Fate plasticity and reprogramming in genetically distinct populations of Danio leucophores.

Understanding genetic and cellular bases of adult form remains a fundamental goal at the intersection of developmental and evolutionary biology. The skin pigment cells of vertebrates, derived from embryonic neural crest, are a useful system for elucidating mechanisms of fate specification, pattern formation, and how particular phenotypes impact organismal behavior and ecology. In a survey of Danio fishes, including the zebrafish Danio rerio, we identified two populations of white pigment cells-leucophores-one of which arises by transdifferentiation of adult melanophores and another of which develops from a yellow-orange xanthophore or xanthophore-like progenitor. Single-cell transcriptomic, mutational, chemical, and ultrastructural analyses of zebrafish leucophores revealed cell-type-specific chemical compositions, organelle configurations, and genetic requirements. At the organismal level, we identified distinct physiological responses of leucophores during environmental background matching, and we showed that leucophore complement influences behavior. Together, our studies reveal independently arisen pigment cell types and mechanisms of fate acquisition in zebrafish and illustrate how concerted analyses across hierarchical levels can provide insights into phenotypes and their evolution.

The ribonucleoside AICAr induces differentiation of myeloid leukemia by activating the ATR/Chk1 via pyrimidine depletion.

Metabolic pathways play important roles in proliferation and differentiation of malignant cells. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr), a precursor in purine biosynthesis and a well-established activator of AMP-activated protein kinase (AMPK), induces widespread metabolic alterations and is commonly used for dissecting the role of metabolism in cancer. We have previously reported that AICAr promotes differentiation and inhibits proliferation of myeloid leukemia cells. Here, using metabolic assays, immunoblotting, flow cytometry analyses, and siRNA-mediated gene silencing in leukemia cell lines, we show that AICAr-mediated differentiation was independent of the known metabolic effects of AMPK, including glucose consumption, but instead depends on the activation of the DNA damage-associated enzyme checkpoint kinase 1 (Chk1) induced by pyrimidine depletion. LC/MS/MS metabolomics analysis revealed that AICAr increases orotate levels and decreases uridine monophosphate (UMP) levels, consistent with inhibition of UMP synthesis at a step downstream of dihydroorotate dehydrogenase (DHODH). AICAr and the DHODH inhibitor brequinar had similar effects on differentiation markers and S-phase arrest, and genetic or pharmacological Chk1 inactivation abrogated both of these effects. Our results delineate an AMPK-independent effect of AICAr on myeloid leukemia differentiation that involves perturbation of pyrimidine biosynthesis and activation of the DNA damage response network.

Discovery of Selective SIRT2 Inhibitors as Therapeutic Agents in B-Cell Lymphoma and Other Malignancies.

Genetic ablation as well as pharmacological inhibition of sirtuin 2 (SIRT2), an NAD+-dependent protein deacylase, have therapeutic effects in various cancers and neurodegenerative diseases. Previously, we described the discovery of a dual SIRT1/SIRT2 inhibitor called cambinol (IC50 56 and 59 µM, respectively), which showed cytotoxic activity against cancer cells in vitro and a marked anti-proliferative effect in a Burkitt lymphoma mouse xenograft model. A number of recent studies have shown a protective effect of SIRT1 and SIRT3 in neurodegenerative and metabolic diseases as well as in certain cancers prompting us to initiate a medicinal chemistry effort to develop cambinol-based SIRT2-specific inhibitors devoid of SIRT1 or SIRT3 modulating activity. Here we describe potent cambinol-based SIRT2 inhibitors, several of which show potency of ~600 nM with >300 to >800-fold selectivity over SIRT1 and 3, respectively. In vitro, these inhibitors are found to be toxic to lymphoma and epithelial cancer cell lines. In particular, compounds 55 (IC50 SIRT2 0.25 µM and <25% inhibition at 50 µM against SIRT1 and SIRT3) and 56 (IC50 SIRT2 0.78 µM and <25% inhibition at 50 µM against SIRT1 and SIRT3) showed apoptotic as well as strong anti-proliferative properties against B-cell lymphoma cells.

Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line.

Cisplatin, one of the most commonly used anticancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analog of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line.

Fish in a Dish: Drug Discovery for Hearing Habilitation.

The majority of hearing loss is caused by the permanent loss of inner ear hair cells. The identification of drugs that modulate the susceptibility to hair cell loss or spur their regeneration is often hampered by the difficulties of assaying for such complex phenomena in mammalian models. The zebrafish has emerged as a powerful animal model for chemical screening in many contexts. Several characteristics of the zebrafish, such as its small size and external location of sensory hair cells, uniquely position it as an ideal model organism for the study of hair cell toxicity, protection, and regeneration. We have used this model to screen for drugs that affect each of these aspects of hair cell biology and have identified compounds that affect each of these processes. The identification of such drugs and drug-like compounds holds promise in the future ability to stem hearing loss in the human population.

Non-specific chemical inhibition of the Fanconi anemia pathway sensitizes cancer cells to cisplatin.

BACKGROUND: Platinum compounds such as cisplatin and carboplatin are DNA crosslinking agents widely used for cancer chemotherapy. However, the effectiveness of platinum compounds is often tempered by the acquisition of cellular drug resistance. Until now, no pharmacological approach has successfully overcome cisplatin resistance in cancer treatment. Since the Fanconi anemia (FA) pathway is a DNA damage response pathway required for cellular resistance to DNA interstrand crosslinking agents, identification of small molecules that inhibit the FA pathway may reveal classes of chemicals that sensitize cancer cells to cisplatin. RESULTS: Through a cell-based screening assay of over 16,000 chemicals, we identified 26 small molecules that inhibit ionizing radiation and cisplatin-induced FANCD2 foci formation, a marker of FA pathway activity, in multiple human cell lines. Most of these small molecules also compromised ionizing radiation-induced RAD51 foci formation and homologous recombination repair, indicating that they are not selective toward the regulation of FANCD2. These compounds include known inhibitors of the proteasome, cathepsin B, lysosome, CHK1, HSP90, CDK and PKC, and several uncharacterized chemicals including a novel proteasome inhibitor (Chembridge compound 5929407).Isobologram analyses demonstrated that half of the identified molecules sensitized ovarian cancer cells to cisplatin. Among them, 9 demonstrated increased efficiency toward FA pathway-proficient, cisplatin-resistant ovarian cancer cells. Six small molecules, including bortezomib (proteasome inhibitor), CA-074-Me (cathepsin B inhibitor) and 17-AAG (HSP90 inhibitor), synergized with cisplatin specifically in FA-proficient ovarian cancer cells (2008 + FANCF), but not in FA-deficient isogenic cells (2008). In addition, geldanamycin (HSP90 inhibitor) and two CHK1 inhibitors (UCN-01 and SB218078) exhibited a significantly stronger synergism with cisplatin in FA-proficient cells when compared to FA-deficient cells, suggesting a contribution of their FA pathway inhibitory activity to cisplatin sensitization. CONCLUSION: Our findings suggest that, despite their lack of specificity, pharmaceutical inhibition of the FA pathway by bortezomib, CA-074-Me, CHK1 inhibitors or HSP90 inhibitors may be a promising strategy to sensitize cisplatin-resistant, FA pathway-proficient tumor cells to cisplatin. In addition, we identified four new small molecules which synergize with cisplatin. Further development of their analogs and evaluation of their combination with cisplatin may lead to the development of efficient cancer treatments.

A proteomic investigation of ligand-dependent HSP90 complexes reveals CHORDC1 as a novel ADP-dependent HSP90-interacting protein.

