The PI3K pathway is commonly activated in breast cancer, with PI3K-AKT pathway inhibitors used clinically. However, mechanisms that limit or enhance the therapeutic effects of PI3K-AKT inhibitors are poorly understood at a genome-wide level. Parallel CRISPR screens in 3 PTEN-null breast cancer cell lines identified genes mediating resistance to capivasertib (AKT inhibitor) and AZD8186 (PI3Kβ inhibitor). The dominant mechanism causing resistance is reactivated PI3K-AKT-mTOR signalling, but not other canonical signalling pathways. Deletion of TSC1/2 conferred resistance to PI3Kβi and AKTi through mTORC1. However, deletion of PIK3R2 and INPPL1 drove specific PI3Kβi resistance through AKT. Conversely deletion of PIK3CA , ERBB2 , ERBB 3 increased PI3Kβi sensitivity while modulation of RRAGC , LAMTOR1 , LAMTOR4 increased AKTi sensitivity. Significantly, we found that Mcl-1 loss enhanced response through rapid apoptosis induction with AKTi and PI3Kβi in both sensitive and drug resistant TSC1/2 null cells. The combination effect was BAK but not BAX dependent. The Mcl-1i + PI3Kβ/AKTi combination was effective across a panel of breast cancer cell lines with PIK3CA and PTEN mutations, and delivered increased anti-tumor benefit in vivo. This study demonstrates that different resistance drivers to PI3Kβi and AKTi converge to reactivate PI3K-AKT or mTOR signalling and combined inhibition of Mcl-1 and PI3K-AKT has potential as a treatment strategy for PI3Kβi/AKTi sensitive and resistant breast tumours.

Arguably, one of the most exciting new technologies for drug discovery in recent years has been that of CRISPR. The ability to efficiently silence or activate gene expression at genome scale using CRISPR/Cas vectors coupled with a lower false-positive rate than previous gene editing approaches has the potential to revolutionise the search for novel drug targets. Here we describe how this technology is transforming target identification, and what the future holds for the integration of genome editing into phenotypic drug discovery. We will guide readers through the process of applying CRISPR technology to phenotypic drug discovery, from project start to completion, describing key considerations and technological advances to maximise success.

Modified messenger RNAs (mRNAs) hold great potential as therapeutics by using the body's own processes for protein production. However, a key challenge is efficient delivery of therapeutic mRNA to the cell cytosol and productive protein translation. Lipid nanoparticles (LNPs) are the most clinically advanced system for nucleic acid delivery; however, a relatively narrow therapeutic index makes them unsuitable for many therapeutic applications. A key obstacle to the development of more potent LNPs is a limited mechanistic understanding of the interaction of LNPs with cells. To address this gap, we performed an arrayed CRISPR screen to identify novel pathways important for the functional delivery of MC3 lipid-based LNP encapsulated mRNA (LNP-mRNA). Here, we have developed and validated a robust, high-throughput screening-friendly phenotypic assay to identify novel targets that modulate productive LNP-mRNA delivery. We screened the druggable genome (7795 genes) and validated 44 genes that either increased (37 genes) or inhibited (14 genes) the productive delivery of LNP-mRNA. Many of these genes clustered into families involved with host cell transcription, protein ubiquitination, and intracellular trafficking. We show that both UDP-glucose ceramide glucosyltransferase and V-type proton ATPase can significantly modulate the productive delivery of LNP-mRNA, increasing and decreasing, respectively, with both genetic perturbation and by small-molecule inhibition. Taken together, these findings shed new light into the molecular machinery regulating the delivery of LNPs into cells and improve our mechanistic understanding of the cellular processes modulating the interaction of LNPs with cells.

Arguably, one of the most exciting new technologies for drug discovery in recent years has been that of CRISPR. The ability to efficiently silence or activate gene expression at genome scale using CRISPR/Cas vectors coupled with a lower false-positive rate than previous gene editing approaches has the potential to revolutionise the search for novel drug targets. Here we describe how this technology is transforming target identification, and what the future holds for the integration of genome editing into phenotypic drug discovery. We will guide readers through the process of applying CRISPR technology to phenotypic drug discovery, from project start to completion, describing key considerations and technological advances to maximise success.

