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Cancer
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Nature
Decay of driver mutations shapes the landscape of intestinal transformation
Colorectal cancer (CRC) has traditionally been thought to develop through stepwise mutation of theAPCtumour suppressor and other driver genes, coupled with expansion of positively selected clones. However, recent publications show that many premalignant lesions comprise multiple clones expressing different mutant APC proteins1,2,3,4. Here, by mediating transformation on different mouse backgrounds containing mutations inKrasor other common CRC driver genes, we establish that the presence of diverse priming events in the normal mouse intestinal epithelium can change the transformation and clonal-selection landscape, permitting the fixation of strong driver mutations inApcandCtnnb1that are otherwise lost due to negative selection. These findings, combined with our demonstration of mutational patterns consistent with similar priming events in human CRC, suggest that the order in which driver mutations occur in intestinal epithelium can determine whether clones are positively or negatively selected and can shape subsequent tumour development.
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Nature
Whole-genome landscapes of 1,364 breast cancers
Breast cancer remains a major global health challenge1. Here, to comprehensively characterize its genomic landscape and the clinical significance of genomic characteristics, we analysed whole-genome sequences from 1,364 clinically annotated breast cancers, with transcriptome data available for most cases. Our study expands the repertoire of oncogenic alterations and identifies novel driver genes, recurrent gene fusions, structural variants and copy number alterations. Timing analyses on copy number alterations suggest that genomic instability emerges decades before tumour diagnosis, and offer insights into early initiation of tumorigenesis. Pattern-driven genomic features, including mutational signatures2, homologous recombination deficiency3, tumour mutational burden and tumour heterogeneity scores4, were associated with clinical outcomes, highlighting their potential utility as predictive biomarkers for clinical evaluation of treatments such as CDK4/6 and HER2 inhibitors, as well as adjuvant and neoadjuvant chemotherapy. These findings highlight the power of large-scale, clinically annotated whole-genome sequencing in advancing our understanding of how genomic alterations shape patient outcomes.
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Nature
NSD2 targeting reverses plasticity and drug resistance in prostate cancer
Lineage plasticity is a cancer hallmark that drives disease progression and treatment resistance1,2. Plasticity is often mediated by epigenetic mechanisms that may be reversible; however, there are few examples of such reversibility. In castration-resistant prostate cancer (CRPC), plasticity mediates resistance to androgen receptor (AR) inhibitors and progression from adenocarcinoma to aggressive subtypes, including neuroendocrine prostate cancer (CRPC-NE)3,4,5. Here we show that plasticity-associated treatment resistance in CRPC can be reversed through the inhibition of NSD2, a histone methyltransferase6. NSD2 upregulation in CRPC-NE correlates with poor survival outcomes, and NSD2-mediated H3K36 dimethylation regulates enhancers of genes associated with neuroendocrine differentiation. In prostate tumour organoids established from genetically engineered mice7that recapitulate the transdifferentiation to neuroendocrine states, and in human CRPC-NE organoids, CRISPR-mediated targeting ofNSD2reverts CRPC-NE to adenocarcinoma phenotypes. Moreover, a canonical AR program is upregulated and responses to the AR inhibitor enzalutamide are restored. Pharmacological inhibition of NSD2 with a first-in-class small molecule reverses plasticity and synergizes with enzalutamide to suppress growth and promote cell death in human patient-derived organoids of multiple CRPC subtypes in culture and in xenografts. Co-targeting of NSD2 and AR may represent a new therapeutic strategy for lethal forms of CRPC that are currently recalcitrant to treatment.