Structural studies of the chaperone HSP90 have revealed that nucleotide and drug ligands induce several distinct conformational states; however, little is known how these conformations affect interactions with co-chaperones and client proteins. Here we use tandem affinity purification and LC-MS/MS to investigate the proteome-wide effects of ATP, ADP, and geldanamycin on the constituents of the human HSP90 interactome. We identified 52 known and novel components of HSP90 complexes that are regulated by these ligands, including several co-chaperones. Interestingly, our results also show that geldanamycin treatment causes HSP90 complexes to become significantly enriched for core transcription machinery, suggesting that HSP90 inhibition may have broad based effects on transcription and RNA processing. We further characterized a novel ADP-dependent HSP90 interaction with the cysteine- and histidine-rich domain (CHORD)-containing protein CHORDC1. We show that this interaction is stimulated by high ADP:ATP ratios in cell lysates and in vitro with purified recombinant proteins. Furthermore, we demonstrate that this interaction is dependent upon the ability of HSP90 to bind nucleotides and requires the presence of a linker region between the CHORD domains in CHORDC1. Together these findings suggest that the HSP90 interactome is dynamic with respect to nucleotide and drug ligands and that pharmacological inhibition of HSP90 may stimulate the formation of specific complexes.

An in vivo Biomarker to Characterize Ototoxic Compounds and Novel Protective Therapeutics.

There are no approved therapeutics for the prevention of hearing loss and vestibular dysfunction from drugs like aminoglycoside antibiotics. While the mechanisms underlying aminoglycoside ototoxicity remain unresolved, there is considerable evidence that aminoglycosides enter inner ear mechanosensory hair cells through the mechanoelectrical transduction (MET) channel. Inhibition of MET-dependent uptake with small molecules or modified aminoglycosides is a promising otoprotective strategy. To better characterize mammalian ototoxicity and aid in the translation of emerging therapeutics, a biomarker is needed. In the present study we propose that neonatal mice systemically injected with the aminoglycosides G418 conjugated to Texas Red (G418-TR) can be used as a histologic biomarker to characterize in vivo aminoglycoside toxicity. We demonstrate that postnatal day 5 mice, like older mice with functional hearing, show uptake and retention of G418-TR in cochlear hair cells following systemic injection. When we compare G418-TR uptake in other tissues, we find that kidney proximal tubule cells show similar retention. Using ORC-13661, an investigational hearing protection drug, we demonstrate in vivo inhibition of aminoglycoside uptake in mammalian hair cells. This work establishes how systemically administered fluorescently labeled ototoxins in the neonatal mouse can reveal important details about ototoxic drugs and protective therapeutics.

Upregulation of RhoB via c-Jun N-terminal kinase signaling induces apoptosis of the human gastric carcinoma NUGC-3 cells treated with NSC12618.

RhoB expression is reduced in most invasive tumors, with loss of RhoB expression correlating significantly with tumor stage. Here, we demonstrate that upregulation of RhoB by the potent anticancer agent NSC126188 induces apoptosis of NUGC-3 human gastric carcinoma cells. The crucial role of RhoB in NSC126188-induced apoptosis is indicated by the rescue of NUGC-3 cells from apoptosis by knockdown of RhoB. In the presence of NSC126188, c-Jun N-terminal kinase (JNK) signaling was activated, and the JNK inhibitor SP600125 reduced RhoB expression and suppressed the apoptosis of NUGC-3 cells. Knockdowns of mitogen-activated protein kinase kinase (MKK) 4/7, JNK1/2 and c-Jun downregulated RhoB expression and rescued cells from apoptotic death in the presence of NSC126188. The JNK inhibitor SP600125 suppressed transcriptional activation of RhoB in the presence of NSC126188, as indicated by a reporter assay that used luciferase under the RhoB promoter. The ability of NSC126188 to increase luciferase activity through both the p300-binding site and the inverted CCAAT sequence (iCCAAT box) suggests that JNK signaling to upregulate RhoB expression is mediated through both the p300-binding site and the iCCAAT box. However, the JNK inhibitor SP600125 did not inhibit the upregulation of RhoB by farnesyltransferase inhibitor (FTI)-277. The p300-binding site did not affect activation of the RhoB promoter by FTI-277 in NUGC-3 cells, suggesting that the transcriptional activation of RhoB by NSC126188 occurs by a different mechanism than that reported for FTIs. Our data indicate that NSC126188 increases RhoB expression via JNK-mediated signaling through a p300-binding site and iCCAAT box resulting in apoptosis of NUGC-3 cells.

