A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity.

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.

A Genome-wide multidimensional RNAi screen reveals pathways controlling MHC class II antigen presentation.

MHC 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 TGFß. 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.

Intra-tumour diversification in colorectal cancer at the single-cell level.

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.

TMEM59 potentiates Wnt signaling by promoting signalosome formation.

Wnt/ß-catenin signaling controls development and adult tissue homeostasis by regulating cell proliferation and cell fate decisions. Wnt binding to its receptors Frizzled (FZD) and low-density lipoprotein-related 6 (LRP6) at the cell surface initiates a signaling cascade that leads to the transcription of Wnt target genes. Upon Wnt binding, the receptors assemble into large complexes called signalosomes that provide a platform for interactions with downstream effector proteins. The molecular basis of signalosome formation and regulation remains elusive, largely due to the lack of tools to analyze its endogenous components. Here, we use internally tagged Wnt3a proteins to isolate and characterize activated, endogenous Wnt receptor complexes by mass spectrometry-based proteomics. We identify the single-span membrane protein TMEM59 as an interactor of FZD and LRP6 and a positive regulator of Wnt signaling. Mechanistically, TMEM59 promotes the formation of multimeric Wnt-FZD assemblies via intramembrane interactions. Subsequently, these Wnt-FZD-TMEM59 clusters merge with LRP6 to form mature Wnt signalosomes. We conclude that the assembly of multiprotein Wnt signalosomes proceeds along well-ordered steps that involve regulated intramembrane interactions.

An Image-Based miRNA Screen Identifies miRNA-135s As Regulators of CNS Axon Growth and Regeneration by Targeting Krüppel-like Factor 4.

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 migration in vitro and in vivo in 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 STATEMENT 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 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.

Epigenetic drug screen identifies the histone deacetylase inhibitor NSC3852 as a potential novel drug for the treatment of pediatric acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease regarding morphology, immunophenotyping, genetic abnormalities, and clinical behavior. The overall survival rate of pediatric AML is 60% to 70%, and has not significantly improved over the past two decades. Children with Down syndrome (DS) are at risk of developing acute megakaryoblastic leukemia (AMKL), which can be preceded by a transient myeloproliferative disorder during the neonatal period. Intensification of current treatment protocols is not feasible due to already high treatment-related morbidity and mortality. Instead, more targeted therapies with less severe side effects are highly needed. PROCEDURE: To identify potential novel therapeutic targets for myeloid disorders in children, including DS-AMKL and non-DS-AML, we performed an unbiased compound screen of 80 small molecules targeting epigenetic regulators in three pediatric AML cell lines that are representative for different subtypes of pediatric AML. Three candidate compounds were validated and further evaluated in normal myeloid precursor cells during neutrophil differentiation and in (pre-)leukemic pediatric patient cells. RESULTS: Candidate drugs LMK235, NSC3852, and bromosporine were effective in all tested pediatric AML cell lines with antiproliferative, proapoptotic, and differentiation effects. Out of these three compounds, the pan-histone deacetylase inhibitor NSC3852 specifically induced growth arrest and apoptosis in pediatric AML cells, without disrupting normal neutrophil differentiation. CONCLUSION: NSC3852 is a potential candidate drug for further preclinical testing in pediatric AML and DS-AMKL.

Aurora A, MCAK, and Kif18b promote Eg5-independent spindle formation.

Inhibition of the microtubule (MT) motor protein Eg5 results in a mitotic arrest due to the formation of monopolar spindles, making Eg5 an attractive target for anti-cancer therapies. However, Eg5-independent pathways for bipolar spindle formation exist, which might promote resistance to treatment with Eg5 inhibitors. To identify essential components for Eg5-independent bipolar spindle formation, we performed a genome-wide siRNA screen in Eg5-independent cells (EICs). We find that the kinase Aurora A and two kinesins, MCAK and Kif18b, are essential for bipolar spindle assembly in EICs and in cells with reduced Eg5 activity. Aurora A promotes bipolar spindle assembly by phosphorylating Kif15, hereby promoting Kif15 localization to the spindle. In turn, MCAK and Kif18b promote bipolar spindle assembly by destabilizing the astral MTs. One attractive way to interpret our data is that, in the absence of MCAK and Kif18b, excessive astral MTs generate inward pushing forces on centrosomes at the cortex that inhibit centrosome separation. Together, these data suggest a novel function for astral MTs in force generation on spindle poles and how proteins involved in regulating microtubule length can contribute to bipolar spindle assembly.