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Nature
Polyclonal origins of human premalignant colorectal lesions
Cancer is generally thought to be caused by expansion of a single mutant cell1. However, analyses of early colorectal cancer lesions suggest that tumors may instead originate from multiple, genetically distinct cell populations2,3. Detecting polyclonal tumor initiation is challenging in patients, as it requires profiling early-stage lesions before clonal sweeps obscure diversity. To investigate this, we analyzed normal colorectal mucosa, benign and dysplastic premalignant polyps, and malignant adenocarcinomas (123 samples) from six individuals with familial adenomatous polyposis (FAP). Individuals with FAP have a germline heterozygousAPCmutation, predisposing them to colorectal cancer and numerous premalignant polyps by early adulthood4. Whole-genome and/or whole-exome sequencing revealed that many premalignant polyps—40% with benign histology and 28% with dysplasia—were composed of multiple genetic lineages that diverged early, consistent with polyclonal origins. This conclusion was reinforced by whole-genome sequencing of single crypts from multiple polyps in additional patients which showed limited sharing of mutations among crypts within the same lesion. In some cases, multiple distinctAPCmutations co-existed in different lineages of a single polyp, consistent with polyclonality. These findings reshape our understanding of early neoplastic events, demonstrating that tumor initiation can arise from the convergence of diverse mutant clones. They also suggest that cell-intrinsic growth advantages alone may not fully explain tumor initiation, highlighting the importance of microenvironmental and tissue-level factors in early cancer evolution.
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Nature
MAPK-driven epithelial cell plasticity drives colorectal cancer therapeutic resistance
The colorectal epithelium is rapidly renewing, with remarkable capacity to regenerate following injury. In colorectal cancer (CRC), this regenerative capacity can be co-opted to drive epithelial plasticity. While oncogenic MAPK signalling in CRC is common, with frequent mutations of bothKRAS(40-50%) andBRAF(10%)1, inhibition of this pathway typically drives resistance clinically. Given the development of KRAS inhibitors, and licensing of BRAF inhibitor combinations2-4, we have interrogated key mechanisms of resistance to these agents in advanced preclinical CRC models. We show that oncogenic MAPK signalling induces epithelial state changesin vivo, driving adoption of a regenerative/revival stem like population, while inhibition leads to rapid transcriptional remodeling of bothKras-andBraf-mutant tumours, favoring a Wnt-associated, canonical stem phenotype. This drives acute therapeutic resistance inKras-and delayed resistance inBraf-driven models. Importantly, where plasticity is restrained, such as in early metastatic disease, or through targeting ligand-dependent Wnt-pathwayRnf43mutations, marked therapeutic responses are observed. This explains the super response to BRAF+EGFR targeted therapies previously observed in a BRAF/RNF43 co-mutant patient population, highlighting the criticality of cellular plasticity in therapeutic response. Together, our data provides clear insight into the mechanisms underpinning resistance to MAPK targeted therapies in CRC. Moreover, strategies that aim to corral stem cell fate, restrict epithelial plasticity or intervene when tumours lack heterogeneity may improve therapeutic efficacy of these agents.
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Nature
Genetic elements promote retention of extrachromosomal DNA in cancer cells
Extrachromosomal DNA (ecDNA) is a prevalent and devastating form of oncogene amplification in cancer1,2. Circular megabase-sized ecDNAs lack centromeres, stochastically segregate during cell division3,4,5,6and persist over many generations. It has been more than 40 years since ecDNAs were first observed to hitchhike on mitotic chromosomes into daughter cell nuclei, but the mechanism underlying this process remains unclear3,7. Here we identify a family of human genomic elements, termed retention elements, that tether episomes to mitotic chromosomes to increase ecDNA transmission to daughter cells. Using Retain-seq, a genome-scale assay that we developed, we reveal thousands of human retention elements that confer generational persistence to heterologous episomes. Retention elements comprise a select set of CpG-rich gene promoters and act additively. Live-cell imaging and chromosome conformation capture show that retention elements physically interact with mitotic chromosomes at regions that are mitotically bookmarked by transcription factors and chromatin proteins. This activity intermolecularly recapitulates promoter–enhancer interactions. Multiple retention elements are co-amplified with oncogenes on individual ecDNAs in human cancers and shape their sizes and structures. CpG-rich retention elements are focally hypomethylated. Targeted cytosine methylation abrogates retention activity and leads to ecDNA loss, which suggests that methylation-sensitive interactions modulate episomal DNA retention. These results highlight the DNA elements and regulatory logic of mitotic ecDNA retention. Amplifications of retention elements promote the maintenance of oncogenic ecDNA across generations of cancer cells, and reveal the principles of episome immortality intrinsic to the human genome.
