TBK1 and IKKε Act Redundantly to Mediate STING-Induced NF-κB Responses in Myeloid Cells.

Stimulator of Interferon Genes (STING) is a critical component of host innate immune defense but can contribute to chronic autoimmune or autoinflammatory disease. Once activated, the cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP) synthase (cGAS)-STING pathway induces both type I interferon (IFN) expression and nuclear factor-κB (NF-κB)-mediated cytokine production. Currently, these two signaling arms are thought to be mediated by a single upstream kinase, TANK-binding kinase 1 (TBK1). Here, using genetic and pharmacological approaches, we show that TBK1 alone is dispensable for STING-induced NF-κB responses in human and mouse immune cells, as well as in vivo. We further demonstrate that TBK1 acts redundantly with IκB kinase ε (IKKε) to drive NF-κB upon STING activation. Interestingly, we show that activation of IFN regulatory factor 3 (IRF3) is highly dependent on TBK1 kinase activity, whereas NF-κB is significantly less sensitive to TBK1/IKKε kinase inhibition. Our work redefines signaling events downstream of cGAS-STING. Our findings further suggest that cGAS-STING will need to be targeted directly to effectively ameliorate the inflammation underpinning disorders associated with STING hyperactivity.

HBO1 is required for the maintenance of leukaemia stem cells.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.

Defining a therapeutic window for kinase inhibitors in leukemia to avoid neutropenia.

Neutropenia represents one of the major dose-limiting toxicities of many current cancer therapies. To circumvent the off-target effects of cytotoxic chemotherapeutics, kinase inhibitors are increasingly being used as an adjunct therapy to target leukemia. In this study, we conducted a screen of leukemic cell lines in parallel with primary neutrophils to identify kinase inhibitors with the capacity to induce apoptosis of myeloid and lymphoid cell lines whilst sparing primary mouse and human neutrophils. We have utilized a high-throughput live cell imaging platform to demonstrate that cytotoxic drugs have limited effects on neutrophil viability but are toxic to hematopoietic progenitor cells, with the exception of the topoisomerase I inhibitor SN-38. The parallel screening of kinase inhibitors revealed that mouse and human neutrophil viability is dependent on cyclin-dependent kinase (CDK) activity but surprisingly only partially dependent on PI3 kinase and JAK/STAT signaling, revealing dominant pathways contributing to neutrophil viability. Mcl-1 haploinsufficiency sensitized neutrophils to CDK inhibition, demonstrating that Mcl-1 is a direct target for CDK inhibitors. This study reveals a therapeutic window for the kinase inhibitors BEZ235, BMS-3, AZD7762, and (R)-BI-2536 to induce apoptosis of leukemia cell lines whilst maintaining immunocompetence and hemostasis.

Cisplatin Increases Sensitivity to FGFR Inhibition in Patient-Derived Xenograft Models of Lung Squamous Cell Carcinoma.

Lung squamous cell carcinoma (SqCC) is a molecularly complex and genomically unstable disease. No targeted therapy is currently approved for lung SqCC, although potential oncogenic drivers of SqCC have been identified, including amplification of the fibroblast growth factor receptor 1 (FGFR1). Reports from a recently completed clinical trial indicate low response rates in patients treated with FGFR tyrosine kinase inhibitors, suggesting inadequacy of FGFR1 amplification as a biomarker of response, or the need for combination treatment. We aimed to develop accurate models of lung SqCC and determine improved targeted therapies for these tumors. We show that detection of FGFR1 mRNA by RNA in situ hybridization is a better predictor of response to FGFR inhibition than FGFR1 gene amplification using clinically relevant patient-derived xenograft (PDX) models of lung SqCC. FGFR1-overexpressing tumors were observed in all histologic subtypes of non-small cell lung cancers (NSCLC) as assessed on a tissue microarray, indicating a broader range of tumors that may respond to FGFR inhibitors. In FGFR1-overexpressing PDX tumors, we observed increased differentiation and reduced proliferation following FGFR inhibition. Combination therapy with cisplatin was able to increase tumor cell death, and dramatically prolonged animal survival compared to single-agent treatment. Our data suggest that FGFR tyrosine kinase inhibitors can benefit NSCLC patients with FGFR1-overexpressing tumors and provides a rationale for clinical trials combining cisplatin with FGFR inhibitors. Mol Cancer Ther; 16(8); 1610-22. ©2017 AACR.

