observed under restricted culture conditions and is influenced by VEGF-A. Such data questions whether neutralising PlGF would have a therapeutic benefit in vivo in the presence of pathological concentrations of VEGF-A.

Selective DNA-PKcs inhibition extends the therapeutic index of localized radiotherapy and chemotherapy.

Potentiating radiotherapy and chemotherapy by inhibiting DNA damage repair is proposed as a therapeutic strategy to improve outcomes for patients with solid tumors. However, this approach risks enhancing normal tissue toxicity as much as tumor toxicity, thereby limiting its translational impact. Using NU5455, a newly identified highly selective oral inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity, we found that it was indeed possible to preferentially augment the effect of targeted radiotherapy on human orthotopic lung tumors without influencing acute DNA damage or a late radiation-induced toxicity (fibrosis) to normal mouse lung. Furthermore, while NU5455 administration increased both the efficacy and the toxicity of a parenterally administered topoisomerase inhibitor, it enhanced the activity of doxorubicin released locally in liver tumor xenografts without inducing any adverse effect. This strategy is particularly relevant to hepatocellular cancer, which is treated clinically with localized drug-eluting beads and for which DNA-PKcs activity is reported to confer resistance to treatment. We conclude that transient pharmacological inhibition of DNA-PKcs activity is effective and tolerable when combined with localized DNA-damaging therapies and thus has promising clinical potential.

Use of a novel Arg-Gly-Asp radioligand, 18F-AH111585, to determine changes in tumor vascularity after antitumor therapy.

UNLABELLED: Despite the recent development of various radiolabeled Arg-Gly-Asp (RGD) peptides for imaging the alphavbeta3 integrin receptor, relatively little attention has been focused on the ability of these radiotracers to monitor changes in tumor vascularity after antitumor therapies. This study describes the favorable in vivo kinetics and tumor-targeting properties of 18F-AH111585, a novel 18F-RGD peptide, and its ability to monitor tumor vascularity noninvasively. METHODS: Mice bearing Lewis lung carcinoma (LLC) tumors or Calu-6 non-small cell lung tumor xenografts were used for in vivo biodistribution and small-animal PET imaging studies. In addition, some animals were treated with either low-dose paclitaxel or the vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor ZD4190. Tumor uptake of 18F-AH111585 and microvessel density were then assessed. RESULTS: Biodistribution of 18F-AH111585 demonstrated rapid clearance from the blood and key background organs and good tumor accumulation, with 1.5 percentage injected dose per gram (%ID/g) present at 2 h after injection in LLC tumors. Small-animal PET imaging of Calu-6 tumors allowed visualization of tumors above background tissue, with mean baseline uptake of 2.2 %ID/g. Paclitaxel therapy reduced the microvessel density in LLC tumor-bearing mice and resulted in significantly reduced 18F-AH111585 tumor uptake (P<0.05). ZD4190 therapy resulted in a significant (31.8%) decrease in 18F-AH111585 uptake in Calu-6 tumors, compared with the vehicle control-treated Calu-6 tumors, which had a 26.9% increase in 18F-AH111585 uptake over the same period (P<0.01). CONCLUSION: 18F-AH111585 is a promising 18F-labeled RGD tracer that offers a new approach to noninvasively image tumor vasculature. This tracer may reveal important information in the assessment of the impact of antitumor therapies, in particular those that predominantly target tumor blood vessels.

Inhibition of vascular endothelial growth factor-a signaling induces hypertension: examining the effect of cediranib (recentin; AZD2171) treatment on blood pressure in rat and the use of concomitant antihypertensive therapy.

