Novel WRN Helicase Inhibitors Selectively Target Microsatellite Unstable Cancer Cells.

Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n-dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumours, and WRN inhibitors are in development. Here, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth In vitro and In vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA-repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair (MMR) alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft (PDX) models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic-lethal targeting of WRN in MSI cancer and tools to dissect WRN biology.

Large-scale Pan-cancer Cell Line Screening Identifies Actionable and Effective Drug Combinations.

Oncology drug combinations can improve therapeutic responses and increase treatment options for patients. The number of possible combinations is vast and responses can be context-specific. Systematic screens can identify clinically relevant, actionable combinations in defined patient subtypes. We present data for 109 anticancer drug combinations from AstraZeneca's oncology small molecule portfolio screened in 755 pan-cancer cell lines. Combinations were screened in a 7 × 7 concentration matrix, with more than 4 million measurements of sensitivity, producing an exceptionally data-rich resource. We implement a new approach using combination Emax (viability effect) and highest single agent (HSA) to assess combination benefit. We designed a clinical translatability workflow to identify combinations with clearly defined patient populations, rationale for tolerability based on tumor type and combination-specific "emergent" biomarkers, and exposures relevant to clinical doses. We describe three actionable combinations in defined cancer types, confirmed in vitro and in vivo, with a focus on hematologic cancers and apoptotic targets. SIGNIFICANCE: We present the largest cancer drug combination screen published to date with 7 × 7 concentration response matrices for 109 combinations in more than 750 cell lines, complemented by multi-omics predictors of response and identification of "emergent" combination biomarkers. We prioritize hits to optimize clinical translatability, and experimentally validate novel combination hypotheses. This article is featured in Selected Articles from This Issue, p. 695.

Establishment and Characterization of an Epstein-Barr Virus-positive Cell Line from a Non-keratinizing Differentiated Primary Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC), a cancer that is etiologically associated with the Epstein-Barr virus (EBV), is endemic in Southern China and Southeast Asia. The scarcity of representative NPC cell lines owing to the frequent loss of EBV episomes following prolonged propagation and compromised authenticity of previous models underscores the critical need for new EBV-positive NPC models. Herein, we describe the establishment of a new EBV-positive NPC cell line, designated NPC268 from a primary non-keratinizing, differentiated NPC tissue. NPC268 can undergo productive lytic reactivation of EBV and is highly tumorigenic in immunodeficient mice. Whole-genome sequencing revealed close similarities with the tissue of origin, including large chromosomal rearrangements, while whole-genome bisulfite sequencing and RNA sequencing demonstrated a hypomethylated genome and enrichment in immune-related pathways, respectively. Drug screening of NPC268 together with six other NPC cell lines using 339 compounds, representing the largest high-throughput drug testing in NPC, revealed biomarkers associated with specific drug classes. NPC268 represents the first and only available EBV-positive non-keratinizing differentiated NPC model, and extensive genomic, methylomic, transcriptomic, and drug response data should facilitate research in EBV and NPC, where current models are limited. SIGNIFICANCE: NPC268 is the first and only EBV-positive cell line derived from a primary non-keratinizing, differentiated nasopharyngeal carcinoma, an understudied but important subtype in Southeast Asian countries. This model adds to the limited number of authentic EBV-positive lines globally that will facilitate mechanistic studies and drug development for NPC.

Benchmark Software and Data for Evaluating CRISPR-Cas9 Experimental Pipelines Through the Assessment of a Calibration Screen.

Genome-wide genetic screens using CRISPR-guide RNA libraries are widely performed in mammalian cells to functionally characterize individual genes and for the discovery of new anticancer therapeutic targets. As the effectiveness of such powerful and precise tools for cancer pharmacogenomics is emerging, tools and methods for their quality assessment are becoming increasingly necessary. Here, we provide an R package and a high-quality reference data set for the assessment of novel experimental pipelines through which a single calibration experiment has been executed: a screen of the HT-29 human colorectal cancer cell line with a commercially available genome-wide library of single-guide RNAs. This package and data allow experimental researchers to benchmark their screens and produce a quality-control report, encompassing several quality and validation metrics. The R code used for processing the reference data set, for its quality assessment, as well as to evaluate the quality of a user-provided screen, and to reproduce the figures presented in this article is available at https://github.com/DepMap-Analytics/HT29benchmark. The reference data is publicly available on FigShare.

scSNV-seq: high-throughput phenotyping of single nucleotide variants by coupled single-cell genotyping and transcriptomics.

