Metastatic disease remains a major cause of cancer-related mortality. Recent studies suggest that dissemination to other organs comes with metabolic changes that allow the metastasizing cancer cells to adapt to new microenvironments. A deeper knowledge of these specific metabolic features and associated vulnerabilities could lead to the development of more effective therapies against metastasis. We used in vivo and ex vivo models of MYC-driven breast tumorigenesis to explore the key metabolic pathways that change when mammary gland tumor cells metastasize to the lung. Stable isotope-resolved metabolomics, mass spectrometry imaging, and single-cell RNA sequencing demonstrated that mammary gland tumor-derived lung metastases have increased synthesis of glutathione fueled by increased cystine uptake. Metastatic cells relied heavily on the availability of extracellular cysteine or cystine, possibly due to downregulated intracellular cysteine synthesis through the transsulfuration pathway. When combined with focal radiotherapy, the amino acid degrader cyst(e)inase effectively reduced metastatic burden in the lungs. Together, these findings show that targeting cystine/cysteine exploits a metabolic dependency that is unique to metastatic cells and acts as a sensitizer to radiotherapy-induced oxidative stress, offering a promising targeted strategy.

Transcription initiation is a highly dynamic and tightly regulated process involving the coordinated action of transcription factors, chromatin remodelers, and RNA polymerase, which determine where and when transcription begins. Accurately mapping and quantifying transcription start sites (TSSs) from nascently transcribed RNAs remains a key area of interest, as it provides critical insights into transcription dynamics. Here, we combine transient transcriptome sequencing with transcription start site sequencing (TT-TSS-seq) to accurately map and quantify transcription initiation sites from nascent transcripts. Because transient metabolic labeling yields low-input RNA, we optimize the TSS-seq protocol to enhance sensitivity and accuracy. Specifically, we refine enzymatic reactions for decapping and RNA ligation and incorporate 5 ' oligonucleotides containing unique molecular identifiers (UMIs) and barcodes to enable accurate quantification and sample multiplexing. The TT-TSS-seq approach detects transcription initiation of unstable transcripts, such as enhancer RNAs. Moreover, we show that a large fraction of genes use multiple transcription initiation sites, yet often produce only a single stable transcript. Overall, TT-TSS-seq provides precise mapping and quantification of transcription initiation sites, offering new insights into transcriptional dynamics and expanding the toolkit for studying gene regulation.

Supplementary Figure 1 Surfactant lipids are enriched in the vicinity of lung metastases from patients with breast cancer; Supplementary Figure 2 AT2 cells and surfactant lipids co-localize in the vicinity of metastases from mice; Supplementary Figure 3 Metastases progression increases the enrichment of AT2 cells; Supplementary Figure 4 The metastasis secretome reprograms AT2 cell lipid metabolism by activating SREBP-1; Supplementary Figure 5 Gpam knockdown in cancer cells does not affect 3D spheroid growth in vitro; Supplementary Figure 6 GPAM and FASN inhibition in cancer cells does not affect metastasis formation in vitro and in vivo

Insertion of fluorescent reporter genes into viral genomes is a powerful tool for monitoring infection. In coronaviruses, this is commonly achieved by replacing accessory ORFs, thereby deleting endogenous gene functions. An alternative strategy is to manipulate viral RNA synthesis by inserting copies of the viral transcription regulatory sequence (TRS), which drives the transcription of viral subgenomic RNAs. However, coronavirus transcription is tightly regulated, and these modifications frequently disrupt native subgenomic RNA synthesis and attenuate viral growth. Here, we describe a reporter coronavirus that overcomes these limitations. Using human coronavirus (HCoV)- OC43 as a model system, we inserted an mNeonGreen reporter between the Spike and ORF5 coding regions, engineering the TRS and surrounding sequence to minimize off- target effects to transcription. This virus is genetically stable, with WT growth kinetics and unaltered subgenomic RNA transcriptional ratios. We developed a flexible reverse genetics system, which allows rapid cloning and virus recovery, supported by optimized HCoV- OC43 culture conditions for high- titre stock generation, and validated analytical reagents. Our reporter virus enabled sensitive detection and isolation of infected cells, facilitating transcriptomic analyses that distinguish host responses in infected and bystander populations based on active viral translation. We found that transcriptional responses to infection of A549 human lung epithelial cells were predominantly inflammatory, rather than interferon- mediated, and that bystander cells upregulated pathways associated with cytokine response signalling and cell-cell contact sensing. Together, these tools expand the experimental utility of HCoVOC43, an important seasonal respiratory pathogen and low- containment model for betacoronavirus biology.

