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Small molecules have played an important role in delineating molecular pathways involved in embryonic development and disease pathology. The need for novel small molecule modulators of biological processes has driven a number of targeted screens on large diverse libraries. However, due to the specific focus of such screens, the majority of the bioactive potential of these libraries remains unharnessed. In order to identify a higher proportion of compounds with interesting biological activities, we screened a diverse synthetic library for compounds that perturb the development of any of the multiple organs in zebrafish embryos. We identified small molecules that affect the development of a variety of structures such as heart, vasculature, brain, and body-axis. We utilized the previously known role of retinoic acid in anterior-posterior (A-P) patterning to identify the target of DTAB, a compound that caused A-P axis shortening in the zebrafish embryo. We show that DTAB is a retinoid with selective activity towards retinoic acid receptors gamma and beta. Thus, conducting zebrafish developmental screens using small molecules will not only enable the identification of compounds with diverse biological activities in a large chemical library but may also facilitate the identification of the target pathways of these biologically active molecules.

Deletions on chromosome 9q are seen in a subset of acute myeloid leukemia (AML) cases and are specifically associated with t(8;21) AML. We previously defined the commonly deleted region in del(9q) AML and characterized the genes in this interval. To determine the critical lost gene(s) that might cooperate with the AML1-ETO fusion gene produced by t(8;21), we developed a set of shRNAs directed against each gene in this region. Within this library, shRNAs to TLE1 and TLE4 were the only shRNAs capable of rescuing AML1-ETO expressing U937T-A/E cells from AML1-ETO–induced cell-cycle arrest and apoptosis. Knockdown of TLE1 or TLE4 levels increased the rate of cell division of the AML1-ETO–expressing Kasumi-1 cell line, whereas forced expression of either TLE1 or TLE4 caused apoptosis and cell death. Knockdown of Gro3, a TLE homolog in zebrafish, cooperated with AML1-ETO to cause an accumulation of noncirculating hematopoietic blast cells. Our data are consistent with a model in which haploinsufficiency of these TLEs overcomes the negative survival and antiproliferative effects of AML1-ETO on myeloid progenitors, allowing preleukemic stem cells to expand into AML. This study is the first to implicate the TLEs as potential tumor suppressor genes in myeloid leukemia.

Identifying the molecular pathways that are required for regeneration remains one of the great challenges of regenerative medicine. Although genetic mutations have been useful for identifying some molecular pathways, small molecule probes of regenerative pathways might offer some advantages, including the ability to disrupt pathway function with precise temporal control. However, a vertebrate regeneration model amenable to rapid throughput small molecule screening is not currently available. We report here the development of a zebrafish early life stage fin regeneration model and its use in screening for small molecules that modulate tissue regeneration. By screening 2000 biologically active small molecules, we identified 17 that specifically inhibited regeneration. These compounds include a cluster of glucocorticoids, and we demonstrate that transient activation of the glucocorticoid receptor is sufficient to block regeneration, but only if activation occurs during wound healing/blastema formation. In addition, knockdown of the glucocorticoid receptor restores regenerative capability to nonregenerative, glucocorticoid-exposed zebrafish. To test whether the classical anti-inflammatory action of glucocorticoids is responsible for blocking regeneration, we prevented acute inflammation following amputation by antisense repression of the Pu.1 gene. Although loss of Pu.1 prevents the inflammatory response, regeneration is not affected. Collectively, these results indicate that signaling from exogenous glucocorticoids impairs blastema formation and limits regenerative capacity through an acute inflammation-independent mechanism. These studies also demonstrate the feasibility of exploiting chemical genetics to define the pathways that govern vertebrate regeneration.

Bone morphogenetic protein (BMP) signals coordinate developmental patterning and have essential physiological roles in mature organisms. Here we describe the first known small-molecule inhibitor of BMP signaling—dorsomorphin, which we identified in a screen for compounds that perturb dorsoventral axis formation in zebrafish. We found that dorsomorphin selectively inhibits the BMP type I receptors ALK2, ALK3 and ALK6 and thus blocks BMP-mediated SMAD1/5/8 phosphorylation, target gene transcription and osteogenic differentiation. Using dorsomorphin, we examined the role of BMP signaling in iron homeostasis. In vitro, dorsomorphin inhibited BMP-, hemojuvelin- and interleukin 6–stimulated expression of the systemic iron regulator hepcidin, which suggests that BMP receptors regulate hepcidin induction by all of these stimuli. In vivo, systemic challenge with iron rapidly induced SMAD1/5/8 phosphorylation and hepcidin expression in the liver, whereas treatment with dorsomorphin blocked SMAD1/5/8 phosphorylation, normalized hepcidin expression and increased serum iron levels. These findings suggest an essential physiological role for hepatic BMP signaling in iron-hepcidin homeostasis.