Operation versus antibiotics--The "appendicitis conundrum" continues: A meta-analysis.

BACKGROUND: Acute appendicitis continues to constitute a diagnostic and therapeutic challenge. The aim of this study was to synthesize evidence from randomized controlled trials (RCTs) comparing nonoperative versus surgical management of uncomplicated acute appendicitis in adult patients. METHODS: A systematic literature search of the PubMed, Cochrane, and Scopus databases was performed with respect to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) statement (end-of-search date: January 29, 2017). Data on the study design, interventions, participants, and outcomes were extracted by two independent reviewers. The random-effects model (DerSimonian-Laird) was used to calculate pooled effect estimates when substantial heterogeneity was encountered; otherwise, the fixed-effects (Mantel-Haenszel) model was implemented. Quality assessment of included RCTs was performed using the modified Jadad scale. RESULTS: Five RCTs were included in this review. Overall, 1,430 adult patients with uncomplicated acute appendicitis underwent either nonoperative (n = 727) or operative management (n = 703). Treatment efficacy at 1-year follow-up was significantly lower (63.8%) for antibiotics compared with the surgery group (93%) (risk ratio [RR], 0.68; 95% confidence interval [CI], 0.60-0.77; p < 0.001). Overall complications were significantly higher in the surgery group (166/703 [23.6%]) compared with the antibiotics group (56/727 [7.7%]) (RR, 0.32; 95% CI, 0.24-0.43; p < 0.001). No difference was found between the two treatment modalities in terms of perforated appendicitis rates (RR, 0.52; 95% CI, 0.14-1.92), length of hospital stay (weighted mean difference [WMD], 0.20; 95% CI, -0.16 to 0.56), duration of pain (WMD, 0.22; 95% CI, -5.30 to -5.73), and sick leave (WMD, -2; 95% CI, -5.2 to 1.1). CONCLUSIONS: Conservative management of uncomplicated appendicitis in adults warrants further study. Addressing patients' expectations via a shared decision-making process is a crucial step in optimizing nonoperative outcomes. LEVEL OF EVIDENCE: Systematic review, level II.

Momelotinib inhibits ACVR1/ALK2, decreases hepcidin production, and ameliorates anemia of chronic disease in rodents.

Patients with myelofibrosis (MF) often develop anemia and frequently become dependent on red blood cell transfusions. Results from a phase 2 study for the treatment of MF with the Janus kinase 1/2 (JAK1/2) inhibitor momelotinib (MMB) demonstrated that MMB treatment ameliorated anemia, which was unexpected for a JAK1/2 inhibitor, because erythropoietin-mediated JAK2 signaling is essential for erythropoiesis. Using a rat model of anemia of chronic disease, we demonstrated that MMB treatment can normalize hemoglobin and red blood cell numbers. We found that this positive effect is driven by direct inhibition of the bone morphogenic protein receptor kinase activin A receptor, type I (ACVR1), and the subsequent reduction of hepatocyte hepcidin production. Of note, ruxolitinib, a JAK1/2 inhibitor approved for the treatment of MF, had no inhibitory activity on this pathway. Further, we demonstrated the effect of MMB is not mediated by direct inhibition of JAK2-mediated ferroportin (FPN1) degradation, because neither MMB treatment nor myeloid-specific deletion of JAK2 affected FPN1 expression. Our data support the hypothesis that the improvement of inflammatory anemia by MMB results from inhibition of ACVR1-mediated hepcidin expression in the liver, which leads to increased mobilization of sequestered iron from cellular stores and subsequent stimulation of erythropoiesis.

