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Background CD40 is a 48 kDa type I transmembrane protein that is constitutively expressed on hematopoietic cells such as dendritic cells, macrophages, and B cells. Engagement of CD40 by CD40L expressed on T cells results in the production of proinflammatory cytokines, induces T helper cell function, and promotes macrophage activation. The involvement of CD40 in chronic immune activation has resulted in CD40 being proposed as a therapeutic target for a range of chronic inflammatory diseases. CD40 antagonists are currently being explored for the treatment of autoimmune diseases and several anti-CD40 agonist mAbs have entered clinical development for oncological indications. Results To better understand the mode of action of anti-CD40 mAbs, we have determined the x-ray crystal structures of the ABBV-323 (anti-CD40 antagonist, ravagalimab) Fab alone, ABBV-323 Fab complexed to human CD40 and FAB516 (anti-CD40 agonist) complexed to human CD40. These three crystals structures 1) identify the conformational CD40 epitope for ABBV-323 recognition 2) illustrate conformational changes which occur in the CDRs of ABBV-323 Fab upon CD40 binding and 3) develop a structural hypothesis for an agonist/antagonist switch in the LCDR1 of this proprietary class of CD40 antibodies. Conclusions The structure of ABBV-323 Fab demonstrates a unique method for antagonism by stabilizing the proposed functional antiparallel dimer for CD40 receptor via novel contacts to LCDR1, namely residue position R32 which is further supported by a closely related agonist antibody FAB516 which shows only monomeric recognition and no contacts with LCDR1 due to a mutation to L32 on LCDR1. These data provide a structural basis for the full antagonist activity of ABBV-323. Electronic supplementary material The online version of this article (10.1186/s12860-019-0213-4) contains supplementary material, which is available to authorized users.

Every cancer originates from a single cell. During expansion of the neoplastic cell population, individual cells acquire genetic and phenotypic differences from each other. Here, to investigate the nature and extent of intra-tumour diversification, we characterized organoids derived from multiple single cells from three colorectal cancers as well as from adjacent normal intestinal crypts. Colorectal cancer cells showed extensive mutational diversification and carried several times more somatic mutations than normal colorectal cells. Most mutations were acquired during the final dominant clonal expansion of the cancer and resulted from mutational processes that are absent from normal colorectal cells. Intra-tumour diversification of DNA methylation and transcriptome states also occurred; these alterations were cell-autonomous, stable, and followed the phylogenetic tree of each cancer. There were marked differences in responses to anticancer drugs between even closely related cells of the same tumour. The results indicate that colorectal cancer cells experience substantial increases in somatic mutation rate compared to normal colorectal cells, and that genetic diversification of each cancer is accompanied by pervasive, stable and inherited differences in the biological states of individual cancer cells.

Breast cancer (BC) comprises multiple distinct subtypes that differ genetically, pathologically, and clinically. Here, we describe a robust protocol for long-term culturing of human mammary epithelial organoids. Using this protocol, >100 primary and metastatic BC organoid lines were generated, broadly recapitulating the diversity of the disease. BC organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. DNA copy number variations as well as sequence changes were consistent within tumor-organoid pairs and largely retained even after extended passaging. BC organoids furthermore populated all major gene-expression-based classification groups and allowed in vitro drug screens that were consistent with in vivo xeno-transplantations and patient response. This study describes a representative collection of well-characterized BC organoids available for cancer research and drug development, as well as a strategy to assess in vitro drug response in a personalized fashion. The heterogeneity of breast cancer subtypes can be captured using organoid cultures that can facilitate drug screens that corroborate with patient responses.

Posttranslational modifications of histones play an important role in the regulation of gene expression and chromatin structure in eukaryotes. The balance between chromatin factors depositing (writers) and removing (erasers) histone marks regulates the steady-state levels of chromatin modifications. Here we describe a novel microscopy-based screening method to identify proteins that regulate histone modification levels in a high-throughput fashion. We named our method CROSS, for Chromatin Regulation Ontology SiRNA Screening. CROSS is based on an siRNA library targeting the expression of 529 proteins involved in chromatin regulation. As a proof of principle, we used CROSS to identify chromatin factors involved in histone H3 methylation on either lysine-4 or lysine-27. Furthermore, we show that CROSS can be used to identify chromatin factors that affect growth in cancer cell lines. Taken together, CROSS is a powerful method to identify the writers and erasers of novel and known chromatin marks and facilitates the identification of drugs targeting epigenetic modifications.

Colorectal cancer (CRC) organoids can be derived from almost all CRC patients and therefore capture the genetic diversity of this disease. We assembled a panel of CRC organoids carrying either wild-type or mutant RAS, as well as normal organoids and tumor organoids with a CRISPR-introduced oncogenic KRAS mutation. Using this panel, we evaluated RAS pathway inhibitors and drug combinations that are currently in clinical trial for RAS mutant cancers. Presence of mutant RAS correlated strongly with resistance to these targeted therapies. This was observed in tumorigenic as well as in normal organoids. Moreover, dual inhibition of the EGFR-MEK-ERK pathway in RAS mutant organoids induced a transient cell-cycle arrest rather than cell death. In vivo drug response of xenotransplanted RAS mutant organoids confirmed this growth arrest upon pan-HER/MEK combination therapy. Altogether, our studies demonstrate the potential of patient-derived CRC organoid libraries in evaluating inhibitors and drug combinations in a preclinical setting. DOI: http://dx.doi.org/10.7554/eLife.18489.001, eLife digest Recent technical advances mean that miniature replicas of many tissues can be grown in the laboratory. These so-called organoids provide scientists with model systems that are not as limited as simple, two-dimensional sheets of cells growing in a petri dish, and less labor and resource intensive than studies using laboratory animals. In particular, organoids grown from tumor cells from cancer patients have been suggested as having numerous advantages over both laboratory-grown cancer cells and mice when it comes to testing potential new anticancer drugs. Mutations in a gene called KRAS are common in many types of cancer including colon cancer. Tumors with these mutations are difficult to treat and so far virtually all attempts to generate compounds that selectively interfere with the KRAS protein encoded by the mutant gene have failed. Instead, drugs that indirectly inhibit this protein’s effects by targeting other proteins in the same signaling pathway are currently being tested on patients. However, there is still a need for better ways to pre-test whether these drugs will be effective in humans without having to expose the patient to side effects or an ineffective drug. Now, Verissimo, Overmeer, Ponsioen et al. have tested clinically-used KRAS pathway inhibitors and drug combinations against normal colon organoids and colon cancer organoids derived from patients with colon cancer. Gene editing techniques were used to introduce KRAS mutations into some of the normal organoids grown from healthy tissue, and into cancer organoids grown from tumors that had a normal copy of the KRAS gene. In all cases, only those organoids with mutant forms of the KRAS gene were resistant to the treatments. Furthermore, when organoids with the KRAS mutation were treated with some combination therapies that are currently being tested in clinical trials, the tumors stopped growing but the tumor cells failed to die. Similar drug treatments on mice carrying human colon cancer organoids confirmed these results, which is in line with previous studies where tumor tissue from human patients was transplanted into mice. These findings show that collections of tumor organoids from multiple patients could help researchers to quickly identify and optimize targeted anticancer therapies before they are incorporated into clinical trials. In the future, clinical studies are needed to verify how accurately the testing of cancer drugs on organoids predicts whether the drug will or will not work in patients. DOI: http://dx.doi.org/10.7554/eLife.18489.002