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Nature
Hepatic zonation determines tumorigenic potential of mutant β-catenin
Oncogenic mutations in phenotypically normal tissue are common across adult organs1,2. This suggests that multiple events need to converge to drive tumorigenesis and that many processes such as tissue differentiation may protect against carcinogenesis. WNT–β-catenin signalling maintains zonal differentiation during liver homeostasis3,4. However, theCTNNB1oncogene—encoding β-catenin—is also frequently mutated in hepatocellular carcinoma, resulting in aberrant WNT signalling that promotes cell growth5,6. Here we investigated the antagonistic interplay between WNT-driven growth and differentiation in zonal hepatocyte populations during liver tumorigenesis. We found that β-catenin mutations co-operate with exogenous MYC expression to drive a proliferative translatome. Differentiation of hepatocytes to an extreme zone 3 fate suppressed this proliferative translatome. Furthermore, a GLUL andLgr5-positive perivenous subpopulation of zone 3 hepatocytes were refractory to WNT-induced and MYC-induced tumorigenesis. However, when mutantCTNNB1andMYCalleles were activated sporadically across the liver lobule, a subset of mutant hepatocytes became proliferative and tumorigenic. These early lesions were characterized by reduced WNT pathway activation and elevated MAPK signalling, which suppresses zone 3 differentiation. The proliferative lesions were also dependent on IGFBP2–mTOR–cyclin D1 pathway signalling, in which inhibition of either IGFBP2 or mTOR suppressed proliferation and tumorigenesis. Therefore, we propose that zonal identity dictates hepatocyte susceptibility to WNT-driven tumorigenesis and that escaping WNT-induced differentiation is essential for liver cancer.
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Nature
Tumour-reactive heterotypic CD8 T cell clusters from clinical samples
Emerging evidence suggests a correlation between CD8+T cell–tumour cell proximity and anti-tumour immune response1,2. However, it remains unclear whether these cells exist as functional clusters that can be isolated from clinical samples. Here, using conventional and imaging flow cytometry, we show that from 21 out of 21 human melanoma metastases, we could isolate heterotypic clusters, comprising CD8+T cells interacting with one or more tumour cells and/or antigen-presenting cells (APCs). Single-cell RNA-sequencing analysis revealed that T cells from clusters were enriched for gene signatures associated with tumour reactivity and exhaustion. Clustered T cells exhibited increased TCR clonality indicative of expansion, whereas TCR-matched T cells showed more exhaustion and co-modulation when conjugated to APCs than when conjugated to tumour cells. T cells that were expanded from clusters ex vivo exerted on average ninefold increased killing activity towards autologous melanomas, which was accompanied by enhanced cytokine production. After adoptive cell transfer into mice, T cells from clusters showed improved patient-derived melanoma control, which was associated with increased T cell infiltration and activation. Together, these results demonstrate that tumour-reactive CD8+T cells are enriched in functional clusters with tumour cells and/or APCs and that they can be isolated and expanded from clinical samples. Typically excluded by single-cell gating in flow cytometry, these distinct heterotypic T cell clusters are a valuable source to decipher functional tumour–immune cell interactions and may also be therapeutically explored.
