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Leptomeningeal metastatic disease (LMD), encompassing entities of 'meningeal carcinomatosis', neoplastic meningitis' and 'leukaemic/lymphomatous meningitis', arises secondary to the metastatic dissemination of cancer cells from extracranial and certain intracranial malignancies into the leptomeninges and cerebrospinal fluid. The clinical burden of LMD has been increasing secondary to more sensitive diagnostics, aggressive local therapies for discrete brain metastases, and improved management of extracranial disease with targeted and immunotherapeutic agents, resulting in improved survival. However, owing to drug delivery challenges and the unique microenvironment of LMD, novel therapies against systemic disease have not yet translated into improved outcomes for these patients. Underdiagnosis and misdiagnosis are common, response assessment remains challenging, and the prognosis associated with this disease of whole neuroaxis remains extremely poor. The dearth of effective therapies is further challenged by the difficulties in studying this dynamic disease state. In this Review, a multidisciplinary group of experts describe the emerging evidence and areas of active investigation in LMD and provide directed recommendations for future research. Drawing upon paradigm-changing advances in mechanistic science, computational approaches, and trial design, the authors discuss domain-specific and cross-disciplinary strategies for optimizing the clinical and translational research landscape for LMD. Advances in diagnostics, multi-agent intrathecal therapies, cell-based therapies, immunotherapies, proton craniospinal irradiation and ongoing clinical trials offer hope for improving outcomes for patients with LMD., Competing Interests: Competing interests: S.A.G. has been a paid consultant for Plus Therapeutics. A.S. has been a paid consultant for Abbvie, BrainLAB, Elekta AB, Merck, Roche and Varian; has received honoraria for past educational seminars from Accuray, AstraZeneca, BrainLAB, Elekta AB, Seagen and Varian; has received research grants from BrainLAB, Elekta AB and Seagen; has received support for travel accommodation/expenses from BrainLAB, Elekta AB and Varian; belongs to the Elekta MR Linac Research Consortium; and is a Clinical Steering Committee Member and chairs the Elekta Oligometastases Group and the Elekta Gamma Knife Icon Group. E.L.R. has received research grants from Bristol Myers Squibb and honoraria for lectures or as an advisory board member from Bayer, Janssen, Leo Pharma, Pierre Fabre, Seattle Genetics and Servier. M.W. has received research grants from Quercis and Versameb and honoraria for lectures, advisory board membership and/or a paid consultancy for Bayer, Curevac, Medac, Merck, Novartis, Novocure, Orbus, Philogen, Roche and Sandoz. T.W. has received research grants from Philogen and honoraria for lectures, advisory board membership and consultancy from Philogen. T.T.B. has received grants from ONO pharmaceuticals through their institution and receives royalties from Oxford University Press and UpToDate, Inc. P.Y.W. has received research grants from AstraZeneca, Black Diamond, Bristol Myers Squibb, Celgene, Chimerix, Eli Lilly, Erasca, Genentech/Roche, Kazia, MediciNova, Merck, Novartis, Nuvation Bio, Servier, Vascular Biogenics, and VBI Vaccines and has been a paid consultant/advisory board member for AstraZeneca, Bayer, Black Diamond, Boehringer Ingelheim, Boston Pharmaceuticals, Celularity, Chimerix, Day One Bio, Genenta, Glaxo Smith Kline, Karyopharm, Merck, Mundipharma, Novartis, Novocure, Nuvation Bio, Prelude Therapeutics, Sapience, Servier, Sagimet, Vascular Biogenics and VBI Vaccines. D.A.H.-K. has had leadership roles in EmpNia, Graegis Pharmaceuticals and TeleRad Oncology; received honoraria from Cincinnati Children’s Hospital DSMC, Defense Finance Service Agent DHHS, General Dynamic Information Tech, and Inc Reviewer; has had consulting or advisory roles at Gerson Lehrman Group and Guidepoint Global; received research funding from Artios; and provided expert testimony to Bendin, Bowman and Brooke, Sumrall and Ladner, and The EMMES Company. M.K. reports grants or contracts from AbbVie, BioNTech, BMS, CNS Pharmaceuticals, Daiichi Sankyo, Immorna Therapeutics, Immvira Therapeutics, JAX lab for genomic research, and Personalis; received consulting fees from AnHeart Therapeutics, Berg Pharma, George Clinical, Menarini Stemline and Servier; received honoraria from GSK; and is on a data safety monitoring board for BPG Bio. R.R. has been an advisory board member and/or received research grants from Bayer, Curevac, Novocure and Servier. C.B. has been a paid consultant for Bionaut Labs, Depuy-Synthes, Haystack Oncology, and Privo Technologies and is a co-founder of Belay Diagnostics and OrisDx. G.F.W. has received grants from Focused Ultrasound Foundation and clinical trial support from Insightec. R.S. has been an advisory board member and/or received research grants from Medacc, Seagen and Servier. P.V. has been a paid consultant for and owns stock in Need Inc.; has received honoraria for lectures from Bayer; and has been an advisory board member for Cercare Medical. V.S. reports grants and/or contracts from AbbVie, AgenSys, Alfa‐Sigma, Altulm, Amgen, Bayer HealthCare Pharmaceuticals, Berghealth, Blueprint Medicines, Boston Biomedical, Boston Pharmaceuticals, Celgene Corporation, Dragonfly Therapeutics, Exelixis, F. Hoffmann‐La Roche AG, FUJIFILM Medical Systems USA, Genentech USA, GlaxoSmithKline, Idera Pharma, Incyte Corporation, Inhibrx, LOXO Oncology, MedImmune, Multivir, Nanocarrier, the National Cancer Institute, the National Comprehensive Cancer Network, Novartis Pharma, OncLive, Oncusp, PERS, Pfizer, PharmaMar, Relay Therapeutics and Takeda Oncology; personal and/or consulting fees from AbbVie, Bayer HealthCare, Pharmaceuticals, F. Hoffmann‐La Roche AG, Illumina, Incyte Corporation, Jazz Pharmaceuticals, LabCorp, Regeneron Pharmaceuticals and Relay Therapeutics; support for other professional activities from Aadi Biosciences, Clinical Care Communications, Illumina, Jazz Pharmaceuticals, Med Learning Group, Medscape, and Phenon Therapeutics; and travel support from ASCO, ESMO, Helsinn Healthcare, and Incyte Corporation outside the submitted work. M.S.A. has received grants from AstraZeneca, Bayer, Bristol Myers Squibb, Incyte, Pharmacyclics, Merck, MimiVax and Novocure; has been a paid consultant for Anheart Therapeutics, Apollomics, Bayer, Cairn Therapeutics, Caris Lifesciences, Cellularity, GSK, Insightec, Janssen, Kiyatec, Novocure, Nuvation, Prelude, Pyramid Biosciences, SDP Oncology, Theraguix, Tocagen, Varian Medical Systems, Viewray, Voyager Therapeutics and Xoft; is a scientific advisory board member for Cairn Therapeutics, Modifi Biosciences and Pyramid Biosciences; and is a stock shareholder in Cytodyn, MedInnovate Advisors and MimiVax. A.O., H.W., D.B., N.M., M.G., L.C.P. and A.M. declare no competing interests., (© 2024. Springer Nature Limited.)

