The NanoBiT Biochemical Assay (NBBA) was designed as a biochemical format of the NanoBiT cellular assay, aiming to screen weak protein-protein interactions (PPIs) in mammalian cell lysates. Here we present a High Throughput Screening (HTS) application of the NBBA to screen small molecule and fragment libraries to identify compounds that block the interaction of KRAS-G12D with phosphatidylinositol 3-kinase (PI3K) p110α. This interaction promotes PI3K activity, resulting in the promotion of cell growth, proliferation and survival, and is required for tumour initiation and growth in mouse lung cancer models, whilst having little effect on the health of normal adult mice, establishing the significance of the p110α/KRAS interaction as an oncology drug target. Despite the weak binding affinity of the p110α/KRAS interaction (KD = 3 μM), the NBBA proved to be robust and displayed excellent Z’-factor statistics during the HTS primary screening of 726,000 compounds, which led to the identification of 8,000 active compounds. A concentration response screen comparing KRAS/p110α with two closely related PI3K isoforms, p110δ and p110γ, identified selective p110α-specific compounds and enabled derivation of an IC50 for these hits. We identified around 30 compounds showing greater than 20-fold selectivity towards p110α versus p110δ and p110γ with IC50 < 2 μM. By using Differential Scanning Fluorimetry (DSF) we confirmed several compounds that bind directly to purified p110α. The most potent hits will be followed up by co-crystallization with p110α to aid further elucidation of the nature of the interaction and extended optimisation of these compounds.

<p>CKAP2L regulates cell migration and its expression correlates with tumour progression of human LuAd</p>

Excel spreadsheet of NUAK1 inhibitor induced phospho-peptide alterations

Merged supplementary Figures & Figure legends and Tables & Table legends

Abstract RAS proteins contribute to the activation of p110α by directly interacting with its RAS binding domain (RBD), resulting in the promotion of cellular functions such as cell growth, proliferation and survival. Previously, we have shown that blocking the interaction of p110α with oncogenic RAS, by introducing specific mutations in the RBD, had a significant impact on tumor initiation and growth in mouse models, whilst having little effect on the health of normal adult mice. These studies highlight the significance of the p110α/KRAS protein-protein interaction (PPI) in tumor progression and maintenance and strongly suggest its importance as a drug target. Thus, we initiated a drug discovery project aiming to identify molecules that bind to p110α and perturb its interaction with KRAS. However, the rather weak binding affinity of the p110α/KRAS interaction (Kd = 3 μM) and the poor solubility of p110α raised significant issues when considering the use of currently available biochemical assays. The NanoLuc® Binary Technology (NanoBiT®) assay was originally developed to detect PPIs in live mammalian cells. However, the assay in its current state was not ideal for high throughput screening (HTS) due to the requirement for large-scale cell based assays. Therefore, we developed the NanoBiT Biochemical Assay (NBBA) as a more suitable and cost-effective approach. The NBBA centres on expressing modified target proteins of interest in mammalian cells and then screening cell lysates rather than live cells. We have previously shown that the NBBA is suitable for detection of both strong and weak PPIs, as exemplified by KRAS/RAF and KRAS/PI3K. Here we present the first application of NBBA for HTS of the KRAS/p110α PPI, using tagged Sm-KRAS-G12D and Lg-p110α. The NBBA proved to be stable and produced good Z’ factor metrics. Our primary screen of more than 720,000 compounds on KRAS/p110α cell lysates produced around 8,000 initial hits. A concentration response screen comparing KRAS/p110α with two other p110 isoforms, p110δ and p110γ, identified selective p110α-specific compounds and enabled derivation of IC50s for these hits. We identified around 30 compounds showing greater than 20-fold selectivity towards p110α versus p110δ and p110γ with IC50 <2μM. Using 1D-NMR (1 – Dimension Nuclear Magnetic Resonance) we identified 9 compounds that binds directly to purified p110α. The positive and most potent hits will be followed up by co-crystallization with p110α to aid further elucidation of the nature of the interaction and extended optimisation of these compounds. Citation Format: Mohamed (Soly) Soliman Ismail, Julian Downward, Jonathan Tart, Gareth Davies, Tiziana Monteverde, David Hancock, Santosh Adhikari, Christopher Stubbs, Carolyn Blackett, Geoff Holdgate, Jason Kettle. High throughput application of the NanoBiT Biochemical Assay for the discovery of selective p110α isoform binders that block its interaction with KRAS [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C038.