Biochemical and Structural Insights into Doublecortin-like Kinase Domain 1.

Doublecortin-like kinase 1 (DCLK1) is a serine/threonine kinase that belongs to the family of microtubule-associated proteins. Originally identified for its role in neurogenesis, DCLK1 has recently been shown to regulate biological processes outside of the CNS. DCLK1 is among the 15 most common putative driver genes for gastric cancers and is highly mutated across various other human cancers. However, our present understanding of how DCLK1 dysfunction leads to tumorigenesis is limited. Here, we provide evidence that DCLK1 kinase activity negatively regulates microtubule polymerization. We present the crystal structure of the DCLK1 kinase domain at 1.7 Å resolution, providing detailed insight into the ATP-binding site that will serve as a framework for future drug design. This structure also allowed for the mapping of cancer-causing mutations within the kinase domain, suggesting that a loss of kinase function may contribute to tumorigenesis.

CIS is a potent checkpoint in NK cell-mediated tumor immunity.

The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory ligands and from cytokines such as IL-15. We identified cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signaling in NK cells. Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFN-γ production and cytotoxicity toward tumors. This was associated with increased JAK-STAT signaling in NK cells in which Cish was deleted. Correspondingly, CIS interacted with the tyrosine kinase JAK1, inhibiting its enzymatic activity and targeting JAK for proteasomal degradation. Cish(-/-) mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity. Our data uncover a potent intracellular checkpoint in NK cell-mediated tumor immunity and suggest possibilities for new cancer immunotherapies directed at blocking CIS function.

Discovery and SAR of novel pyrazolo[1,5-a]pyrimidines as inhibitors of CDK9.

The serine-threonine kinase CDK9 is a target of emerging interest for the development of anti-cancer drugs. There are multiple lines of evidence linking CDK9 activity to cancer, including the essential role this kinase plays in transcriptional regulation through phosphorylation of the C-terminal domain (CTD) of RNA polymerase II. Indeed, inhibition of CDK9 has been shown to result in a reduction of short-lived proteins such as the pro-survival protein Mcl-1 in malignant cells leading to the induction of apoptosis. In this work we report our initial studies towards the discovery of selective CDK9 inhibitors, starting from the known multi-kinase inhibitor PIK-75 which possesses potent CDK9 activity. Our series is based on a pyrazolo[1,5-a]pyrimidine nucleus and, importantly, the resultant lead compound 18b is devoid of the structural liabilities present in PIK-75 and possesses greater selectivity.

BET inhibitors induce apoptosis through a MYC independent mechanism and synergise with CDK inhibitors to kill osteosarcoma cells.

Osteosarcoma (OS) survival rates have plateaued in part due to a lack of new therapeutic options. Here we demonstrate that bromodomain inhibitors (BETi), JQ1, I-BET151, I-BET762, exert potent anti-tumour activity against primary and established OS cell lines, mediated by inhibition of BRD4. Strikingly, unlike previous observations in long-term established human OS cell lines, the antiproliferative activity of JQ1 in primary OS cells was driven by the induction of apoptosis, not cell cycle arrest. In further contrast, JQ1 activity in OS was mediated independently of MYC downregulation. We identified that JQ1 suppresses the transcription factor FOSL1 by displacement of BRD4 from its locus. Loss of FOSL1 phenocopied the antiproliferative effects of JQ1, identifying FOSL1 suppression as a potential novel therapeutic approach for OS. As a monotherapy JQ1 demonstrated significant anti-tumour activity in vivo in an OS graft model. Further, combinatorial treatment approaches showed that JQ1 increased the sensitivity of OS cells to doxorubicin and induced potent synergistic activity when rationally combined with CDK inhibitors. The greater level of activity achieved with the combination of BETi with CDK inhibitors demonstrates the efficacy of this combination therapy. Taken together, our studies show that BET inhibitors are a promising new therapeutic for OS.

HSP90 inhibition suppresses lipopolysaccharide-induced lung inflammation in vivo.