CRISPR screens with single-cell transcriptomic readouts are a valuable tool to understand the effect of genetic perturbations including single nucleotide variants (SNVs) associated with diseases. Interpretation of these data is currently limited as genotypes cannot be accurately inferred from guide RNA identity alone. scSNV-seq overcomes this limitation by coupling single-cell genotyping and transcriptomics of the same cells enabling accurate and high-throughput screening of SNVs. Analysis of variants across the JAK1 gene with scSNV-seq demonstrates the importance of determining the precise genetic perturbation and accurately classifies clinically observed missense variants into three functional categories: benign, loss of function, and separation of function.

Haplotype-specific assembly of shattered chromosomes in esophageal adenocarcinomas.

The epigenetic landscape of cancer is regulated by many factors, but primarily it derives from the underlying genome sequence. Chromothripsis is a catastrophic localized genome shattering event that drives, and often initiates, cancer evolution. We characterized five esophageal adenocarcinoma organoids with chromothripsis using long-read sequencing and transcriptome and epigenome profiling. Complex structural variation and subclonal variants meant that haplotype-aware de novo methods were required to generate contiguous cancer genome assemblies. Chromosomes were assembled separately and scaffolded using haplotype-resolved Hi-C reads, producing accurate assemblies even with up to 900 structural rearrangements. There were widespread differences between the chromothriptic and wild-type copies of chromosomes in topologically associated domains, chromatin accessibility, histone modifications, and gene expression. Differential epigenome peaks were most enriched within 10 kb of chromothriptic structural variants. Alterations in transcriptome and higher-order chromosome organization frequently occurred near differential epigenetic marks. Overall, chromothripsis reshapes gene regulation, causing coordinated changes in epigenetic landscape, transcription, and chromosome conformation.

Single-cell transcriptomic analysis of human pleura reveals stromal heterogeneity and informs in vitro models of mesothelioma.

The pleural lining of the thorax regulates local immunity, inflammation and repair. A variety of conditions, both benign and malignant, including pleural mesothelioma, can affect this tissue. A lack of knowledge concerning the mesothelial and stromal cells comprising the pleura has hampered the development of targeted therapies. Here, we present the first comprehensive single-cell transcriptomic atlas of the human parietal pleura and demonstrate its utility in elucidating pleural biology. We confirm the presence of known universal fibroblasts and describe novel, potentially pleural-specific, fibroblast subtypes. We also present transcriptomic characterisation of multiple in vitro models of benign and malignant mesothelial cells, and characterise these through comparison with in vivo transcriptomic data. While bulk pleural transcriptomes have been reported previously, this is the first study to provide resolution at the single-cell level. We expect our pleural cell atlas will prove invaluable to those studying pleural biology and disease. It has already enabled us to shed light on the transdifferentiation of mesothelial cells, allowing us to develop a simple method for prolonging mesothelial cell differentiation in vitro.

A comprehensive clinically informed map of dependencies in cancer cells and framework for target prioritization.

Genetic screens in cancer cell lines inform gene function and drug discovery. More comprehensive screen datasets with multi-omics data are needed to enhance opportunities to functionally map genetic vulnerabilities. Here, we construct a second-generation map of cancer dependencies by annotating 930 cancer cell lines with multi-omic data and analyze relationships between molecular markers and cancer dependencies derived from CRISPR-Cas9 screens. We identify dependency-associated gene expression markers beyond driver genes, and observe many gene addiction relationships driven by gain of function rather than synthetic lethal effects. By combining clinically informed dependency-marker associations with protein-protein interaction networks, we identify 370 anti-cancer priority targets for 27 cancer types, many of which have network-based evidence of a functional link with a marker in a cancer type. Mapping these targets to sequenced tumor cohorts identifies tractable targets in different cancer types. This target prioritization map enhances understanding of gene dependencies and identifies candidate anti-cancer targets for drug development.

Tissue Organoid Cultures Metabolize Dietary Carcinogens Proficiently and Are Effective Models for DNA Adduct Formation.