CRISPR/Cas9 is increasingly being used as a tool to prosecute functional genomic screens. However, it is not yet possible to apply the approach at scale across a full breadth of cell types and endpoints. In order to address this, we developed a novel and robust workflow for array-based lentiviral CRISPR/Cas9 screening. We utilized a β-lactamase reporter gene assay to investigate mediators of TNF-α-mediated NF-κB signaling. The system was adapted for CRISPR/Cas9 through the development of a cell line stably expressing Cas9 and application of a lentiviral gRNA library comprising mixtures of four gRNAs per gene. We screened a 743-gene kinome library whereupon hits were independently ranked by percent inhibition, Z′ score, strictly standardized mean difference, and T statistic. A consolidated and optimized ranking was generated using Borda-based methods. Screening data quality was above acceptable limits (Z′ ≥ 0.5). In order to determine the contribution of individual gRNAs and to better understand false positives and negatives, a subset of gRNAs, against 152 genes, were profiled in singlicate format. We highlight the use of known reference genes and high-throughput, next-generation amplicon and RNA sequencing to assess screen data quality. Screening with singlicate gRNAs was more successful than screening with mixtures at identifying genes with known regulatory roles in TNF-α-mediated NF-κB signaling and was found to be superior to previous RNAi-based methods. These results add to the available data on TNF-α-mediated NF-κB signaling and establish a high-throughput functional genomic screening approach, utilizing a vector-based arrayed gRNA library, applicable across a wide variety of endpoints and cell types at a genome-wide scale.

The PI3K-AKT-mTOR pathway is frequently hyper-activated in breast cancer and several inhibitors targeting the PI3K pathway, including AKT (Capivasertib; AZD5363) and PI3Kβ/δ (AZD8186), are currently in clinical development (phase I/II) targeting tumours with mutational activation of the pathway including those with loss of PTEN. A better understanding of the mechanisms of resistance/sensitization to Capivasertib and AZD8186 in PTEN-null breast cancer is critical to fully exploit the anti-tumour activity of these compounds and to develop combination strategies for more effective therapy. Here we performed genome-wide CRISPR-Cas9 knockout screens to identify genes that when targeted promote resistance (gRNA enrichment) or sensitivity (gRNA depletion) to Capivasertib and AZD8186 in three PTEN-null breast cancer cell lines. Our screens identified five genes (NPRL2, DEPDC5, DDIT4, HNRNPD and ZC3H4) whose inactivation promoted resistance to Capivasertib; two resistance genes (PIK3R2 and INPPL1) for AZD8186 and three resistance genes (TSC2, TSC1 and FIBP) for both compounds across all three cell lines. While this result strongly suggests that mTOR pathway reactivation is the major resistance mechanism, we also identified novel resistance genes such as FIBP. Our screens also identified a number of genes that when inactivated sensitize cells to Capivasertib and AZD8186 treatment. Eighteen of these sensitizers have inhibitors in clinical development and we have performed a combination screen to identify compounds that give the best synergy with Capivasertib/AZD8186. The most striking combination effect was detected with the Mcl-1 inhibitor, AZD5991. Mechanistic work is ongoing and our data suggest that Capivasertib/AZD8186 prime cells for apoptosis by a previously undescribed mechanism and combined inhibition with AZD5991 drives a rapid apoptotic response. Importantly, the combination with AZD5991 remains effective in breast cancer cells with acquired resistance to Capivasertib/AZD8186. These combinations showed synergistic tumour suppressive effect in a PTEN-null TNBC breast cancer xenograft model (AZD5991 + AZD8186/Capivasertib) and activity in a patient-derived xenograft model (AZD5991 + Capivasertib). Overall, our CRISPR screening data provide new insights into the genes and pathways that drive resistance to inhibitors of the PI3K pathway in PTEN-null breast cancer and have identified novel synergistic combinations to maximize therapeutic response. Citation Format: Shanade Dunn, Jason Yu, Albert Gris-Oliver, James Pilling, Philip Hopcroft, Urs Yelland, Natalie Cureton, Anna Staniszewsla, Yan Zi Au, Swee Hoe Ong, Beverley Isherwood, Violeta Serra, Simon Barry, Barry R. Davies, James T. Lynch, Kosuke Yusa. Genome-wide CRISPR screens identify combination strategies for Capivasertib (AZD5363; AKT) and AZD8186 (PI3Kβ/δ) in PTEN-null breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 321.

During aging, there is a decreased ability to maintain skeletal muscle mass and function (sarcopenia). Such changes in skeletal muscle are also co-morbidities of diseases including cancer, congestive heart failure and chronic obstructive pulmonary disease. The loss of muscle mass results in decreased strength and exercise tolerance and reduced ability to perform daily activities. Pharmacological agents addressing these pathologies could have significant clinical impact, but their identification requires understanding of mechanisms driving myotube formation (myogenesis) and atrophy and provision of relevant assays. The aim of this study was to develop robust in vitro methods to study human myogenesis.

