Kieron May

Cancer cell lines remain foundational for research and drug discovery, yet they incompletely capture tumour diversity, lack linked patient context, and have undergone adaptation to culture. Tumour organoids are three-dimensional cultures derived from patient tissue that offer a powerful complement to cell lines ¹ . Here we derived and characterized 256 clinically annotated tumour organoids directly from colorectal, oesophageal, ovarian, pancreatic and gastric cancers as renewable, genetically stable models. Extensive characterization of each model and matched patient tumour samples included whole-genome and transcriptome sequencing, and genome-wide CRISPR–Cas9 screens across 162 organoids mapped gene dependencies. Integrative analyses revealed genomic and clinical markers of dependency across common and rare subtypes, identified organoid-specific essential genes, and revealed targetable vulnerabilities following tumour evolution in paired pre- and post-treatment samples. In colorectal cancer, functional and pharmacological interrogation of the EGFR–RAS–MAPK axis uncovered differential effects of KRAS variant alleles. This open, publicly available resource provides a systematic map of gene dependencies in patient-derived organoids, expanding the model diversity and mechanistic insight needed to advance precision oncology.

Osimertinib is the current standard-of-care for treatment-naive patients with EGFR mutation-positive advanced/metastatic non-small cell lung cancer (NSCLC), however resistance inevitably emerges. Osimertinib does not eradicate all cancer cells even in culture, leaving a long-lasting sub-population of Drug Tolerant Persister (DTP) cells that is common to many chemotherapeutics. The DTP population is non-proliferative and seemingly dormant, but resistant clones eventually emerge from the DTP state. Here we show that extensive DNA replication occurs in the DTP state and cells frequently progress through the cell cycle, though cell death is also frequent, such that cell division and cell loss are balanced and the population remains approximately constant. Cell cycling occurs with aberrant gene expression and abnormal DNA replication pattern, leading to DNA damage, extrachromosomal circular DNA formation and mitotic defects, such that replicating DTP cells are hypersensitive to low doses of ATM and ATR inhibitors. Our findings suggest that the DTP state is highly mutagenic and that targeting DNA repair in DTP cells has the potential to prevent the emergence of resistance through de novo mutations.

This file contain information on the Cell lines and organoids used in this study

This file contains details of the BE sgRNA library

Supplementary Figure 1. Generating base editor-expressing MSI cell lines for screening. Supplementary Figure 2. Quality control for base-editor screens in MSI cell lines. Supplementary Figure 3. Discovery and characterization of novel inhibitors of WRN helicase. Supplementary Figure 4. Correlation of WRN inhibitor sensitivity with genetic and molecular markers in MSI models. Supplementary Figure 5. Comparative analysis of DNA damage in MSI and MSS cells treated with WRN inhibitors and genetic inactivation. Supplementary Figure 6: Effects of GSK_WRN4 on MSI and MSS colorectal cancer xenografts.

This table lists the results from Base editing screen

TrAEL-seq is a robust method for profiling DNA replication genome-wide that works in unsynchronized cells and does not require drugs or nucleotide analogues. Here, we provide an updated method for TrAEL-seq that improves sample quality and includes multiplexing of up to six samples which dramatically improves throughput, and we validate TrAEL-seq in multiple mammalian cell lines. The updated protocol is straightforward and robust yet provides excellent resolution comparable to OK-seq in mammalian cell samples. High resolution replication profiles can be obtained across large panels of samples and in dynamic systems, for example during the progressive onset of oncogene induced senescence. In addition to mapping zones where replication initiates and terminates, TrAEL-seq is sensitive to replication fork speed, revealing effects of both transcription and proximity to replication Initiation Zones on fork progression. Although forks move more slowly through transcribed regions, this does not have a significant impact on the broader dynamics of replication fork progression, and instead replication forks accelerate across the first ∼1 Mb of travel irrespective of local transcriptional activity. We propose that this is a consequence of fewer replication forks being active later in S-phase when these distal regions replicate and there being less competition for replication factors.

This table lists the results from Base editing screen

Supplementary Figure 1. Generating base editor-expressing MSI cell lines for screening. Supplementary Figure 2. Quality control for base-editor screens in MSI cell lines. Supplementary Figure 3. Discovery and characterization of novel inhibitors of WRN helicase. Supplementary Figure 4. Correlation of WRN inhibitor sensitivity with genetic and molecular markers in MSI models. Supplementary Figure 5. Comparative analysis of DNA damage in MSI and MSS cells treated with WRN inhibitors and genetic inactivation. Supplementary Figure 6: Effects of GSK_WRN4 on MSI and MSS colorectal cancer xenografts.

This file contain information on the Cell lines and organoids used in this study