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Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low RNA quality. In this study, leveraging a recent advance in single-cell genomic technology, we introduce snPATHO-seq, a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples. We benchmarked the performance of the snPATHO-seq workflow against existing 10x 3' and Flex assays designed for frozen or fresh samples and highlighted the consistency in snRNA-seq data produced by all workflows. The snPATHO-seq workflow also demonstrated high robustness when tested across a wide range of healthy and diseased FFPE tissue samples. When combined with FFPE spatial transcriptomic technologies such as FFPE Visium, the snPATHO-seq provides a multi-modal sampling approach for FFPE samples, allowing more comprehensive transcriptomic characterization., (© 2024. The Author(s).)

The quality of standard single-cell experiments often depends on the immediate processing of cells or tissues post-harvest to preserve fragile and vulnerable cell populations, unless the samples are adequately fixed and stored. Despite the recent rise in popularity of probe-based and aldehyde-fixed RNA assays, these methods face limitations in species and target availability and are not suitable for immunoprofiling or assessing chromatin accessibility. Recently, a reversible fixation strategy known as FixNCut has been successfully deployed to separate sampling from downstream applications in a reproducible and robust manner, avoiding stress or necrosis-related artifacts. In this article, we present an optimized and robust practical guide to the FixNCut protocol to aid the end-to-end adaptation of this versatile method. This protocol not only decouples tissue or cell harvesting from single-cell assays but also enables a flexible and decentralized workflow that unlocks the potential for single-cell analysis as well as unconventional study designs that were previously considered unfeasible. Key features • Reversible fixation: Preserves cellular and molecular structures with the option to later reverse the fixation for downstream applications, maintaining cell integrity • Compatibility with single-cell assays: Supports single-cell genomic assays such as scRNA-seq and ATAC-seq, essential for high-resolution analysis of cell function and gene expression • Flexibility in sample handling: Allows immediate fixation post-collection, decoupling sample processing from analysis, beneficial in settings where immediate processing is impractical • Preservation of RNA and DNA integrity: Effectively preserves RNA and DNA, reducing degradation to ensure accurate transcriptomic and genomic profiling • Suitability for various biological samples: Applicable to a wide range of biological samples, including tissues and cell suspensions, whether freshly isolated or post-dissociated • Enables multi-center studies: Facilitates collaborative research across multiple centers by allowing sample fixation at the point of collection, enhancing research scale and diversity • Avoidance of artifacts: Minimizes stress or necrosis-related artifacts, preserving the natural cellular physiology for accurate genomic and transcriptomic analysis., Competing Interests: Competing interestsH.H. is a co-founder and shareholder of Omniscope, a scientific advisory board member of MiRXES and Nanostring, and a consultant to Moderna and Singularity. L.G.M is an advisor and shareholder of Omniscope, and advisor for ArgenTAG and BioScryb. Omniscope has filed a patent related to the application of the FixNCut protocol. All other authors declare no competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY-NC license.)

Single-cell assay for transposase-accessible chromatin by sequencing (scATAC-seq) has emerged as a powerful tool for dissecting regulatory landscapes and cellular heterogeneity. However, an exploration of systemic biases among scATAC-seq technologies has remained absent. In this study, we benchmark the performance of eight scATAC-seq methods across 47 experiments using human peripheral blood mononuclear cells (PBMCs) as a reference sample and develop PUMATAC, a universal preprocessing pipeline, to handle the various sequencing data formats. Our analyses reveal significant differences in sequencing library complexity and tagmentation specificity, which impact cell-type annotation, genotype demultiplexing, peak calling, differential region accessibility and transcription factor motif enrichment. Our findings underscore the importance of sample extraction, method selection, data processing and total cost of experiments, offering valuable guidance for future research. Finally, our data and analysis pipeline encompasses 169,000 PBMC scATAC-seq profiles and a best practices code repository for scATAC-seq data analysis, which are freely available to extend this benchmarking effort to future protocols., (© 2023. The Author(s).)

The use of single-cell technologies for clinical applications requires disconnecting sampling from downstream processing steps. Early sample preservation can further increase robustness and reproducibility by avoiding artifacts introduced during specimen handling. We present FixNCut, a methodology for the reversible fixation of tissue followed by dissociation that overcomes current limitations. We applied FixNCut to human and mouse tissues to demonstrate the preservation of RNA integrity, sequencing library complexity, and cellular composition, while diminishing stress-related artifacts. Besides single-cell RNA sequencing, FixNCut is compatible with multiple single-cell and spatial technologies, making it a versatile tool for robust and flexible study designs., (© 2024. Crown.)

