Secreted proteins regulate the balance between cellular proliferation and G0 arrest and therefore play important roles in tumour dormancy. Tumour dormancy presents a significant clinical challenge for breast cancer patients, where non-proliferating, G0-arrested cancer cells remain at metastatic sites, below the level of clinical detection, some of which can re-enter proliferation and drive tumour relapse. Knowing which secreted proteins can regulate entry into and exit from G0 allows us to manipulate their signalling to prevent tumour relapse. To identify novel secreted proteins that can promote breast cancer G0 arrest, we performed a secretome-wide, image-based screen for proteins that increase the fraction of cells in G0 arrest. From a secretome library of 1282 purified proteins, we identified 29 candidates that promote G0 arrest in non-transformed and transformed breast epithelial cells. The assay we have developed can be adapted for use in other perturbation screens in other cell types. All datasets have been made available for re-analysis and our candidate proteins are presented for alternative bioinformatic refinement or further experimental follow up.

Supplementary Table 1. Mass Spectrometer and UPLC system parameters, Supplementary Table 2. Optimization parameters for mass spectrometry analysis, Supplementary Table 3. Estimated mouse PK model parameter values for AZD2811, Supplementary Table 4. Summary table of the pIC50 and nuclear area, Supplementary Table 5: PK parameters summary, Supplementary Figure 1. Representative bioluminescent images of the disseminated U2932Luc2 DLBCL xenograft model in vivo taken at day 18 post treatment initiation, Supplementary Figure 2. 96h post treatment, AZD2811 nanoparticle treatment reduces pHH3 levels with no evidence of polyploidy in the OCI-LY19 xenograft model in vivo, Supplementary Figure 3. In SCID mice AZD2811 nanoparticle is well tolerated at the different doses and schedules explored

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.

Supplemental data figure legends

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/ .

Cellular senescence is believed to be a key driver of several age-related diseases, including COPD and IPF. Therefore, senothereapeutics represent an attractive area of research with potential for future therapies against these debilitating diseases. To identify novel targets of senescence in an unbiased manner, we set up an arrayed whole genome CRISPR knock-out screen in primary human lung fibroblasts (NHLFs) assessing etoposide-induced nuclear p21 expression as a marker of senescence using confocal microscopy. The NHLF screen achieved a hit rate of 1.6%. To identify genes that also modulate senescence in a cell model relevant to COPD, we followed-up the top 250 hits from the NHLF screen with a validation screen in small airway epithelial cells (SAECs). Again, the assay used etoposide-induced nuclear p21 expression as well as modulated morphology endpoints such as nuclear- and cellular area, all of which are characteristics of senescent cells. The SAEC screen identified 38 targets that significantly reduced nuclear p21. Of those, we triaged 12 hits that were also effective in reducing cell and nuclear area. In silico pathway analysis showed involvement of these targets in exocytosis, DNA damage response, splicing, protein synthesis and growth hormone signaling; the role of these processes in driving or maintaining cellular senescence is largely unknown. In conclusion, we have successfully identified novel targets involved in senescence. These targets will be further investigated to understand their role in disease and pursued for the development of new therapeutic approaches for targeting senescence.