Phenotypic screening seeks to identify substances that modulate phenotypes in a desired manner with the aim of progressing first-in-class agents. Successful campaigns require physiological relevance, robust screening, and an ability to deconvolute perturbed pathways. High-content analysis (HCA) is increasingly used in cell biology and offers one approach to prosecution of phenotypic screens, but challenges exist in exploitation where data generated are high volume and complex. We combine development of an organotypic model with novel HCA tools to map phenotypic responses to pharmacological perturbations. We describe implementation for angiogenesis, a process that has long been a focus for therapeutic intervention but has lacked robust models that recapitulate more completely mechanisms involved. The study used human primary endothelial cells in co-culture with stromal fibroblasts to model multiple aspects of angiogenic signaling: cell interactions, proliferation, migration, and differentiation. Multiple quantitative descriptors were derived from automated microscopy using custom-designed algorithms. Data were extracted using a bespoke informatics platform that integrates processing, statistics, and feature display into a streamlined workflow for building and interrogating fingerprints. Ninety compounds were characterized, defining mode of action by phenotype. Our approach for assessing phenotypic outcomes in complex assay models is robust and capable of supporting a range of phenotypic screens at scale.

Dynamic regulation of specific molecular processes and cellular phenotypes in live cell systems reveal unique insights into cell fate and drug pharmacology that are not gained from traditional fixed endpoint assays. Recent advances in microscopic imaging platform technology combined with the development of novel optical biosensors and sophisticated image analysis solutions have increased the scope of live cell imaging applications in drug discovery. We highlight recent literature examples where live cell imaging has uncovered novel insight into biological mechanism or drug mode-of-action. We survey distinct types of optical biosensors and associated analytical methods for monitoring molecular dynamics, in vitro and in vivo. We describe the recent expansion of live cell imaging into automated target validation and drug screening activities through the development of dedicated brightfield and fluorescence kinetic imaging platforms. We provide specific examples of how temporal profiling of phenotypic response signatures using such kinetic imaging platforms can increase the value of in vitro high-content screening. Finally, we offer a prospective view of how further application and development of live cell imaging technology and reagents can accelerate preclinical lead optimization cycles and enhance the in vitro to in vivo translation of drug candidates.

We report an automated optically sectioning fluorescence lifetime imaging (FLIM) multiwell plate reader for high content analysis (HCA) in drug discovery and accelerated research in cell biology. The system utilizes a Nipkow disc confocal microscope and performs unsupervised FLIM with autofocus, automatic setting of acquisition parameters and automated localisation of cells in the field of view. We demonstrate its applications to test dye solutions, fixed and live cells and FLIM‐FRET. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Hepatitis C virus C, E1, E2 and p7 proteins are cleaved from a viral polyprotein by host signal peptidases. Cleavage at the E2/p7 site is incomplete in genotype 1a strain H (resulting in E2, p7 and E2p7 species), although it has been reported to be more efficient in genotype 1b strain BK. Here, the proteolytic processing and transmembrane topology of genotype 1a strain H77c p7 was investigated when expressed in the context of E2p7. Partial processing was seen at the E2/p7 site in mammalian cells, the efficiency of which improved in the presence of nucleotide sequences downstream of p7. In insect cells, no processing at the E2/p7 site occurred and the uncleaved E2p7 species was incorporated into virus-like particles when expressed in the context of CE1E2p7c-myc. E2p7c-myc formed a heterodimer with E1, indicating that, like the well-characterized E1-E2 complex, the E1-E2p7 heterodimer may also play a functional role in virus replication. Comparison of the p7 signal peptide sequences of strains BK and H77c revealed 3 aa differences (positions 720, 733 and 742). Mutational analysis showed that the V720L change in the H77c sequence substantially increased processivity at the E2/p7 site. The p7 protein adopts a double membrane-spanning topology with both its N and C termini orientated luminally in the endoplasmic reticulum. The transmembrane topology of E2p7 species was examined by two independent means. In both cases, the C terminus of p7 in E2p7 was found to be cytoplasmically orientated, indicating that p7 adopts a dual transmembrane topology.

ABSTRACT The G and P genotypes of 3,601 rotavirus strains collected in the United Kingdom between 1995 and 1999 were determined (M. Iturriza-Gómara et al., J. Clin. Microbiol. 38:4394–4401, 2000). In 95.4% of the strains the most common G and P combinations, G1P[8], G2P[4], G3P[8], and G4P[8], were found. A small but significant number (2%) of isolates from the remaining strains were reassortants of the most common cocirculating strains, e.g., G1P[4] and G2P[8]. Rotavirus G9P[6] and G9P[8] strains, which constituted 2.7% of all viruses, were genetically closely related in their G components, but the P components of the G9P[8] strains were very closely related to those of cocirculating strains of the more common G types (G1, G3, and G4). In conclusion, genetic interaction by reassortment among cocirculating rotaviruses is not a rare event and contributes significantly to their overall diversity.