Signal Inhibitory Receptor on Leukocytes-1 Limits the Formation of Neutrophil Extracellular Traps, but Preserves Intracellular Bacterial Killing.

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.

Screening of a Library of FDA-Approved Drugs Identifies Several Enterovirus Replication Inhibitors That Target Viral Protein 2C.

Enteroviruses (EVs) represent many important pathogens of humans. Unfortunately, no antiviral compounds currently exist to treat infections with these viruses. We screened the Prestwick Chemical Library, a library of approved drugs, for inhibitors of coxsackievirus B3, identified pirlindole as a potent novel inhibitor, and confirmed the inhibitory action of dibucaine, zuclopenthixol, fluoxetine, and formoterol. Upon testing of viruses of several EV species, we found that dibucaine and pirlindole inhibited EV-B and EV-D and that dibucaine also inhibited EV-A, but none of them inhibited EV-C or rhinoviruses (RVs). In contrast, formoterol inhibited all enteroviruses and rhinoviruses tested. All compounds acted through the inhibition of genome replication. Mutations in the coding sequence of the coxsackievirus B3 (CV-B3) 2C protein conferred resistance to dibucaine, pirlindole, and zuclopenthixol but not formoterol, suggesting that 2C is the target for this set of compounds. Importantly, dibucaine bound to CV-B3 protein 2C in vitro, whereas binding to a 2C protein carrying the resistance mutations was reduced, providing an explanation for how resistance is acquired.

Intracellular bacterial growth is controlled by a kinase network around PKB/AKT1.

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.

Ultrahigh contrast from a frequency-doubled chirped-pulse-amplification beamline.

This paper describes frequency-doubled operation of a high-energy chirped-pulse-amplification beamline. Efficient type-I second-harmonic generation was achieved using a 3 mm thick 320 mm aperture KDP crystal. Shots were fired at a range of energies achieving more than 100 J in a subpicosecond, 527 nm laser pulse with a power contrast of 10(14).

Overview of laser systems for the Orion facility at the AWE.

The commissioning of the Orion laser facility at the Atomic Weapons Establishment (AWE) in the UK has recently been completed. The facility is a twelve beam Nd:glass-based system for studying high energy density physics. It consists of ten frequency-tripled beam-lines operating with nanosecond pulses, synchronized with two beam-lines with subpicosecond pulses, each capable of delivering 500 J to target. One of the short pulse beams has the option of frequency doubling, at reduced aperture, to yield up to 100 J at 527 nm in a subpicosecond pulse with high temporal contrast. An extensive array of target diagnostics is provided. This article describes the laser design and commissioning and presents key performance data of the facility's laser systems.

Boronic acid-based inhibitor of autotaxin reveals rapid turnover of LPA in the circulation.

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) approximately 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.

Identification of F-box only protein 7 as a negative regulator of NF-kappaB signalling.

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.

Modelling the Compaction Step of a Platform Direct Compression Process.

The ability to predict formulation behaviour at production scale during formulation design can reduce the time to market and decrease product development costs. However, it is challenging to extrapolate compaction settings for direct compression formulations between tablet press models during scale-up and transfer from R&D to commercial production. The aim of this study was to develop statistical process models to predict tablet tensile strength, porosity and disintegration time from compaction parameters (pre-compression and main compression force, and press speed), for three formulations, with differing deformation characteristics (plastic, brittle and elastic), on three tablet press models (one pilot-scale tablet press (KG RoTab) and two production-scale presses (Fette 1200i and GEA Modul P)). The deformation characteristics of yield pressure and elastic recovery were determined for the model placebo formulations investigated. To facilitate comparison of dwell time settings between tablet press models, the design of experiments (DoE) approach was 9 individual 16-run response surface DoEs (3 formulation × 3 press models), whose results were combined to create a polynomial regression model for each tablet property. These models predicted tablet tensile strength, porosity and disintegration time and enabled the construction of design spaces to produce tablets with specified target properties, for each formulation on each press. The models were successfully validated. This modelling approach provides an understanding of the compaction behaviour of formulations with varying deformation behaviour on development and commercial tablet press models. This understanding can be applied to inform achievable production rates at a commercial scale, during the formulation development.

Small Molecule Inhibitor Targeting CDT1/Geminin Protein Complex Promotes DNA Damage and Cell Death in Cancer Cells.

DNA replication initiation requires the loading of MCM2-7 complexes at the origins of replication during G1. Replication licensing renders chromatin competent for DNA replication and its tight regulation is essential to prevent aberrant DNA replication and genomic instability. CDT1 is a critical factor of licensing and its activity is controlled by redundant mechanisms, including Geminin, a protein inhibitor of CDT1. Aberrant CDT1 and Geminin expression have been shown to promote tumorigenesis in vivo and are also evident in multiple human tumors. In this study, we developed an in vitro AlphaScreen™ high-throughput screening (HTS) assay for the identification of small-molecule inhibitors targeting the CDT1/Geminin protein complex. Biochemical characterization of the most potent compound, AF615, provided evidence of specific, dose-dependent inhibition of Geminin binding to CDT1 both in-vitro and in cells. Moreover, compound AF615 induces DNA damage, inhibits DNA synthesis and reduces viability selectively in cancer cell lines, and this effect is CDT1-dependent. Taken together, our data suggest that AF615 may serve as a useful compound to elucidate the role of CDT1/Geminin protein complex in replication licensing and origin firing as well as a scaffold for further medicinal chemistry optimisation.

Small molecules to regulate the GH/IGF1 axis by inhibiting the growth hormone receptor synthesis.

Growth hormone (GH) and insulin-like growth factor-1 (IGF1) play an important role in mammalian development, cell proliferation and lifespan. Especially in cases of tumor growth there is an urgent need to control the GH/IGF1 axis. In this study we screened a 38,480-compound library, and in two consecutive rounds of analogues selection, we identified active lead compounds based on the following criteria: inhibition the GH receptor (GHR) activity and its downstream effectors Jak2 and STAT5, and inhibition of growth of breast and colon cancer cells. The most active small molecule (BM001) inhibited both the GH/IGF1 axis and cell proliferation with an IC50 of 10-30 nM of human cancer cells. BM001 depleted GHR in human lymphoblasts. In preclinical xenografted experiments, BM001 showed a strong decrease in tumor volume in mice transplanted with MDA-MB-231 breast cancer cells. Mechanistically, the drug acts on the synthesis of the GHR. Our findings open the possibility to inhibit the GH/IGF1 axis with a small molecule.

Process Model Approach to Predict Tablet Weight Variability for Direct Compression Formulations at Pilot and Production Scale.

Optimizing processing conditions to achieve a critical quality attribute (CQA) is an integral part of pharmaceutical quality by design (QbD). It identifies combinations of material and processing parameters ensuring that processing conditions achieve a targeted CQA. Optimum processing conditions are formulation and equipment-dependent. Therefore, it is challenging to translate a process design between formulations, pilot-scale and production-scale equipment. In this study, an empirical model was developed to determine optimum processing conditions for direct compression formulations with varying flow properties, across pilot- and production-scale tablet presses. The CQA of interest was tablet weight variability, expressed as percentage relative standard deviation. An experimental design was executed for three model placebo blends with varying flow properties. These blends were compacted on one pilot-scale and two production-scale presses. The process model developed enabled the optimization of processing parameters for each formulation, on each press, with respect to a target tablet weight variability of <1%RSD. The model developed was successfully validated using data for additional placebo and active formulations. Validation formulations were benchmarked to formulations used for model development, employing permeability index values to indicate blend flow.