The nuclear factor κB (NF-κB) signalling pathway controls important cellular events such as cell proliferation, differentiation, apoptosis and immune responses. Pathway activation occurs rapidly upon TNFα stimulation and is highly dependent on ubiquitination events. Using cytoplasmic to nuclear translocation of the NF-κB transcription factor family member p65 as a read-out, we screened a synthetic siRNA library targeting enzymes involved in ubiquitin conjugation and de-conjugation for modifiers of regulatory ubiquitination events in NF-κB signalling. We identified F-box protein only 7 (FBXO7), a component of Skp, Cullin, F-box (SCF)-ubiquitin ligase complexes, as a negative regulator of NF-κB signalling. F-box protein only 7 binds to, and mediates ubiquitin conjugation to cIAP1 and TRAF2, resulting in decreased RIP1 ubiquitination and lowered NF-κB signalling activity.

SummaryMHC class II molecules (MHC-II) present peptides to T helper cells to facilitate immune responses and are strongly linked to autoimmune diseases. To unravel processes controlling MHC-II antigen presentation, we performed a genome-wide flow cytometry-based RNAi screen detecting MHC-II expression and peptide loading followed by additional high-throughput assays. All data sets were integrated to answer two fundamental questions: what regulates tissue-specific MHC-II transcription, and what controls MHC-II transport in dendritic cells? MHC-II transcription was controlled by nine regulators acting in feedback networks with higher-order control by signaling pathways, including TGFβ. MHC-II transport was controlled by the GTPase ARL14/ARF7, which recruits the motor myosin 1E via an effector protein ARF7EP. This complex controls movement of MHC-II vesicles along the actin cytoskeleton in human dendritic cells (DCs). These genome-wide systems analyses have thus identified factors and pathways controlling MHC-II transcription and transport, defining targets for manipulation of MHC-II antigen presentation in infection and autoimmunity.

BACKGROUND: The anti-apoptotic protein B cell lymphoma/leukaemia 2 (BCL-2) is highly expressed in the majority of all neuroblastomas. In previous preclinical studies, we have shown that treatment of BCL-2-dependent neuroblastoma with BCL-2 inhibitors leads to programmed cell death. These results have contributed to the initiation of a phase I trial to study the safety and pharmacokinetics of Venetoclax in children with relapsed or refractory neuroblastoma. AIM: The current study aims to identify and validate targeted combination strategies to prevent or overcome neuroblastoma resistance to Venetoclax. METHODS: Targeted drug candidates for combination treatment with Venetoclax were identified by high-throughput drug screening of non-resistant and Venetoclax-resistant BCL-2-dependent neuroblastoma cell lines. Top hits were subsequently tested more extensively in vitro and in vivo to validate the screening results. RESULTS: High-throughput drug screens identified the MDM2 inhibitor Idasanutlin as one of the strongest re-sensitizers for Venetoclax in Venetoclax-resistant BCL-2-dependent neuroblastoma cells with wild-type p53. Subsequent in vitro validation showed that Idasanutlin induced cyclin dependent kinase inhibitor 1 (p21)-mediated growth arrest in non-resistant neuroblastoma cells, but induced a BCL-2-associated X protein (BAX)-mediated apoptotic response in Venetoclax-resistant neuroblastoma cells in the presence of Venetoclax. In vivo combination of Venetoclax with Idasanultin resulted in a remarkably improved anticancer effect compared to single agent therapy, with very good partial and complete responses in BCL-2-dependent neuroblastoma xenografts. Combination screens additionally revealed that the clinically studied targeted inhibitors CUDC-907 (PI3K/HDAC), Prexasertib (CHK1) and Talazoparib (PARP1/2) improved the efficacy of Venetoclax more potently than ALK inhibitors in BCL-2-dependent neuroblastoma cells harboring ALK mutations. CONCLUSION: Our findings suggest that the clinical use of Venetoclax for the treatment of children with BCL-2-dependent neuroblastoma tumors can be improved by combination therapy with targeted inhibitors. The presence of additional neuroblastoma driving genomic events is not always predictive of the optimal combination strategy for BCL-2-dependent neuroblastoma subgroups. These findings should guide the design of combination Phase 2 trials of Venetoclax with other targeted compounds such as Idasanutlin, CUDC-907, Prexasertib and Talazoparib. Citation Format: M. Emmy M. Dolman, Laurel T. Bate-Eya, Lindy Vernooij, Bianca Koopmans, Lindy K. Alles, Anke H. Essing, Daphne Lelieveld, David A. Egan, Mark Kerstjens, Ronald W. Stam, Huib N. Caron, Jan J. Molenaar. Preclinical identification of Venetoclax combination strategies with Idasanutlin, CUDC-907, Prexasertib or Talazoparib for neuroblastoma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2630.