Dinoxin B, a withanolide from Datura inoxia leaves with specific cytotoxic activities.

A new withanolide, dinoxin B (12,21-dihydroxy-1-oxowitha-2,5,24-trienolide-27-O-ß-D-glucopyranoside, 1), was isolated from a methanol extract of Datura inoxia leaves, using bioassay-guided fractionation. The structure was determined by spectroscopic techniques, including (1)H, (13)C, and 2D NMR experiments as well as by HRMS. Extracts and the purified compound were tested for their antiproliferative activities toward a panel of human normal and cancer cell lines. Dinoxin B (1) and its aglycone (2) exhibited submicromolar IC(50) values against multiple human cancer cell lines. Among the most sensitive were several breast cancer cell lines. Dinoxin B (1) was found only in D. inoxia and was not detected in D. metel or D. stramonium. The accumulation of this compound was limited largely to leaf tissue, with little to none detected in extracts from the flowers, fruits, roots, or stems of D. inoxia.

Sirtuin modulators.

Members of the sirtuin family including the founding protein Sir2 in Saccharomyces cerevisiae have been linked to lifespan extension in simple organisms. This finding prompted evaluation of the role of Sir2 orthologues in many aging-associated conditions including neurodegeneration, type II diabetes and cancer. These studies have demonstrated that genetic and pharmacologic manipulation of sirtuin activity have beneficial effects in a surprisingly broad spectrum of aging-associated conditions suggesting that the Sir2-family of enzymes presents an attractive target for the development of pharmacological agents. While the initial model favored pharmacological activators of sirtuins as calorie restriction mimetics, it now appears that either activation or inhibition of sirtuins may be desirable for ameliorating disease depending on the pathological condition and the target tissue. In this chapter we review the development of pharmacological small molecule activators and inhibitors of the sirtuin family of enzymes.

Identification of genetic and chemical modulators of zebrafish mechanosensory hair cell death.

Inner ear sensory hair cell death is observed in the majority of hearing and balance disorders, affecting the health of more than 600 million people worldwide. While normal aging is the single greatest contributor, exposure to environmental toxins and therapeutic drugs such as aminoglycoside antibiotics and antineoplastic agents are significant contributors. Genetic variation contributes markedly to differences in normal disease progression during aging and in susceptibility to ototoxic agents. Using the lateral line system of larval zebrafish, we developed an in vivo drug toxicity interaction screen to uncover genetic modulators of antibiotic-induced hair cell death and to identify compounds that confer protection. We have identified 5 mutations that modulate aminoglycoside susceptibility. Further characterization and identification of one protective mutant, sentinel (snl), revealed a novel conserved vertebrate gene. A similar screen identified a new class of drug-like small molecules, benzothiophene carboxamides, that prevent aminoglycoside-induced hair cell death in zebrafish and in mammals. Testing for interaction with the sentinel mutation suggests that the gene and compounds may operate in different pathways. The combination of chemical screening with traditional genetic approaches is a new strategy for identifying drugs and drug targets to attenuate hearing and balance disorders.

Chloroquine kills hair cells in zebrafish lateral line and murine cochlear cultures: Implications for ototoxicity.