Previous mode of action studies identified methionine aminopeptidase 2 (MetAP-2) as the target of the anti-angiogenic natural product fumagillin and its drug candidate analog, TNP-470. We report here that TNP-470-mediated MetAP-2 inhibition blocks noncanonical Wnt signaling, which plays a critical role in development, cell differentiation, and tumorigenesis. Consistent with this finding, antisense MetAP-2 morpholino oligonucleotide injection in zebrafish embryos pheno-copies gastrulation defects seen in noncanonical Wnt5 loss-of-function zebrafish mutants. MetAP-2 inhibition or depletion blocks signaling downstream of the Wnt receptor Frizzled, but upstream of Calmodulin-dependent Kinase II, RhoA, and c-Jun N-terminal Kinase. Moreover, we demonstrate that TNP-470 does not block the canonical Wnt/β-catenin pathway. Thus, TNP-470 selectively regulates noncanonical over canonical Wnt signaling and provides a unique means to explore and dissect the biological systems mediated by these pathways.

Normal heart function is critically dependent on the timing and coordination provided by a complex network of specialized cells: the cardiac conduction system. We have employed functional assays in zebrafish to explore early steps in the patterning of the conduction system that previously have been inaccessible. We demonstrate that a ring of atrioventricular conduction tissue develops at 40 hours post-fertilization in the zebrafish heart. Analysis of the mutant cloche reveals a requirement for endocardial signals in the formation of this tissue. The differentiation of these specialized cells, unlike that of adjacent endocardial cushions and valves, is not dependent on blood flow or cardiac contraction. Finally, both neuregulin and notch1b are necessary for the development of atrioventricular conduction tissue. These results are the first demonstration of the endocardial signals required for patterning central `slow' conduction tissue, and they reveal the operation of distinct local endocardial-myocardial interactions within the developing heart tube.

The zebrafish has become a widely used model organism because of its fecundity, its morphological and physiological similarity to mammals, the existence of many genomic tools and the ease with which large, phenotype-based screens can be performed. Because of these attributes, the zebrafish might also provide opportunities to accelerate the process of drug discovery. By combining the scale and throughput of in vitro screens with the physiological complexity of animal studies, the zebrafish promises to contribute to several aspects of the drug development process, including target identification, disease modelling, lead discovery and toxicology.

SPECIFIC AIMSHeart failure represents the end stage of familial or acquired cardiac diseases where compensatory mechanisms are no longer able to restore cardiac function and can further accelerate the disease process. A wide array of genes has been shown to be altered in that process and a number of them have been explored in human and animal models of heart failure. Analysis of transcriptional reprogramming in heart failure enables us to explore the events that trigger the cascade of changes that progress to heart failure as well as the compensatory mechanisms that take place to preserve cardiac function.Beyond the many structural changes, failing hearts show a permanent decline in contractile function. At a cellular level, the functional defect is detected as a reduction in the amplitude of cell shortening, velocities of shortening and relaxation, as well as abnormal intracellular Ca2+ homeostasis. At the molecular level, despite differences in the triggering event, a defect in protein level and functio...

We used zebrafish to screen and identify small molecules that affect the process of vertebrate hematopoietic development. Zebrafish embryos were exposed to a library of 5000 synthetic compounds and screened for defects in primitive erythropoiesis. Here, we present the characterization of hemolytic anemia induced in zebrafish by the small molecule 5115318 (3-[5-methyl-furan 2-yl]-propionic acid N'-phenyl-hydrazide). This compound is capable of generating hemoglobin aggregates and Heinz bodies in red cells in vivo only. The induced anemia is reversible and treated fish recover in about 4 days. This study shows the feasibility of using zebrafish to screen for small molecules that can modulate the specific process of erythropoiesis.

Publisher Summary This chapter describes the discovery and use of small molecules for probing biological processes in zebrafish. The chapter illustrates that the zebrafish has become established as a powerful tool for genetic experimentation. In contrast to the other mainstays of metazoan genetics, Drosophila, C. elegans and mouse, it has proved equally amenable to chemical screens. In contrast to the other mainstays of metazoan genetics, Drosophila, C. elegans, and mouse, it has proved equally amenable to chemical screens. Whether the goal is discovery of conditional probes for basic research or discovery of therapeutic targets and lead compounds, the zebrafish enables high-throughput assessment of the biological effects of small molecules. Several challenges remain, particularly in the area of identifying small molecule targets. However, the ability to discover specific, interesting, and useful compounds using zebrafish is clear, and zebrafish-based small molecule screens can provide a novel and accessible approach for launching projects, both in pathway dissection and drug discovery.