Chronic lymphocytic leukemia is a malignant lymphoproliferative disorder for which primary or acquired drug resistance represents a major challenge. To investigate the underlying molecular mechanisms, we generate a mouse model of ibrutinib resistance, in which, after initial treatment response, relapse under therapy occurrs with an aggressive outgrowth of malignant cells, resembling observations in patients. A comparative analysis of exome, transcriptome and proteome of sorted leukemic murine cells during treatment and after relapse suggests alterations in the proteasome activity as a driver of ibrutinib resistance. Preclinical treatment with the irreversible proteasome inhibitor carfilzomib administered upon ibrutinib resistance prolongs survival of mice. Longitudinal proteomic analysis of ibrutinib-resistant patients identifies deregulation in protein post-translational modifications. Additionally, cells from ibrutinib-resistant patients effectively respond to several proteasome inhibitors in co-culture assays. Altogether, our results from orthogonal omics approaches identify proteasome inhibition as potentially attractive treatment for chronic lymphocytic leukemia patients resistant or refractory to ibrutinib., Competing Interests: Competing interests: S.S.S. has received honoraria from AbbVie, AstraZeneca, BeiGene, and Janssen, and research support from BeiGene and TG Therapeutics outside of this work. E.T. has received honoraria from Abbvie, Janssen, AstraZeneca, BeiGene and research support by Abbvie, Hofmann-La Roche and Janssen-Cilag. StSt has received advisory board honoraria, research support, travel support, speaker fees from AbbVie, AstraZeneca, BeiGene, BMS, Gilead, GSK, Hoffmann-La Roche, Janssen, Novartis, and Sunesis. JD has received research funding from Roche/Genentech. MS has received advisory board honoraria from AbbVie and research support from Bayer AG. The remaining authors declare no competing interests., (© 2025. The Author(s).)

Background: Physical activity supports weight regulation and metabolic health, but its timing in relation to obesity and diabetes remains unclear. We aimed to assess the diurnal timing of physical activity and its association with obesity and diabetes., Methods: We cross-sectionally analyzed hip-worn accelerometry data from 61,116 participants aged 20-75 in the German National Cohort between 2015 and 2019. We divided physical activity into sex- and age-standardized quartiles of total morning (06:00-11:59), afternoon (12:00-17:59), evening (18:00-23:59), and nighttime (00:00-06:00) physical activity. Using multivariable logistic regression, we estimated associations of physical activity timing with obesity (BMI ≥ 30.0 kg/m 2 ) and diabetes (self-reported or HbA1c ≥ 6.5%). We accounted for sex, age, study region, education, employment, risky alcohol use, smoking, night shift work, and sleep duration., Results: High afternoon (top vs. bottom quartile, OR: 0.36, 95% CI: 0.33-0.38) and evening physical activity (OR: 0.45, 95% CI: 0.42-0.48) showed lower obesity odds than high morning activity (OR: 0.71, 95% CI: 0.66-0.76), whereas nighttime activity increased obesity odds (OR: 1.58, 95% CI: 1.48-1.68). Associations were similar for diabetes, with the lowest odds for afternoon (OR: 0.47, 95% CI: 0.42-0.53), followed by evening (OR: 0.56, 95% CI: 0.50-0.62) and morning activity (OR: 0.80, 95% CI: 0.71-0.89), and higher odds for nighttime activity (OR: 1.43, 95% CI: 1.29-1.58). Findings were not modified by employment status, night shift work, and sleep duration., Conclusions: Our cross-sectional findings require longitudinal corroboration but suggest afternoon and evening activity provide greater metabolic health benefits than morning activity, while nighttime activity is discouraged., Competing Interests: Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: The NAKO obtained ethics approval from all local committees of the study centers (original ethics approval of the leading ethics committee of the Bayerische Landesaerztekammer with protocol code 13023). All participants provided written informed consent. This study was conducted in line with the Declaration of Helsinki and the Strengthening the Reporting of Observational studies in Epidemiology (STROBE) guideline for cross-sectional studies., (© 2025. The Author(s).)

Accurate melanoma diagnosis is crucial for patient outcomes and reliability of AI diagnostic tools. We assess interrater variability among eight expert pathologists reviewing histopathological images and clinical metadata of 792 melanoma-suspicious lesions prospectively collected at eight German hospitals. Moreover, we provide access to the largest panel-validated dataset featuring dermoscopic and histopathological images with metadata. Complete agreement is achieved in 53.5% of cases (424/792), and a majority vote ( ≥ five pathologists) in 90.9% (720/792). Considerable discordance is observed for non-invasive melanomas (complete agreement in only 10/73 cases). The expert panel disagrees with the local pathologists' and dermatologists' diagnoses in 14.9% and 33.5% of cases, respectively. This variability highlights the diagnostic challenges of early-stage melanomas and the need to reconsider how ground truth is established in routine care and AI research. Including at least two pathologists or virtual panels may contribute to more consistent diagnostic results., Competing Interests: Competing interests: Prof Utikal reported personal fees from Amgen, Bristol Myers Squibb, GSK, Immunocore, LEO Pharma, Merck Sharp & Dohme, Novartis, Pierre Fabre, Rheacell, Roche, and Sanofi outside the submitted work. Dr Wohlfeil received honoraria from Bristol Myers Squibb, Novartis, and Sun Pharma outside the submitted work. Prof Meier reported grants from Novartis and Roche and others (travel support or/and speaker’s fees or/and advisor’s honoraria) from BMS, MSD, and Pierre Fabre outside the submitted work. Dr Hobelsberger reported clinical trial support and advisor’s honoraria from Almirall, speaker’s honoraria from Almirall, UCB, and AbbVie, and travel support from UCB, Janssen Cilag, Almirall, Novartis, Lilly, LEO Pharma, and AbbVie outside the submitted work. Prof Heinzerling reported other (clinical studies) from BMS, MSD, Pierre Fabre, Replimune, and Sanofi; personal fees from Biomedx, BMS, MSD, Sun, Pierre Fabre, Novartis, and Sanofi; and grants from Therakos outside the submitted work. Prof Haferkamp reported speaker’s fees and advisor’s honoraria from BMS, MSD, Novartis, and Pierre Fabre outside the submitted work. Dr Schlaak reported personal fees from BMS, Novartis, Immunocore, Sun Pharma, MSD, Recordati, and Sanofi Aventis outside the submitted work. Prof Berking reported personal fees from BMS, MSD, InflaRx, Novartis, Sanofi, LEO Pharma, Almirall Hermal, Pierre Fabre, Immunocore, and Delcath outside the submitted work. Dr Erdmann received travel support and speaker’s honoraria from Immunocore, Novartis, Pierre Fabre, and Sanofi outside the submitted work. Dr Sondermann reported grants from Almirall, Novartis, and Medi GmbH; and personal fees from AbbVie, Almirall, BMS, Boehringer Ingelheim, Celgene, Janssen, LEO Pharma, Lilly, Novartis, Pfizer, Sanofi Genzyme, and UCB outside the submitted work. Prof Goebeler reported grants for clinical studies from Argenx, Novartis, Janssen, and Galderma; personal fees from Almirall (consulting), Janssen (advisory board, speaker), GSK (advisory board, speaker), and Lilly (speaker) outside the submitted work. Dr Llamas-Velasco reported fees as an advisory board member, consultant, research support, participation in clinical trials, and honorary for speaking with the following pharmaceutical companies: Abbvie, Almirall, Amgen, Boehringer, BMS, Celgene, Janssen, Leo, Lilly, Kyowa kirin, Novartis and UCB, outside the submitted work. Dr. Kather declares consulting services for Owkin, France; DoMore Diagnostics, Norway; Panakeia, UK; AstraZeneca, UK; Scailyte, Switzerland; Mindpeak, Germany; and MultiplexDx, Slovakia. Furthermore, he holds shares in StratifAI GmbH, Germany, has received a research grant from GSK, and has received honoraria from AstraZeneca, Bayer, Eisai, Janssen, MSD, BMS, Roche, Pfizer, and Fresenius. Dr Gabriela Poch received travel support and speaker’s honoraria from MSD, BMS, Novartis, Sun Pharma, and Amgen outside the submitted work. Dr Brinker reported being the owner of Smart Health Heidelberg GmbH outside the submitted work. The remaining authors declare no competing interests., (© 2025. The Author(s).)

