A semi-automated pipeline for quantifying drusen-like deposits in human induced pluripotent stem cell-derived retinal pigment epithelium cells.

Age-Related Macular Degeneration (AMD) is a highly prevalent form of retinal disease amongst Western communities over 50 years of age. A hallmark of AMD pathogenesis is the accumulation of drusen underneath the retinal pigment epithelium (RPE), a biological process also observable in vitro. The accumulation of drusen has been shown to predict the progression to advanced AMD, making accurate characterisation of drusen in vitro models valuable in disease modelling and drug development. More recently, deposits above the RPE in the subretinal space, called reticular pseudodrusen (RPD) have been recognized as a sub-phenotype of AMD. While in vitro imaging techniques allow for the immunostaining of drusen-like deposits, quantification of these deposits often requires slow, low throughput manual counting of images. This further lends itself to issues including sampling biases, while ignoring critical data parameters including volume and precise localization. To overcome these issues, we developed a semi-automated pipeline for quantifying the presence of drusen-like deposits in vitro, using RPE cultures derived from patient-specific induced pluripotent stem cells (iPSCs). Using high-throughput confocal microscopy, together with three-dimensional reconstruction, we developed an imaging and analysis pipeline that quantifies the number of drusen-like deposits, and accurately and reproducibly provides the location and composition of these deposits. Extending its utility, this pipeline can determine whether the drusen-like deposits locate to the apical or basal surface of RPE cells. Here, we validate the utility of this pipeline in the quantification of drusen-like deposits in six iPSCs lines derived from patients with AMD, following their differentiation into RPE cells. This pipeline provides a valuable tool for the in vitro modelling of AMD and other retinal disease, and is amenable to mid and high throughput screenings.

Loss of Integrin-Linked Kinase Sensitizes Breast Cancer to SRC Inhibitors.

SRC is a nonreceptor tyrosine kinase with key roles in breast cancer development and progression. Despite this, SRC tyrosine kinase inhibitors have so far failed to live up to their promise in clinical trials, with poor overall response rates. We aimed to identify possible synergistic gene-drug interactions to discover new rational combination therapies for SRC inhibitors. An unbiased genome-wide CRISPR-Cas9 knockout screen in a model of triple-negative breast cancer revealed that loss of integrin-linked kinase (ILK) and its binding partners α-Parvin and PINCH-1 sensitizes cells to bosutinib, a clinically approved SRC/ABL kinase inhibitor. Sensitivity to bosutinib did not correlate with ABL dependency; instead, bosutinib likely induces these effects by acting as a SRC tyrosine kinase inhibitor. Furthermore, in vitro and in vivo models showed that loss of ILK enhanced sensitivity to eCF506, a novel and highly selective inhibitor of SRC with a unique mode of action. Whole-genome RNA sequencing following bosutinib treatment in ILK knockout cells identified broad changes in the expression of genes regulating cell adhesion and cell-extracellular matrix. Increased sensitivity to SRC inhibition in ILK knockout cells was associated with defective adhesion, resulting in reduced cell number as well as increased G1 arrest and apoptosis. These findings support the potential of ILK loss as an exploitable therapeutic vulnerability in breast cancer, enhancing the effectiveness of clinical SRC inhibitors. SIGNIFICANCE: A CRISPR-Cas9 screen reveals that loss of integrin-linked kinase synergizes with SRC inhibition, providing a new opportunity for enhancing the clinical effectiveness of SRC inhibitors in breast cancer.

Discovery of AL-GDa62 as a Potential Synthetic Lethal Lead for the Treatment of Gastric Cancer.

Diffuse gastric cancer and lobular breast cancer are aggressive malignancies that are frequently associated with inactivating mutations in the tumor suppressor gene CDH1. Synthetic lethal (SL) vulnerabilities arising from CDH1 dysfunction represent attractive targets for drug development. Recently, SLEC-11 (1) emerged as a SL lead in E-cadherin-deficient cells. Here, we describe our efforts to optimize 1. Overall, 63 analogues were synthesized and tested for their SL activity toward isogenic mammary epithelial CDH1-deficient cells (MCF10A-CDH1-/-). Among the 26 compounds with greater cytotoxicity, AL-GDa62 (3) was four-times more potent and more selective than 1 with an EC50 ratio of 1.6. Furthermore, 3 preferentially induced apoptosis in CDH1-/- cells, and Cdh1-/- mammary and gastric organoids were significantly more sensitive to 3 at low micromolar concentrations. Thermal proteome profiling of treated MCF10A-CDH1-/- cell protein lysates revealed that 3 specifically inhibits TCOF1, ARPC5, and UBC9. In vitro, 3 inhibited SUMOylation at low micromolar concentrations.