During embryonic development, axons extend over long distances to establish functional connections. In contrast, axon regeneration in the adult mammalian CNS is limited in part by a reduced intrinsic capacity for axon growth. Therefore, insight into the intrinsic control of axon growth may provide new avenues for enhancing CNS regeneration. Here, we performed one of the first miRNome-wide functional miRNA screens to identify miRNAs with robust effects on axon growth. High-content screening identified miR-135a and miR-135b as potent stimulators of axon growth and cortical neuron migrationin vitroandin vivoin male and female mice. Intriguingly, both of these developmental effects of miR-135s relied in part on silencing of Krüppel-like factor 4 (KLF4), a well known intrinsic inhibitor of axon growth and regeneration. These results prompted us to test the effect of miR-135s on axon regeneration after injury. Our results show that intravitreal application of miR-135s facilitates retinal ganglion cell (RGC) axon regeneration after optic nerve injury in adult mice in part by repressing KLF4. In contrast, depletion of miR-135s further reduced RGC axon regeneration. Together, these data identify a novel neuronal role for miR-135s and the miR-135–KLF4 pathway and highlight the potential of miRNAs as tools for enhancing CNS axon regeneration.SIGNIFICANCE STATEMENTAxon regeneration in the adult mammalian CNS is limited in part by a reduced intrinsic capacity for axon growth. Therefore, insight into the intrinsic control of axon growth may provide new avenues for enhancing regeneration. By performing an miRNome-wide functional screen, our studies identify miR-135s as stimulators of axon growth and neuron migration and show that intravitreal application of these miRNAs facilitates CNS axon regeneration after nerve injury in adult mice. Intriguingly, these developmental and regeneration-promoting effects rely in part on silencing of Krüppel-like factor 4 (KLF4), a well known intrinsic inhibitor of axon regeneration. Our data identify a novel neuronal role for the miR-135–KLF4 pathway and support the idea that miRNAs can be used for enhancing CNS axon regeneration.

MicroRNAs (miRNAs) are single-stranded, noncoding RNAs that are important in many biological processes1,2. Although the oncogenic and tumour-suppressive functions of several miRNAs have been characterized, the role of miRNAs in mediating tumour metastasis was addressed only recently3 and still remains largely unexplored4,5. To identify potential metastasis-promoting miRNAs, we set up a genetic screen using a non-metastatic, human breast tumour cell line that was transduced with a miRNA-expression library and subjected to a trans-well migration assay. We found that human miR-373 and miR-520c stimulated cancer cell migration and invasion in vitro and in vivo, and that certain cancer cell lines depend on endogenous miR-373 activity to migrate efficiently. Mechanistically, the migration phenotype of miR-373 and miR-520c can be explained by suppression of CD44. We found significant upregulation of miR-373 in clinical breast cancer metastasis samples that correlated inversely with CD44 expression. Taken together, our findings indicate that miRNAs are involved in tumour migration and invasion, and implicate miR-373 and miR-520c as metastasis-promoting miRNAs.

Recombinant human erythropoietin (rHu-EPO) is used to treat anemia by activating the erythropoietin receptor (EPOR) in erythroid progenitor cells, leading to proliferation and differentiation into mature red blood cells. To allow less frequent dosing, a hyperglycosylated version of EPO has been developed with a longer half-life. In principle, an agonistic antibody targeting EPOR would offer an even longer half-life, support robust monthly dosing, and, unlike EPO products, reduce the risk of pure red cell aplasia. The efficiency of signaling and corresponding potency of previously reported antibody mimics are generally suboptimal compared with EPO and not suitable for clinical use. Here we describe a potent, fully human, agonistic antibody (ABT007) targeting EPOR that supports potent, more sustained, and less pulsatile elevation of hematocrit in a human EPOR–expressing transgenic mouse model compared with standard doses of rHu-EPO while requiring less frequent dosing. Resolution of the crystal structure of the EPOR extracellular domain (ECD) complexed to the ABT007 Fab fragment, determined at 0.32 nm, identifies a binding site that is consistent with a novel mechanism of receptor activation based on a unique antibody-imposed conformational change. These results demonstrate that a symmetric molecule can serve as a potent activator of the EPOR.

The Farnesoid X receptor (FXR) regulates bile salt, glucose and cholesterol homeostasis by binding to DNA response elements, thereby activating gene expression (direct transactivation). FXR also inhibits the immune response via tethering to NF-κB (tethering transrepression). FXR activation therefore has therapeutic potential for liver and intestinal inflammatory diseases. We aim to identify and develop gene-selective FXR modulators, which repress inflammation, but do not interfere with its metabolic capacity. In a high-throughput reporter-based screen, mometasone furoate (MF) was identified as a compound that reduced NF-κB reporter activity in an FXR-dependent manner. MF reduced mRNA expression of pro-inflammatory cytokines and induction of direct FXR target genes in HepG2-GFP-FXR cells and intestinal organoids was minor. Computational studies disclosed three putative binding modes of the compound within the ligand binding domain of the receptor. Interestingly, mutation of W469A residue within the FXR ligand binding domain abrogated the decrease in NF-κB activity. Finally, we show that MF-bound FXR inhibits NF-κB subunit p65 recruitment to the DNA of pro-inflammatory genes CXCL2 and IL8. Although MF is not suitable as selective anti-inflammatory FXR ligand due to nanomolar affinity for the glucocorticoid receptor, we show that separation between metabolic and anti-inflammatory functions of FXR can be achieved.