Inflammation is an important component of cancer diathesis and treatment-refractory inflammation is a feature of many chronic degenerative lung diseases. HSP90 is a 90kDa protein which functions as an ATP-dependent molecular chaperone that regulates the signalling conformation and expression of multiple protein client proteins especially oncogenic mediators. HSP90 inhibitors are in clinical development as cancer therapies but the myeleosuppressive and neutropenic effect of first generation geldanamycin-class inhibitors has confounded studies on the effects on HSP90 inhibitors on inflammation. To address this we assessed the ability of Ganetespib, a non-geldanamycin HSP90 blocker, to suppress lipopolysaccharide (LPS)-induced cellular infiltrates, proteases and inflammatory mediator and transcriptional profiles. Ganetespib (10-100 mg/kg, i.v.) did not directly cause myelosuppression, as assessed by video micrography and basal blood cell count, but it strongly and dose-dependently suppressed LPS-induced neutrophil mobilization into blood and neutrophil- and mononuclear cell-rich steroid-refractory lung inflammation. Ganetespib also suppressed B cell and NK cell accumulation, inflammatory cytokine and chemokine induction and MMP9 levels. These data identify non-myelosuppresssive HSP90 inhibitors as potential therapies for inflammatory diseases refractory to conventional therapy, in particular those of the lung.

A laparoscopic swine model of noncompressible torso hemorrhage.

BACKGROUND: Hemorrhage persists as the leading cause of potentially preventable civilian and military death. Noncompressible torso hemorrhage (NCTH) is a particularly lethal injury complex, with few contemporary prehospital interventions available. Various porcine models of hemorrhage have been developed for civilian and military trauma research. However, the predominant contemporary models lack key physiologic characteristics including the natural tamponade provided by an intact abdominal wall.To improve physiologic and clinical relevance, we developed a laparoscopic model of NCTH. This approach maintains both the integrity of the peritoneum and the natural tamponade effect of an intact abdominal wall while preserving the intrinsic physiologic responses to hemorrhage. Furthermore, we present data quantifying the contribution of the swine contractile spleen in the context of uncontrolled hemorrhage. METHODS: Anesthetized adult male Yorkshire swine underwent a laparoscopic Grade V liver injury, with or without open preinjury splenectomy. Animals were observed without intervention for a total of 120 minutes after injury to simulate point of injury, transport time, and arrival at hospital. RESULTS: Shed blood-to-body weight ratio did not differ among groups; however, mortality was higher in splenectomized animals (67% vs. 33%). Cox regression modeling demonstrated a critical time point of 45 minutes and blood pressure as significant predictors of mortality. CONCLUSION: This study describes a model of NCTH that reflects clinically relevant physiology in trauma and uncontrolled hemorrhage. In addition, it quantitatively assesses the role of the swine contractile spleen in the described model.

Inhibition of the JAK2/STAT3 pathway in ovarian cancer results in the loss of cancer stem cell-like characteristics and a reduced tumor burden.