A Conformation Selective Mode of Inhibiting SRC Improves Drug Efficacy and Tolerability.

Despite the approval of several multikinase inhibitors that target SRC and the overwhelming evidence of the role of SRC in the progression and resistance mechanisms of many solid malignancies, inhibition of its kinase activity has thus far failed to improve patient outcomes. Here we report the small molecule eCF506 locks SRC in its native inactive conformation, thereby inhibiting both enzymatic and scaffolding functions that prevent phosphorylation and complex formation with its partner FAK. This mechanism of action resulted in highly potent and selective pathway inhibition in culture and in vivo. Treatment with eCF506 resulted in increased antitumor efficacy and tolerability in syngeneic murine cancer models, demonstrating significant therapeutic advantages over existing SRC/ABL inhibitors. Therefore, this mode of inhibiting SRC could lead to improved treatment of SRC-associated disorders. SIGNIFICANCE: Small molecule-mediated inhibition of SRC impairing both catalytic and scaffolding functions confers increased anticancer properties and tolerability compared with other SRC/ABL inhibitors.

A high-throughput screen to identify novel synthetic lethal compounds for the treatment of E-cadherin-deficient cells.

The cell-cell adhesion protein E-cadherin (CDH1) is a tumor suppressor that is required to maintain cell adhesion, cell polarity and cell survival signalling. Somatic mutations in CDH1 are common in diffuse gastric cancer (DGC) and lobular breast cancer (LBC). In addition, germline mutations in CDH1 predispose to the autosomal dominant cancer syndrome Hereditary Diffuse Gastric Cancer (HDGC). One approach to target cells with mutations in specific tumor suppressor genes is synthetic lethality. To identify novel synthetic lethal compounds for the treatment of cancers associated with E-cadherin loss, we have undertaken a high-throughput screening campaign of ~114,000 lead-like compounds on an isogenic pair of human mammary epithelial cell lines - with and without CDH1 expression. This unbiased approach identified 12 novel compounds that preferentially harmed E-cadherin-deficient cells. Validation of these compounds using both real-time and end-point viability assays identified two novel compounds with significant synthetic lethal activity, thereby demonstrating that E-cadherin loss creates druggable vulnerabilities within tumor cells. In summary, we have identified novel synthetic lethal compounds that may provide a new strategy for the prevention and treatment of both sporadic and hereditary LBC and DGC.

E-cadherin-deficient cells have synthetic lethal vulnerabilities in plasma membrane organisation, dynamics and function.

BACKGROUND: The E-cadherin gene (CDH1) is frequently mutated in diffuse gastric cancer and lobular breast cancer, and germline mutations predispose to the cancer syndrome Hereditary Diffuse Gastric Cancer. We are taking a synthetic lethal approach to identify druggable vulnerabilities in CDH1-mutant cancers. METHODS: Density distributions of cell viability data from a genome-wide RNAi screen of isogenic MCF10A and MCF10A-CDH1-/- cells were used to identify protein classes affected by CDH1 mutation. The synthetic lethal relationship between selected protein classes and E-cadherin was characterised by drug sensitivity assays in both the isogenic breast MCF10A cells and CDH1-isogenic gastric NCI-N87. Endocytosis efficiency was quantified using cholera toxin B uptake. Pathway metagene expression of 415 TCGA gastric tumours was statistically correlated with CDH1 expression. RESULTS: MCF10A-CDH1-/- cells showed significantly altered sensitivity to RNAi inhibition of groups of genes including the PI3K/AKT pathway, GPCRs, ion channels, proteosomal subunit proteins and ubiquitinylation enzymes. Both MCF10A-CDH1-/- and NCI-N87-CDH1-/- cells were more sensitive than wild-type cells to compounds that disrupted plasma membrane composition and trafficking, but showed contrasting sensitivities to inhibitors of actin polymerisation and the chloride channel inhibitor NS3728. The MCF10A-CDH1-/- cell lines showed reduced capacity to endocytose cholera toxin B. Pathway metagene analysis identified 20 Reactome pathways that were potentially synthetic lethal in tumours. Genes involved in GPCR signalling, vesicle transport and the metabolism of PI3K and membrane lipids were strongly represented amongst the candidate synthetic lethal genes. CONCLUSIONS: E-cadherin loss leads to disturbances in receptor signalling and plasma membrane trafficking and organisation, creating druggable vulnerabilities.

A Comparison of Real-Time and Endpoint Cell Viability Assays for Improved Synthetic Lethal Drug Validation.