In response to microbial invasion, neutrophils release neutrophil extracellular traps (NETs) to trap and kill extracellular microbes. Alternatively, NET formation can result in tissue damage in inflammatory conditions and may perpetuate autoimmune disease. Intervention strategies that are aimed at modifying pathogenic NET formation should ideally preserve other neutrophil antimicrobial functions. We now show that signal inhibitory receptor on leukocytes-1 (SIRL-1) attenuates NET release by human neutrophils in response to distinct triggers, including opsonized Staphylococcus aureus and inflammatory danger signals. NET release has different kinetics depending on the stimulus, and rapid NET formation is independent of NADPH oxidase activity. In line with this, we show that NET release and reactive oxygen species production upon challenge with opsonized S. aureus require different signaling events. Importantly, engagement of SIRL-1 does not affect bacterially induced production of reactive oxygen species, and intracellular bacterial killing by neutrophils remains intact. Thus, our studies define SIRL-1 as an intervention point of benefit to suppress NET formation in disease while preserving intracellular antimicrobial defense.

Daxx was first identified as a protein that binds the cytosolic domain of Fas and links this receptor to an apoptosis pathway involving activation of Jun N-terminal kinase (JNK). We show here that cells overexpressing the human homolog of Daxx (hDaxx) display enhanced sensitivity to apoptosis induced by Fas but not by several other cell death stimuli. hDaxx-mediated enhancement of Fas-induced apoptosis was correlated with accelerated activation of caspases but not with JNK induction. Although specifically enhancing Fas function, hDaxx does not bind Fas and instead is found in the nucleus where it localizes to PML oncogenic domains (PODs). Moreover, the hDaxx protein also exhibits the ability to repress transcription. Mutagenesis studies demonstrated a correlation between the localization of hDaxx to PODs and its ability to enhance Fas-induced cell death. Arsenic trioxide (As(2)O(3)), an agent that accentuates POD formation, collaborated synergistically with overexpression of hDaxx to increase cellular sensitivity to Fas-induced apoptosis. Taken together, these findings argue that hDaxx promotes sensitivity to Fas from a nuclear location, probably by modulating the transcription of genes involved in Fas-induced caspase activation and apoptosis.

Disaggregation of the spherical nuclear bodies termed promyelocytic (PML) oncogenic domains (PODs) is a characteristic of acute promyelocytic leukemia. Here, we demonstrate that the cAMP enhancer binding protein (CREB)-binding protein (CBP) associates with PML in vitro and is recruited to the PODs in vivo . Through its association with CBP, wild-type PML dramatically stimulates nuclear receptor transcriptional activity. These results demonstrate that a fraction of CBP is compartmentalized to the POD through its association with PML and thus suggest that PML and other POD-associated proteins may play an unexpectedly broad role in aspects of transcriptional regulation and human disease.

Elongation-factor-3 (EF-3) is an essential factor of the fungal protein synthesis machinery. In this communication the structure of EF-3 from Saccharomyces cerevisiae is characterized by differential scanning calorimetry (DSC), ultracentrifugation, and limited tryptic digestion. DSC shows a major transition at a relatively low temperature of 39 degrees C, and a minor transition at 58 degrees C. Ultracentrifugation shows that EF-3 is a monomer; thus, these transitions could not reflect the unfolding or dissociation of a multimeric structure. EF-3 forms small aggregates, however, when incubated at room temperature for an extended period of time. Limited proteolysis of EF-3 with trypsin produced the first cleavage at the N-side of Gln775, generating a 90-kDa N-terminal fragment and a 33-kDa C-terminal fragment. The N-terminal fragment slowly undergoes further digestion generating two major bands, one at approximately 75 kDa and the other at approximately 55 kDa. The latter was unusually resistant to further tryptic digestion. The 33-kDa C-terminal fragment was highly sensitive to tryptic digestion. A 30-min tryptic digest showed that the N-terminal 60% of EF-3 was relatively inaccessible to trypsin, whereas the C-terminal 40% was readily digested. These results suggest a tight structure of the N-terminus, which may give rise to the 58 degrees C transition, and a loose structure of the C-terminus, giving rise to the 39 degrees C transition. Three potentially functional domains of the protein were relatively resistant to proteolysis: the supposed S5-homologous domain (Lys102-Ile368), the N-terminal ATP-binding cassette (Gly463-Lys622), and the aminoacyl-tRNA-synthase homologous domain (Glu820-Gly865). Both the basal and ribosome-stimulated ATPase activities were inactivated by trypsin, but the ribosome-stimulated activity was inactivated faster.

The emergence of resistance in a tumor population is most often associated with a disregulation of gene expression, usually at the level of transcription. A major goal in the field of cancer chemotherapy is to define the mechanisms underlying transcriptional regulation of drug resistance genes in an effort to identify targets for therapeutic intervention. Recently, considerable progress has been made in identifying the molecular mechanisms involved in the transcriptional regulation of the P-glycoprotein (Pgp) gene. When overexpressed in tumor cells, Pgp confers resistance to a variety of chemotherapeutic agents; this resistance has been termed MDR (multidrug resistance). Moreover, Pgp is a normal component of a variety of highly differentiated cell types and, as such, is regulated by both internal and external environmental stimuli. In this review, we will discuss the current knowledge regarding the DNA elements and protein factors involved in both constitutive and inducible regulation of Pgp transcription in normal and tumor cells.

High-content screening (HCS) uses high-capacity automated fluorescence imaging for the quantitative analysis of single cells and cell populations. Here, we developed an HCS assay for rapid screening of non-viral gene delivery systems as exemplified by the screening of a small library of peptide-based transfectants. These peptides were simultaneously screened for transfection efficiency, cytotoxicity, induction of cell permeability and the capacity to transfect non-dividing cells. We demonstrated that HCS is a valuable extension to the already existing screening methods for the in vitro evaluation of non-viral gene delivery systems with the added value that multiple parameters can be screened in parallel thereby obtaining more information from a single screening event, which will accelerate the development of novel gene delivery systems.