Short hairpin RNAs (shRNAs) used to generate knockdown cell lines

Transcription start site (TSS) selection diversifies the transcriptome and proteome, yet how alternative TSSs regulate development remains unclear. We show that the chromatin regulator ASH2L undergoes developmentally regulated alternative TSS switching in differentiating mouse cells, generating distinct mRNA and protein isoforms: a full-length ASH2L and a truncated form lacking an intrinsically disordered region (IDR). While both ASH2L isoforms are conserved across mammals, their regulation in mouse pluripotent stem cells is uniquely driven by a mouse-specific retrotransposon. This element suppresses transcription from the downstream TSS through a transcription interference mechanism involving SETD2-directed histone H3 lysine 36 methylation. The resulting stem cell-specific truncated ASH2L isoform primes developmental gene promoters for histone H3 lysine 4 methylation, establishing a chromatin state required for embryogenesis and motor neuron differentiation. These findings show that co-option of a mouse-specific retrotransposon rewires Ash2l TSS and protein isoform usage to control developmental timing and cell fate decisions.

Cancer cells that seed in the lung require lipids often produced by alveolar type II (AT2) cells. However, whether overt metastases depend on AT2 cell-derived lipids and whether AT2 cells can be targeted to reduce metastasis growth remains unknown. We discovered that breast cancer-derived lung metastases stimulate the proliferation of AT2 cells in their vicinity and reprogram them into lipid feeder cells in mice and patients using spatial analysis. Mechanistically, the metastasis secretome activates the transcription factor sterol regulatory element-binding transcription factor 1 (SREBP-1) in AT2 cells, enhancing the expression of key de novo lipid synthesis genes, including fatty acid synthase (FASN) and glycerol-3-phosphate acyltransferase 1 (GPAM). Deleting Fasn selectively in AT2 cells or targeting FASN and GPAM systemically significantly impairs lung metastasis growth in mice. In summary, we discovered that overt metastases reprogram AT2 cells and that targeting the lipid metabolism of AT2 cells impairs metastasis growth. SIGNIFICANCE:Current therapies in oncology targeting the cancer or immune cell compartment of tumors show limited efficacy against breast cancer-derived metastases. We discovered that decreasing the lipid metabolism of lung resident AT2 cells is sufficient to impair lung metastasis growth in mice without apparent adverse effects.

Radiotherapy is known to cause changes in the tumour stroma which can undermine treatment efficacy. Our understanding of this process has historically centred around effects driven by Transforming Growth Factor-beta (TGF-β) and alpha-smooth muscle actin (α-SMA)+ fibroblasts. Here, we identified a rapid expansion of podoplanin (PDPN)+ fibroblasts following radiotherapy in breast, head and neck and melanoma tumours. This fibrosis was not dependent on TGF-β, but was downstream of a radiotherapy-induced adaptive immune response. CD8+ T-cells entering the tumour after radiation were sequestered at the interface between residual tumour cells and PDPN+ fibroblasts and failed to enter the tumour core. Genetic deletion of PDPN in fibroblasts impacted their cytoskeleton and ability to organise extracellular matrix. This was associated with increased CD8+ T-cell entry and spontaneous tumour regression. Overall, we identify a mechanism whereby PDPN+ fibrosis limits immune-mediated radiation cell kill and demonstrate that disruption of PDPN signalling favours tumour control. Significance In this study we show that rapid podoplanin (PDPN)+ fibroblast expansion following radiotherapy limits immune-mediated radiation cell kill. Targeting PDPN and associated downstream signalling improves tumour control and is a promising strategy in combination with radiotherapy. ![Figure][1] ### Competing Interest Statement A.W. reports research funding from Artera AI, AstraZeneca, Roche Genentech, Veracyte and speaker honoraria from Johnson and Johnson. E.S. reports grants from Novartis, Merck Sharp Dohme, AstraZeneca and personal fees from Phenomic outside the submitted work. GG receives funding from Merck Sharp & Dohme Corp, New Jersey, USA (LKR190557). AB receives PhD funding from AstraZeneca. BOL reports research funding from Pfizer, consultancy and/or speaker honoraria from Pfizer, Merck Serono, Merck, Eisai, Oliver Wyman, Outrun and funding for travel/meeting attendance from Merck Serono and Pfizer. Cancer Research UK, https://ror.org/054225q67, RCCCSF-Nov24/100003, CRUK-A19763, RCCSCF-May22\100001., CC2040, RMH/ICR CRUK RadNet, City of London CRUK RadNet Medical Research Council, https://ror.org/03x94j517, CC2040 Wellcome Trust, https://ror.org/029chgv08, CC2040 European Research Council, https://ror.org/0472cxd90, ERC Advanced Grant CAN_ORGANISE, Grant agreement number 101019366 [1]: pending:yes