Background— Drug-induced QT prolongation and torsades de pointes remain significant and often unpredictable clinical problems. Current in vitro preclinical assays are limited by biological simplicity, and in vivo models suffer from expense and low throughput. Methods and Results— During a screen for the effects of 100 small molecules on the heart rate of the zebrafish, Danio rerio , we found that drugs that cause QT prolongation in humans consistently caused bradycardia and AV block in the zebrafish. Of 23 such drugs tested, 18 were positive in this initial screen. Poor absorption explained 4 of 5 false-negative results, as demonstrated by microinjection. Overall, 22 of 23 compounds that cause repolarization abnormalities were positive in this assay. Antisense “knockdown” of the zebrafish KCNH2 ortholog yielded bradycardia in a dose dependent manner confirming the effects of reduction of repolarizing potassium current in this model. Classical drug-drug interactions between erythromycin and cisapride, as well as cimetidine and terfenadine, were also reproduced. Conclusion— This simple high-throughput assay is a promising addition to the repertoire of preclinical tests for drug-induced repolarization abnormalities. The genetic tractability of the zebrafish will allow the exploration of heritable modifiers of such drug effects.

To the Editor: Miller et al. recently described a TALE nuclease architecture for performing efficient genome editing1. The authors demonstrated that TALE nucleases, composed of an engineered array of TALE repeats fused to the non-specific FokI cleavage domain, could be used to introduce targeted double-stranded breaks (DSBs) in human cells with high efficiency. Repair of these DSBs by normal DNA repair mechanisms such as non-homologous end-joining (NHEJ) or homologous recombination (HR) enables introduction of alterations at or near the site of the break. A single 34 amino acid TALE repeat binds to one bp of DNA and repeats that bind each of the four DNA bases have been described2, 3. These modules can be assembled into arrays capable of binding extended DNA sequences. TALE nucleases may have advantages over engineered zinc finger nucleases (ZFNs) due to the relative ease with which they can be designed and their potential ability to be targeted to a wide range of sequences (with target sites reported to be as frequent as 1 in 35 bps of random DNA sequence4). We sought to determine whether the TALE nuclease framework described by Miller et al. could also be used to efficiently modify endogenous genes in zebrafish. Previous studies have shown that error-prone repair of ZFN-induced DSBs by NHEJ can result in the efficient introduction of small insertions or deletions (indels) at cleavage sites in endogenous zebrafish genes5–7. These indels frequently result in frameshift knockout mutations that can be passed through the germline to create mutant fish5–9. ZFN technology has enabled reverse genetics studies to be performed in zebrafish, a capability that did not previously exist. However, engineering ZFNs can be challenging due to the need to account for context-dependent effects among individual fingers in an array. In addition, although many zebrafish genes can be targeted with ZFNs made by publicly available methods that account for context-dependence7, 10, it can in some instances be difficult to target within some genes in zebrafish due to the currently limited targeting range of publicly available ZFN engineering platforms. Thus, if TALE nucleases could be used to introduce targeted mutations in zebrafish, this platform would provide an important additional capability for this model organism. To test the ability of TALE nucleases to function in zebrafish, we targeted DNA sequences in two endogenous zebrafish genes gria3a and hey2 (Figure 1). To avoid confounding effects that might affect binding and cleavage of DNA sites by TALE nucleases (e.g.--chromatin structure or DNA methylation), we chose to target sequences that we had efficiently altered previously in zebrafish using engineered ZFNs (Supplementary Figures 1 and 2). Using an iterative assembly approach (Supplementary Methods), we constructed four TALE nuclease monomers to partially overlapping sites in the gria3a gene and two TALE nuclease monomers to a site in the hey2 gene (Figure 1 and Supplementary Figure 3). These six TALE nuclease monomers all harbor the wild-type FokI cleavage domain (Supplementary Figures 4 and 5) and can be paired in combinations to make three TALE nuclease dimers to the gria3a gene and one TALE nuclease dimer to the hey2 gene (Figure 1). We injected RNAs encoding the various TALE nuclease pairs into one-cell stage zebrafish embryos and determined the frequency of NHEJ-mediated mutagenesis at the target site by sequence analysis of alleles from pooled injected embryos (Supplementary Methods, Supplementary Figs. 6–10 and Supplementary Table 1). As shown in Figure 1, we found that all four pairs of TALE nucleases induced targeted indels with high mutation frequencies ranging from 11 to 33%. These frequencies are comparable to what we obtained with ZFNs targeted to DNA sequences in the same vicinity of the gene (Supplementary Figure 1); however, we note that TALE nucleases harbor wild-type FokI domains whereas the ZFNs harbor obligate heterodimeric FokI domains11. Although small indels were typically observed with the TALE nucleases, some large deletions (up to 303 basepairs) were also found (Figure 1). Figure 1 Target sequences, frequencies of mutations, and sequences of mutations induced by TALE nucleases in embryonic zebrafish cells To assess the toxicity of our engineered TALE nucleases, we scored the percentages of dead and deformed embryos that resulted from mRNA microinjections (Supplementary Figure 11). Although we cannot directly compare these results with the microinjections of ZFNs due to the differences between the FokI endonuclease domains used (EL/KK heterodimeric FokI11 for ZFNs versus wild-type FokI for the TALE nucleases) and the specific sequences targeted, the toxicity we observed with injection of 600 pg of TALE nuclease mRNAs (ranging between 40–80%) appears similar to that observed with 400–500 pg of mRNAs encoding ZFNs targeted to sequences in the same vicinity and to other genes (Supplementary Figure 12 and Reference 7). An important future experiment will be to demonstrate germline transmission of TALE nuclease-induced mutations. Given that the frequencies of mutation and the extent of toxicities we observe are similar to what we have seen with ZFNs, we expect that TALE nuclease-induced mutations should be efficiently passed through the germline to progeny and we are currently conducting experiments to test this prediction. Successful germline transmission of these mutations will be critical for using TALE nucleases to perform reverse genetics in zebrafish. Progeny fish bearing TALE nuclease-induced mutations, unlike founder F0 fish, will not be mosaic (i.e.--these fish will have uniform mutation of all cells in the organism); such mutant fish will enable determination of whether both mono-allelic and bi-allelic alterations of a gene are possible and will provide a more straightforward background on which to perform analysis of off-target effects. In summary, we show that the TALE nuclease framework described by Miller et al. can be used to efficiently introduce targeted indel mutations in endogenous zebrafish genes at the somatic cell level. Although in this study we chose two genomic loci that have been successfully targeted with ZFNs before, all six TALE nuclease monomers we constructed showed high mutagenesis activities when tested in various pairwise combinations, suggesting that the TALE nuclease framework is also highly robust and effective in zebrafish. As is the case with ZFNs, the complete genome-wide spectrum of off-target mutations introduced by TALE nucleases remains unknown. However, expression of the TALE nucleases we made in zebrafish does not show toxicity substantially different from that observed with expression of ZFNs, suggesting that the magnitude of off-target effects may be comparable with the two types of nucleases. In principle, off-target mutations made by TALE nucleases can be removed by out-crossing the founder assuming that they are not tightly linked to the intended mutation. In addition, mutant phenotypes could also be confirmed by generation of a second mutant allele using nucleases targeted to a different site. For mutagenesis of genes in zebrafish (and other model organisms such as C. elegans12), TALE nucleases may offer potential advantages over ZFNs because they can be easily and quickly assembled in a modular fashion and they can potentially target a greater range of DNA sequences. Thus, we expect that the ability to utilize both ZFNs and TALE nucleases should enable any researcher to rapidly and easily create targeted mutations in their endogenous zebrafish gene of interest.