DNA methylation (DNAm) is a key epigenetic mark that shows profound alterations in cancer. Read-level methylomes enable more in-depth analyses, due to their broad genomic coverage and preservation of rare cell-type signals, compared to summarized data such as 450K/EPIC microarrays. Here, we propose MethylBERT, a Transformer-based model for read-level methylation pattern classification. MethylBERT identifies tumour-derived sequence reads based on their methylation patterns and local genomic sequence, and estimates tumour cell fractions within bulk samples. In our evaluation, MethylBERT outperforms existing deconvolution methods and demonstrates high accuracy regardless of methylation pattern complexity, read length and read coverage. Moreover, we show its applicability to cell-type deconvolution as well as non-invasive early cancer diagnostics using liquid biopsy samples. MethylBERT represents a significant advancement in read-level methylome analysis and enables accurate tumour purity estimation. The broad applicability of MethylBERT will enhance studies on both tumour and non-cancerous bulk methylomes., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Human cancer cell lines are the mainstay of cancer research. Recent reports showed that highly mutated adult carcinoma cell lines (mainly HeLa and MCF-7) present striking diversity across laboratories and that long-term continuous culturing results in genomic/transcriptomic heterogeneity with strong phenotypical implications. Here, we hypothesize that oligomutated pediatric sarcoma cell lines mainly driven by a fusion transcription factor, such as Ewing sarcoma (EwS), are genetically and phenotypically more stable than the previously investigated adult carcinoma cell lines. A comprehensive molecular and phenotypic characterization of multiple EwS cell line strains, together with a simultaneous analysis during 12 months of continuous cell culture show that fusion-driven pediatric sarcoma cell line strains are genomically more stable than adult carcinoma strains, display remarkably stable and homogenous transcriptomes, and exhibit uniform and stable drug response. Additionally, the analysis of multiple EwS cell lines subjected to long-term continuous culture reveals that variable degrees of genomic/transcriptomic/phenotypic changes among fusion-driven cell lines, further exemplifying that the potential for reproducibility of in vitro scientific results may be rather understood as a spectrum, even within the same tumor entity., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Colorectal cancer (CRC) is stratified into four consensus molecular subtypes (CMS1-4). CMS3 represents the metabolic subtype, but its wiring remains largely undefined. To identify the underlying tumorigenesis of CMS3, organoids derived from 16 genetically engineered mouse models are analyzed. Upon in vitro Cre-recombinase activation, transformation is established and transcriptional profiling reveals that distinct CMSs (CMS2-4) are modeled with different organoids. CMS3-like, metabolic signature-positive, organoids are induced by KRAS mutations. Interestingly, metabolic signatures are subsequently shown to result from enterocyte-like differentiation both in organoids and human cancers. Further analysis reveals carbamoyl-phosphate synthase 1 (CPS1) and sucrase-isomaltase (SI) as signature proteins. More importantly, CPS1 is crucial for de novo pyrimidine synthesis in CMS3 and its inhibition targets proliferation and stemness, facilitating enterocyte-like differentiation, while CMS2 and CMS4 models are not affected. Our data point to an enterocyte-like differentiation of CMS3 CRCs and reveal a selective vulnerability of this subtype through CPS1 inhibition., Competing Interests: Competing interests: The Authors declare no competing interests. Inclusion & ethics: Inclusion & ethics statement One or more of the authors of this paper self-identifies as an unrepresented ethnic minority in science. One or more of the authors of this paper self-identifies as a gender minority in their field of research. One or more of the authors of this paper self-identifies as a member of the LGBTQIA+ community., (© 2024. The Author(s).)

Genome editing using CRISPR-Cas systems is a promising avenue for the treatment of genetic diseases. However, cellular and humoral immunogenicity of genome editing tools, which originate from bacteria, complicates their clinical use. Here we report reduced immunogenicity (Red)(i)-variants of two clinically relevant nucleases, SaCas9 and AsCas12a. Through MHC-associated peptide proteomics (MAPPs) analysis, we identify putative immunogenic epitopes on each nuclease. Using computational modeling, we rationally design these proteins to evade the immune response. SaCas9 and AsCas12a Redi variants are substantially less recognized by adaptive immune components, including reduced binding affinity to MHC molecules and attenuated generation of cytotoxic T cell responses, yet maintain wild-type levels of activity and specificity. In vivo editing of PCSK9 with SaCas9.Redi.1 is comparable in efficiency to wild-type SaCas9, but significantly reduces undesired immune responses. This demonstrates the utility of this approach in engineering proteins to evade immune detection., Competing Interests: Competing interests: F.Z., R.R., R.K.M., Y.S., L.N., and I.K. are co-inventors on a patent (“ENGINEERED TYPE II CAS POLYNUCLEOTIDES WITH REDUCED IMMUNOGENICITY AND USES THEREOF” (PCT/US2024/022898)) related to this work filed by the Broad Institute, MIT, and Cyrus Biotechnology. L.N. and Y.S. are executives at and co-founders of, and I.K. a senior scientist at, Cyrus Biotechnology. F.Z. is a scientific advisor and cofounder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, Aera Therapeutics, and Moonwalk Biosciences. F.Z. is a scientific advisor for Octant. M.J.F. reports speaker honoraria from Pfizer, Roche, and Kerna Ventures. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Competing Interests: Competing interests: J.Z. is a founder, shareholder and scientific advisor of Quantro Therapeutics GmbH, a shareholder and scientific advisor of Mirimus Inc., and receives research funding from Boehringer Ingelheim. W.P. declares no competing interests.

Fibroblast heterogeneity is increasingly recognised across cancer conditions. Given their important contribution to disease progression, mapping fibroblasts' heterogeneity is critical to devise effective anti-cancer therapies. Cancer-associated fibroblasts (CAFs) represent the most abundant cell population in pancreatic ductal adenocarcinoma (PDAC). Whether CAF phenotypes are differently specified by PDAC cell lineages remains to be elucidated. Here, we reveal an important role for the MAPK signalling pathway in defining PDAC CAF phenotypes. We show that epithelial MAPK activity promotes the myofibroblastic differentiation of CAFs by sustaining the expression and secretion of TGF-β1. We integrate single-cell profiling of post-perturbation transcriptional responses from mouse models with cellular and spatial profiles of human tissues to define a MAPK high CAF (mapCAF) phenotype. We show that this phenotype associates with basal-like tumour cells and reduced frequency of CD8 + T cells. In addition to elevated MAPK activity, this mapCAF phenotype is characterized by TGF-β signalling, hypoxia responsive signatures, and immunoregulatory gene programs. Furthermore, the mapCAF signature is enriched in myofibroblastic CAFs from various cancer conditions and correlates with reduced response to immune checkpoint inhibition in melanoma. Altogether, our data expand our knowledge on CAF phenotype heterogeneity and reveal a potential strategy for targeting myofibroblastic CAFs in vivo., Competing Interests: Competing interests: J.S. receives honoraria as consultant or for continuing medical education presentations from AstraZeneca, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Immunocore, MSD Sharp Dohme, Novartis, Roche/Genentech and Servier. His institution receives research funding from Abalos Therapeutics, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Eisbach Bio and Roche/Genentech; he holds ownership in FAPI Holding (<3%), all outside the submitted work. The other authors declare no competing interests., (© 2024. The Author(s).)