Autotaxin (ATX) is a secreted nucleotide pyrophosphatase/phosphodiesterase that functions as a lysophospholipase D to produce the lipid mediator lysophosphatidic acid (LPA), a mitogen, chemoattractant, and survival factor for many cell types. The ATX-LPA signaling axis has been implicated in angiogenesis, chronic inflammation, fibrotic diseases and tumor progression, making this system an attractive target for therapy. However, potent and selective nonlipid inhibitors of ATX are currently not available. By screening a chemical library, we have identified thiazolidinediones that selectively inhibit ATX-mediated LPA production both in vitro and in vivo. Inhibitor potency was approximately 100-fold increased (IC 50 ∼ 30 nM) after the incorporation of a boronic acid moiety, designed to target the active-site threonine (T210) in ATX. Intravenous injection of this inhibitor into mice resulted in a surprisingly rapid decrease in plasma LPA levels, indicating that turnover of LPA in the circulation is much more dynamic than previously appreciated. Thus, boronic acid-based small molecules hold promise as candidate drugs to target ATX.

Purpose: Hereditary breast cancer is partly explained by germline mutations in BRCA1 and BRCA2. Although patients carry heterozygous mutations, their tumors have typically lost the remaining wild-type allele. Selectively targeting BRCA deficiency may therefore constitute an important therapeutic approach. Clinical trials applying this principle are underway, but it is unknown whether the compounds tested are optimal. It is therefore important to identify alternative compounds that specifically target BRCA deficiency and to test new combination therapies to establish optimal treatment strategies.Experimental Design: We did a high-throughput pharmaceutical screen on BRCA2-deficient mouse mammary tumor cells and isogenic controls with restored BRCA2 function. Subsequently, we validated positive hits in vitro and in vivo using mice carrying BRCA2-deficient mammary tumors.Results: Three alkylators—chlorambucil, melphalan, and nimustine—displayed strong and specific toxicity against BRCA2-deficient cells. In vivo, these showed heterogeneous but generally strong BRCA2-deficient antitumor activity, with melphalan and nimustine doing better than cisplatin and the poly-(ADP-ribose)-polymerase inhibitor olaparib (AZD2281) in this small study. In vitro drug combination experiments showed synergistic interactions between the alkylators and olaparib. Tumor intervention studies combining nimustine and olaparib resulted in recurrence-free survival exceeding 330 days in 3 of 5 animals tested.Conclusions: We generated and validated a platform for identification of compounds with specific activity against BRCA2-deficient cells that translates well to the preclinical setting. Our data call for the re-evaluation of alkylators, especially melphalan and nimustine, alone or in combination with the poly-(ADP-ribose)-polymerase inhibitors, for the treatment of breast cancers with a defective BRCA pathway. Clin Cancer Res; 16(1); 99–108

The human peptidyl-prolyl isomerase FK-binding protein (FKBP) was cloned as a fusion partner with CMP-KDO synthetase (CKS), and the resultant construct was characterized as an improved high-expression source for FKBP. The CKS-FKBP fusion was expressed as a soluble protein at levels approaching 1 gm/L in Escherichia coli fermentations. The fusion protein was purified to near homogeneity by a one-step ammonium sulfate fractionation of whole cell lysate. After selective cleavage, the fusion precursor produced yields approaching 300 mg of purified FKBP per liter of harvested culture, a approximately 30 to 60-fold increase over that observed for a nonfusion construct. Selective cleavage of the fusion partners was accomplished using either hydroxylamine or specific, limited proteolysis. Once separated from the CKS fusion partner, the FKBP was isolated in a single step by either reversed-phase HPLC or chromatography on Q-Sepharose. For comparison of physical and chemical properties, a nonfusion construct of recombinant human FKBP was expressed in E. coli and isolated. The purified FKBPs exhibited expected SDS-PAGE molecular weights and N-terminal sequences. The proteins had similar proton NMR spectra and binding to [3H]FK-506. The fusion construct, CKS-FKBP, was also found to bind [3H]FK-506. These data indicate that FKBP fused to the C-terminus of CKS folds independently of the fusion partner and suggests the fused FKBP adopts a conformation resembling that of the native protein.

We describe the development of a series of soluble, potent, and bioavailable nonpeptide renin inhibitors. These inhibitors derived from a series of novel nonpeptide renin inhibitors which were recently identified in our laboratories, by alteration of the nature of the C-terminus (P2') of the molecules. Introduction of basic substituents into modified hydroxyethylene dipeptide isosteres gave inhibitors with improved solubility as well as improved potency against human plasma renin. In addition, these modifications produced inhibitors which displayed markedly improved intraduodenal bioavailability in both the ferret and cynomolgus monkey. We also present data which demonstrate excellent efficacy in the monkey for A-74273 (65), with an intraduodenal bioavailability of 16 +/- 4% in the monkey, compared to 1.7 +/- 0.5% for the dipeptide renin inhibitor enalkiren (A-64662, 75). A-74273 is an example of a nonpeptide inhibitor which possesses a good balance of the desirable properties of potency, solubility, and lipophilicity and which is well absorbed into the intestine.

Recombinant major histocompatibility complex (MHC) class I molecules complexed with pathogen-specific or other disease-associated antigens have become essential reagents for the analysis of adaptive T-cell responses. However, conventional techniques for the production of recombinant peptide-MHC (pMHC) complexes are highly involved and thereby limit the use of pMHC complexes in terms of antigen diversity. To make pMHC-based techniques suitable for high-throughput analyses we developed an MHC peptide exchange technology based on the use of conditional MHC ligands. This technology enables the parallel production of thousands of different pMHC complexes within hours, allowing the development of high-throughput MHC-based assay systems to identify MHC ligands and cytotoxic T-cell responses. These high-throughput assays should prove valuable for the screening of entire disease-associated proteomes, including pathogen-encoded proteomes, tumor-associated antigens, and autoimmune antigens.