BACKGROUND: Current treatment of ovarian cancer patients with chemotherapy leaves behind a residual tumor which results in recurrent ovarian cancer within a short time frame. We have previously demonstrated that a single short-term treatment of ovarian cancer cells with chemotherapy in vitro resulted in a cancer stem cell (CSC)-like enriched residual population which generated significantly greater tumor burden compared to the tumor burden generated by control untreated cells. In this report we looked at the mechanisms of the enrichment of CSC-like residual cells in response to paclitaxel treatment. METHODS: The mechanism of survival of paclitaxel-treated residual cells at a growth inhibitory concentration of 50% (GI50) was determined on isolated tumor cells from the ascites of recurrent ovarian cancer patients and HEY ovarian cancer cell line by in vitro assays and in a mouse xenograft model. RESULTS: Treatment of isolated tumor cells from the ascites of ovarian cancer patients and HEY ovarian cancer cell line with paclitaxel resulted in a CSC-like residual population which coincided with the activation of Janus activated kinase 2 (JAK2) and signal transducer and activation of transcription 3 (STAT3) pathway in paclitaxel surviving cells. Both paclitaxel-induced JAK2/STAT3 activation and CSC-like characteristics were inhibited by a low dose JAK2-specific small molecule inhibitor CYT387 (1 µM) in vitro. Subsequent, in vivo transplantation of paclitaxel and CYT387-treated HEY cells in mice resulted in a significantly reduced tumor burden compared to that seen with paclitaxel only-treated transplanted cells. In vitro analysis of tumor xenografts at protein and mRNA levels demonstrated a loss of CSC-like markers and CA125 expression in paclitaxel and CYT387-treated cell-derived xenografts, compared to paclitaxel only-treated cell-derived xenografts. These results were consistent with significantly reduced activation of JAK2 and STAT3 in paclitaxel and CYT387-treated cell-derived xenografts compared to paclitaxel only-treated cell derived xenografts. CONCLUSIONS: This proof of principle study demonstrates that inhibition of the JAK2/STAT3 pathway by the addition of CYT387 suppresses the 'stemness' profile in chemotherapy-treated residual cells in vitro, which is replicated in vivo, leading to a reduced tumor burden. These findings have important implications for ovarian cancer patients who are treated with taxane and/or platinum-based therapies.

Simplified silvestrol analogues with potent cytotoxic activity.

The complex natural products silvestrol (1) and episilvestrol (2) are inhibitors of translation initiation through binding to the DEAD-box helicase eukaryotic initiation factor 4A (eIF4A). Both compounds are potently cytotoxic to cancer cells in vitro, and 1 has demonstrated efficacy in vivo in several xenograft cancer models. Here we show that 2 has limited plasma membrane permeability and is metabolized in liver microsomes in a manner consistent with that reported for 1. In addition, we have prepared a series of analogues of these compounds where the complex pseudo-sugar at C6 has been replaced with chemically simpler moieties to improve drug-likeness. Selected compounds from this work possess excellent activity in biochemical and cellular translation assays with potent activity against leukemia cell lines.

Targeting BCL-2 with the BH3 mimetic ABT-199 in estrogen receptor-positive breast cancer.

The prosurvival protein BCL-2 is frequently overexpressed in estrogen receptor (ER)-positive breast cancer. We have generated ER-positive primary breast tumor xenografts that recapitulate the primary tumors and demonstrate that the BH3 mimetic ABT-737 markedly improves tumor response to the antiestrogen tamoxifen. Despite abundant BCL-XL expression, similar efficacy was observed with the BCL-2 selective inhibitor ABT-199, revealing that BCL-2 is a crucial target. Unexpectedly, BH3 mimetics were found to counteract the side effect of tamoxifen-induced endometrial hyperplasia. Moreover, BH3 mimetics synergized with phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitors in eliciting apoptosis. Importantly, these two classes of inhibitor further enhanced tumor response in combination therapy with tamoxifen. Collectively, our findings provide a rationale for the clinical evaluation of BH3 mimetics in therapy for breast cancer.

The Use of JAK-specific inhibitors as chemical biology tools.

The JAK family of protein tyrosine kinases are now recognized as important participants in a wide range of pathologies, from cancer to inflammatory diseases. In the last decade, the drive to develop drugs targeting members of this family has begun to deliver a panel of small molecule inhibitors of JAK family members, with a range of potencies and specificities. A number of these compounds have already found widespread use as biochemical tools in the elucidation of JAK activity in specific signaling and disease processes; however, many of the first generation compounds are poorly characterized with suboptimal potencies and selectivities.Herein, we present the data for those small molecule JAK inhibitors that have been described in the peer-reviewed literature and the benefits and potential issues that may be associated with the use of these tool compounds.

Synthesis and biological evaluation of a potent salicylihalamide A lactam analogue.