Neoantigen-specific T cell receptors (neoTCRs) promise safe, personalized anti-tumor immunotherapy. However, detailed assessment of neoTCR-characteristics affecting therapeutic efficacy is mostly missing. Previously, we identified diverse neoTCRs restricted to different neoantigens in a melanoma patient. In this work, we now combine single-cell TCR-sequencing and RNA-sequencing after neoantigen-specific restimulation of peripheral blood-derived CD8 + T cells of this patient. We detect neoTCRs with specificity for the previously detected neoantigens and perform fine-characterization of neoTCR-transgenic (tg) T cells in vitro and in vivo. We describe a heterogeneous spectrum of TCR-intrinsic activation patterns in response to a shared neoepitope ranging from previously detected more highly frequent neoTCRs with moderate activation to rare ones with initially stronger activation. Experimental restimulation of adoptively transferred neoTCR-tg T cells in a xenogeneic rechallenge tumor model demonstrates superior anti-tumor responses of moderate neoTCR-tg T cells upon repeated tumor contact. These insights have significant implications for the selection of TCRs for therapeutic engineering of TCR-tg T cells., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Paediatric medulloblastomas with chromothripsis are characterised by high genomic instability and are among the tumours with the worst prognosis. However, the molecular makeup and the determinants of the aggressiveness of chromothriptic medulloblastoma are not well understood. Here, we apply spatial transcriptomics to profile a cohort of 13 chromothriptic and non-chromothriptic medulloblastomas from the same molecular subgroup. Our data reveal a higher extent of spatial intra-tumour heterogeneity in chromothriptic medulloblastomas compared to non-chromothripictic tumours, which is associated with increased proliferation and stemness, but lower immune infiltration and differentiation. Spatial mapping of genetic subclones of the same tumour identify a regionally distinct architecture and clone-specific phenotypic features, with distinct degrees of differentiation, proliferation and immune infiltration between clones. We conduct temporal profiling of 11 samples from patient-derived xenografts from a patient with chromothriptic medulloblastoma, covering the transition from the minimal residual disease stage to treatment-resistant regrown tumours. In chromothriptic medulloblastoma, an ecosystem of cells from multiple genetic clones resist treatment and lead to relapse. Finally, we identify tumour microtubes in chromothriptic medulloblastoma, calling for exploration of cell network communication as a putative target., Competing Interests: Competing interests: O.S. is a paid advisor of Insitor.INC. The remaining authors declare no competing interests., (© 2024. The Author(s).)

The long-term consequences of cancer and its therapy on the patients' immune system years after cancer-free survival remain poorly understood. Here, we present an in-depth characterization of the bone marrow immune ecosystem of multiple myeloma long-term survivors, from initial diagnosis up to 17 years following a single therapy line and cancer-free survival. Using comparative single-cell analyses combined with molecular, genomic, and functional approaches, we demonstrate that multiple myeloma long-term survivors exhibit pronounced alterations in their bone marrow microenvironment associated with impaired immunity. These immunological alterations were frequently linked to an inflammatory immune circuit fueled by the long-term persistence or resurgence of residual myeloma cells. Notably, even in the complete absence of any detectable residual disease for decades, sustained changes in the immune system were observed, suggesting an irreversible 'immunological scarring' caused by the initial exposure to the cancer and therapy. Collectively, our study provides key insights into the molecular and cellular bone marrow ecosystem of long-term survivors of multiple myeloma, revealing both reversible and irreversible alterations in the immune compartment., Competing Interests: Competing interests: C.M.-T. received research funding from Bioline RX and Pfizer. M.B. has served as a consultant for Takeda and Novartis; received research funding from Novartis and travel support from Celgene, Amgen, and Janssen. H.G. received funding from the International Myeloma Foundation – Black Swan and the Dietmar-Hopp-Foundation. The other authors declare no competing interests related to this study., (© 2024. The Author(s).)

Aberrant translation causes ribosome stalling, which leads to the ubiquitination of ribosomal proteins and induces ribosome-associated quality control. As part of this quality control process, the E3 ubiquitin ligase RNF10 monoubiquitinates ribosomal protein RPS3. Here, we demonstrate that RNF10-mediated RPS3 monoubiquitination antagonizes ribosomal half-mer formation by promoting dissociation of 40S subunits from ribosomes stalled during translation elongation. Interestingly, RNF10 also promotes dissociation of 40S subunits stalled during aberrant translation initiation. Moreover, RNF10 levels are tightly coupled to the amount of 40S subunits. Knockdown of RPS proteins, which abrogates 40S ribosome biogenesis, results in proteasomal degradation of RNF10. Vice versa, knockdown of RPL proteins, which abrogates 60S biogenesis, leads to the accumulation of stalled initiating 40S subunits, increased RNF10 levels, and RPS3 monoubiquitination. As a factor required for the resolution of stalled translation events, RNF10 is part of a fundamental mechanism by which cells respond to imbalances in ribosomal subunit stoichiometry., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Targeted (nano-)drug delivery is essential for treating respiratory diseases, which are often confined to distinct lung regions. However, spatio-temporal profiling of drugs or nanoparticles (NPs) and their interactions with lung macrophages remains unresolved. Here, we present LungVis 1.0, an AI-powered imaging ecosystem that integrates light sheet fluorescence microscopy with deep learning-based image analysis pipelines to map NP deposition and dosage holistically and quantitatively across bronchial and alveolar (acinar) regions in murine lungs for widely-used bulk-liquid and aerosol-based delivery methods. We demonstrate that bulk-liquid delivery results in patchy NP distribution with elevated bronchial doses, whereas aerosols achieve uniform deposition reaching distal alveoli. Furthermore, we reveal that lung tissue-resident macrophages (TRMs) are dynamic, actively patrolling and redistributing NPs within alveoli, contesting the conventional paradigm of TRMs as static entities. LungVis 1.0 provides an advanced framework for exploring pulmonary delivery dynamics and deepening insights into TRM-mediated lung immunity., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Recent successes in developing small molecule degraders that act through the ubiquitin system have spurred efforts to extend this technology to other mechanisms, including the autophagosomal-lysosomal pathway. Therefore, reports of autophagosome tethering compounds (ATTECs) have received considerable attention from the drug development community. ATTECs are based on the recruitment of targets to LC3/GABARAP, a family of ubiquitin-like proteins that presumably bind to the autophagosome membrane and tether cargo-loaded autophagy receptors into the autophagosome. In this work, we rigorously tested the target engagement of the reported ATTECs to validate the existing LC3/GABARAP ligands. Surprisingly, we were unable to detect interaction with their designated target LC3 using a diversity of biophysical methods. Intrigued by the idea of developing ATTECs, we evaluated the ligandability of LC3/GABARAP by in silico docking and large-scale crystallographic fragment screening. Data based on approximately 1000 crystal structures revealed that most fragments bound to the HP2 but not to the HP1 pocket within the LIR docking site, suggesting a favorable ligandability of HP2. Through this study, we identified diverse validated LC3/GABARAP ligands and fragments as starting points for chemical probe and ATTEC development., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Chromothripsis is a frequent form of genome instability, whereby a presumably single catastrophic event generates extensive genomic rearrangements of one or multiple chromosome(s). However, little is known about the heterogeneity of chromothripsis across different clones from the same tumour, as well as changes in response to treatment. Here we analyse single-cell genomic and transcriptomic alterations linked with chromothripsis in human p53-deficient medulloblastoma and neural stem cells (n = 9). We reconstruct the order of somatic events, identify early alterations likely linked to chromothripsis and depict the contribution of chromothripsis to malignancy. We characterise subclonal variation of chromothripsis and its effects on extrachromosomal circular DNA, cancer drivers and putatively druggable targets. Furthermore, we highlight the causative role and the fitness consequences of specific rearrangements in neural progenitors., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Fibrinolysis influences the mobilization of hematopoietic stem cells from their bone marrow microenvironment (BMM). Here we show that activation of plasmin, a key fibrinolytic agent, by annexin A2 (ANXA2) distinctly impacts progression of BCR-ABL1 + B-cell acute lymphoblastic leukemia (B-ALL) via modulation of the extracellular matrix (ECM) in the BMM. The dense ECM in a BMM with decreased plasmin activity entraps insulin-like growth factor (IGF) 1 and reduces mTORC2-dependent signaling and proliferation of B-ALL cells. Conversely, B-ALL conditions the BMM to induce hepatic generation of plasminogen, the plasmin precursor. Treatment with ε-aminocaproic acid (EACA), which inhibits plasmin activation, reduces tumor burden and prolongs survival, including in xenogeneic models via increased fibronectin in the BMM. Human data confirm that IGF1 and fibronectin staining in trephine biopsies are correlated. Our studies suggest that fibrinolysis-mediated ECM remodeling and subsequent growth factor release influence B-ALL progression and inhibition of this process by EACA may be beneficial as adjunct therapy., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: General obesity commonly represented by body mass index (BMI) is an established risk factor for colorectal cancer (CRC). However, it is unclear to what extent this association is accounted for by central obesity. We aimed to evaluate the associations between BMI, waist-to-hip ratio (WHR), and waist circumference (WC) with CRC risk and to investigate if and to what extent these associations are independent from each other., Methods: Data from more than 500,000 male and female participants aged 40-69, recruited in the UK Biobank study between 2006 and 2010, were analyzed. Multivariable Cox proportional hazards models were fitted and hazard ratios (HR) and their 95% confidence intervals (CI) were calculated., Results: During a median follow-up of 12.5 years, of 460,784 participants, 5,977 developed CRC. Multivariable adjusted HRs (95% CIs) per standard deviation increase of BMI, WHR, and WC were 1.10 (1.07-1.13), 1.18 (1.14-1.22), and 1.14 (1.11-1.18), respectively. After mutual adjustment, the association with CRC was substantially attenuated for BMI (1.04 (1.01-1.07)), and remained substantially stronger for WHR (1.15 (1.11-1.20)). Furthermore, WHR showed strong, statistically significant associations with CRC risk within all BMI categories, whereas associations of BMI with CRC risk were weak and not statistically significant within WHR categories. BMI was also not associated with CRC risk in women and with rectal cancer after mutual adjustment. Conversely, WHR was strongly associated with CRC risk in both sexes and with both colon and rectal cancer risk before and after adjustment for BMI. BMI and WC could not be mutually adjusted for due to their high correlation., Conclusion: Central obesity is a much stronger predictor of CRC and may account for most of the CRC risk linked to obesity. Our findings also emphasize the need for incorporating measures such as WHR alongside BMI in clinical practice to improve obesity prevention and management., Competing Interests: Competing interests The authors declare no competing interests. Ethics approval and consent to participate The UK Biobank was approved by the North West Multi center Research Ethics Committee (MREC) as a Research Tissue Bank (RTB) approval (renewed approval in 2021:21/NW/0157) and has collected signed electronic informed consent from all participants. The study was performed in accordance with the Declaration of Helsinki. This research has been conducted using the UK Biobank Resource under application No 66591., (© 2024. The Author(s).)