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Nature
Cytosolic acetyl-coenzyme A is a signalling metabolite to control mitophagy
Acetyl-coenzyme A (AcCoA) sits at the nexus of nutrient metabolism and shuttles between the canonical and non-canonical tricarboxylic acid cycle1,2, which is dynamically regulated by nutritional status, such as fasting3. Here we find that mitophagy is triggered after a reduction in cytosolic AcCoA levels through short-term fasting and through inhibition of ATP-citrate lyase (encoded byACLY), mitochondrial citrate/malate antiporter (encoded bySLC25A1) or acyl-CoA synthetase short chain family member 2 (encoded byACSS2), and the mitophagy can be counteracted by acetate supplementation. Notably, NOD-like receptor (NLR) family member X1 (NLRX1) mediates this effect. Disrupting NLRX1 abolishes cytosolic AcCoA reduction-induced mitophagy both in vitro and in vivo. Mechanically, the mitochondria outer-membrane-localized NLRX1 directly binds to cytosolic AcCoA within a conserved pocket on its leucine-rich repeat (LRR) domain. Moreover, AcCoA binds to the LRR domain and enhances its interaction with the nucleotide-binding and oligomerization (NACHT) domain, which helps to maintain NLRX1 in an autoinhibited state and prevents the association between NLRX1 and light chain 3 (LC3). Furthermore, we find that the AcCoA–NLRX1 axis underlies the KRAS-inhibitor-induced mitophagy response and promotes drug resistance, providing a metabolic mechanism of KRAS inhibitor resistance. Thus, cytosolic AcCoA is a signalling metabolite that connects metabolism to mitophagy through its receptor NLRX1.
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Nature
Anti-progestin therapy targets hallmarks of breast cancer risk
Breast cancer is the leading cause of cancer-related death in women worldwide1. Here, in the Breast Cancer-Anti-Progestin Prevention Study 1 (BC-APPS1; NCT02408770 ), we assessed whether progesterone receptor antagonism with ulipristal acetate for 12 weeks reduces surrogate markers of breast cancer risk in 24 premenopausal women. We used multilayered OMICs and live-cell approaches as readouts for molecular features alongside clinical imaging and tissue micromechanics correlates. Ulipristal acetate reduced epithelial proliferation (Ki67) and the proportion, proliferation and colony formation capacity of luminal progenitor cells, the putative cell of origin of aggressive breast cancers2. MRI scans showed reduction in fibroglandular volume with treatment, whereas single-cell RNA sequencing, proteomics, histology and atomic force microscopy identified extracellular matrix remodelling with reduced collagen organization and tissue stiffness. Collagen VI was the most significantly downregulated protein after ulipristal acetate treatment, and we uncovered an unanticipated spatial association between collagen VI and SOX9high luminal progenitor cell localization, establishing a link between collagen organization and luminal progenitor activity. Culture of primary human breast epithelial cells in a stiff environment increased luminal progenitor activity, which was antagonized by anti-progestin therapy, strengthening this mechanistic link. This study offers a template for biologically informed early-phase therapeutic cancer prevention trials and demonstrates the potential for premenopausal breast cancer prevention with progesterone receptor antagonists through stromal remodelling and luminal progenitor suppression. Results of an early-phase breast cancer prevention trial demonstrate the potential for breast cancer prevention in premenopausal women with anti-progestin therapy by inducing epithelial–stromal remodelling and suppression of luminal progenitors.
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Nature
Lymph node environment drives FSP1 targetability in metastasizing melanoma
Ferroptosis has emerged as an actionable target to eliminate therapy-resistant and metastatic cancers1. However, which ferroptosis surveillance systems may offer a therapeutic window to leverage redox maladaptation in cancer remains unclear. In melanoma, glutathione peroxidase 4 (GPX4) impedes ferroptosis during haematogenous metastasis, but is dispensable during lymphatic metastasis2. Here, using a metastatic mouse melanoma model selected for lymph node metastasis, we show that lymph-node-derived metastatic cells exhibit markedly diminished expression of glutamate–cysteine ligase (GCLC) and reduced glutathione (GSH) levels relative to their parental counterparts. This metabolic shift occurs within the hypoxic lymphatic niche. Under comparable low-oxygen conditions, GPX4 undergoes ubiquitination and proteasomal degradation. In response, lymph node metastatic cells acquire increased reliance on ferroptosis suppressor protein 1 (FSP1), which is localized with perinuclear lysosomes. These findings reveal that the reduced reliance on the GPX4 axis enables melanoma cells to shift toward FSP1 dependency. Notably, intratumoural monotherapy with selective FSP1 inhibitors (viFSP1 and FSEN1) effectively suppresses melanoma growth in lymph nodes, but not in subcutaneous tumours, emphasizing a microenvironment-specific dependency on FSP1. Thus, targeting FSP1 in the lymph nodes holds strong potential for blocking melanoma progression.