Dot1 methylates histone H3 lysine 79 (H3K79) on the nucleosome core and is involved in Sir protein-mediated silencing. Previous studies suggested that H3K79 methylation within euchromatin prevents nonspecific binding of the Sir proteins, which in turn facilitates binding of the Sir proteins in unmethylated silent chromatin. However, the mechanism by which the Sir protein binding is influenced by this modification is unclear. We performed genome-wide synthetic genetic array (SGA) analysis and identified interactions of DOT1 with SIR1 and POL32. The synthetic growth defects found by SGA analysis were attributed to the loss of mating type identity caused by a synthetic silencing defect. By using epistasis analysis, DOT1, SIR1, and POL32 could be placed in different pathways of silencing. Dot1 shared its silencing phenotypes with the NatA N-terminal acetyltransferase complex and the conserved N-terminal bromo adjacent homology (BAH) domain of Sir3 (a substrate of NatA). We classified all of these as affecting a common silencing process, and we show that mutations in this process lead to nonspecific binding of Sir3 to chromatin. Our results suggest that the BAH domain of Sir3 binds to histone H3K79 and that acetylation of the BAH domain is required for the binding specificity of Sir3 for nucleosomes unmethylated at H3K79.

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from approximately 1-5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr approximately 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence and pH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P6-P'3 sequence of human angiotensinogen.

MicroRNAs (miRNAs) are potent post-transcriptional regulators of protein coding genes. Patterns of misexpression of miRNAs in cancer suggest key functions of miRNAs in tumorigenesis. However, current bioinformatics tools do not entirely support the identification and characterization of the mode of action of such miRNAs. Here, we used a novel functional genetic approach and identified miR-221 and miR-222 (miR-221&222) as potent regulators of p27(Kip1), a cell cycle inhibitor and tumor suppressor. Using miRNA inhibitors, we demonstrate that certain cancer cell lines require high activity of miR-221&222 to maintain low p27(Kip1) levels and continuous proliferation. Interestingly, high levels of miR-221&222 appear in glioblastomas and correlate with low levels of p27(Kip1) protein. Thus, deregulated expression of miR-221&222 promotes cancerous growth by inhibiting the expression of p27(Kip1).

With the emergence of multidrug resistant (MDR) bacteria, it is imperative to develop new intervention strategies. Current antibiotics typically target pathogen rather than host-specific biochemical pathways. Here we have developed kinase inhibitors that prevent intracellular growth of unrelated pathogens such as Salmonella typhimurium and Mycobacterium tuberculosis. An RNA interference screen of the human kinome using automated microscopy revealed several host kinases capable of inhibiting intracellular growth of S. typhimurium. The kinases identified clustered in one network around AKT1 (also known as PKB). Inhibitors of AKT1 prevent intracellular growth of various bacteria including MDR-M. tuberculosis. AKT1 is activated by the S. typhimurium effector SopB, which promotes intracellular survival by controlling actin dynamics through PAK4, and phagosome-lysosome fusion through the AS160 (also known as TBC1D4)-RAB14 pathway. AKT1 inhibitors counteract the bacterial manipulation of host signalling processes, thus controlling intracellular growth of bacteria. By using a reciprocal chemical genetics approach, we identified kinase inhibitors with antibiotic properties and their host targets, and we determined host signalling networks that are activated by intracellular bacteria for survival.

Kringle 5 (K5) of human plasminogen has been shown to inhibit angiogenesis by inducing the apoptosis of proliferating endothelial cells. Peptide regions around the lysine-binding pocket of K5 largely mediate these effects, particularly the peptide PRKLYDY, which we show to compete with K5 for the binding to endothelial cells. The cell surface binding site for K5 that mediates these effects has not been defined previously. Here, we report that glucose-regulated protein 78, exposed on cell surfaces of proliferating endothelial cells as well as on stressed tumor cells, plays a key role in the antiangiogenic and antitumor activity of K5. We also report that recombinant K5-induced apoptosis of stressed HT1080 fibrosarcoma cells involves enhanced activity of caspase-7, consistent with the disruption of glucose-regulated protein 78-procaspase-7 complexes. These results establish recombinant K5 as an inhibitor of a stress response pathway, which leads to both endothelial and tumor cell apoptosis.

The identity of the physiological metal cofactor for human methionine aminopeptidase-2 (MetAP2) has not been established. To examine this question, we first investigated the effect of eight divalent metal ions, including Ca(2+), Co(2+), Cu(2+), Fe(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+), on recombinant human methionine aminopeptidase apoenzymes in releasing N-terminal methionine from three peptide substrates: MAS, MGAQFSKT, and (3)H-MASK(biotin)G. The activity of MetAP2 on either MAS or MGAQFSKT was enhanced 15-25-fold by Co(2+) or Mn(2+) metal ions in a broad concentration range (1-1000 microM). In the presence of reduced glutathione to mimic the cellular environment, Co(2+) and Mn(2+) were also the best stimulators (approximately 30-fold) for MetAP2 enzyme activity. To determine which metal ion is physiologically relevant, we then tested inhibition of intracellular MetAP2 with synthetic inhibitors selective for MetAP2 with different metal cofactors. A-310840 below 10 microM did not inhibit the activity of MetAP2-Mn(2+) but was very potent against MetAP2 with other metal ions including Co(2+), Fe(2+), Ni(2+), and Zn(2+) in the in vitro enzyme assays. In contrast, A-311263 inhibited MetAP2 with Mn(2+), as well as Co(2+), Fe(2+), Ni(2+), and Zn(2+). In cell culture assays, A-310840 did not inhibit intracellular MetAP2 enzyme activity and did not inhibit cell proliferation despite its ability to permeate and accumulate in cytosol, while A-311263 inhibited both intracellular MetAP2 and proliferation in a similar concentration range, indicating cellular MetAP2 is functioning as a manganese enzyme but not as a cobalt, zinc, iron, or nickel enzyme. We conclude that MetAP2 is a manganese enzyme and that therapeutic MetAP2 inhibitors should inhibit MetAP2-Mn(2+).

The remarkable success of retinoic acid in the treatment of acute promyelocytic leukemias and the subsequent discovery that mutant forms of a retinoid acid receptor (RARalpha) are invariably associated with this disease has generated considerable interest among both clinicians and basic scientists. Studies both in cell culture and in transgenic animals suggest that mutant RARs interfere with normal retinoid-mediated transactivation and granulocytic differentiation. More recently, a pivotal link between transcriptional silencing, the oncogenic functions of RAR mutants, and hormonal responses in APL patients has been established. These studies have greatly advanced our understanding of the molecular changes involved in leukemogenesis, have helped to reveal new aspects of cellular differentiation, and might lead to improved treatment strategies for human leukemias.