Identification of lysosome-targeting drugs with anti-inflammatory activity as potential invasion inhibitors of treatment resistant HER2 positive cancers.

PURPOSE: Most HER2 positive invasive cancers are either intrinsic non-responsive or develop resistance when treated with 1st line HER2 targeting drugs. Both 1st and 2nd line treatments of HER2 positive cancers are aimed at targeting the HER2 receptor directly, thereby strongly limiting the treatment options of HER2/ErbB2 inhibition resistant invasive cancers. METHODS: We used phenotypic high throughput microscopy screening to identify efficient inhibitors of ErbB2-induced invasion using 1st line HER2 inhibitor trastuzumab- and pertuzumab-resistant, p95-ErbB2 expressing breast cancer cells in conjunction with the Prestwick Chemical Library®. The screening entailed a drug's ability to inhibit ErbB2-induced, invasion-promoting positioning of lysosomes at the cellular periphery, a phenotype that defines their invasiveness. In addition, we used high throughput microscopy and biochemical assays to assess the effects of the drugs on lysosomal membrane permeabilization (LMP) and autophagy, two features connected to cancer treatment. Using 2nd line HER2 inhibitor lapatinib resistant 3-dimensional model systems, we assessed the effects of the drugs on ErbB2 positive breast cancer spheroids and developed a high-throughput invasion assay for HER2 positive ovarian cancer organoids for further evaluation. RESULTS: We identified Auranofin, Colchicine, Monensin, Niclosamide, Podophyllotoxin, Quinacrine and Thiostrepton as efficient inhibitors of invasive growth of 2nd line HER2 inhibitor lapatinib resistant breast cancer spheroids and ovarian cancer organoids. We classified these drugs into four groups based on their ability to target lysosomes by inducing autophagy and/or LMP, i.e., drugs inducing early LMP, early autophagy with late LMP, late LMP, or neither. CONCLUSIONS: Our results indicate that targetable lysosome-engaging cellular pathways downstream of ErbB2 contribute to invasion. They support lysosomal trafficking as an attractive target for therapy aiming at preventing the spreading of cancer cells. Since these drugs additionally possess anti-inflammatory activities, they could serve as multipurpose drugs simultaneously targeting infection/inflammation and cancer spreading.

High-Throughput Screening Identifies Idasanutlin as a Resensitizing Drug for Venetoclax-Resistant Neuroblastoma Cells.

Neuroblastoma tumors frequently overexpress the anti-apoptotic protein B-cell lymphoma/leukemia 2 (BCL-2). We previously showed that treating BCL-2-dependent neuroblastoma cells with the BCL-2 inhibitor venetoclax results in apoptosis, but unfortunately partial therapy resistance is observed. The current study describes the identification of drugs capable of resensitizing venetoclax-resistant neuroblastoma cells to venetoclax. To examine these effects, venetoclax resistance was induced in BCL-2-dependent neuroblastoma cell lines KCNR and SJNB12 by continuous exposure to high venetoclax concentrations. Non-resistant and venetoclax-resistant neuroblastoma cell lines were exposed to a 209-compound library in the absence and presence of venetoclax to identify compounds that were more effective in the venetoclax-resistant cell lines under venetoclax pressure. Top hits were further validated in combination with venetoclax using BCL-2-dependent neuroblastoma model systems. Overall, high-throughput drug screening identified the MDM2 inhibitor idasanutlin as a promising resensitizing agent for venetoclax-resistant neuroblastoma cell lines. Idasanutlin treatment induced BAX-mediated apoptosis in venetoclax-resistant neuroblastoma cells in the presence of venetoclax, whereas it caused p21-mediated growth arrest in control cells. In vivo combination treatment showed tumor regression and superior efficacy over single-agent therapies in a BCL-2-dependent neuroblastoma cell line xenograft and a patient-derived xenograft. However, xenografts less dependent on BCL-2 were not sensitive to venetoclax-idasanutlin combination therapy. This study demonstrates that idasanutlin can overcome resistance to the BCL-2 inhibitor venetoclax in preclinical neuroblastoma model systems, which supports clinical development of a treatment strategy combining the two therapies.