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Nature
Targeting FSP1 triggers ferroptosis in lung cancer
Emerging evidence indicates that cancer cells are susceptible to ferroptosis, a form of cell death that is triggered by uncontrolled lipid peroxidation1,2,3. Despite broad enthusiasm about harnessing ferroptosis as a novel anti-cancer strategy, whether ferroptosis is a barrier to tumorigenesis and can be leveraged therapeutically remains unknown4,5. Here, using genetically engineered mouse models of lung adenocarcinoma, we performed tumour-specific loss-of-function studies of two key ferroptosis suppressors, GPX46,7and ferroptosis suppressor protein 1 (FSP1)8,9, and observed increased lipid peroxidation and robust suppression of tumorigenesis, suggesting that lung tumours are highly sensitive to ferroptosis. Furthermore, across multiple pre-clinical models, we found that FSP1 was required for ferroptosis protection in vivo, but not in vitro, underscoring a heightened need to buffer lipid peroxidation under physiological conditions. Lipidomic analyses revealed thatFsp1-knockout tumours had an accumulation of lipid peroxides, and inhibition of ferroptosis with genetic, dietary or pharmacological approaches effectively restored the growth ofFsp1-knockout tumours in vivo. UnlikeGPX4, expression ofFSP1(also known asAIFM2) was prognostic for disease progression and poorer survival in patients with lung adenocarcinoma, highlighting its potential as a viable therapeutic target. To this end, we demonstrated that pharmacologic inhibition of FSP1 had significant therapeutic benefit in pre-clinical lung cancer models. Our studies highlight the importance of ferroptosis suppression in vivo and pave the way for FSP1 inhibition as a therapeutic strategy for patients with lung cancer.
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Nature
Evidence for improved DNA repair in long-lived bowhead whale
At more than 200 years, the maximum lifespan of the bowhead whale exceeds that of all other mammals. The bowhead is also the second-largest animal on Earth1, reaching over 80,000 kg. Despite its very large number of cells and long lifespan, the bowhead is not highly cancer-prone, an incongruity termed Peto’s paradox2. Here, to understand the mechanisms that underlie the cancer resistance of the bowhead whale, we examined the number of oncogenic hits required for malignant transformation of whale primary fibroblasts. Unexpectedly, bowhead whale fibroblasts required fewer oncogenic hits to undergo malignant transformation than human fibroblasts. However, bowhead whale cells exhibited enhanced DNA double-strand break repair capacity and fidelity, and lower mutation rates than cells of other mammals. We found the cold-inducible RNA-binding protein CIRBP to be highly expressed in bowhead fibroblasts and tissues. Bowhead whale CIRBP enhanced both non-homologous end joining and homologous recombination repair in human cells, reduced micronuclei formation, promoted DNA end protection, and stimulated end joining in vitro. CIRBP overexpression inDrosophilaextended lifespan and improved resistance to irradiation. These findings provide evidence supporting the hypothesis that, rather than relying on additional tumour suppressor genes to prevent oncogenesis3,4,5, the bowhead whale maintains genome integrity through enhanced DNA repair. This strategy, which does not eliminate damaged cells but faithfully repairs them, may be contributing to the exceptional longevity and low cancer incidence in the bowhead whale.