An altered gut microbiota is associated with type 1 diabetes (T1D), affecting the production of short-chain fatty acids (SCFA) and glucose homeostasis. We previously demonstrated that enhancing serum acetate and butyrate using a dietary supplement (HAMSAB) improved glycemia in non-obese diabetic (NOD) mice and patients with established T1D. The effects of SCFA on immune-infiltrated islet cells remain to be clarified. Here, we performed single-cell RNA sequencing on islet cells from NOD mice fed an HAMSAB or control diet. HAMSAB induced a regulatory gene expression profile in pancreas-infiltrated immune cells. Moreover, HAMSAB maintained the expression of β-cell functional genes and decreased cellular stress. HAMSAB-fed mice showed preserved pancreatic endocrine cell identity, evaluated by decreased numbers of poly-hormonal cells. Finally, SCFA increased insulin levels in human β-like cells and improved transplantation outcome in NOD/SCID mice. Our findings support the use of metabolite-based diet as attractive approach to improve glucose control in T1D., Competing Interests: E.M. is an inventor on a patent WO2018027274A1 submitted by Monash University that covers methods and compositions of metabolites for treatment and prevention of autoimmune disease related to this paper and stock ownership for ImmunoBiota Therapeutics Pty Ltd. E.N.G. declares that there are no other relationships or activities that might bias, or be perceived to bias, the present work., (© 2023 The Author(s).)

TP53 and RB1 loss-of-function mutations are common in osteosarcoma. During development, combined loss of TP53 and RB1 function leads to downregulation of autophagy and the aberrant formation of primary cilia, cellular organelles essential for the transmission of canonical Hedgehog (Hh) signaling. Excess cilia formation then leads to hypersensitivity to Hedgehog (Hh) ligand signaling. In mouse and human models, we now show that osteosarcomas with mutations in TP53 and RB1 exhibit enhanced ligand-dependent Hh pathway activation through Smoothened (SMO), a transmembrane signaling molecule required for activation of the canonical Hh pathway. This dependence is mediated by hypersensitivity to Hh ligand and is accompanied by impaired autophagy and increased primary cilia formation and expression of Hh ligand in vivo. Using a conditional genetic mouse model of Trp53 and Rb1 inactivation in osteoblast progenitors, we further show that deletion of Smo converts the highly malignant osteosarcoma phenotype to benign, well differentiated bone tumors. Conversely, conditional overexpression of SHH ligand, or a gain-of-function SMO mutant in committed osteoblast progenitors during development blocks terminal bone differentiation. Finally, we demonstrate that the SMO antagonist sonidegib (LDE225) induces growth arrest and terminal differentiation in vivo in osteosarcomas that express primary cilia and Hh ligand combined with mutations in TP53. These results provide a mechanistic framework for aberrant Hh signaling in osteosarcoma based on defining mutations in the tumor suppressor, TP53., (© 2023. The Author(s).)

Meiotic crossovers are required for accurate chromosome segregation and producing new allelic combinations. Meiotic crossover numbers are tightly regulated within a narrow range, despite an excess of initiating DNA double-strand breaks. Here, we reveal the tumor suppressor FANCM as a meiotic anti-crossover factor in mammals. We use unique large-scale crossover analyses with both single-gamete sequencing and pedigree-based bulk-sequencing datasets to identify a genome-wide increase in crossover frequencies in Fancm -deficient mice. Gametogenesis is heavily perturbed in Fancm loss-of-function mice, which is consistent with the reproductive defects reported in humans with biallelic FANCM mutations. A portion of the gametogenesis defects can be attributed to the cGAS-STING pathway after birth. Despite the gametogenesis phenotypes in Fancm mutants, both sexes are capable of producing offspring. We propose that the anti-crossover function and role in gametogenesis of Fancm are separable and will inform diagnostic pathways for human genomic instability disorders., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Approximately 20% of acute myeloid leukemia (AML) patients carry mutations in IDH1 or IDH2 that result in over-production of the oncometabolite D-2-hydroxyglutarate (2-HG). Small molecule inhibitors that block 2-HG synthesis can induce complete morphological remission; however, almost all patients eventually acquire drug resistance and relapse. Using a multi-allelic mouse model of IDH1-mutant AML, we demonstrate that the clinical IDH1 inhibitor AG-120 (ivosidenib) exerts cell-type-dependent effects on leukemic cells, promoting delayed disease regression. