Hit screening with multivariate robust outlier detection.

Hit screening, which involves the identification of compounds or targets capable of modulating disease-relevant processes, is an important step in drug discovery. Some assays, such as image-based high-content screenings, produce complex multivariate readouts. To fully exploit the richness of such data, advanced analytical methods that go beyond the conventional univariate approaches should be employed.

An aptamer-mediated base editing platform for simultaneous knockin and multiple gene knockout for allogeneic CAR-T cells generation.

Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. In conventional gene editing platforms that rely on nuclease activity, such as clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells.

Highly scalable arrayed CRISPR mediated gene silencing in primary lung small airway epithelial cells.

Small airway epithelial cells (SAECs) play a central role in the pathogenesis of lung diseases and are now becoming a crucial cellular model for target identification and validation in drug discovery. However, primary cell lines such as SAECs are often difficult to transfect using traditional lipofection methods; therefore, gene editing using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is often carried out through ribonucleoprotein (RNP) electroporation. Here we have established a robust, scalable, and automated arrayed CRISPR nuclease (CRISPRn) screening workflow for SAECs which can be combined with a myriad of disease-specific endpoint assays.

Development and Characterization of a Modular CRISPR and RNA Aptamer Mediated Base Editing System.

Conventional CRISPR approaches for precision genome editing rely on the introduction of DNA double-strand breaks (DSB) and activation of homology-directed repair (HDR), which is inherently genotoxic and inefficient in somatic cells. The development of base editing (BE) systems that edit a target base without requiring generation of DSB or HDR offers an alternative. Here, we describe a novel BE system called Pin-point that recruits a DNA base-modifying enzyme through an RNA aptamer within the gRNA molecule.

A 'synthetic-sickness' screen for senescence re-engagement targets in mutant cancer backgrounds.

Senescence is a universal barrier to immortalisation and tumorigenesis. As such, interest in the use of senescence-induction in a therapeutic context has been gaining momentum in the past few years; however, senescence and immortalisation remain underserved areas for drug discovery owing to a lack of robust senescence inducing agents and an incomplete understanding of the signalling events underlying this complex process. In order to address this issue we undertook a large-scale morphological siRNA screen for inducers of senescence phenotypes in the human melanoma cell line A375P.

Pre-clinical use of isogenic cell lines and tumours in vitro and in vivo for predictive biomarker discovery; impact of KRAS and PI3KCA mutation status on MEK inhibitor activity is model dependent.

Studies to identify predictive biomarkers can be carried out in isogenic cancer cell lines, which enable interrogation of the effect of a specific mutation. We assessed the effects of four drugs, the PI3K-mammalian target of rapamycin inhibitor dactolisib, the PI3K inhibitor pictrelisib, and the MEK (MAPK/ERK Kinase) inhibitors PD 0325901 and selumetinib, in isogenic DLD1 parental, KRAS(+/-), KRAS(G13D/-), PIK3CA(+/-) and PIK3CA(E545K/-) colorectal carcinoma cell lines. Importantly, we found substantial differences in the growth of these cells and in their drug sensitivity depending on whether they were studied under 2D (standard tissue culture on plastic) or 3D (in vitro soft agar and in vivo xenograft) conditions.

BIM(EL), an intrinsically disordered protein, is degraded by 20S proteasomes in the absence of poly-ubiquitylation.

BIM-extra long (BIM(EL)), a pro-apoptotic BH3-only protein and part of the BCL-2 family, is degraded by the proteasome following activation of the ERK1/2 signalling pathway. Although studies have demonstrated poly-ubiquitylation of BIM(EL) in cells, the nature of the ubiquitin chain linkage has not been defined. Using ubiquitin-binding domains (UBDs) specific for defined ubiquitin chain linkages, we show that BIM(EL) undergoes K48-linked poly-ubiquitylation at either of two lysine residues.

Refining the minimal sequence required for ERK1/2-dependent poly-ubiquitination and proteasome-dependent turnover of BIM.

The pro-apoptotic protein BIM(EL) is phosphorylated by ERK1/2 and this targets the protein for poly-ubiquitination and degradation by the proteasome as a survival mechanism. To define in greater detail the sequence determinants required for BIM(EL) turnover we have compared various BIM splice variants and truncation mutants. Of the naturally occurring splice variants BIMbeta1, which lacks the C-terminal hydrophobic domain, the BH3 domain and is cytosolic, exhibited the fastest turnover rate.

Apoptosis and autophagy: BIM as a mediator of tumour cell death in response to oncogene-targeted therapeutics.

The BCL-2 homology domain 3 (BH3)-only protein, B-cell lymphoma 2 interacting mediator of cell death (BIM) is a potent pro-apoptotic protein belonging to the B-cell lymphoma 2 protein family. In recent years, advances in basic biology have provided a clearer picture of how BIM kills cells and how BIM expression and activity are repressed by growth factor signalling pathways, especially the extracellular signal-regulated kinase 1/2 and protein kinase B pathways. In tumour cells these oncogene-regulated pathways are used to counter the effects of BIM, thereby promoting tumour cell survival.

c-Cbl is not required for ERK1/2-dependent degradation of BimEL.

Bim(EL) the most abundant Bim splice variant, is subject to ERK1/2-catalysed phosphorylation, which targets it for ubiquitination and proteasome-dependent destruction. In contrast, inactivation of ERK1/2, following withdrawal of survival factors, promotes stabilization of Bim(EL). It has been proposed that the RING finger protein Cbl binds to Bim(EL) and serves as its E3 ubiquitin ligase.

Bim and the pro-survival Bcl-2 proteins: opposites attract, ERK repels.

Bim (Bcl-2-interacting mediator of cell death) is a BH3-only protein (BOP), a pro-apoptotic member of the Bcl-2 protein family. The Bim mRNA undergoes alternate splicing to give rise to the short, long and extra long protein variants (Bim(S), Bim(L) and Bim(EL)). These proteins have distinct potency in promoting death and distinct modes of regulation conferred by their interaction with other proteins.

ERK1/2-dependent phosphorylation of BimEL promotes its rapid dissociation from Mcl-1 and Bcl-xL.

The proapoptotic protein Bim is expressed de novo following withdrawal of serum survival factors. Here, we show that Bim-/- fibroblasts and epithelial cells exhibit reduced cell death following serum withdrawal in comparison with their wild-type counterparts. In viable cells, Bax associates with Bcl-2, Bcl-x(L) and Mcl-1.