Centriole integrity, vital for cilia formation and chromosome segregation, is crucial for human health. The inner scaffold within the centriole lumen composed of the proteins POC1B, POC5 and FAM161A is key to this integrity. Here, we provide an understanding of the function of inner scaffold proteins. We demonstrate the importance of an interaction network organised by POC1A-POC1B heterodimers within the centriole lumen, where the WD40 domain of POC1B localises close to the centriole wall, while the POC5-interacting WD40 of POC1A resides in the centriole lumen. The POC1A-POC5 interaction and POC5 tetramerization are essential for inner scaffold formation and centriole stability. The microtubule binding proteins FAM161A and MDM1 by binding to POC1A-POC1B, likely positioning the POC5 tetramer near the centriole wall. Disruption of POC1A or POC1B leads to centriole microtubule defects and deletion of both genes causes centriole disintegration. These findings provide insights into organisation and function of the inner scaffold., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Transition of cytosine to thymine in CpG dinucleotides is the most frequent type of mutation in cancer. This increased mutability is commonly attributed to the spontaneous deamination of 5-methylcytosine (5mC), which is normally repaired by the base-excision repair (BER) pathway. However, the contribution of 5mC deamination in the increasing diversity of cancer mutational signatures remains poorly explored. We integrate mutational signatures analysis in a large series of tumor whole genomes with lineage-specific epigenomic data to draw a detailed view of 5mC deamination in cancer. We uncover tumor type-specific patterns of 5mC deamination signatures in CpG and non-CpG contexts. We demonstrate that the BER glycosylase MBD4 preferentially binds to active chromatin and early replicating DNA, which correlates with lower mutational burden in these domains. We validate our findings by modeling BER deficiencies in isogenic cell models. Here, we establish MBD4 as the main actor responsible for 5mC deamination repair in humans., Competing Interests: Competing interests D. Rieke reports advisory agreement with BeiGene and Bayer, honoraria from Bristol Myers Squibb, Bayer and Roche, research support from Seagen, and personal fees from Bayer and Johnson & Johnson, all outside the submitted work. A. Picca reports personal fees from AstraZeneca and Servier, all outside the submitted work. F. Bielle reports funding of research from Abbvie, service agreement for research contracted between his institution and Treefrog Therapeutics as well as Owkin, personal fees from Bristol Myers Squibb and a next-of-kin employed by Bristol Myers Squibb, all outside the submitted work. M.L. Yaspo is COO/CSO and shareholder of Alacris Theranostics without conflict of interest with the submitted work. M. Rodrigues reports non-financial support from AstraZeneca and Merck Sharp and Dohme, grants from Daiichi Sankyo, personal fees from AstraZeneca, Immunocore, Merck Sharp and Dohme and GlaxoSmithKline, all outside the submitted work. M.-H. Stern reports grants from Immunocore and Bionano, and royalties from Myriad Genetics, all outside the submitted work. The remaining authors have no conflict of interest to declare., (© 2024. The Author(s).)

Genetic variation at the 19q13.3 KLK locus is linked with prostate cancer susceptibility in men. The non-synonymous KLK3 single nucleotide polymorphism (SNP), rs17632542 (c.536 T > C; Ile163Thr-substitution in PSA) is associated with reduced prostate cancer risk, however, the functional relevance is unknown. Here, we identify that the SNP variant-induced change in PSA biochemical activity mediates prostate cancer pathogenesis. The 'Thr' PSA variant leads to small subcutaneous tumours, supporting reduced prostate cancer risk. However, 'Thr' PSA also displays higher metastatic potential with pronounced osteolytic activity in an experimental metastasis in-vivo model. Biochemical characterisation of this PSA variant demonstrates markedly reduced proteolytic activity that correlates with differences in in-vivo tumour burden. The SNP is associated with increased risk for aggressive disease and prostate cancer-specific mortality in three independent cohorts, highlighting its critical function in mediating metastasis. Carriers of this SNP allele have reduced serum total PSA and a higher free/total PSA ratio that could contribute to late biopsy decisions and delay in diagnosis. Our results provide a molecular explanation for the prominent 19q13.3 KLK locus, rs17632542 SNP, association with a spectrum of prostate cancer clinical outcomes., (© 2024. The Author(s).)