Supplementary Table results

The PI3K pathway is commonly activated in breast cancer, with PI3K-AKT pathway inhibitors used clinically. However, mechanisms that limit or enhance the therapeutic effects of PI3K-AKT inhibitors are poorly understood at a genome-wide level. Parallel CRISPR screens in 3 PTEN-null breast cancer cell lines identified genes mediating resistance to capivasertib (AKT inhibitor) and AZD8186 (PI3Kβ inhibitor). The dominant mechanism causing resistance is reactivated PI3K-AKT-mTOR signalling, but not other canonical signalling pathways. Deletion of TSC1/2 conferred resistance to PI3Kβi and AKTi through mTORC1. However, deletion of PIK3R2 and INPPL1 drove specific PI3Kβi resistance through AKT. Conversely deletion of PIK3CA , ERBB2 , ERBB 3 increased PI3Kβi sensitivity while modulation of RRAGC , LAMTOR1 , LAMTOR4 increased AKTi sensitivity. Significantly, we found that Mcl-1 loss enhanced response through rapid apoptosis induction with AKTi and PI3Kβi in both sensitive and drug resistant TSC1/2 null cells. The combination effect was BAK but not BAX dependent. The Mcl-1i + PI3Kβ/AKTi combination was effective across a panel of breast cancer cell lines with PIK3CA and PTEN mutations, and delivered increased anti-tumor benefit in vivo. This study demonstrates that different resistance drivers to PI3Kβi and AKTi converge to reactivate PI3K-AKT or mTOR signalling and combined inhibition of Mcl-1 and PI3K-AKT has potential as a treatment strategy for PI3Kβi/AKTi sensitive and resistant breast tumours.

Abstract Drug resistance is ultimately the cause of death for most cancer patients – even initially strong responses to treatment are usually followed by the emergence of resistance over time. This suggests the existence of residual or persistent cancer cells, creating a reservoir that ultimately gives rise to stable resistance. These drug tolerant persisters (‘the deadly survivors’) have been described for over a decade in numerous studies; they are often present as a minor fraction of the total tumour population and may exploit non-genetic (transcriptional) programs to allow the cells to survive drug treatment. 10-20% of lung adenocarcinoma patients harbour activating mutations in EGFR. Although treatment with the EGFR kinase inhibitor osimertinib has improved overall survival in such patients, almost all patients ultimately develop drug resistance. We carried out parallel genome-wide CRISPR gain and loss of function screens in EGFR mutant lung cancer cell lines treated with EGFR inhibitors, to identify the genes and pathways that may be important in enabling the survival of persister cells. We observed recurrent resistant genes in previously identified resistance pathways including PI3K (PTEN, TSC1, TSC2), MAPK (KRAS, NF1, MET), cell death (BCL2L11, BAX), the mediator complex (MED24, MED19) and ubiquitination (KCTD5, KEAP1). A secondary screen of 63 resistance genes that combined high content microscopy with CRISPR gene knockout demonstrated that 21% (13/63) of genes were associated with increased nuclear localisation of YAP1/WWTR1, key activators of the Hippo pathway. A closer review of the CRISPR screen data confirmed that many resistance hits are members of this pathway - upstream regulators (NF2, AMOTL2), core signalling genes (LATS1, LATS2), main effectors (WWTR1, YAP1), transcriptional co-effectors (TEAD3, FOSL1, VGLL4) and the SWI/SNF complex (ARID2, SMARCA4, SMARCB1, PBRM1). Hippo signalling is mediated through YAP1 and WWTR1 which bind to TEAD transcription factors and activate transcriptional programs affecting cell proliferation and apoptosis. We confirmed using CRISPR that knockout (NF2) or overexpression (YAP1, WWTR1) of key Hippo genes in the EGFR mutant lung cancer cell lines PC-9, HCC827 and HCC4006 resulted in up to 60-fold increased resistance to osimertinib and increased expression of canonical Hippo transcriptional targets. We therefore reasoned that the Hippo pathway might be involved in maintaining the survival of drug tolerant persister cells in this setting. Acute treatment of EGFR mutant cell lines with osimertinib was associated with increased nuclear localisation of YAP1 and WWTR1 and increased expression of canonical Hippo transcriptional targets. Furthermore, the combination of osimertinib and a TEAD inhibitor (K-975) almost completely abolished the survival of drug tolerant persister cells following treatment, indicating that this pathway is an important survival mechanism following drug treatment. Consequently, we propose Hippo signalling as an important target mechanism for the prevention of resistance to osimertinib. Citation Format: Matthias Pfeifer, Jonathan Brammeld, Stacey Price, Matthew Martin, Hannah Thorpe, Aurelie Bornot, Ercia Banks, Nin Guan, Shanade Dunn, Maria Lisa Guerriero, Daniel O'Neill, James Pilling, Davide Gianni, James Brownell, Paul Smith, Ultan McDermott. Gain and loss of function genome-wide CRISPR screens identify Hippo signalling as an important driver of resistance in EGFR mutant lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P066.