A systematic evaluation of structure-absorption relationships using a high throughput intraduodenal rat screening model has led to the delineation of a set of structural parameters that appear to govern bioavailability in a series of peptide-based renin inhibitors. Optimum structures, exemplified by 25 and 41, incorporated a single, solubilizing substituent at the C- or N-terminus combined with a lipophilic P2-site residue. Both inhibitors gave unprecedented plasma drug levels upon intraduodenal administration to monkeys, and the calculated bioavailability for 41 (14 +/- 4%) is the highest reported for any peptidic renin inhibitor.

Acute promyelocytic leukemia (APL) is characterized by a variety of chromosomal translocations into the retinoic acid receptor-α (RARα) gene. The first of these cloned was t(15;17), a translocation that results in fusion of the PML gene with RARα. This translocation is also the most prevalent, found in 95% of APL cases. Other translocations associated with fusion proteins in variant APLs were subsequently discovered: t(11;17) PLZF-RARα (found in less than 5% of cases), another t(11;17) translocation, NuMA-RARα (less than 1%), and t(5;17) NPM-RARα (less than 1%). All of these, with the exception of the PLZF-RARα translocation, are associated with a disease that responds to high doses of retinoids (1). The realization that all of these translocations involved RARα led to the proposal that the disease is caused by dysfunctional regulation of retinoid-inducible genes critical to myeloid differentiation. Recent work supporting this idea has shown that PML-RARα has a reduced ability to release transcriptional corepressors such as SMRT and NCoR in response to retinoids and that the PLZF portion of the PLZF-RARα fusion constitutively binds these repressors (2, 3). This would suggest that in APL cells, RARα target genes may be transcriptionally super-repressed, and may also explain why PML-RARα APL responds to high doses of retinoids, whereas PLZF-RARα APL is retinoid resistant. The ultimate proof of the hypothesis, however, has awaited the identification of critical target genes, the regulation of which is disrupted by the fusion proteins. In this issue of the JCI, Park et al. propose that the gene for the myeloid-specific transcription factor C/EBPe may be such a target (4). Previous studies have shown that C/EBPe transcription is induced by retinoids during granulocytic, but not monocytic, differentiation (5). Mice in which the gene is deleted exhibit impaired granulopoiesis (6). Therefore C/EBPe has many of the predicted characteristics of a critical target gene in APL. It remained to be demonstrated that its promoter contained a functional retinoic acid response element (RARE) and that overexpression of C/EBPe alone was sufficient to induce granulocytic differentiation. These key experiments are presented in this issue. The authors identified a RARE in the C/EBPe promoter and showed it to be functional in transfection assays. They demonstrated that C/EBPe expression could be induced in a ligand-dependent manner by PML-RARα (a fusion product found in a retinoid-sensitive APL) but not by PLZF-RARα (a fusion product found in a retinoid-resistant APL). Overexpression of C/EBPe in U937 cells was then shown to be sufficient for the induction of granulopoiesis, as evidenced by morphological changes and an increase in the expression of granulocytic markers such as CD11b, CD66b, lactoferrin, and collagenase. The expected decrease in proliferation and increase in apoptosis were also observed. Therefore, the authors are justified in proposing that C/EBPe may be an important downstream target of RARα in myeloid differentiation. What makes C/EBPe especially intriguing in this role are the findings, outlined in an earlier paper from the Koeffler laboratory, that suggest that C/EBPe expression may be a node at which cross-talk between regulatory pathways occurs. The differentiation agents DMSO and HMBA were shown to induce C/EBPe mRNA in the NB4 APL cell line, but only after the cells were primed by a transient 30-minute incubation with retinoic acid (7). The characterization of the pathways that cooperate with retinoids in the induction of C/EBPe, and indeed independent pathways that regulate C/EBPe expression or function, will now be a priority. Knowledge of such pathways may lead to the development of therapeutic strategies whereby C/EBPe transcription can be induced in the absence of retinoids. These strategies might be especially useful for the treatment of retinoid-resistant APLs, such those resulting from the t(11;17) PLZF-RARα translocation, or APLs in which retinoid resistance is mediated by mutations in the PML-RARα ligand-binding domain. It will be a challenging task, however, to unravel the complexities of the gene’s regulation. It is already known, for example, that the C/EBPe gene can be transcribed from alternate promoters (8). Identification of the genes regulated by C/EPBe will give a more complete understanding of its role in myeloid differentiation; the application of recently developed DNA array technologies may accelerate this process. C/EBPe is a member of a growing family of transcription factors; in addition to C/EBPe, at least 3 of these factors (C/EBPα, β, and δ) are expressed during hematopoiesis (9). The various C/EBP family members are known to be capable of both homo- and heterodimerization, and because the expression of C/EBPα and C/EBPe are known to overlap, it is probable that they functionally interact during myeloid differentiation. This will make an analysis of C/EBPe target gene regulation quite challenging. To make matters even more complicated, the C/EBPe gene, through the use of alternative splicing and alternative promoters, encodes 3 different proteins. All of these have identical dimerization and DNA-binding domains, but different NH2-terminal regions. The functional significance of these differences is unknown (8). Is the suppression of C/EBPe gene expression the common theme in all APL variants? One experiment presented by Park et al. would suggest that it is not. The overexpression of PLZF-RARα in U937 cells in the absence of retinoids did not repress C/EBPe expression, whereas the overexpression of PML-RARα did. This would suggest that PLZF-RARα and PML-RARα might act through different pathways, with different target specificity. Now that we are beginning to identify downstream targets of the retinoid receptors, we can look forward to a great increase in our understanding of the molecular basis of myeloid differentiation. This, allied with recent studies that have shed light on the function of PML in cell growth, apoptosis, and transcriptional regulation (1), should pave the way for a deeper understanding of the molecular events that are involved in the development of APL.