Infectious diseases and cancer evade immune surveillance using similar mechanisms. Targeting immune mechanisms using common strategies thus represents a promising avenue to improve prevention and treatment. Synthetic immunology can provide such strategies by applying engineering principles from synthetic biology to immunology. Synthetic biologists engineer cells by top-down genetic manipulation or bottom-up assembly from nanoscale building blocks. Recent successes in treating advanced tumours and diseases using genetically engineered immune cells highlight the power of the top-down synthetic immunology approach. However, genetic immune engineering is mostly limited to ex vivo applications and is subject to complex counter-regulation inherent to immune functions. Bottom-up synthetic biology can harness the rich nanotechnology toolbox to engineer molecular and cellular systems from scratch and equip them with desired functions. These are beginning to be tailored to perform targeted immune functions and should hence allow intervention strategies by rational design. In this Perspective we conceptualize bottom-up synthetic immunology as a new frontier field that uses nanotechnology for crucial innovations in therapy and the prevention of infectious diseases and cancer., Competing Interests: Competing interests M.P. is an inventor on patent applications describing the identification of tumour-reactive TCRs and a founder of Tcelltech GmbH., (© 2024. Springer Nature Limited.)

Gallbladder cancer (GBC) presents as an aggressive malignancy with poor patient outcome. Like other epithelial cancers, the mechanisms of GBC cancer progression remain vague and efforts in finding targeted therapies fall below expectations. This study combined proteomic analysis of formalin-fixed paraffin-embedded (FFPE) GBC samples, functional and molecular characterization of potential oncogenes and identification of potential therapeutic strategies for GBC. We identified Carcinoembryonic Antigen-related Cell Adhesion Molecule 6 (CEACAM6) as one of the significantly most upregulated proteins in GBC. CEACAM6 overexpression has been observed in other cancer entities but the molecular function remains unclear. Our functional analyses in vitro and in vivo mouse models revealed that CEACAM6 supported the initial steps of cancer progression and metastasis by decreasing cell adhesion and promoting migration and invasion of GBC cells. Conversely, CEACAM6 knockdown abolished GBC aggressiveness by increasing cell adhesion while reducing cell migration, cell proliferation, and colony formation. BirA-BioID followed by mass-spectrometry revealed Integrin Beta-1 (ITGB1) and Protein Kinase C Delta (PRKCD) as direct molecular and functional partners of CEACAM6 supporting GBC cell migration. ERK and AKT signaling and their downstream target genes were regulated by CEACAM6 and thus the treatment with AKT inhibitor capivasertib or ERK inhibitor ulixertinib mitigated the CEACAM6-induced migration. These findings demonstrate that CEACAM6 is crucially involved in gallbladder cancer progression by promoting migration and inhibiting cell adhesion through ERK and AKT signaling providing specific options for treatment of CEACAM6-positive cancers., (© 2024. The Author(s).)

The Four Core Genotypes (FCG) is a mouse model system used to disentangle the function of sex chromosomes and hormones. We report that a copy of a 3.2 MB region of the X chromosome has translocated to the Y Sry- chromosome and thus increased the expression of X-linked genes including the single-stranded RNA sensor and autoimmune disease mediator Tlr7. This previously-unreported X-Y translocation complicates the interpretation of studies reliant on C57BL/6J FCG mice., (© 2024. The Author(s).)

Immune checkpoint inhibitors (ICI) have significantly improved overall survival in melanoma patients. However, 60% experience severe adverse events and early response markers are lacking. Circulating tumour DNA (ctDNA) is a promising biomarker for treatment-response and recurrence detection. The prospective PET/LIT study included 104 patients with palliative combined or adjuvant ICI. Tumour-informed sequencing panels to monitor 30 patient-specific variants were designed and 321 liquid biopsies of 87 patients sequenced. Mean sequencing depth after deduplication using UMIs was 6000x and the error rate of UMI-corrected reads was 2.47×10 -4 . Variant allele fractions correlated with PET/CT MTV (rho=0.69), S100 (rho=0.72), and LDH (rho=0.54). A decrease of allele fractions between T1 and T2 was associated with improved PFS and OS in the palliative cohort (p = 0.008 and p < 0.001). ctDNA was detected in 76.9% of adjuvant patients with relapse (n = 10/13), while all patients without progression (n = 9) remained ctDNA negative. Tumour-informed liquid biopsies are a reliable tool for monitoring treatment response and early relapse in melanoma patients with ICI., (© 2024. The Author(s).)

Tumour mutational burden (TMB), defined as the total number of somatic non-synonymous mutations present within the cancer genome, varies across and within cancer types. A first wave of retrospective and prospective research identified TMB as a predictive biomarker of response to immune-checkpoint inhibitors and culminated in the disease-agnostic approval of pembrolizumab for patients with TMB-high tumours based on data from the Keynote-158 trial. Although the applicability of outcomes from this trial to all cancer types and the optimal thresholds for TMB are yet to be ascertained, research into TMB is advancing along three principal avenues: enhancement of TMB assessments through rigorous quality control measures within the laboratory process, including the mitigation of confounding factors such as limited panel scope and low tumour purity; refinement of the traditional TMB framework through the incorporation of innovative concepts such as clonal, persistent or HLA-corrected TMB, tumour neoantigen load and mutational signatures; and integration of TMB with established and emerging biomarkers such as PD-L1 expression, microsatellite instability, immune gene expression profiles and the tumour immune contexture. Given its pivotal functions in both the pathogenesis of cancer and the ability of the immune system to recognize tumours, a profound comprehension of the foundational principles and the continued evolution of TMB are of paramount relevance for the field of oncology., (© 2024. Springer Nature Limited.)

Chimeric antigen receptor (CAR)-modified natural killer (NK) cells show antileukemic activity against acute myeloid leukemia (AML) in vivo. However, NK cell-mediated tumor killing is often impaired by the interaction between human leukocyte antigen (HLA)-E and the inhibitory receptor, NKG2A. Here, we describe a strategy that overcomes CAR-NK cell inhibition mediated by the HLA-E-NKG2A immune checkpoint. We generate CD33-specific, AML-targeted CAR-NK cells (CAR33) combined with CRISPR/Cas9-based gene disruption of the NKG2A-encoding KLRC1 gene. Using single-cell multi-omics analyses, we identified transcriptional features of activation and maturation in CAR33-KLRC1 ko -NK cells, which are preserved following exposure to AML cells. Moreover, CAR33-KLRC1 ko -NK cells demonstrate potent antileukemic killing activity against AML cell lines and primary blasts in vitro and in vivo. We thus conclude that NKG2A-deficient CAR-NK cells have the potential to bypass immune suppression in AML., (© 2024. The Author(s).)

The peptide hormone glucagon is a fundamental metabolic regulator that is also being considered as a pharmacotherapeutic option for obesity and type 2 diabetes. Despite this, we know very little regarding how glucagon exerts its pleiotropic metabolic actions. Given that the liver is a chief site of action, we performed in situ time-resolved liver phosphoproteomics to reveal glucagon signaling nodes. Through pathway analysis of the thousands of phosphopeptides identified, we reveal "membrane trafficking" as a dominant signature with the vesicle trafficking protein SEC22 Homolog B (SEC22B) S137 phosphorylation being a top hit. Hepatocyte-specific loss- and gain-of-function experiments reveal that SEC22B was a key regulator of glycogen, lipid and amino acid metabolism, with SEC22B-S137 phosphorylation playing a major role in glucagon action. Mechanistically, we identify several protein binding partners of SEC22B affected by glucagon, some of which were differentially enriched with SEC22B-S137 phosphorylation. In summary, we demonstrate that phosphorylation of SEC22B is a hepatocellular signaling node mediating the metabolic actions of glucagon and provide a rich resource for future investigations on the biology of glucagon action., (© 2024. The Author(s).)