Human tissue three-dimensional (3D) organoid cultures have the potential to reproduce in vitro the physiological properties and cellular architecture of the organs from which they are derived. The ability of organoid cultures derived from human stomach, liver, kidney, and colon to metabolically activate three dietary carcinogens, aflatoxin B1 (AFB1), aristolochic acid I (AAI), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was investigated. In each case, the response of a target tissue (liver for AFB1; kidney for AAI; colon for PhIP) was compared with that of a nontarget tissue (gastric). After treatment cell viabilities were measured, DNA damage response (DDR) was determined by Western blotting for p-p53, p21, p-CHK2, and γ-H2AX, and DNA adduct formation was quantified by mass spectrometry. Induction of the key xenobiotic-metabolizing enzymes (XMEs) CYP1A1, CYP1A2, CYP3A4, and NQO1 was assessed by qRT-PCR. We found that organoids from different tissues can activate AAI, AFB1, and PhIP. In some cases, this metabolic potential varied between tissues and between different cultures of the same tissue. Similarly, variations in the levels of expression of XMEs were observed. At comparable levels of cytotoxicity, organoids derived from tissues that are considered targets for these carcinogens had higher levels of adduct formation than a nontarget tissue.

Highlights from the 1st European cancer dependency map symposium and workshop.

The systematic identification of tumour vulnerabilities through perturbational experiments on cancer models, including genome editing and drug screens, is playing a crucial role in combating cancer. This collective effort is known as the Cancer Dependency Map (DepMap). The 1st European Cancer Dependency Map Symposium (EuroDepMap), held in Milan last May, featured talks, a roundtable discussion, and a poster session, showcasing the latest discoveries and future challenges related to the DepMap. The symposium aimed to facilitate interactions among participants across Europe, encourage idea exchange with leading experts, and present their work and future projects. Importantly, it sparked discussions on future endeavours, such as screening more complex cancer models and accounting for tumour evolution.

A landscape of response to drug combinations in non-small cell lung cancer.

Combination of anti-cancer drugs is broadly seen as way to overcome the often-limited efficacy of single agents. The design and testing of combinations are however very challenging. Here we present a uniquely large dataset screening over 5000 targeted agent combinations across 81 non-small cell lung cancer cell lines. Our analysis reveals a profound heterogeneity of response across the tumor models. Notably, combinations very rarely result in a strong gain in efficacy over the range of response observable with single agents. Importantly, gain of activity over single agents is more often seen when co-targeting functionally proximal genes, offering a strategy for designing more efficient combinations. Because combinatorial effect is strongly context specific, tumor specificity should be achievable. The resource provided, together with an additional validation screen sheds light on major challenges and opportunities in building efficacious combinations against cancer and provides an opportunity for training computational models for synergy prediction.

RAF1 contributes to cell proliferation and STAT3 activation in colorectal cancer independently of microsatellite and KRAS status.

More than 30% of all human cancers are driven by RAS mutations and activating KRAS mutations are present in 40% of colorectal cancer (CRC) in the two main CRC subgroups, MSS (Microsatellite Stable) and MSI (Microsatellite Instable). Studies in RAS-driven tumors have shown essential roles of the RAS effectors RAF and specifically of RAF1, which can be dependent or independent of RAF's ability to activate the MEK/ERK module. In this study, we demonstrate that RAF1, but not its kinase activity, plays a crucial role in the proliferation of both MSI and MSS CRC cell line-derived spheroids and patient-derived organoids, and independently of KRAS mutation status. Moreover, we could define a RAF1 transcriptomic signature which includes genes that contribute to STAT3 activation, and could demonstrate that RAF1 ablation decreases STAT3 phosphorylation in all CRC spheroids tested. The genes involved in STAT3 activation as well as STAT3 targets promoting angiogenesis were also downregulated in human primary tumors expressing low levels of RAF1. These results indicate that RAF1 could be an attractive therapeutic target in both MSI and MSS CRC regardless of their KRAS status and support the development of selective RAF1 degraders rather than RAF1 inhibitors for clinical use in combination therapies.

Base editing screens map mutations affecting interferon-γ signaling in cancer.