Hutchinson-Gilford Progeria Syndrome (HGPS), characterised by accelerated ageing, causes cardiovascular defects resembling aspects of cardiovascular ageing. We used human left ventricle cardiomyocytes (CMs) derived from HGPS-induced pluripotent stem cells (iPSCs), and their isogenic-corrected controls, to investigate HGPS-CM dysfunction and identify potential therapies. Our results revealed that HGPS-iPSC-CMs exhibit greater maturity and associated elevated oxidative stress compared to controls, which they could not contend with, leading to cellular senescence. Increased senescence was also observed in cardiac tissue from mouse and human physiologically-aged and HGPS individuals. Functionally, HGPS-iPSC-CMs showed dysregulated mitochondrial respiration and calcium handling. Amongst the six drugs tested, rapamycin and lonafarnib were the most effective against HGPS-cardiac phenotypes. Although lonafarnib raised safety concerns, it partially reverted the cardiac senescent phenotype by inducing cellular autophagy and decreasing progerin expression in progeroid mice. Our study supports the use of HGPS-iPSC-CMs to identify novel biomarkers and therapies for HGPS, and potentially cardiac physiological-ageing. ### Competing Interest Statement The Francis Crick Institute has filed a patent application related to the left ventricle differentiation protocol used in this work (WO 2020/245612), and A.S.B. is listed as an inventor. The Francis Crick Institute has granted an exclusive license to Axol Bioscience to commercialise the protocol for the generation and sale of cardiomyocytes for R&D and the provision of contract research services. N.D., J.C.S., and A.S.B. may benefit from this license. The rest of the authors have no conflict of interest to disclose. FCT, 2022.07615.PTDC, PTDC/BTM-SAL/5174/2020 FEDER, Program COMPETE European projects, RESETaging (ref. 952266), CHAngeing - Connected Hubs in Ageing: Healthy Living to Protect Cerebrovascular Function” (Ref. 101087071), DREAMs - Drug Repurposing with Artificial Intelligence for Muscular Disorders” (Ref: 101080229), PRR project “HfPT” Wellcome Trust, https://ror.org/029chgv08, 210987/Z/18/Z Francis Crick Institute, Cancer Research UK (FC001157), UK Medical Research Council (FC001157), Wellcome Trust (FC001157) Germany

The tRNA nuclease SLFN11 is epigenetically silenced in ∼50% of treatment-naive tumours and is the strongest predictor of chemoresistance but why it is frequently inactivated in cancer is unknown. To acquire immortality, cancer cells can activate alternative lengthening of telomeres (ALT), typically accompanied by ATRX loss. Here, we implicate SLFN11 in sensing telomere replication stress, triggering eradication of ATRX deficient cells prior to ALT establishment. Whereas progressive telomere shortening of cells lacking telomerase and ATRX leads to telomere crisis and cell death, SLFN11 loss confers tolerance to PML-BLM dependent ALT intermediates, permitting emergence of ALT survivors. We propose that during tumorigenesis SLFN11 inactivation is selected as means to tolerate endogenous replication stress following telomere crisis, leading to the development of therapy resistant tumours before treatment. ### Competing Interest Statement S.S-B., A.I.I., S.L. and S.J.B. are inventors on patent WO2024/240908 that relates to the treatment and/or prevention of ALT-positive cancers. S.J.B. is a co-founder and shareholder at Artios Pharma Ltd. The other authors declare no competing interests.

The transcription factor FOXL2 is required in ovarian somatic cells for female fertility. Differential timing of Foxl2 deletion, in embryonic versus adult mouse ovary, leads to distinctive outcomes, suggesting different roles across development. Here, we comprehensively investigated FOXL2's role through a multi-omics approach to characterize gene expression dynamics and chromatin accessibility changes, coupled with genome-wide identification of FOXL2 targets and on-chromatin interacting partners in somatic cells across ovarian development. We found that FOXL2 regulates more targets postnatally, through interaction with factors regulating primordial follicle formation and steroidogenesis. Deletion of one interactor, ubiquitin-specific protease 7 (Usp7), results in impairment of somatic cell differentiation, germ cell nest breakdown, and ovarian development, leading to sterility. Our datasets constitute a comprehensive resource for exploration of the molecular mechanisms of ovarian development and causes of female infertility.