Proteome analysis by data-independent acquisition (DIA) has become a powerful approach to obtain deep proteome coverage, and has gained recent traction for label-free analysis of single cells. However, optimal experimental design for DIA-based single-cell proteomics has not been fully explored, and performance metrics of subsequent data analysis tools remain to be evaluated. Therefore, we here formalize and comprehensively evaluate a DIA data analysis strategy that exploits the co-analysis of low-input samples with a so-called matching enhancer (ME) of higher input, to increase sensitivity, proteome coverage, and data completeness. We assess the matching specificity of DIA-ME by a two-proteome model, and demonstrate that false discovery and false transfer are maintained at low levels when using DIA-NN software, while preserving quantification accuracy. We apply DIA-ME to investigate the proteome response of U-2 OS cells to interferon gamma (IFN-γ) in single cells, and recapitulate the time-resolved induction of IFN-γ response proteins as observed in bulk material. Moreover, we uncover co- and anti-correlating patterns of protein expression within the same cell, indicating mutually exclusive protein modules and the co-existence of different cell states. Collectively our data show that DIA-ME is a powerful, scalable, and easy-to-implement strategy for single-cell proteomics., (© 2024. The Author(s).)

Intravital 2P-microscopy enables the longitudinal study of brain tumor biology in superficial mouse cortex layers. Intravital microscopy of the white matter, an important route of glioblastoma invasion and recurrence, has not been feasible, due to low signal-to-noise ratios and insufficient spatiotemporal resolution. Here, we present an intravital microscopy and artificial intelligence-based analysis workflow (Deep3P) that enables longitudinal deep imaging of glioblastoma up to a depth of 1.2 mm. We find that perivascular invasion is the preferred invasion route into the corpus callosum and uncover two vascular mechanisms of glioblastoma migration in the white matter. Furthermore, we observe morphological changes after white matter infiltration, a potential basis of an imaging biomarker during early glioblastoma colonization. Taken together, Deep3P allows for a non-invasive intravital investigation of brain tumor biology and its tumor microenvironment at subcortical depths explored, opening up opportunities for studying the neuroscience of brain tumors and other model systems., (© 2024. The Author(s).)

HPV-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC) are recognized as distinct entities. There remains uncertainty surrounding the causal effects of smoking and alcohol on the development of these two cancer types. Here we perform multivariable Mendelian randomization (MR) to evaluate the causal effects of smoking and alcohol on the risk of HPV-positive and HPV-negative HNSCC in 3431 cases and 3469 controls. Lifetime smoking exposure, as measured by the Comprehensive Smoking Index (CSI), is associated with increased risk of both HPV-negative HNSCC (OR = 3.03, 95%CI:1.75-5.24, P = 7.00E-05) and HPV-positive HNSCC (OR = 2.73, 95%CI:1.39-5.36, P = 0.003). Drinks Per Week is also linked with increased risk of both HPV-negative HNSCC (OR = 7.72, 95%CI:3.63-16.4, P = 1.00E-07) and HPV-positive HNSCC (OR = 2.66, 95%CI:1.06-6.68, P = 0.038). Smoking and alcohol independently increase the risk of both HPV-positive and HPV-negative HNSCC. These findings have important implications for understanding the modifying risk factors between HNSCC subtypes., (© 2024. The Author(s).)

Determining the label to target ratio, also known as the degree of labeling (DOL), is crucial for quantitative fluorescence microscopy and a high DOL with minimal unspecific labeling is beneficial for fluorescence microscopy in general. Yet robust, versatile and easy-to-use tools for measuring cell-specific labeling efficiencies are not available. Here we present a DOL determination technique named protein-tag DOL (ProDOL), which enables fast quantification and optimization of protein-tag labeling. With ProDOL various factors affecting labeling efficiency, including substrate type, incubation time and concentration, as well as sample fixation and cell type can be easily assessed. We applied ProDOL to investigate how human immunodeficiency virus-1 pathogenesis factor Nef modulates CD4 T cell activation measuring total and activated copy numbers of the adapter protein SLP-76 in signaling microclusters. ProDOL proved to be a versatile and robust tool for labeling calibration, enabling determination of labeling efficiencies, optimization of strategies and quantification of protein stoichiometry., (© 2024. The Author(s).)

Chronic liver disease and cancer are global health challenges. The role of the circadian clock as a regulator of liver physiology and disease is well established in rodents, however, the identity and epigenetic regulation of rhythmically expressed genes in human disease is less well studied. Here we unravel the rhythmic transcriptome and epigenome of human hepatocytes using male human liver chimeric mice. We identify a large number of rhythmically expressed protein coding genes in human hepatocytes of male chimeric mice, which includes key transcription factors, chromatin modifiers, and critical enzymes. We show that hepatitis C virus (HCV) infection, a major cause of liver disease and cancer, perturbs the transcriptome by altering the rhythmicity of the expression of more than 1000 genes, and affects the epigenome, leading to an activation of critical pathways mediating metabolic alterations, fibrosis, and cancer. HCV-perturbed rhythmic pathways remain dysregulated in patients with advanced liver disease. Collectively, these data support a role for virus-induced perturbation of the hepatic rhythmic transcriptome and pathways in cancer development and may provide opportunities for cancer prevention and biomarkers to predict HCC risk., (© 2024. The Author(s).)

Cellular senescence is characterized by a permanent growth arrest and is associated with tissue aging and cancer. Senescent cells secrete a number of different cytokines referred to as the senescence-associated secretory phenotype (SASP), which impacts the surrounding tissue and immune response. Here, we find that senescent cells exhibit higher rates of protein synthesis compared to proliferating cells and identify eIF5A as a crucial regulator of this process. Polyamine metabolism and hypusination of eIF5A play a pivotal role in sustaining elevated levels of protein synthesis in senescent cells. Mechanistically, we identify a p53-dependent program in senescent cells that maintains hypusination levels of eIF5A. Finally, we demonstrate that functional eIF5A is required for synthesizing mitochondrial ribosomal proteins and monitoring the immune clearance of premalignant senescent cells in vivo. Our findings establish an important role of protein synthesis during cellular senescence and suggest a link between eIF5A, polyamine metabolism, and senescence immune surveillance., (© 2024. The Author(s).)

B-cell malignancies, such as chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), remain incurable, with MM particularly prone to relapse. Our study introduces a novel mouse model with active RANK signaling and the TCL1 oncogene, displaying both CLL and MM phenotypes. In younger mice, TCL1 and RANK expression expands CLL-like B1-lymphocytes, while MM originates from B2-cells, becoming predominant in later stages and leading to severe disease progression and mortality. The induced MM mimics human disease, exhibiting features like clonal plasma cell expansion, paraproteinemia, anemia, and kidney and bone failure, as well as critical immunosurveillance strategies that promote a tumor-supportive microenvironment. This research elucidates the differential impacts of RANK activation in B1- and B2-cells and underscores the distinct roles of single versus combined oncogenes in B-cell malignancies. We also demonstrate that human MM cells express RANK and that inhibiting RANK signaling can reduce MM progression in a xenotransplantation model. Our study provides a rationale for further investigating the effects of RANK signaling in B-cell transformation and the shaping of a tumor-promoting microenvironment., (© 2024. The Author(s).)

