An Isogenic Cell Line Panel for Sequence-based Screening of Targeted Anti-cancer Drugs

We describe the creation and characterization of an isogenic cell line panel representing common cancer pathways, with multiple features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cells were generated using CRISPR-technologies. Surprisingly, we discovered most of these lines did not result in complete gene inactivation, despite integration of sgRNA at the desired genomic site. However, a subset of the lines harbored true, biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterize lines covering 19 pathways frequently subject to loss of function in cancers. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a multiplexed high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation of TP53. These cell lines and screening approach should prove useful for researchers studying a variety of cellular and biochemical phenomena.

Human Cell Line Panel With Human/Mouse Artificial Chromosomes for Functional Analyses of Desired Genes

Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) are non-integrating chromosomal gene delivery vectors for molecular biology research. Recently, microcell-mediated chromosome transfer of HACs/MACs has been achieved into various human cells including human immortalised mesenchymal stem cells (hiMSCs) and human induced pluripotent stem cells (hiPSCs). However, the conventional strategy of gene-introduction with HAC/MAC required laborious and time-consuming stepwise isolation of clones for gene loading into HACs/MACs in donor cell lines (CHO and A9) and then transferring the HAC/MAC into cells via microcell-mediated chromosome transfer (MMCT). To overcome these limitations and accelerate chromosome vector based functional assay in human cells, we established various human cell lines (HEK293, HT1080, hiMSCs, and hiPSCs) with HACs/MACs that harbour a gene-loading site via MMCT. Model genes, such as tdTomato, TagBFP2, and ELuc, were introduced into the premade HAC/MAC-introduced cell lines via the Cre-loxP system or simultaneous insertion of multiple gene-loading vectors (SIM system). The model genes on the HACs/MACs were stably expressed and the HACs/MACs were stably maintained in the cell lines. Thus, our strategy using the HAC/MAC-containing cell line panel has dramatically simplified and accelerated gene introduction via HACs/MACs, thereby facilitating functional analyses of introduced genes.

A human isogenic iPSC-derived cell line panel identifies major regulators of aberrant astrocyte proliferation in Down syndrome

Astrocytes exert adverse effects on the brains of individuals with Down syndrome (DS). Although a neurogenic-to-gliogenic shift in the fate-specification step has been reported, the mechanisms and key regulators underlying the accelerated proliferation of astrocyte precursor cells (APCs) in DS remain elusive. Here, we established a human isogenic cell line panel based on DS-specific induced pluripotent stem cells, the XIST-mediated transcriptional silencing system in trisomic chromosome 21, and genome/chromosome-editing technologies to eliminate phenotypic fluctuations caused by genetic variation. The transcriptional responses of genes observed upon XIST induction and/or downregulation are not uniform, and only a small subset of genes show a characteristic expression pattern, which is consistent with the proliferative phenotypes of DS APCs. Comparative analysis and experimental verification using gene modification reveal dose-dependent proliferation-promoting activity of DYRK1A and PIGP on DS APCs. Our collection of human isogenic cell lines provides a comprehensive set of cellular models for further DS investigations.

Design and synthesis of new pyrazolylbenzimidazoles as sphingosine 1-kinase inhibitors

Sphingosine-1 Kinase (SphK-1) is one of the important enzymes of phospholipids and it is inhibition is one of the therapeutic strategies for different diseases. SphK1 over expression is observed in different types of cancer that indicating its important role in tumor growth. In search of effective SphK-1 inhibitors, a new series of pyrazolylbenzimidazoles was synthesized and evaluated as sphingosine1-kinase (Sphk-1) inhibitors. In order to evaluate the binding affinities of all the synthesized compounds, all compounds were subjected to docking analysis and fluorescence quenching. The results indicated that there is a consistency between the docking and the fluorescence quenching results which revealed that compounds 47 and 48 exhibited significant decrease in the fluorescence intensity of SphK1 as well as they formed stable protein–ligand complexes. In addition, Enzyme inhibition assay was performed and showed effective inhibitory potential towards SphK-1. Moreover, IC50 values was calculated and displayed that compounds 47 and 48 were the most promising compounds. In addition, antiproliferation study for all the synthesized compounds was performed against NCI 60-cell line panel. The target compounds 47 and 48 demonstrated effective antitumor activity inhibitory potential to the SphK-1. Most of these compounds fit well into the ATP-binding site of SphK1 and form significant hydrogen-bonding interactions with catalytically relevant residues as predicted by molecular docking. In this article, insight has been given for the importance of pyrazolylbenzimidazoles as Sphk1 inhibitors and the perspectives that they hold for future research.

Data variability in standardised cell culture experiments

Despite much debate about a perceived ‘reproducibility crisis’ in the life sciences, it remains unclear what level of replicability is technically possible [1,2]. Here, we analysed the variation among drug response data of the NCI60 project, which for decades has tested anti-cancer agents in a 60-cell line panel following a standardised protocol [3]. In total, 2.8 million compound/cell line experiments are available in the NCI60 resource CellMiner [4]. The largest fold change between the lowest and highest GI50 (concentration that reduces cell viability by 50%) in a compound/cell line combination was 3.16 x 1010. All compound/cell line combinations with >100 experiments displayed maximum GI50 fold changes >5, 99.7% maximum fold changes >10, 87.3% maximum fold changes >100, and 70.5% maximum fold changes >1000. FDA-approved drugs and experimental agents displayed similar variation. The variability remained very high after removal of outliers and among experiments performed in the same month. Hence, our analysis shows that high variability is an intrinsic feature of experimentation in biological systems, even among highly standardised experiments in a world-leading research environment. Thus, a narrow focus on experiment standardisation does not ensure a high level of replicability on its own.

BAX and SMAC regulate bistable properties of the apoptotic caspase system

The initiation of apoptosis is a core mechanism in cellular biology by which organisms control the removal of damaged or unnecessary cells. The irreversible activation of caspases is essential for apoptosis, and mathematical models have demonstrated that the process is tightly regulated by positive feedback and a bistable switch. BAX and SMAC are often dysregulated in diseases such as cancer or neurodegeneration and are two key regulators that interact with the caspase system generating the apoptotic switch. Here we present a mathematical model of how BAX and SMAC control the apoptotic switch. Formulated as a system of ordinary differential equations, the model summarises experimental and computational evidence from the literature and incorporates the biochemical mechanisms of how BAX and SMAC interact with the components of the caspase system. Using simulations and bifurcation analysis, we find that both BAX and SMAC regulate the time-delay and activation threshold of the apoptotic switch. Interestingly, the model predicted that BAX (not SMAC) controls the amplitude of the apoptotic switch. Cell culture experiments using siRNA mediated BAX and SMAC knockdowns validated this model prediction. We further validated the model using data of the NCI-60 cell line panel using BAX protein expression as a cell-line specific parameter and show that model simulations correlated with the cellular response to DNA damaging drugs and established a defined threshold for caspase activation that could distinguish between sensitive and resistant melanoma cells. In summary, we present an experimentally validated dynamic model that summarises our current knowledge of how BAX and SMAC regulate the bistable properties of irreversible caspase activation during apoptosis.