Hearing and balance deficits have been reported during and following treatment with the antimalarial drug chloroquine. However, experimental work examining the direct actions of chloroquine on mechanoreceptive hair cells in common experimental models is lacking. This study examines the effects of chloroquine on hair cells using two common experimental models: the zebrafish lateral line and neonatal mouse cochlear cultures. Zebrafish larvae were exposed to varying concentrations of chloroquine phosphate or hydroxychloroquine for 1 h or 24 h, and hair cells assessed by antibody staining. A significant, dose-dependent reduction in the number of surviving hair cells was seen across conditions for both exposure periods. Hydroxychloroquine showed similar toxicity. In mouse cochlear cultures, chloroquine damage was specific to outer hair cells in tissue from the cochlear basal turn, consistent with susceptibility to other ototoxic agents. These findings suggest a need for future studies employing hearing and balance monitoring during exposure to chloroquine and related compounds, particularly with interest in these compounds as therapeutics against viral infections including coronavirus.

A potent peptide-steroid conjugate accumulates in cartilage and reverses arthritis without evidence of systemic corticosteroid exposure.

On-target, off-tissue toxicity limits the systemic use of drugs that would otherwise reduce symptoms or reverse the damage of arthritic diseases, leaving millions of patients in pain and with limited physical mobility. We identified cystine-dense peptides (CDPs) that rapidly accumulate in cartilage of the knees, ankles, hips, shoulders, and intervertebral discs after systemic administration. These CDPs could be used to concentrate arthritis drugs in joints. A cartilage-accumulating peptide, CDP-11R, reached peak concentration in cartilage within 30 min after administration and remained detectable for more than 4 days. Structural analysis of the peptides by crystallography revealed that the distribution of positive charge may be a distinguishing feature of joint-accumulating CDPs. In addition, quantitative whole-body autoradiography showed that the disulfide-bonded tertiary structure is critical for cartilage accumulation and retention. CDP-11R distributed to joints while carrying a fluorophore imaging agent or one of two different steroid payloads, dexamethasone (dex) and triamcinolone acetonide (TAA). Of the two payloads, the dex conjugate did not advance because the free drug released into circulation was sufficient to cause on-target toxicity. In contrast, the CDP-11R-TAA conjugate alleviated joint inflammation in the rat collagen-induced model of rheumatoid arthritis while avoiding toxicities that occurred with nontargeted steroid treatment at the same molar dose. This conjugate shows promise for clinical development and establishes proof of concept for multijoint targeting of disease-modifying therapeutic payloads.

Sir2 flexes its muscle.

A new report reveals a role for the mammalian NAD-dependent deacetylase Sir2 in repressing the muscle cell differentiation program and implicates the cellular redox state as a critical determinant of transcriptional activity of differentiation-specific genes.

Telomere length as a quantitative trait: genome-wide survey and genetic mapping of telomere length-control genes in yeast.

Telomere length-variation in deletion strains of Saccharomyces cerevisiae was used to identify genes and pathways that regulate telomere length. We found 72 genes that when deleted confer short telomeres, and 80 genes that confer long telomeres relative to those of wild-type yeast. Among identified genes, 88 have not been previously implicated in telomere length control. Genes that regulate telomere length span a variety of functions that can be broadly separated into telomerase-dependent and telomerase-independent pathways. We also found 39 genes that have an important role in telomere maintenance or cell proliferation in the absence of telomerase, including genes that participate in deoxyribonucleotide biosynthesis, sister chromatid cohesion, and vacuolar protein sorting. Given the large number of loci identified, we investigated telomere lengths in 13 wild yeast strains and found substantial natural variation in telomere length among the isolates. Furthermore, we crossed a wild isolate to a laboratory strain and analyzed telomere length in 122 progeny. Genome-wide linkage analysis among these segregants revealed two loci that account for 30%-35% of telomere length-variation between the strains. These findings support a general model of telomere length-variation in outbred populations that results from polymorphisms at a large number of loci. Furthermore, our results laid the foundation for studying genetic determinants of telomere length-variation and their roles in human disease.

A small-molecule inhibitor of Tcf/beta-catenin signaling down-regulates PPARgamma and PPARdelta activities.