The Wnt signalling pathway balances the opposing activities of two proteins to transmit signals within cells. An inhibitor that stabilizes one of these proteins reveals a new target for anticancer drug development.

Wnt signals are seemingly ubiquitous in biology, controlling processes as diverse as bristle patterning in flies and tissue regeneration in humans. A new report describes the discovery of small molecules that inhibit Wnt signaling by two unprecedented mechanisms, paving the way for fundamental studies and perhaps improved treatment of colon cancer.

The identification of PDK1 as a kinase that phosphorylates the AGC family of kinases led to a hunt for ‘PDK2’, a hypothetical regulated kinase(s) that would be required for full activation of the AGC kinases. Recent findings suggest that the elusive PDK2 may actually be a familiar kinase with an atypical associate.

Protein phosphatases participate in the regulation of a variety of cellular processes. Control of their enzymatic activity and specificity is made possible largely by an array of regulatory subunits. Novel serine/threonine phosphatases--PP4 and PP6 in human cells--have been discovered recently, for which regulatory subunits are yet to be identified. We report here the identification of a potential regulatory subunit of these phosphatases. Using the yeast two-hybrid system, we have found that alpha 4, a previously identified phosphoprotein, associates constitutively with the catalytic subunits of PP4, PP6, and both isoforms of PP2A. These interactions have been confirmed by direct binding and do not require phosphorylation of alpha 4, although it is unclear whether alpha 4 phosphorylation has any effect on its association with the phosphatases. The binding activity appears to reside in the N-terminal 50 amino acids of the phosphatases, consistent with a previous observation that the first 55 residues of PPV, a Drosophila homolog of PP6, may harbor the element for regulation. alpha 4 shares 37% sequence homology with Tap42, an S. cerevisiae protein that has been reported to associate with PP2A and Sit4 (yeast homolog of PP6) and comprises a regulatory component in the rapamycin-sensitive Tor signalling pathway. By analogy, alpha 4 and its associated phosphatases may participate in the mammalian rapamycin-sensitive pathway mediated by FRAP.