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Nature
SARS-CoV-2 mRNA vaccines sensitize tumours to immune checkpoint blockade
Immune checkpoint inhibitors (ICIs) extend survival in many patients with cancer but are ineffective in patients without pre-existing immunity1,2,3,4,5,6,7,8,9. Although personalized mRNA cancer vaccines sensitize tumours to ICIs by directing immune attacks against preselected antigens, personalized vaccines are limited by complex and time-intensive manufacturing processes10,11,12,13,14. Here we show that mRNA vaccines targeting SARS-CoV-2 also sensitize tumours to ICIs. In preclinical models, SARS-CoV-2 mRNA vaccines led to a substantial increase in type I interferon, enabling innate immune cells to prime CD8+T cells that target tumour-associated antigens. Concomitant ICI treatment is required for maximal efficacy in immunologically cold tumours, which respond by increasing PD-L1 expression. Similar correlates of vaccination response are found in humans, including increases in type I interferon, myeloid–lymphoid activation in healthy volunteers and PD-L1 expression on tumours. Moreover, receipt of SARS-CoV-2 mRNA vaccines within 100 days of initiating ICI is associated with significantly improved median and three-year overall survival in multiple large retrospective cohorts. This benefit is similar among patients with immunologically cold tumours. Together, these results demonstrate that clinically available mRNA vaccines targeting non-tumour-related antigens are potent immune modulators capable of sensitizing tumours to ICIs.
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Nature
Parity and lactation induce T cell mediated breast cancer protection
Parity and breastfeeding reduce the risk of breast cancer, particularly triple-negative breast cancer (TNBC)1,2, yet the immunological mechanisms underlying this protection remain unclear. Here, we show that parity induces an accumulation of CD8+T cells, including cells with a tissue-resident memory (TRM)-like phenotype within human normal breast tissue. In murine models, pregnancy followed by lactation and involution drove the accumulation of CD8⁺ T cells in the mammary gland, coinciding with reduced tumour growth and increased intratumoural immune cell infiltration, effects that were abrogated by CD8⁺ T cell depletion. Importantly, this CD8+T cell dependent tumour control was only observed following a complete cycle of lactation and involution. Consistent with this, primary TNBCs from parous women exhibited greater T cell infiltration and improved clinical outcomes. Together these findings, spanning preclinical models and over 1000 patient samples, provide new insight into how reproductive history shapes breast immunity, positioning CD8⁺ T cells as key mediators of parity-associated protection and informing novel strategies for both prevention and treatment of breast cancer.
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Nature
Neoadjuvant immunotherapy in mismatch-repair-proficient colon cancers
Immune checkpoint blockade (ICB) has led to paradigm shifts in the treatment of various tumour types1-4, yet limited efficacy has been observed in patients with metastatic mismatch-repair proficient (pMMR) colorectal cancer5. Here we report clinical results and in-depth analysis of patients with early-stage pMMR colon cancer from the phase II NICHE study (ClinicalTrials.gov: NCT03026140). A total of 31 patients received neoadjuvant treatment of nivolumab plus ipilimumab followed by surgery. The response rate was 26% and included six patients with a major pathological response (≤10% residual viable tumour). One patient with an ongoing clinical complete response did not undergo surgery. Circulating tumour DNA (ctDNA) was positive in 26/31 patients at baseline, and clearance was observed in 5/6 responders prior to surgery, while 19/20 non-responders remained ctDNA+. Responses were observed despite a low tumour mutational burden in all tumours, while chromosomal genomic instability scores were significantly higher in responders compared to non-responders. Furthermore, responding tumours had significantly higher baseline expression of proliferation signatures and TCF1, and imaging mass cytometry revealed a higher percentage of Ki-67+cancer and Ki-67+CD8+T cells in responders compared to non-responders. These results provide a comprehensive analysis of response to neoadjuvant ICB in early-stage pMMR colon cancers and identify potential biomarkers for patient selection.