The biological purpose of Integrator and RNA polymerase II (RNAPII) promoter-proximal pausing remains uncertain. Here, we show that loss of INTS6 in human cells results in increased interaction of RNAPII with proteins that can mediate its dissociation from the DNA template, including the CRL3ARMC5 E3 ligase, which ubiquitylates CTD serine5-phosphorylated RPB1 for degradation. ARMC5-dependent RNAPII ubiquitylation is activated by defects in factors acting at the promoter-proximal pause, including Integrator, DSIF, and capping enzyme. This ARMC5 checkpoint normally curtails a sizeable fraction of RNAPII transcription, and ARMC5 knockout cells produce more uncapped transcripts. When both the Integrator and CRL3ARMC5 turnover mechanisms are compromised, cell growth ceases and RNAPII with high pausing propensity disperses from the promoter-proximal pause site into the gene body. These data support a model in which CRL3ARMC5 functions alongside Integrator in a checkpoint mechanism that removes faulty RNAPII complexes at promoter-proximal pause sites to safeguard transcription integrity.

Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and transcription factor IIH (TFIIH) around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryoelectron microscopy (cryo-EM) and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.

Supplementary Materials and Methods Supplementary Table S1 - Sequencing coverage for tumor regions and germline samples Supplementary Table S2 - Clinical characteristics of patients with EAC Supplementary Table S4 - Annotation of chromosomal segments that overlap with TCGA ESCA recurrent amplifications and tumor regions in our EAC cohort Supplementary Figure S1 - Mutations identified using M-seq compared with a single biopsy Supplementary Figure S2 - Relationship of intratumor heterogeneity and response to NAC treatment Supplementary Figure S3 - Tumor Phylograms Supplementary Figure S4 - Copy number events lead to mutational heterogeneity Supplementary Figure S5 - Heatmap of the all driver mutations identified across all tumor regions Supplementary Figure S6 - Driver mutations identified using M-seq compared with a single biopsy Supplementary Figure S7 - Copy number states across the genome for each tumor region Supplementary Figure S8 - Chromosome view of chromosome 19, tumor sample EAC017, region R1, demonstrating chromothripsis Supplementary Figure S9 - wGII scores for the TCGA ESCA cohort and M-seq EAC cohort Supplementary Figure S10 - The trinucleotide context for temporally dissected combined EAC cohort Supplementary Figure S11 - Assessing copy number heterogeneity using a minimum consecutive segment method

During embryonic development, mutually antagonistic signaling cascades determine gonadal fate toward a testicular or ovarian identity. Errors in this process result in disorders of sex development (DSDs), characterized by discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells toward gonadal progenitors. Transcriptomic analysis reveals that the in vitro–derived murine gonadal cells are equivalent to embryonic day 11.5 in vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete anti-Müllerian hormone, migrate, and form tubular structures. Cells derived from 46,XY DSD female hiPSCs, carrying an NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR-Cas9–mediated variant correction rescued the phenotype. This is a robust tool to understand mechanisms of sex determination and model DSDs.

The evolution of established cancers is driven by selection of cells with enhanced fitness. Subclonal mutations in numerous epigenetic regulator genes are common across cancer types, yet their functional impact has been unclear. Here, we show that disruption of the epigenetic regulatory network increases the tolerance of cancer cells to unfavorable environments experienced within growing tumors by promoting the emergence of stress-resistant subpopulations. Disruption of epigenetic control does not promote selection of genetically defined subclones or favor a phenotypic switch in response to environmental changes. Instead, it prevents cells from mounting an efficient stress response via modulation of global transcriptional activity. This "transcriptional numbness" lowers the probability of cell death at early stages, increasing the chance of long-term adaptation at the population level. Our findings provide a mechanistic explanation for the widespread selection of subclonal epigenetic-related mutations in cancer and uncover phenotypic inertia as a cellular trait that drives subclone expansion.

RNA polymerase II (RNAPII) transcription involves initiation from a promoter, transcriptional elongation through the gene, and termination in the terminator region. In bacteria, terminators often contain specific DNA elements provoking polymerase dissociation, but RNAPII transcription termination is thought to be driven entirely by protein co-factors. We used biochemical reconstitution, single-molecule studies, and genome-wide analysis in yeast to study RNAPII termination. Transcription into natural terminators by pure RNAPII results in spontaneous termination at specific sequences containing T-tracts. Single-molecule analysis indicates that termination involves pausing without backtracking. The "torpedo"Rat1-Rai1 exonuclease (XRN2 in humans) greatly stimulates spontaneous termination but is ineffectual on other paused RNAPIIs. By contrast, elongation factor Spt4-Spt5 (DSIF) suppresses termination. Genome-wide analysis further indicates that termination occurs by transcript cleavage at the poly(A) site exposing a new 50 RNA-end that allows Rat1-Rai1 loading, which then catches up with destabilized RNAPII at specific termination sites to end

PDF file, 20K, Results of DAVID bioinformatic analysis.