The first synthesis of a lactam analogue of salicylihalamide A (1) is reported. A key step in the approach was a photochemical acylation coupling between amine 10 and dioxinone 9 to form the amide 19. Acetylation followed by RCM with Grubbs 1st generation catalyst gave the desired E-lactam 23 (E : Z ratio 87 : 13) as the major compound. Conversion of macrolactam 23 into the vinyl iodide 26 followed by Cu catalysed cross coupling with the diene amide 7 gave aza-salicylihalamide analogue 3 in good yield. This compound demonstrated potent activity against several human leukaemia cell lines.

Prehospital interventions performed in a combat zone: a prospective multicenter study of 1,003 combat wounded.

BACKGROUND: Battlefield care given to a casualty before hospital arrival impacts clinical outcomes. To date, the published data regarding care given in the prehospital setting of a combat zone are limited. The purpose of this study was to describe the incidence and efficacy of specific prehospital lifesaving interventions (LSIs; interventions that could affect the outcome of the casualty), consistent with the Tactical Combat Casualty Care paradigm, performed during the resuscitation of casualties in a combat zone. METHODS: We performed a prospective observational study between November 2009 and November 2011. Casualties were enrolled as they were treated at six US surgical facilities in Afghanistan. Descriptive data were collected on a standardized data collection form and included mechanism of injury, airway management, chest and hemorrhage interventions, vascular access, type of fluid administered, and hypothermia prevention. On arrival to the military hospital, the treating physician determined whether an intervention was performed correctly and whether an intervention was not performed that should have been performed (missed LSI). RESULTS: A total of 1,003 patients met the inclusion criteria. Their mean (SD) age was 25 (8.5) years and 97% were male. The mechanism of injury was explosion in 60% of patients, penetrating in 24% of patients, blunt in 15% of patients, and burn in 0.8% of patients. The most commonly performed LSIs included hemorrhage control (n = 599), hypothermia prevention (n = 429), and vascular access (n = 388). Of the missed LSIs, 252 were identified with the highest percentage of missed opportunities being composed of endotracheal intubation, chest needle decompression, and hypotensive resuscitation. In contrast, tourniquet application had the lowest percentage of missed opportunities. CONCLUSIONS: In our prospective study of prehospital LSIs performed in a combat zone, we observed a higher rate of incorrectly performed and missed LSIs in airway and chest (breathing) interventions than hemorrhage control interventions. The most commonly performed LSIs had lower incorrect and missed LSI rates.

The microtubule depolymerizing agent CYT997 causes extensive ablation of tumor vasculature in vivo.

The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2-yl)phenyl)urea (CYT997), reported previously by us (Bioorg Med Chem Lett 19:4639-4642, 2009; Mol Cancer Ther 8:3036-3045, 2009), is potently cytotoxic to a variety of cancer cell lines in vitro and shows antitumor activity in vivo. In addition to its cytotoxic activity, CYT997 possesses antivascular effects on tumor vasculature. To further characterize the vascular disrupting activity of CYT997 in terms of dose and temporal effects, we studied the activity of the compound on endothelial cells in vitro and on tumor blood flow in vivo by using a variety of techniques. In vitro, CYT997 is shown to potently inhibit the proliferation of vascular endothelial growth factor-stimulated human umbilical vein endothelial cells (IC(50) 3.7 ± 1.8 nM) and cause significant morphological changes at 100 nM, including membrane blebbing. Using the method of corrosion casting visualized with scanning electron microscopy, a single dose of CYT997 (7.5 mg/kg i.p.) in a metastatic cancer model was shown to cause destruction of tumor microvasculature in metastatic lesions. Furthermore, repeat dosing of CYT997 at 10 mg/kg and above (intraperitoneally, b.i.d.) was shown to effectively inhibit development of liver metastases. The time and dose dependence of the antivascular effects were studied in a DLD-1 colon adenocarcinoma xenograft model using the fluorescent dye Hoechst 33342. CYT997 demonstrated rapid and dose-dependent vascular shutdown, which persists for more than 24 h after a single oral dose. Together, the data demonstrate that CYT997 possesses potent antivascular activity and support continuing development of this promising compound.