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Nature
Sex and smoking bias in the selection of somatic mutations in human bladder
Men are at higher risk of several cancer types than women1. For bladder cancer the risk is four times higher for reasons that are not clear2. Smoking is also a principal risk factor for several tumour types, including bladder cancer3. As tumourigenesis is driven by somatic mutations, we wondered whether the landscape of clones in the normal bladder differs by sex and smoking history. Using ultradeep duplex DNA sequencing (approximately 5,000×), we identified thousands of clonal driver mutations in 16 genes across 79 normal bladder samples from 45 people. Men had significantly more truncating driver mutations inRBM10,CDKN1AandARID1Athan women, despite similar levels of non-protein-affecting mutations. This result indicates stronger positive selection on driver truncating mutations in these genes in the male urothelium. We also found activatingTERTpromoter mutations driving clonal expansions in the normal bladder that were associated strongly with age and smoking. These findings indicate that bladder cancer risk factors, such as sex and smoking, shape the clonal landscape of the normal urothelium. The high number of mutations identified by this approach offers a new strategy to study the functional effect of thousands of mutations in vivo—natural saturation mutagenesis—that can be extended to other human tissues.
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Nature
Somatic mutation and selection at population scale
As we age, many tissues become colonized by microscopic clones carrying somatic driver mutations1,2,3,4,5,6,7. Some of these clones represent a first step towards cancer whereas others may contribute to ageing and other diseases. However, our understanding of this phenomenon remains limited due to the challenge of detecting mutations in small clones. Here we introduce a new version of nanorate sequencing (NanoSeq)8, a duplex sequencing method with an error rate lower than five errors per billion base pairs, which is compatible with whole-exome and targeted capture. Deep sequencing of polyclonal samples with single-molecule sensitivity simultaneously profiles large numbers of clones, providing accurate mutation rates, signatures and driver frequencies in any tissue. Applying targeted NanoSeq to 1,042 non-invasive samples of oral epithelium and 371 blood samples from a twin cohort, we report an extremely rich selection landscape, with 46 genes under positive selection in oral epithelium, more than 62,000 driver mutations and evidence of negative selection in essential genes. High-resolution maps of selection across coding and non-coding sites are obtained for many genes: a form of in vivo saturation mutagenesis. Multivariate regression models enable mutational epidemiology studies on how exposures and cancer risk factors, such as age, tobacco or alcohol, alter the acquisition or selection of somatic mutations. Accurate single-molecule sequencing provides a powerful tool to study early carcinogenesis, cancer prevention and the role of somatic mutations in ageing and disease.
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Nature
Tracking clonal evolution during treatment in ovarian cancer using cell-free DNA
Emergence of drug resistance is the main cause of therapeutic failure in patients with high-grade serous ovarian cancer (HGSOC)1. To study drug resistance in patients, we developed CloneSeq-SV, which combines single-cell whole-genome sequencing2with targeted deep sequencing of clone-specific genomic structural variants in time-series cell-free DNA. CloneSeq-SV exploits tumour clone-specific structural variants as highly sensitive endogenous cell-free DNA markers, enabling the relative abundance measurements and evolutionary analysis of co-existing clonal populations over the therapeutic time course. Here, using this approach, we studied 18 patients with HGSOC over a multi-year period from diagnosis to recurrence and showed that drug resistance typically arose from selective expansion of a single or small subset of clones present at diagnosis. Drug-resistant clones frequently showed interpretable and distinctive genomic features, including chromothripsis, whole-genome doubling, and high-level amplifications of oncogenes such asCCNE1,RAB25,MYCandNOTCH3. Phenotypic analysis of matched single-cell RNA sequencing data3indicated pre-existing and clone-specific transcriptional states such as upregulation of epithelial-to-mesenchymal transition and VEGF pathways, linked to drug resistance. In one notable case, clone-specificERBB2amplification affected the efficacy of a secondary targeted therapy with a positive patient outcome. Together, our findings indicate that drug-resistant states in HGSOC pre-exist at diagnosis, leading to positive selection and reduced clonal complexity at relapse. We suggest these findings motivate investigation of evolution-informed adaptive treatment regimens to ablate drug resistance in future HGSOC studies.