Human development relies on the correct replication, maintenance and segregation of our genetic blueprints. How these processes are monitored across embryonic lineages, and why genomic mosaicism varies during development remain unknown. Using pluripotent stem cells, we identify that several patterning signals-including WNT, BMP, and FGF-converge into the modulation of DNA replication stress and damage during S-phase, which in turn controls chromosome segregation fidelity in mitosis. We show that the WNT and BMP signals protect from excessive origin firing, DNA damage and chromosome missegregation derived from stalled forks in pluripotency. Cell signalling control of chromosome segregation declines during lineage specification into the three germ layers, but re-emerges in neural progenitors. In particular, we find that the neurogenic factor FGF2 induces DNA replication stress-mediated chromosome missegregation during the onset of neurogenesis, which could provide a rationale for the elevated chromosomal mosaicism of the developing brain. Our results highlight roles for morphogens and cellular identity in genome maintenance that contribute to somatic mosaicism during mammalian development., (© 2024. The Author(s).)

Risk of progression of monoclonal gammopathy of undetermined significance (MGUS) into multiple myeloma and related plasma cell disorders can be determined by three major risk stratification models, namely Mayo2005, Sweden2014, and NCI2019. This retrospective study of 427 patients with MGUS diagnosed according to the 2014 International Myeloma Working Group criteria aimed to describe and analyze the longitudinal applicability of these risk models. In all three models, the majority of patients remained at their baseline risk group, whereas small numbers of patients migrated to a different risk group. Proportions of patients among risk groups remained stable over time (e.g. Mayo2005 model, low-risk group, at baseline: 43%, after 1, 2, 3, 4, 5, and 8 years: 40%, 37%, 37%, 43%, 44%, and 43%). All three risk models reliably distinguished risk of progression at baseline, upon yearly reassessment (e.g. 1 year from diagnosis) and in time-dependent analyses. Upstaging to a high-risk category was associated with an increased risk of progression in all three models (Mayo2005: hazard ratio [HR] = 5.43, 95% confidence interval [95% CI] 1.21-24.39, p = 0.027; Sweden2014: HR = 13.02, 95% CI 5.25-32.28, p < 0.001; NCI2019: HR = 5.85, 95% CI 2.49-13.74, p < 0.001). Our study shows that MGUS risk stratification models can be applied longitudinally to repeatedly determine and improve individual risk of progression. Patient migration to higher risk categories during follow up should prompt more frequent monitoring in clinical routine., (© 2024. The Author(s).)

Genome-wide association studies (GWASs) based on common single nucleotide polymorphisms (SNPs) have identified several loci associated with the risk of monoclonal gammopathy of unknown significance (MGUS), a precursor condition for multiple myeloma (MM). We hypothesized that analyzing haplotypes might be more useful than analyzing individual SNPs, as it could identify functional chromosomal units that collectively contribute to MGUS risk. To test this hypothesis, we used data from our previous GWAS on 992 MGUS cases and 2910 controls from three European populations. We identified 23 haplotypes that were associated with the risk of MGUS at the genome-wide significance level (p < 5 × 10 -8 ) and showed consistent results among all three populations. In 10 genomic regions, strong promoter, enhancer and regulatory element-related histone marks and their connections to target genes as well as genome segmentation data supported the importance of these regions in MGUS susceptibility. Several associated haplotypes affected pathways important for MM cell survival such as ubiquitin-proteasome system (RNF186, OTUD3), PI3K/AKT/mTOR (HINT3), innate immunity (SEC14L1, ZBP1), cell death regulation (BID) and NOTCH signaling (RBPJ). These pathways are important current therapeutic targets for MM, which may highlight the advantage of the haplotype approach homing to functional units., (© 2024. The Author(s).)

Follicular lymphoma (FL) exhibits considerable variability in biological features and clinical trajectories across patients. To dissect the diversity of FL, we utilized a Bernoulli mixture model to identify genetic subtypes in 713 pre-treatment tumor tissue samples. Our analysis revealed the existence of five subtypes with unique genetic profiles that correlated with clinicopathological characteristics. The clusters were enriched in specific mutations as follows: CS (CREBBP and STAT6), TT (TNFAIP3 and TP53), GM (GNA13 and MEF2B), Q (quiescent, for low mutation burden), and AR (mutations of mTOR pathway-related genes). The subtype Q was enriched for patients with stage I disease and associated with a lower proliferative history than the other subtypes. The AR subtype was unique in its enrichment for IgM-expressing FL cases and was associated with advanced-stage and more than 4 nodal sites. The existence of subtypes was validated in an independent cohort of 418 samples from the GALLIUM trial. Notably, patients assigned to the TT subtype consistently experienced inferior progression-free survival when treated with immunochemotherapy. Our findings offer insight into core pathways distinctly linked with each FL cluster and are expected to be informative in the era of targeted therapies., (© 2024. The Author(s).)

Translation initiation is a highly regulated step needed for protein synthesis. Most cell-based mechanistic work on translation initiation has been done using non-stressed cells growing in medium with sufficient nutrients and oxygen. This has yielded our current understanding of 'canonical' translation initiation, involving recognition of the mRNA cap by eIF4E1 followed by successive recruitment of initiation factors and the ribosome. Many cells, however, such as tumor cells, are exposed to stresses such as hypoxia, low nutrients or proteotoxic stress. This leads to inactivation of mTORC1 and thereby inactivation of eIF4E1. Hence the question arises how cells translate mRNAs under such stress conditions. We study here how mRNAs are translated in an eIF4E1-independent manner by blocking eIF4E1 using a constitutively active version of eIF4E-binding protein (4E-BP). Via ribosome profiling we identify a subset of mRNAs that are still efficiently translated when eIF4E1 is inactive. We find that these mRNAs preferentially release eIF4E1 when eIF4E1 is inactive and bind instead to eIF3d via its cap-binding pocket. eIF3d then enables these mRNAs to be efficiently translated due to its cap-binding activity. In sum, our work identifies eIF3d-dependent translation as a major mechanism enabling mRNA translation in an eIF4E-independent manner., (© 2024. The Author(s).)

Somatic copy number alterations (CNAs) are major mutations that contribute to the development and progression of various cancers. Despite a few computational methods proposed to detect CNAs from single-cell transcriptomic data, the technical sparsity of such data makes it challenging to identify allele-specific CNAs, particularly in complex clonal structures. In this study, we present a statistical method, XClone, that strengthens the signals of read depth and allelic imbalance by effective smoothing on cell neighborhood and gene coordinate graphs to detect haplotype-aware CNAs from scRNA-seq data. By applying XClone to multiple datasets with challenging compositions, we demonstrated its ability to robustly detect different types of allele-specific CNAs and potentially indicate whole genome duplication, therefore enabling the discovery of corresponding subclones and the dissection of their phenotypic impacts., (© 2024. The Author(s).)

Multiple myeloma (MM) is an incurable malignancy of plasma cells. Epidemiological studies indicate a substantial heritable component, but the underlying mechanisms remain unclear. Here, in a genome-wide association study totaling 10,906 cases and 366,221 controls, we identify 35 MM risk loci, 12 of which are novel. Through functional fine-mapping and Mendelian randomization, we uncover two causal mechanisms for inherited MM risk: longer telomeres; and elevated levels of B-cell maturation antigen (BCMA) and interleukin-5 receptor alpha (IL5RA) in plasma. The largest increase in BCMA and IL5RA levels is mediated by the risk variant rs34562254-A at TNFRSF13B. While individuals with loss-of-function variants in TNFRSF13B develop B-cell immunodeficiency, rs34562254-A exerts a gain-of-function effect, increasing MM risk through amplified B-cell responses. Our results represent an analysis of genetic MM predisposition, highlighting causal mechanisms contributing to MM development., (© 2024. The Author(s).)