Cell viability assays fulfill a central role in drug discovery studies. It is therefore important to understand the advantages and disadvantages of the wide variety of available assay methodologies. In this study, we compared the performance of three endpoint assays (resazurin reduction, CellTiter-Glo, and nuclei enumeration) and two real-time systems (IncuCyte and xCELLigence). Of the endpoint approaches, both the resazurin reduction and CellTiter-Glo assays showed higher cell viabilities when compared directly to stained nuclei counts. The IncuCyte and xCELLigence real-time systems were comparable, and both were particularly effective at tracking the effects of drug treatment on cell proliferation at sub-confluent growth. However, the real-time systems failed to evaluate contrasting cell densities between drug-treated and control-treated cells at full growth confluency. Here, we showed that using real-time systems in combination with endpoint assays alleviates the disadvantages posed by each approach alone, providing a more effective means to evaluate drug toxicity in monolayer cell cultures. Such approaches were shown to be effective in elucidating the toxicity of synthetic lethal drugs in an isogenic pair of MCF10A breast cell lines.

Synthetic Lethal Screens Identify Vulnerabilities in GPCR Signaling and Cytoskeletal Organization in E-Cadherin-Deficient Cells.

The CDH1 gene, which encodes the cell-to-cell adhesion protein E-cadherin, is frequently mutated in lobular breast cancer (LBC) and diffuse gastric cancer (DGC). However, because E-cadherin is a tumor suppressor protein and lost from the cancer cell, it is not a conventional drug target. To overcome this, we have taken a synthetic lethal approach to determine whether the loss of E-cadherin creates druggable vulnerabilities. We first conducted a genome-wide siRNA screen of isogenic MCF10A cells with and without CDH1 expression. Gene ontology analysis demonstrated that G-protein-coupled receptor (GPCR) signaling proteins were highly enriched among the synthetic lethal candidates. Diverse families of cytoskeletal proteins were also frequently represented. These broad classes of E-cadherin synthetic lethal hits were validated using both lentiviral-mediated shRNA knockdown and specific antagonists, including the JAK inhibitor LY2784544, Pertussis toxin, and the aurora kinase inhibitors alisertib and danusertib. Next, we conducted a 4,057 known drug screen and time course studies on the CDH1 isogenic MCF10A cell lines and identified additional drug classes with linkages to GPCR signaling and cytoskeletal function that showed evidence of E-cadherin synthetic lethality. These included multiple histone deacetylase inhibitors, including vorinostat and entinostat, PI3K inhibitors, and the tyrosine kinase inhibitors crizotinib and saracatinib. Together, these results demonstrate that E-cadherin loss creates druggable vulnerabilities that have the potential to improve the management of both sporadic and familial LBC and DGC.

E-cadherin loss alters cytoskeletal organization and adhesion in non-malignant breast cells but is insufficient to induce an epithelial-mesenchymal transition.

BACKGROUND: E-cadherin is an adherens junction protein that forms homophilic intercellular contacts in epithelial cells while also interacting with the intracellular cytoskeletal networks. It has roles including establishment and maintenance of cell polarity, differentiation, migration and signalling in cell proliferation pathways. Its downregulation is commonly observed in epithelial tumours and is a hallmark of the epithelial to mesenchymal transition (EMT). METHODS: To improve our understanding of how E-cadherin loss contributes to tumorigenicity, we investigated the impact of its elimination from the non-tumorigenic breast cell line MCF10A. We performed cell-based assays and whole genome RNAseq to characterize an isogenic MCF10A cell line that is devoid of CDH1 expression due to an engineered homozygous 4 bp deletion in CDH1 exon 11. RESULTS: The E-cadherin-deficient line, MCF10A CDH1-/- showed subtle morphological changes, weaker cell-substrate adhesion, delayed migration, but retained cell-cell contact, contact growth inhibition and anchorage-dependent growth. Within the cytoskeleton, the apical microtubule network in the CDH1-deficient cells lacked the radial pattern of organization present in the MCF10A cells and F-actin formed thicker, more numerous stress fibres in the basal part of the cell. Whole genome RNAseq identified compensatory changes in the genes involved in cell-cell adhesion while genes involved in cell-substrate adhesion, notably ITGA1, COL8A1, COL4A2 and COL12A1, were significantly downregulated. Key EMT markers including CDH2, FN1, VIM and VTN were not upregulated although increased expression of proteolytic matrix metalloprotease and kallikrein genes was observed. CONCLUSIONS: Overall, our results demonstrated that E-cadherin loss alone was insufficient to induce an EMT or enhance transforming potential in the non-tumorigenic MCF10A cells but was associated with broad transcriptional changes associated with tissue remodelling.