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Nature
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Neuroblastoma is a highly lethal childhood tumour derived from differentiation-arrested neural crest cells1,2. Like all cancers, its growth is fuelled by metabolites obtained from either circulation or local biosynthesis3,4. Neuroblastomas depend on local polyamine biosynthesis, and the inhibitor difluoromethylornithine has shown clinical activity5. Here we show that such inhibition can be augmented by dietary restriction of upstream amino acid substrates, leading to disruption of oncogenic protein translation, tumour differentiation and profound survival gains in theTh-MYCNmouse model. Specifically, an arginine- and proline-free diet decreases the amount of the polyamine precursor ornithine and enhances tumour polyamine depletion by difluoromethylornithine. This polyamine depletion causes ribosome stalling, unexpectedly specifically at codons with adenosine in the third position. Such codons are selectively enriched in cell cycle genes and low in neuronal differentiation genes. Thus, impaired translation of these codons, induced by combined dietary and pharmacological intervention, favours a pro-differentiation proteome. These results suggest that the genes of specific cellular programmes have evolved hallmark codon usage preferences that enable coherent translational rewiring in response to metabolic stresses, and that this process can be targeted to activate differentiation of paediatric cancers.
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Nature
Systematic discovery of CRISPR-boosted CAR T cell immunotherapies
Chimeric antigen receptor (CAR) T cell therapy has shown remarkable success in treating blood cancers, but CAR T cell dysfunction remains a common cause of treatment failure1. Here we present CELLFIE, a CRISPR screening platform for enhancing CAR T cells across multiple clinical objectives. We performed genome-wide screens in human primary CAR T cells, with readouts capturing key aspects of T cell biology, including proliferation, target cell recognition, activation, apoptosis and fratricide, and exhaustion. Screening hits were prioritized using a new in vivo CROP-seq2 method in a xenograft model of human leukaemia, establishing several gene knockouts that boost CAR T cell efficacy. Most notably, we discovered that RHOG knockout is a potent and unexpected CAR T cell enhancer, both individually and together with FAS knockout, which was validated across multiple in vivo models, CAR designs and sample donors, and in patient-derived cells. Demonstrating the versatility of the CELLFIE platform, we also conducted combinatorial CRISPR screens to identify synergistic gene pairs and saturation base-editing screens to characterize RHOG variants. In summary, we discovered, validated and biologically characterized CRISPR-boosted CAR T cells that outperform standard CAR T cells in widely used benchmarks, establishing a foundational resource for optimizing cell-based immunotherapies. CELLFIE, a CRISPR platform for optimizing cell-based immunotherapies, identifies gene knockouts that enhance CAR T cell efficacy using in vitro and in vivo screens.
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Nature
Basal cell of origin resolves neuroendocrine–tuft lineage plasticity in cancer
Neuroendocrine and tuft cells are rare chemosensory epithelial lineages defined by the expression of ASCL1 and POU2F3 transcription factors, respectively. Neuroendocrine cancers, including small cell lung cancer (SCLC), frequently display tuft-like subsets, a feature linked to poor patient outcomes1,2,3,4,5,6,7,8,9. The mechanisms driving neuroendocrine–tuft tumour heterogeneity and the origins of tuft-like cancers are unknown. Using multiple genetically engineered animal models of SCLC, we demonstrate that a basal cell of origin (but not the accepted neuroendocrine origin) generates neuroendocrine–tuft-like tumours that highly recapitulate human SCLC. Single-cell clonal analyses of basal-derived SCLC further uncovered unexpected transcriptional states, including anAtoh1+state, and lineage trajectories underlying neuroendocrine–tuft plasticity. Uniquely in basal cells, the introduction of genetic alterations enriched in human tuft-like SCLC, including high MYC, PTEN loss and ASCL1 suppression, cooperates to promote tuft-like tumours. Transcriptomics of 944 human SCLCs revealed a basal-like subset and a tuft–ionocyte-like state that altogether demonstrate notable conservation between cancer states and normal basal cell injury response mechanisms10,11,12,13. Together, these data indicate that the basal cell is a probable origin for SCLC and other neuroendocrine–tuft cancers that can explain neuroendocrine–tuft heterogeneity, offering new insights for targeting lineage plasticity.