Background: The solid tumor microvasculature is characterized by structural immaturity and functional abnormality, and mediates several deleterious aspects of tumor behavior. The vascular endothelial protein tyrosine phosphatase (VE-PTP) attenuates the activity of the endothelial cell (EC) Tie-2 receptor tyrosine kinase, a key mediator of vessel maturation. Here we determine the role of VE-PTP in the vasculature of primary and metastatic mammary carcinomas. Methods: AKB-9778 is a first-in-class pharmacologic VE-PTP inhibitor. We systematically examined its effects in vitro and in vivo. First we studied the effects of AKB-9778 on Tie-2 signaling in two endothelial cell lines in vitro, and also measured its impact on Tie-2 activation in normal and tumor ECs in mice. We next used embryonic zebrafish assays to determine the effects of AKB-9778 on embryonic angiogenesis. We also studied the impact of AKB-9778 therapy on the tumor vasculature, tumor growth and metastatic progression using orthotopic models of murine mammary carcinoma as well as spontaneous and experimental metastasis models (4T1, E0771, P0008, MMTV-PyMT). Finally, we used endothelial nitric oxide synthase (eNOS) deficient mice to establish the role of eNOS in mediating the effects of VE-PTP inhibition. Results: AKB-9778 induced ligand-independent Tie-2 activation in ECs in vitro and in vivo, and impaired embryonic zebrafish angiogenesis. In mouse models of breast cancer, AKB-9778 (i) delayed the early phase of mammary tumor growth by maintaining vascular maturity; (ii) slowed the growth of spontaneous micrometastases by preventing extravasation of tumor cells into distant organ parenchyma (prolonging survival above adjuvant chemotherapy alone); and (iii) matured established primary tumor blood vessels (increased pericyte coverage, reduced permeability) in turn enhancing tumor perfusion, reducing hypoxia, and improving radiation response. Experiments using eNOS knockout mice showed that the effects of AKB-9778 on tumor vessels were mediated in part by eNOS activation. Conclusions: Phosphatase inhibition is a relatively unexplored field in cancer, and this is to our knowledge the first demonstration of the effects of VE-PTP inhibition in any disease. Our results demonstrate that pharmacological VE-PTP inhibition can normalize the structure and function of tumor vessels through Tie-2 activation, which delays tumor growth, slows metastatic progression, and enhances response to concomitant cytotoxic treatments. Furthermore we provide genetic evidence for the causal role of eNOS in mediating the beneficial effects of VEPTP inhibition in established tumors. By using models of adjuvant therapy (combined with chemotherapy) and radiation treatment, our results indicate possible avenues for further studying the clinical translatability of VE-PTP inhibition in cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C1. Citation Format: Shom Goel, Nisha Gupta, Brian P. Walcott, Matija Snuderl, Cristina T. Kesler, Benjamin J. Vakoc, Randall T. Peterson, Timothy P. Padera, Dan G. Duda, Dai Fukumura, Rakesh K. Jain. Functional normalization of the breast cancer vasculature through activation of Tie2 using a vascular-endothelial protein tyrosine phosphatase inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C1.

We have previously reported a simple and customizable CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided Cas9 nuclease (RGN) system that can be used to efficiently and robustly introduce somatic indel mutations in endogenous zebrafish genes. Here we demonstrate that RGN-induced mutations are heritable, with efficiencies of germline transmission reaching as high as 100%. In addition, we extend the power of the RGN system by showing that these nucleases can be used with single-stranded oligodeoxynucleotides (ssODNs) to create precise intended sequence modifications, including single nucleotide substitutions. Finally, we describe and validate simple strategies that improve the targeting range of RGNs from 1 in every 128 basepairs (bps) of random DNA sequence to 1 in every 8 bps. Together, these advances expand the utility of the CRISPR-Cas system in the zebrafish beyond somatic indel formation to heritable and precise genome modifications.

Hypoxia Inducible Factor (HIF) target genes are involved in several cellular processes including angiogenesis, erythropoiesis, metabolism and stem cell proliferation. The von Hippel-Lindau (VHL) tumor suppressor protein targets HIF for degradation in conditions of normal oxygen tension. Loss-of-VHL leads to constitutive expression of HIF. We have discovered small molecules that repress HIF-2α translation by enhancing the binding of Iron-Regulatory Protein 1 (IRP1) to the Iron-Responsive Element (IRE) within the 5’-UTR of HIF-2α mRNA and showed that these small molecules specifically down-regulate HIF-2α expression in vitro. To validate the HIF-2α inhibitors in vivo, first we tested their efficacy to inhibit acute hif induction in wild-type (wt) zebrafish embryos challenged with the hypoxia mimetic DMOG. Treatment of wt embryos with 100μM of DMOG up-regulated the expression of zebrafish HIF-target genes, such as vascular endothelial growth factor (vegf), erythropoietin (epo) and prolyl-hydroxylase 3 (phd3). Pre-treatment of embryos with the small molecule HIF-2α inhibitor 76 (10nM) significantly decreased the DMOG-induced up-regulation of HIF target genes and resulted in decrease of DMOG-induced erythrocytosis and blood vessel sprouting. Furthermore, we tested the efficacy of this HIF-2α inhibitor in a vhl zebrafish model (homozygougous mutants harbouring inactivating vhl mutations), which recapitulates aspects of human VHL disease (erythrocytosis, abnormal vascular proliferation in sites such as retina and brain, early lethality). Treatment with inhibitor 76 (10nM, 100nM and 1μM) down regulated the expression of the HIF-target genes phd3, epo and vegf in a dose response manner and rescued the erythrocytosis and sprouting phenotype of vhl embryos. To image and quantify the blood vessel abnormalities of vhl embryos, we generated a Fli-GFP::vhl+/- line and showed that compound 76 improves the abnormal connections between the intersegmental vessels of the Fli-GFP::vhl embryo tail, as well as their human hemangioblastoma-resembling brain vessel proliferation. Finally, we showed that treatment of vhl embryos with compound 76 rescues early lethality. Our data demonstrate that HIF-2α inhibitor 76 is active in vivo at low concentrations and encourage further development and preclinical testing of this HIF-2α inhibitor for treatment of VHL-associated lesions, such as renal cell carcinoma, hemangioblastoma and neuroendocrine tumors. Citation Format: Ana Metelo, Li Xiang, Rania Baker, Lee Kamentsky, Anne Carpenter, Randall Peterson. Small molecule HIF-2a inhibitors down regulate its activity in vivo and ameliorate the vhl zebrafish phenotype. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1578. doi:10.1158/1538-7445.AM2013-1578