Activation of the Wnt/beta-catenin signaling pathway occurs in several types of cancers and thus it is an attractive target for anticancer drug development. To identify compounds that inhibit this pathway, we screened a chemical library using a cell-based beta-catenin/Tcf-responsive reporter. We identified FH535, a compound that suppresses both Wnt/beta-catenin and peroxisome proliferator-activated receptor (PPAR) signaling. FH535 antagonizes both PPARgamma and PPARdelta ligand-dependent activation and shows structural similarity to GW9662, a known PPARgamma antagonist. The effect of FH535 on beta-catenin/Tcf activity is reduced in cells carrying a deletion of the PPARdelta gene, as well as by the PPARgamma agonist lysophosphatidic acid. Mechanistically, FH535 inhibits recruitment of the coactivators beta-catenin and GRIP1 but not the corepressors NCoR and SMRT. Its repression of beta-catenin recruitment, in comparison with GW9662, is linked to FH535's unique capability to inhibit the Wnt/beta-catenin signaling pathway. The antiproliferation effect of the compound observed on some transformed colon lung and liver cell lines is suggestive of its potential therapeutic value in the treatment of cancer.

Identification and characterization of small-molecule inhibitors of hepsin.

Hepsin is a type II transmembrane serine protease overexpressed in the majority of human prostate cancers. We recently demonstrated that hepsin promotes prostate cancer progression and metastasis and thus represents a potential therapeutic target. Here we report the identification of novel small-molecule inhibitors of hepsin catalytic activity. We utilized purified human hepsin for high-throughput screening of established drug and chemical diversity libraries and identified sixteen inhibitory compounds with IC(50) values against hepsin ranging from 0.23-2.31 microM and relative selectivity of up to 86-fold or greater. Two compounds are orally administered drugs established for human use. Four compounds attenuated hepsin-dependent pericellular serine protease activity in a dose dependent manner with limited or no cytotoxicity to a range of cell types. These compounds may be used as leads to develop even more potent and specific inhibitors of hepsin to prevent prostate cancer progression and metastasis.

Long-term tracking demonstrates effectiveness of a partnership-led training program to advance the careers of biomedical researchers from underrepresented groups.

The demographic profile of the biomedical workforce in the U.S. does not reflect the population at large, raising concerns that there will be insufficient trained researchers in the future, and the scope of research interests will not be sufficiently broad. To diversify and expand the pool of researchers trained to conduct research on cancer and cancer health disparities, a series of training activities to recruit and train primarily Hispanic students at both the undergraduate and graduate level were developed. The strengths of both a Hispanic Serving Institution and an NIH-designated Comprehensive Cancer Center were leveraged to develop appropriate research training and professional development activities. The career progression of the participants and degree completion rates was tracked, along with persistent interest in biomedical research in general and cancer and cancer health disparities research in particular for these underrepresented individuals. Finally, this report demonstrates that these training activities increased general knowledge about cancer among participants.

ORC-13661 protects sensory hair cells from aminoglycoside and cisplatin ototoxicity.

Aminoglycoside (AG) antibiotics are widely used to prevent life-threatening infections, and cisplatin is used in the treatment of various cancers, but both are ototoxic and result in loss of sensory hair cells from the inner ear. ORC-13661 is a new drug that was derived from PROTO-1, a compound first identified as protective in a large-scale screen utilizing hair cells in the lateral line organs of zebrafish larvae. Here, we demonstrate, in zebrafish larvae and in mouse cochlear cultures, that ORC-13661 provides robust protection of hair cells against both ototoxins, the AGs and cisplatin. ORC-13661 also prevents both hearing loss in a dose-dependent manner in rats treated with amikacin and the loading of neomycin-Texas Red into lateral line hair cells. In addition, patch-clamp recordings in mouse cochlear cultures reveal that ORC-13661 is a high-affinity permeant blocker of the mechanoelectrical transducer (MET) channel in outer hair cells, suggesting that it may reduce the toxicity of AGs by directly competing for entry at the level of the MET channel and of cisplatin by a MET-dependent mechanism. ORC-13661 is therefore a promising and versatile protectant that reversibly blocks the hair cell MET channel and operates across multiple species and toxins.