CRISPR/Cas9 is increasingly being used as a tool to prosecute functional genomic screens. However, it is not yet possible to apply the approach at scale across a full breadth of cell types and endpoints. In order to address this, we developed a novel and robust workflow for array-based lentiviral CRISPR/Cas9 screening. We utilized a β-lactamase reporter gene assay to investigate mediators of TNF-α-mediated NF-κB signaling. The system was adapted for CRISPR/Cas9 through the development of a cell line stably expressing Cas9 and application of a lentiviral gRNA library comprising mixtures of four gRNAs per gene. We screened a 743-gene kinome library whereupon hits were independently ranked by percent inhibition, Z′ score, strictly standardized mean difference, and T statistic. A consolidated and optimized ranking was generated using Borda-based methods. Screening data quality was above acceptable limits (Z′ ≥ 0.5). In order to determine the contribution of individual gRNAs and to better understand false positives and negatives, a subset of gRNAs, against 152 genes, were profiled in singlicate format. We highlight the use of known reference genes and high-throughput, next-generation amplicon and RNA sequencing to assess screen data quality. Screening with singlicate gRNAs was more successful than screening with mixtures at identifying genes with known regulatory roles in TNF-α-mediated NF-κB signaling and was found to be superior to previous RNAi-based methods. These results add to the available data on TNF-α-mediated NF-κB signaling and establish a high-throughput functional genomic screening approach, utilizing a vector-based arrayed gRNA library, applicable across a wide variety of endpoints and cell types at a genome-wide scale.

Abstract Barasertib (AZD1152), a pro-drug of the highly potent and selective Aurora B kinase inhibitor AZD2811, showed promising clinical activity in relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients administered as a 4-day infusion. To improve potential therapeutic benefit of Aurora B kinase inhibition, a nanoparticle formulation of AZD2811 has been developed to address limitations of repeated intravenous infusion. One of the challenges with the use of nanoparticles for chronic treatment of tumors is optimizing dose and schedule required to enable repeat administration to sustain tumor growth inhibition. AZD2811 gives potent cell growth inhibition across a range of DLBCL cells lines in vitro. In vivo, repeat administration of the AZD2811 nanoparticle gave antitumor activity at half the dose intensity of AZD1152. Compared with AZD1152, a single dose of AZD2811 nanoparticle gave less reduction in pHH3, but increased apoptosis and reduction of cells in G1 and G2–M, albeit at later time points, suggesting that duration and depth of target inhibition influence the nature of the tumor cell response to drug. Further exploration of the influence of dose and schedule on efficacy revealed that AZD2811 nanoparticle can be used flexibly with repeat administration of 25 mg/kg administered up to 7 days apart being sufficient to maintain equivalent tumor control. Timing of repeat administration could be varied with 50 mg/kg every 2 weeks controlling tumor control as effectively as 25 mg/kg every week. AZD2811 nanoparticle can be administered with very different doses and schedules to inhibit DLBCL tumor growth, although maximal tumor growth inhibition was achieved with the highest dose intensities.

