Biology subjects

Graham, R. E.

Publications and source records attributed to Graham, R. E..

4 recordsLinked to original sources

bioRxiv2024

Development of a pipeline to evaluate YAP-TEAD inhibitor potential - direct TEAD inhibition represses cancerous growth in a NF2-mutant model of mesothelioma

As the core, tumorigenic downstream effectors of the Hippo signalling pathway, YAP/TAZ and the TEAD family of transcription factors represent attractive targets for drug discovery efforts within cancer research. This is particularly true within the context of pleural mesothelioma, in which there are many recent preclinical developments and clinical trials evaluating the efficacy of TEAD inhibitors. The range of inhibitors have shown great promise, but comparisons of their performances are so far limited. Here we develop a high content pipeline that enables a comparative analysis of currently developed YAP/TAZ-TEAD inhibitors. We take advantage of isogenic cellular models that enable us to examine inhibitor specificity. We identify genetic compensation of the Hippo pathway transcriptional module, with implications for therapeutic targeting, and implement Cell Painting to develop a detailed morphological profiling pipeline that enables further characterisation, quantification, and analysis of off-target effects. Our pipeline is scalable and allows us to establish specificity and comparative potency within cancer relevant assays in a clinically relevant cellular model.

cancer biology↗

bioRxiv2024

Compound screening in primary human airway basal cells identifies Wnt pathway activators as potential pro-regenerative therapies

Regeneration of the airway epithelium restores barrier function and mucociliary clearance following lung injury and infection. Basal cells are tissue-resident airway stem cells that enact regeneration, yet the mechanisms regulating their proliferation and differentiation remain incompletely understood. To identify compounds that promote primary human airway basal cell proliferation, we performed phenotype-based compound screening of 1,429 compounds (from the ENZO and Prestwick Chemical libraries) in 384-well format using primary cells transduced with lentiviral luciferase. 16 pro-proliferative compounds validated in independent donor cell cultures, with several hit compounds activating the Wnt signalling pathway. The effects of compounds on proliferation were further explored in concentration-response, colony formation and 3D organoid assays. Structurally and functionally-related compounds that more potently induced both Wnt activation and basal cell proliferation were investigated. One such compound, 1-azakenpaullone, induced Wnt target gene activation and basal cell proliferation in mice in the absence of tracheal injury. Our results demonstrate the pro-proliferative effect of small-molecule Wnt activators on airway basal cells. These findings contribute to the rationale to develop novel approaches to modulate Wnt signalling during airway epithelial repair. Summary statementIshii, Orr and colleagues perform a high-throughput screen of 1,429 compounds in primary human airway epithelial cells, identifying Wnt activating compounds as promoters of proliferation.

cell biology↗

bioRxiv2023

Single-cell morphological tracking of liver cell states to identify small-molecule modulators of liver differentiation

Alternative therapeutic strategies are urgently required to treat liver disease, which is responsible for 2 million deaths anually. By combining Cell Painting, a morphological profiling assay that captures diverse cellular states, with the bi-potent HepaRG(R) liver progenitor cell line, we have developed a high-throughput, single-cell technique, to track liver cell fate and map small-molecule induced changes using a morphological atlas of bi-lineage liver cell differentiation. To our knowledge this is the first-time single-cell trajectory inference has been applied to image-based Cell Painting data and leveraged for drug screening. The overarching goal of this new method is to aid research into understanding liver cell regeneration mechanisms and facilitate the development of cell-based and small-molecule therapies. Using this approach, we have identified a class of small-molecule SRC family kinase inhibitors that promote differentiation of HepaRG(R) single-cells towards the hepatocyte-like lineage and promotes differentiation of primary human hepatic progenitor cells towards a hepatocyte-like phenotype in vitro.

cell biology↗

bioRxiv2023

A cautionary note on the use of N-acetylcysteine as a reactive oxygen species antagonist to assess copper mediated cell death.

A new form of cell death has recently been proposed involving copper-induced cell death, termed cuproptosis. This new form of cell death has been widely studied in relation to a novel class of copper ionophores, including elesclomol and disulfiram. However, the exact mechanism leading to cell death remains contentious. The oldest and mostly widely accepted biological mechanism is that the accumulated intracellular copper leads to excessive build-up of reactive oxygen species and that this is what ultimately leads to cell death. Most of this evidence is largely based on studies using N-acetylcysteine (NAC), an antioxidant, to relieve the oxidative stress and prevent cell death. However, here we have demonstrated using inductively coupled mass-spectrometry, that NAC pretreatment significantly reduces intracellular copper uptake triggered by the ionophores, elesclomol and disulfiram, suggesting that reduction in copper uptake, rather than the antioxidant activity of NAC, is responsible for the diminished cell death. We present further data showing that key mediators of reactive oxygen species are not upregulated in response to elesclomol treatment, and further that sensitivity of cancer cell lines to reactive oxygen species does not correlate with sensitivity to these copper ionophores. Our findings are in line with several recent studies proposing the mechanism of cuproptosis is instead via copper mediated aggregation of proteins, resulting in proteotoxic stress leading to cell death. Overall, it is vital to disseminate this key piece of information regarding NACs activity on copper uptake since new research attributing the effect of NAC on copper ionophore activity to quenching of reactive oxygen species is being published regularly and our studies suggest their conclusions may be misleading.

biochemistry↗