CD36 is an endothelial cell (EC) receptor that mediates angiostatic activity and oxidative stress, which are important in ischemic disease and tumor progression. CD36 interacts with TSR domain proteins including thrombospondin-1. This results in a signal that is likely to initiate an antiangiogenic “switch," subsequently converting a growth factor-mediated proangiogenic response to an antiangiogenic response. However, in pathologic settings including malignant tumors, robust angiogenesis occurs despite the abundance of TSR-containing proteins in the microenvironment. This suggests that there is a mechanism by which TSR-mediated antiangiogenesis could be blunted via the localized downregulation of EC CD36 transcription. We previously reported that lysophosphatidic acid (LPA), a bioactive signaling phospholipid mediator, down-regulates microvascular EC (MVEC) CD36 transcription by protein kinase PKD-1 signaling via G protein coupled receptor LPA1,3. However, further elucidating the CD36 transcriptional mechanisms is critical to understanding the antiangiogenic switch. To this end, we recently discovered that obesity-derived LPA inhibited CD36 expression in tumor-associated ECs (TAECs). Similarly, inducing the constitutively active PKD-1 or PKD-CA expression also resulted in CD36 downregulation. Mechanistically, LPA treatment led to nuclear accumulation of PKD-1, HDAC7 and FoxO1. Furthermore, LPA-mediated PKD-1 signaling enhanced FoxO1-HDAC7 interaction in the nucleus. This interaction was attenuated with PKD-1 silencing. Intriguingly, doxycycline induced PKD-CA expression increased ephrin B2 expression and ERK activation, which are two critical “molecular signatures” involved in arteriogenesis. Functionally, FGF-2 induced a potent proangiogenic response even in the presence of TSP-1 in ECs pre-treated with LPA. Doxycycline induced PKD-CA expression stimulated robust arterial EC branching in a 3D spheroid assay. This angiogenic response was inhibited by a novel PKD-1 specific inhibitor. Moreover, in a mouse Lewis lung carcinoma model, we detected a minimal level of endothelial CD36 expression with robust tumor angiogenesis. The role of PKD-1 signaling in CD36 transcriptional repression and tumor arteriogenesis is a critical yet underexplored and underappreciated field. We propose that the LPA-PKD-FoxO1 signaling axis is essential for CD36 transcriptional repression and arteriogenic signaling and responses. The PKD-1 stimulated nuclear signaling network is critical to epigenetic suppression of CD36 transcription and silencing of CD36 antiangiogenic switch, subsequently turning on the angiogenic switch and stimulating the arteriogenic responses. Targeting the LPA-PKD1-FoxO1 signaling axis could have therapeutic potential in the ischemic disease and malignant tumors. Citation Format: Jacob D. Kohlenberg, Yiliang Chen, Brad Best, Peter Storz, Randall T. Peterson, Roy Silverstein, Bin Ren. A novel LPA-PKD1-FoxO1 pathway in endothelial cells provides an angiogenic switch via down-regulation of CD36 transcription and induction of arteriogenic responses . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-338. doi:10.1158/1538-7445.AM2013-LB-338