The PI3K-AKT-mTOR pathway is frequently hyper-activated in breast cancer and several inhibitors targeting the PI3K pathway, including AKT (Capivasertib; AZD5363) and PI3Kβ/δ (AZD8186), are currently in clinical development (phase I/II) targeting tumours with mutational activation of the pathway including those with loss of PTEN. A better understanding of the mechanisms of resistance/sensitization to Capivasertib and AZD8186 in PTEN-null breast cancer is critical to fully exploit the anti-tumour activity of these compounds and to develop combination strategies for more effective therapy. Here we performed genome-wide CRISPR-Cas9 knockout screens to identify genes that when targeted promote resistance (gRNA enrichment) or sensitivity (gRNA depletion) to Capivasertib and AZD8186 in three PTEN-null breast cancer cell lines. Our screens identified five genes (NPRL2, DEPDC5, DDIT4, HNRNPD and ZC3H4) whose inactivation promoted resistance to Capivasertib; two resistance genes (PIK3R2 and INPPL1) for AZD8186 and three resistance genes (TSC2, TSC1 and FIBP) for both compounds across all three cell lines. While this result strongly suggests that mTOR pathway reactivation is the major resistance mechanism, we also identified novel resistance genes such as FIBP. Our screens also identified a number of genes that when inactivated sensitize cells to Capivasertib and AZD8186 treatment. Eighteen of these sensitizers have inhibitors in clinical development and we have performed a combination screen to identify compounds that give the best synergy with Capivasertib/AZD8186. The most striking combination effect was detected with the Mcl-1 inhibitor, AZD5991. Mechanistic work is ongoing and our data suggest that Capivasertib/AZD8186 prime cells for apoptosis by a previously undescribed mechanism and combined inhibition with AZD5991 drives a rapid apoptotic response. Importantly, the combination with AZD5991 remains effective in breast cancer cells with acquired resistance to Capivasertib/AZD8186. These combinations showed synergistic tumour suppressive effect in a PTEN-null TNBC breast cancer xenograft model (AZD5991 + AZD8186/Capivasertib) and activity in a patient-derived xenograft model (AZD5991 + Capivasertib). Overall, our CRISPR screening data provide new insights into the genes and pathways that drive resistance to inhibitors of the PI3K pathway in PTEN-null breast cancer and have identified novel synergistic combinations to maximize therapeutic response. Citation Format: Shanade Dunn, Jason Yu, Albert Gris-Oliver, James Pilling, Philip Hopcroft, Urs Yelland, Natalie Cureton, Anna Staniszewsla, Yan Zi Au, Swee Hoe Ong, Beverley Isherwood, Violeta Serra, Simon Barry, Barry R. Davies, James T. Lynch, Kosuke Yusa. Genome-wide CRISPR screens identify combination strategies for Capivasertib (AZD5363; AKT) and AZD8186 (PI3Kβ/δ) in PTEN-null breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 321.

Pharmaceutical agents despite their efficacy to treat disease can cause additional unwanted cardiovascular side effects. Cardiotoxicity is characterized by changes in either the function and/or structure of the myocardium. Over recent years, functional cardiotoxicity has received much attention, however morphological damage to the myocardium and/or loss of viability still requires improved detection and mechanistic insights. A human 3D cardiac microtissue containing human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), cardiac endothelial cells and cardiac fibroblasts was used to assess their suitability to detect drug induced changes in cardiac structure. Histology and clinical pathology confirmed these cardiac microtissues were morphologically intact, lacked a necrotic/apoptotic core and contained all relevant cell constituents. High-throughput methods to assess mitochondrial membrane potential, endoplasmic reticulum integrity and cellular viability were developed and 15 FDA approved structural cardiotoxins and 14 FDA approved non-structural cardiotoxins were evaluated. We report that cardiac microtissues provide a high-throughput experimental model that is both able to detect changes in cardiac structure at clinically relevant concentrations and provide insights into the phenotypic mechanisms of this liability.