The binding of a 13C-labeled cyclosporin A (CsA) analog to cyclophilin (peptidyl prolyl isomerase) was examined by means of isotope-edited nuclear magnetic resonance (NMR) techniques. A trans 9,10 peptide bond was adopted when CsA was bound to cyclophilin, in contrast to the cis 9,10 peptide bond found in the crystalline and solution conformations of CsA. Furthermore, nuclear Overhauser effects (NOEs) were observed between the zeta 3 and epsilon 3 protons of the methylleucine (MeLeu) residue at position 9 of CsA and tryptophan121 (Trp121) and phenylalanine (Phe) protons of cyclophilin, suggesting that the MeLeu9 residue of CsA interacts with cyclophilin. These results illustrate the power of isotope-edited NMR techniques for rapidly providing useful information about the conformations and active site environment of inhibitors bound to their target enzymes.

Incorporation of nonreactive polar functionalities at the C- and N-termini of renin inhibitors led to the development of a subnanomolar compound (21) with millimolar solubility. This inhibitor demonstrated excellent efficacy and a long duration of action upon intravenous administration to monkeys. While activity was also observed intraduodenally, a comparison of the blood pressure responses indicated low bioavailability. Subsequent experiments in rats showed that, although the compound was absorbed from the gastrointestinal tract, extensive liver extraction severely limited bioavailability.

The synthesis of a series of renin inhibitors in which the P2 and P3 amino acids are replaced with the hydroxyethylene dipeptide isostere is reported. In vitro evaluation of the inhibitors has revealed that this isostere is an acceptable amide-bond replacement in which activity is maintained and stability is enhanced. Structure-activity relationships of this series resemble but do not parallel those of the corresponding dipeptide-containing inhibitors.

Highlights • Design of a high throughput toxicity assay for spheroid-type tumor cell cultures. • Cell viability, rather than a proxy thereof, is measured. • Cell viability is measured on a per-cell, rather than per-well, basis. • The method can be implemented on standard automated microscopes. • It provides a low-cost approach for accurate and reproducible toxicity screens., Novel spheroid-type tumor cell cultures directly isolated from patients’ tumors preserve tumor characteristics better than traditionally grown cell lines. However, such cultures are not generally used for high-throughput toxicity drug screens. In addition, the assays that are commonly used to assess drug-induced toxicity in such screens usually measure a proxy for cell viability such as mitochondrial activity or ATP-content per culture well, rather than actual cell death. This generates considerable assay-dependent differences in the measured toxicity values. To address this problem we developed a robust method that documents drug-induced toxicity on a per-cell, rather than on a per-well basis. The method involves automated drug dispensing followed by paired image- and FACS-based analysis of cell death and cell cycle changes. We show that the two methods generate toxicity data in 96-well format which are highly concordant. By contrast, the concordance of these methods with frequently used well-based assays was generally poor. The reported method can be implemented on standard automated microscopes and provides a low-cost approach for accurate and reproducible high-throughput toxicity screens in spheroid type cell cultures. Furthermore, the high versatility of both the imaging and FACS platforms allows straightforward adaptation of the high-throughput experimental setup to include fluorescence-based measurement of additional cell biological parameters.

The synthesis of diol-containing renin inhibitors has revealed that a simple vicinal diol functionality corresponding to the scissile Leu-Val bond in human angiotensinogen is capable of imparting inhibitory activity at a comparable or higher level than either the corresponding aldehyde or hydroxymethyl functionality (compare inhibitors 2a-c or 3a-c). This finding has led to the further optimization of a series of small transition-state analogue inhibitors by the inclusion of a second hydroxyl group in the Leu-Val surrogate to give compounds that inhibited human renin in the 200-700-pM range (e.g. 43, 45, 63, 66). The magnitude of effect of the second hydroxyl group on potency is not only dictated by the absolute stereochemistry of the diol but also by the side chain of the P1 residue. Molecular modeling of the diol-containing inhibitors suggests that one of the hydroxyl groups hydrogen bonds to Asp 32 and Asp 215, while the second hydrogen bonds to Asp 215. These diol inhibitors are extremely selective for human renin over the related enzymes cathepsin D, pepsin, and gastricsin. At high concentrations, compounds containing a leucine or phenylalanine rather than a histidine at the P2 position gave only minor amounts of inhibition of the other enzymes. Inhibitor 43 suppressed plasma renin activity completely and lowered mean blood pressure in monkeys after both intravenous and intraduodenal administration, but the blood pressure drop lasted less than 1 h. Monitoring the blood levels of 43 by enzyme inhibition assay after intraduodenal administration to monkeys or oral administration to rats revealed low absorption and rapid clearance. While intratracheal administration to dogs gave approximately 50% bioavailability, rapid clearance was still a problem. After examination of inhibitor 45 in a sensitive primate model in which monkeys were rendered both hypertensive and hyperreninemic, the effects on lowering systolic but not diastolic pressure were apparent even after 22 h postdosing. Details on the synthesis, in vitro structure-activity relationships, molecular modeling, in vivo activity, and metabolism of these inhibitors are described.

Native human renin, produced from the culture of human chorionic trophoblasts, has been purified to homogeneity on a milligram scale using a five-step purification scheme. The chorion cells secrete 50–200 milliGoldblatt Units of trypsin-activatable prorenin per ml into the medium. The pro-enzyme is partially purified by ammonium sulfate fractionation and chromatographies on QAE-Sephadex and cibracon blue-agarose. Following conversion of prorenin to the active enzyme by porcine trypsin, the renin is purified to homogeneity by affinity chromatography and gel filtration. Chorionic prorenin has a molecular weight of 43 000; the active enzyme 40 000. Both proteins exist as a single polypeptide chain as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions. The average specific activity of six different preparations was found to be 1072 Goldblatt Units/mg. The amino acid composition and N-terminal sequence of the active enzyme has been determined and is identical to the human kidney enzyme. Microheterogeneity of chorionic renin was demonstrated by isoelectrofocusing analysis. The physical characterization of chorionic renin is compared with that reported for the human kidney enzyme.