PURPOSE: Inhibition of vascular endothelial growth factor-A (VEGF) signaling is a key therapeutic approach in oncology given the role of VEGF in angiogenesis and vascular permeability in solid tumors. Clinical trials examining VEGF signaling inhibitors commonly report hypertension. We examined the effect of cediranib, a highly potent VEGF signaling inhibitor, on the blood pressure of rats and the ability of standard antihypertensive agents to modulate the consequences of VEGF signaling inhibition. EXPERIMENTAL DESIGN: The ability of cediranib to induce hypertensive changes and the effect of giving antihypertensive therapy were investigated in conscious, unrestrained telemetered rats. Two antihypertensive agents were studied: captopril, an angiotensin-converting enzyme inhibitor, and nifedipine, a dihydropyridine calcium channel blocker. The antitumor activity of cediranib, alone and in combination with nifedipine, was also evaluated in a LoVo human colorectal tumor xenograft model in nude rats. All treatments were given orally. RESULTS: Administration of 0.1 to 1.5 mg/kg/d of cediranib for 4 consecutive days induced a relatively mild hypertensive effect, elevating diastolic blood pressure by 10 to 14 mmHg. Dosing 3 mg/kg/d cediranib for 4 days induced a marked hypertension of 35 to 50 mmHg. Captopril (30 mg/kg, qd) was effective at lowering a 10 mmHg increase in blood pressure but not a 35 to 50 mmHg increase. However, the latter was rapidly reversed by administration of nifedipine (10 mg/kg, bd). Coadministration of nifedipine did not negatively affect the antitumor activity of cediranib (1.5 mg/kg/d). CONCLUSIONS: Hypertension is a direct consequence of inhibiting VEGF signaling but can be controlled with appropriately selected, standard antihypertensive medication.

The use and refinement of rodent models in anti-cancer drug discovery: a review.

This review describes the changing use of tumour models in rodents (predominantly mice) as employed over the last four decades in anti-cancer drug discovery, and the refinements in the experimental methods used. Such models are required to examine the complexities of cancer biology (e.g. tumour angiogenesis, invasion and metastasis, host immunity factors) and the impact of potential therapies (e.g. drug pharmacokinetics, pharmacodynamics and therapeutic index), and they have produced efficacious human therapeutics. Animal welfare considerations have driven refinements to animal models of cancer over time, with the most dramatic refinements being facilitated by the move away from inherently cytotoxic therapeutic approaches toward targeted inhibitors of disease-related processes. Whereas, four decades ago, the impact of disease burden was used as an endpoint in the absence of defined mechanistic parameters, acute pharmacodynamic measures are now increasingly used to minimise the adverse effects of disease and experimental procedures in a given animal. The changes in the UK guidelines on the use of rodents in preclinical cancer testing are also used as an illustration of the progressive refinement in tumour models and drug testing.

Dual inhibition of VEGFR and EGFR signaling reduces the incidence and size of intestinal adenomas in Apc(Min/+) mice.

Both the epidermal growth factor (EGF) and the vascular endothelial growth factor (VEGF) pathways are associated with intestinal cancer, and therapeutic approaches targeting either EGF receptor (EGFR) or VEGF receptor (VEGFR) signaling have recently been approved for patients with advanced colorectal cancer. The Apc(Min/+) mouse is a well-characterized in vivo model of intestinal tumorigenesis, and animals with this genetic mutation develop macroscopically detectable adenomas from approximately 6 weeks of age. Previous work in the Apc(Min/+) mouse has shown that therapeutic approaches targeting either VEGFR or EGFR signaling affect predominantly the size or number of adenomas, respectively. In this study, we have assessed the effect of inhibiting both these key pathways simultaneously using ZD6474 (Vandetanib, ZACTIMA), a selective inhibitor of VEGFR and EGFR tyrosine kinases. To assess the effects of ZD6474 on early- and later-stage disease, treatment was initiated in 6- and 10-week-old Apc(Min/+) mice for 28 days. ZD6474 markedly reduced both the number and the size of polyps when administered at either an early or a later stage of polyp development. This reduction in both adenoma number and size resulted in a total reduction in tumor burden in the small intestine of nearly 75% in both studies (P < 0.01). The current data build on the concept that EGFR-dependent tumor cell proliferation and VEGF/VEGFR2-dependent angiogenesis and survival are distinct key mechanisms in polyp development. Pharmacologic inhibition of both signaling pathways has significant antitumor effects at both early and late stages of polyp development. Therefore, targeting both VEGFR- and EGFR-dependent signaling may be a beneficial strategy in early intestinal cancer.