Hypothesis: Asbestos-driven inflammation contributes to malignant pleural mesothelioma beyond the acquisition of rate-limiting mutations. Methods: Genetically modified conditional allelic mice that were previously shown to develop mesothelioma in the absence of exposure to asbestos were induced with lentiviral vector expressing Cre recombinase with and without intrapleural injection of amosite asbestos and monitored until symptoms required euthanasia. Resulting tumours were examined histologically and by immunohistochemistry for expression of lineage markers and immune cell infiltration. Results: Injection of asbestos dramatically accelerated disease onset and end-stage tumour burden. Tumours developed in the presence of asbestos showed increased macrophage infiltration. Pharmacological suppression of macrophages in mice with established tumours failed to extend survival or to enhance response to chemotherapy. Conclusion: Asbestos-driven inflammation contributes to the severity of mesothelioma beyond the acquisition of rate-limiting mutations, however, targeted suppression of macrophages in established epithelioid mesothelioma showed no therapeutic benefit.

<p>Characterization of CKAP2L role in cell proliferation</p>

In image-based profiling, software extracts thousands of morphological features of cells from multi-channel fluorescence microscopy images, yielding single-cell profiles that can be used for basic research and drug discovery. Powerful applications have been proven, including clustering chemical and genetic perturbations on the basis of their similar morphological impact, identifying disease phenotypes by observing differences in profiles between healthy and diseased cells and predicting assay outcomes by using machine learning, among many others. Here, we provide an updated protocol for the most popular assay for image-based profiling, Cell Painting. Introduced in 2013, it uses six stains imaged in five channels and labels eight diverse components of the cell: DNA, cytoplasmic RNA, nucleoli, actin, Golgi apparatus, plasma membrane, endoplasmic reticulum and mitochondria. The original protocol was updated in 2016 on the basis of several years’ experience running it at two sites, after optimizing it by visual stain quality. Here, we describe the work of the Joint Undertaking for Morphological Profiling Cell Painting Consortium, to improve upon the assay via quantitative optimization by measuring the assay’s ability to detect morphological phenotypes and group similar perturbations together. The assay gives very robust outputs despite various changes to the protocol, and two vendors’ dyes work equivalently well. We present Cell Painting version 3, in which some steps are simplified and several stain concentrations can be reduced, saving costs. Cell culture and image acquisition take 1–2 weeks for typically sized batches of ≤20 plates; feature extraction and data analysis take an additional 1–2 weeks. This protocol is an update to Nat. Protoc. 11, 1757–1774 (2016): https://doi.org/10.1038/nprot.2016.105 We provide an updated protocol for image-based profiling with Cell Painting. A detailed procedure, with standardized conditions for the assay, is presented, along with a comprehensive description of parameters to be considered when optimizing the assay.

6 supplementary Figures with legends under each; 3 Supplementary Tables; Supplementary Methods and References

<p>CKAP2L binding partners were identified by LC-MS</p>

<p>Differentially expressed genes in CKAP2L-depleted cells</p>

<p>unprocessed original images of western blots</p>

Correlation analysis between LuAd gene expression and CIN

<p>Functional annotation analysis of genes showing RPB1 reduced binding upon CKAP2L downregulation</p>

List of all primers used in the study

Image-based profiling has emerged as a powerful technology for various steps in basic biological and pharmaceutical discovery, but the community has lacked a large, public reference set of data from chemical and genetic perturbations. Here we present data generated by the Joint Undertaking for Morphological Profiling (JUMP)-Cell Painting Consortium, a collaboration between 10 pharmaceutical companies, six supporting technology companies, and two non-profit partners. When completed, the dataset will contain images and profiles from the Cell Painting assay for over 116,750 unique compounds, over-expression of 12,602 genes, and knockout of 7,975 genes using CRISPR-Cas9, all in human osteosarcoma cells (U2OS). The dataset is estimated to be 115 TB in size and capturing 1.6 billion cells and their single-cell profiles. File quality control and upload is underway and will be completed over the coming months at the Cell Painting Gallery: https://registry.opendata.aws/cellpainting-gallery . A portal to visualize a subset of the data is available at https://phenaid.ardigen.com/jumpcpexplorer/ .

Spreadsheet of genes co-regulated by MYC & Miz1 in KMC PDAC cells

Downregulation of MYC and KRAS signalling

<p>Effect of CKAP2L depletion on RNA Pol II distribution</p>