Abstract Barasertib (AZD1152), a highly potent and selective aurora kinase B inhibitor, gave promising clinical activity in elderly acute myeloid leukemia (AML) patients. However, clinical utility was limited by the requirement for a 7-day infusion. Here we assessed the potential of a nanoparticle formulation of the selective Aurora kinase B inhibitor AZD2811 (formerly known as AZD1152-hQPA) in preclinical models of AML. When administered to HL-60 tumor xenografts at a single dose between 25 and 98.7 mg/kg, AZD2811 nanoparticle treatment delivered profound inhibition of tumor growth, exceeding the activity of AZD1152. The improved antitumor activity was associated with increased phospho-histone H3 inhibition, polyploidy, and tumor cell apoptosis. Moreover, AZD2811 nanoparticles increased antitumor activity when combined with cytosine arabinoside. By modifying dose of AZD2811 nanoparticle, therapeutic benefit in a range of preclinical models was further optimized. At high-dose, antitumor activity was seen in a range of models including the MOLM-13 disseminated model. At these higher doses, a transient reduction in bone marrow cellularity was observed demonstrating the potential for the formulation to target residual disease in the bone marrow, a key consideration when treating AML. Collectively, these data establish that AZD2811 nanoparticles have activity in preclinical models of AML. Targeting Aurora B kinase with AZD2811 nanoparticles is a novel approach to deliver a cell-cycle inhibitor in AML, and have potential to improve on the clinical activity seen with cell-cycle agents in this disease. Mol Cancer Ther; 16(6); 1031–40. ©2017 AACR.

Abstract Purpose: PTEN-null tumors become dependent on the PI3Kβ isoform and can be targeted by molecules such as the selective PI3Kβ inhibitor AZD8186. However, beyond the modulation of the canonical PI3K pathway, the consequences of inhibiting PI3Kβ are poorly defined. Experimental Design: To determine the broader impact of AZD8186 in PTEN-null tumors, we performed a genome-wide RNA-seq analysis of PTEN-null triple-negative breast tumor xenografts treated with AZD8186. Mechanistic consequences of AZD8186 treatment were examined across a number of PTEN-null cell lines and tumor models. Results: AZD8186 treatment resulted in modification of transcript and protein biomarkers associated with cell metabolism. We observed downregulation of cholesterol biosynthesis genes and upregulation of markers associated with metabolic stress. Downregulation of cholesterol biosynthesis proteins, such as HMGCS1, occurred in PTEN-null cell lines and tumor xenografts sensitive to AZD8186. Therapeutic inhibition of PI3Kβ also upregulated PDHK4 and increased PDH phosphorylation, indicative of reduced carbon flux into the TCA cycle. Consistent with this, metabolomic analysis revealed a number of changes in key carbon pathways, nucleotide, and amino acid biosynthesis. Conclusions: This study identifies novel mechanistic biomarkers of PI3Kβ inhibition in PTEN-null tumors supporting the concept that targeting PI3Kβ may exploit a metabolic dependency that contributes to therapeutic benefit in inducing cell stress. Considering these additional pathways will guide biomarker and combination strategies for this class of agents. Clin Cancer Res; 23(24); 7584–95. ©2017 AACR.

Cardiotoxicity is a common cause of attrition in preclinical and clinical drug development. Current in vitro approaches have two main limitations, they either are limited to low throughput methods not amendable to drug discovery or lack the physiological responses to allow an integrated risk assessment. A human 3D cardiac microtissue containing humaninduced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), cardiac endothelial cells and cardiac fibroblast were used to assess their suitability to detect drug induced changes in cardiomyocyte contraction. These cardiac microtissues, have a uniform size, spontaneously beat, lack a hypoxic core, and contain key markers of each cell type. Application of field stimulation and measurement of cardiac contraction confirm cardiac microtissues to be a suitable model to investigate drug-induced changes in cardiomyocyte contractility. Using a bespoke image acquisition work flow and optical flow analysis method to test 29 inotroptic and 13 non-inotroptic compounds in vivo. We report that cardiac microtissues provide a high-throughput experimental model that is both able to detect changes in cardiac contraction with a sensitivity and specificity of 80 and 91%, respectively, and provide insight into the direction of the inotropic response. Allowing improved in vitro cardiac contractility risk assessment. Moreover, our data provide evidence of the detection of this liability at therapeutically relevant concentrations with a throughput amenable to drug discovery.