Hypoxia-inducible factor (HIF) is a central regulator of the cellular response to hypoxia. Hypoxia promotes HIF activity by increasing its stability, transcriptional activity and translation. HIF target genes involved in several cellular processes including proliferation, angiogenesis, metabolism and promotion of the “stem-cell” phenotype. In normoxic conditions, HIF-α is recognized for degradation by the von Hippel-Lindau tumor suppressor protein (VHL). Loss-of-VHL function leads to constitutive expression of HIF regulatory subunits and consequent over-expression of HIF-target genes. We and others have shown that HIF2a is necessary and sufficient for the development of VHL-related renal cell carcinoma. We have developed small molecules that repress HIF-2α translation, in vitro, by enhancing the binding of Iron-Regulatory Protein 1 (IRP1) to the Iron-Responsive Element (IRE) within the 5′-UTR of HIF-2α mRNA (Zimmer M. et al. Molecular Cell 2008;32: 828-848). We showed that these small molecules down-regulate HIF2a expression in vitro, in all renal cell carcinoma cell lines. To validate these small molecules in vivo, we tested their efficacy on wild-type zebrafish embryos. Treatment of 3 days post fertilization zebrafish embryos with 100microM of the hypoxic mimetic DMOG or hypoxia dramatically upregulated the expression of HIF-target genes, such as Vascular Endothelial Growth Factor (Vegf), erythropoietin (Epo) and prolyl-hydroxylase 3 (Phd3). Pre-treatment of embryos with the small molecule HIF2a inhibitor 76 potently decreased the DMOG-induced upregulation of HIF target genes. The IC50 values of these small molecules were significantly less in vivo than in vitro. We are currently testing the efficacy of HIF2a inhibitors in rescuing the VHL zebrafish phenotype and prolonging survival of VHL mutant zebrafish embryos. In addition, data on the efficacy of these inhibitors on the growth of human renal cell carcinoma cells as tumors in the nude mouse xenograft assay will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4253. doi:1538-7445.AM2012-4253

Abstract 686 Introduction: Anemia of chronic disease (ACD), the second most prevalent form of anemia, is commonly associated with chronic inflammatory, infectious, or neoplastic conditions. ACD is characterized by high hepcidin levels that decrease serum iron levels by inducing degradation of the iron exporter ferroportin. In contrast, a relative deficiency of hepcidin leads to ferroportin overexpression and iron overload. Hepcidin is transcriptionally regulated by interleukin-6 (IL-6) and bone morphogenic protein (BMP) signaling. Binding of BMP ligands to type II and type I BMP receptors induces the type II receptor to phosphorylate and activate one of four type I receptors. We sought to identify the type I BMP receptor that participates in the ability of IL-6 to induce hepatic hepcidin gene expression. Methods: The four type I BMP receptors are Alk1, Alk2, Alk3, and Alk6. Alk1 is predominantly expressed in the endothelium. Alk6 is expressed at low levels in murine liver. In contrast, Alk2 and Alk3 are abundantly expressed in hepatocytes. Global deficiency of Alk2 or Alk3 is embryonic lethal. To selectively delete Alk2 or Alk3 in hepatocytes, we studied mice homozygous for Alk2 or Alk3 sequences flanked by loxP sites (Alk2fl/fl and Alk3fl/fl, respectively) that also carried a transgene specifying Cre recombinase under the control of the albumin gene promoter (Alb-Cre). Eight- to 12-week-old male mice (Alk2fl/fl, Alk2fl/fl; Alb-Cre, Alk3fl/fl, Alk3fl/fl; Alb-Cre) on a standard, iron-replete diet were injected via the tail vein with an adenovirus specifying IL-6 (Ad.IL-6) or an adenovirus specifying green fluorescent protein (GFP; Ad.GFP), as a control (1010 particles per ml for both). Seventy-two hours later, mice were euthanized, and blood was obtained for measurement of serum iron levels and transferrin saturations. Livers were harvested, and RNA was extracted. Hepatic levels of mRNAs encoding Alk2, Alk3, hepcidin, heme oxygenase-1 (HO-1, a transcriptional target of IL-6), and Id-1 (a BMP gene target) were measured by qRT-PCR. Hepatic STAT3 phosphorylation (a marker of IL-6 receptor activation) was measured using immunoblot techniques. Results: Liver-specific deletion of Alk2 or Alk3 caused mild and severe iron overload, respectively. Injection of Ad.IL-6, but not Ad.GFP, decreased serum iron levels and transferrin saturations in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice. In contrast, infection of Alk3fl/fl; Alb-Cre mice with Ad.IL-6 did not alter serum iron levels and only modestly reduced transferrin saturations. Infection with Ad.IL-6 induced of hepatic hepcidin gene expression in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice. Hepatic hepcidin mRNA levels were markedly reduced in Ad.GFP-infected Alk3fl/fl; Alb-Cre mice, and infection with Ad.IL-6 failed to increase hepcidin mRNA levels in this genotype. Ad.IL-6 infection induced hepatic Id-1 mRNA levels in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice, but not in Alk3fl/fl; Alb-Cre mice. Infection with Ad.IL-6 induced hepatic STAT-3 phosphorylation and HO-1 gene expression in all 4 genotypes of mice. Conclusions: Taken together, these results demonstrate that the response to IL-6, as reflected by STAT-3 phosphorylation and induction of HO-1 gene expression, does not require Alk2 or Alk3. In contrast, BMP signaling, predominantly via Alk3, is essential for the induction of hepcidin gene expression by IL-6. These results suggest that selective inhibition of Alk3 may represent a novel therapeutic approach to the treatment of ACD. Disclosures: Peterson: Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties. Yu:Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties. Bloch:Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties.