Interferon-γ (IFN-γ) signaling mediates host responses to infection, inflammation and anti-tumor immunity. Mutations in the IFN-γ signaling pathway cause immunological disorders, hematological malignancies, and resistance to immune checkpoint blockade (ICB) in cancer; however, the function of most clinically observed variants remains unknown. Here, we systematically investigate the genetic determinants of IFN-γ response in colorectal cancer cells using CRISPR-Cas9 screens and base editing mutagenesis. Deep mutagenesis of JAK1 with cytidine and adenine base editors, combined with pathway-wide screens, reveal loss-of-function and gain-of-function mutations, including causal variants in hematological malignancies and mutations detected in patients refractory to ICB. We functionally validate variants of uncertain significance in primary tumor organoids, where engineering missense mutations in JAK1 enhanced or reduced sensitivity to autologous tumor-reactive T cells. We identify more than 300 predicted missense mutations altering IFN-γ pathway activity, generating a valuable resource for interpreting gene variant function.

AKT-mTORC1 reactivation is the dominant resistance driver for PI3Kß/AKT inhibitors in PTEN-null breast cancer and can be overcome by combining with Mcl-1 inhibitors.

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 (PI3Kß 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 PI3Kßi and AKTi through mTORC1. However, deletion of PIK3R2 and INPPL1 drove specific PI3Kßi resistance through AKT. Conversely deletion of PIK3CA, ERBB2, ERBB3 increased PI3Kßi 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 PI3Kßi in both sensitive and drug resistant TSC1/2 null cells. The combination effect was BAK but not BAX dependent. The Mcl-1i + PI3Kß/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 PI3Kßi 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 PI3Kßi/AKTi sensitive and resistant breast tumours.

Pan-cancer proteomic map of 949 human cell lines.

The proteome provides unique insights into disease biology beyond the genome and transcriptome. A lack of large proteomic datasets has restricted the identification of new cancer biomarkers. Here, proteomes of 949 cancer cell lines across 28 tissue types are analyzed by mass spectrometry. Deploying a workflow to quantify 8,498 proteins, these data capture evidence of cell-type and post-transcriptional modifications. Integrating multi-omics, drug response, and CRISPR-Cas9 gene essentiality screens with a deep learning-based pipeline reveals thousands of protein biomarkers of cancer vulnerabilities that are not significant at the transcript level. The power of the proteome to predict drug response is very similar to that of the transcriptome. Further, random downsampling to only 1,500 proteins has limited impact on predictive power, consistent with protein networks being highly connected and co-regulated. This pan-cancer proteomic map (ProCan-DepMapSanger) is a comprehensive resource available at https://cellmodelpassports.sanger.ac.uk.

Can Drug Repurposing Accelerate Precision Oncology?

Ongoing new insights in the field of cancer diagnostics, genomic profiling, and cancer behavior have raised the demand for novel, personalized cancer treatments. As the development of new cancer drugs is a challenging, costly, and time-consuming endeavor, drug repurposing is regarded as an attractive alternative to potentially accelerate this. In this review, we describe strategies for drug repurposing of anticancer agents, translation of preclinical findings in novel trial designs, and associated challenges. Furthermore, we provide suggestions to further utilize the potential of drug repurposing within precision oncology, with a focus on combinatorial approaches. SIGNIFICANCE: Oncologic drug development is a timely and costly endeavor, with only few compounds progressing to meaningful therapy options. Although repurposing of existing agents for novel, oncologic indications provides an opportunity to accelerate this process, it is not without challenges.

A suspension technique for efficient large-scale cancer organoid culturing and perturbation screens.

Organoid cell culture methodologies are enabling the generation of cell models from healthy and diseased tissue. Patient-derived cancer organoids that recapitulate the genetic and histopathological diversity of patient tumours are being systematically generated, providing an opportunity to investigate new cancer biology and therapeutic approaches. The use of organoid cultures for many applications, including genetic and chemical perturbation screens, is limited due to the technical demands and cost associated with their handling and propagation. Here we report and benchmark a suspension culture technique for cancer organoids which allows for the expansion of models to tens of millions of cells with increased efficiency in comparison to standard organoid culturing protocols. Using whole-genome DNA and RNA sequencing analyses, as well as medium-throughput drug sensitivity testing and genome-wide CRISPR-Cas9 screening, we demonstrate that cancer organoids grown as a suspension culture are genetically and phenotypically similar to their counterparts grown in standard conditions. This culture technique simplifies organoid cell culture and extends the range of organoid applications, including for routine use in large-scale perturbation screens.

Effective drug combinations in breast, colon and pancreatic cancer cells.