Abstract 2043 Introduction: Anemia of chronic disease (ACD) describes anemia associated with diverse chronic inflammatory, infectious, or neoplastic processes. These conditions are frequently associated with increased circulating levels of inflammatory cytokines such as interleukin 6 (IL-6). IL-6 regulates expression of the hormone hepcidin, which inhibits the release of iron from hepatocytes, macrophages, and enterocytes into the circulation. In addition to IL-6, hepcidin gene expression is known to be transcriptionally regulated by bone morphogenetic protein (BMP) signaling. Hypothesis: We hypothesized that BMP signaling is required for the induction of hepcidin gene expression by IL-6 and plays a critical role in the pathogenesis of ACD. Methods: We used a turpentine-dependent model of ACD in mice. Mice were challenged with weekly subcutaneous injections of turpentine, which induces anemia in an IL-6 dependent manner. This model was studied to determine hepcidin gene expression and rescue ACD using BMP inhibition. Moreover, we examined hepcidin gene expression in zebrafish injected with Pseudomonas aeruginosa, and in transgenic zebrafish overexpressing human IL-6. The regulation of hepcidin gene expression was also studied in the human hepatocarcinoma cell line (HepG2). Results: Injections of mice with IL-6 (0.8 μg/g ip) increased hepatic hepcidin mRNA levels expression at 24 hours and decreased serum iron concentrations. Both effects were prevented by a small molecule BMP type I receptor kinase inhibitor, LDN-193189, or protein BMP antagonists. Weekly turpentine injections induced microcytic anemia after 3 weeks with a decrease in hemoglobin levels from 12.8±0.3 to 9.7±1.7 g/dL (*p In zebrafish, microinjection with Pseudomonas aeruginosa or overexpression of human IL-6 induced hepatic hepcidin expression, an effect which was blocked by LDN-193189. Incubation of HepG2 cells with IL-6 (100 ng/ml) increased hepcidin mRNA levels 2 to 5 fold. Pretreatment with LDN-193189, or recombinant protein BMP antagonists such as noggin, abrogated the induction of hepcidin expression by IL-6. Incubation of HepG2 cells with BMP6 (2.5 to 10 ng/ml) modestly increased hepcidin mRNA levels. However, the combination of IL-6 and BMP6 synergistically increased hepcidin gene expression (*p Conclusion: BMP signaling appears to play a critical role in the pathogenesis of anemia in a mouse ACD model. Our findings support the concept that BMP signaling is required for the induction of hepcidin gene expression by IL-6 in vitro and in vivo. Moreover, manipulation of BMP signaling represents a potentially novel therapeutic approach to the treatment of anemia associated with inflammation. Disclosures: Steinbicker: Deutsche Forschungsgemeinschaft DFG: Research Funding. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Peterson:Massachusetts General Hospital Executive Committee on Research and NIDDK 1R01DK082971: Research Funding. Bloch:Massachusetts General Hospital Executive Committee on Research and NIDDK 1R01DK082971: Research Funding. Yu:Harvard Stem Cell Institute Seed Grant and the Howard Hughes Medical Institute Early Career Physician-Scientist Award: Honoraria, Research Funding; NHLBI 5K08HL079943: Research Funding.

Nat. Med. 14, 1363–1369 (2008); published online 30 November 2008; corrected after print 4 December 2008 In the version of this article initially published, the title included a misspelling—'heterotropic' should have been 'heterotopic'. Additionally, the fourth and fifth sentences of the abstract were incorrectly worded and have been corrected to state more clearly the role of Ad.

The earliest examples of small molecule discovery involved serendipitous phenotypic observations in whole organisms, but this organism-based process has given way in recent decades to systematic, high-throughput assays using purified proteins, cells, or cell extracts. In vitro screens have been successful at identifying modifiers of well-understood biological processes, but they are limited in their ability to discover modifiers of processes that are poorly understood or occur only in an integrated physiological context. Small model organisms, especially the zebrafish, make it possible to combine the advantages of organism-based small molecule discovery with the technologies and throughput of modern screening. The combination of model organisms with high-throughput screening is likely to extend small molecule discovery efforts to fields of study such as developmental biology and to broaden the range of diseases for which drug screening can be performed.

The identification of 3-phosphoinositide-dependent kinase 1 (PDK1) as one of the elusive 70 kDa S6 kinase kinases has filled a gap in the signaling pathway by which extracellular receptors regulate translation. Will it cause us to reconsider the relationships between previously identified members of the pathway?

Phenotypic screens and the target identification that follows can lead to surprising new connections between small molecules and targets. In this issue of Chemistry & Biology, Zhou and colleagues seek to understand the pigmentation of fish skin and end up uncovering an interesting clinical hypothesis about the trypanocidal compound nifurtimox.