Combinations of anti-cancer drugs can overcome resistance and provide new treatments1,2. The number of possible drug combinations vastly exceeds what could be tested clinically. Efforts to systematically identify active combinations and the tissues and molecular contexts in which they are most effective could accelerate the development of combination treatments. Here we evaluate the potency and efficacy of 2,025 clinically relevant two-drug combinations, generating a dataset encompassing 125 molecularly characterized breast, colorectal and pancreatic cancer cell lines. We show that synergy between drugs is rare and highly context-dependent, and that combinations of targeted agents are most likely to be synergistic. We incorporate multi-omic molecular features to identify combination biomarkers and specify synergistic drug combinations and their active contexts, including in basal-like breast cancer, and microsatellite-stable or KRAS-mutant colon cancer. Our results show that irinotecan and CHEK1 inhibition have synergistic effects in microsatellite-stable or KRAS-TP53 double-mutant colon cancer cells, leading to apoptosis and suppression of tumour xenograft growth. This study identifies clinically relevant effective drug combinations in distinct molecular subpopulations and is a resource to guide rational efforts to develop combinatorial drug treatments.

Functional Genomic Identification of Predictors of Sensitivity and Mechanisms of Resistance to Multivalent Second-Generation TRAIL-R2 Agonists.

Multivalent second-generation TRAIL-R2 agonists are currently in late preclinical development and early clinical trials. Herein, we use a representative second-generation agent, MEDI3039, to address two major clinical challenges facing these agents: lack of predictive biomarkers to enable patient selection and emergence of resistance. Genome-wide CRISPR knockout screens were notable for the lack of resistance mechanisms beyond the canonical TRAIL-R2 pathway (caspase-8, FADD, BID) as well as p53 and BAX in TP53 wild-type models, whereas a CRISPR activatory screen identified cell death inhibitors MCL-1 and BCL-XL as mechanisms to suppress MEDI3039-induced cell death. High-throughput drug screening failed to identify genomic alterations associated with response to MEDI3039; however, transcriptomics analysis revealed striking association between MEDI3039 sensitivity and expression of core components of the extrinsic apoptotic pathway, most notably its main apoptotic effector caspase-8 in solid tumor cell lines. Further analyses of colorectal cell lines and patient-derived xenografts identified caspase-8 expression ratio to its endogenous regulator FLIP(L) as predictive of sensitivity to MEDI3039 in several major solid tumor types and a further subset indicated by caspase-8:MCL-1 ratio. Subsequent MEDI3039 combination screening of TRAIL-R2, caspase-8, FADD, and BID knockout models with 60 compounds with varying mechanisms of action identified two inhibitor of apoptosis proteins (IAP) that exhibited strong synergy with MEDI3039 that could reverse resistance only in BID-deleted models. In summary, we identify the ratios of caspase-8:FLIP(L) and caspase-8:MCL-1 as potential predictive biomarkers for second-generation TRAIL-R2 agonists and loss of key effectors such as FADD and caspase-8 as likely drivers of clinical resistance in solid tumors.

High-content CRISPR screening.

CRISPR screens are a powerful source of biological discovery, enabling the unbiased interrogation of gene function in a wide range of applications and species. In pooled CRISPR screens, various genetically encoded perturbations are introduced into pools of cells. The targeted cells proliferate under a biological challenge such as cell competition, drug treatment or viral infection. Subsequently, the perturbation-induced effects are evaluated by sequencing-based counting of the guide RNAs that specify each perturbation. The typical results of such screens are ranked lists of genes that confer sensitivity or resistance to the biological challenge of interest. Contributing to the broad utility of CRISPR screens, adaptations of the core CRISPR technology make it possible to activate, silence or otherwise manipulate the target genes. Moreover, high-content read-outs such as single-cell RNA sequencing and spatial imaging help characterize screened cells with unprecedented detail. Dedicated software tools facilitate bioinformatic analysis and enhance reproducibility. CRISPR screening has unravelled various molecular mechanisms in basic biology, medical genetics, cancer research, immunology, infectious diseases, microbiology and other fields. This Primer describes the basic and advanced concepts of CRISPR screening and its application as a flexible and reliable method for biological discovery, biomedical research and drug development - with a special emphasis on high-content methods that make it possible to obtain detailed biological insights directly as part of the screen.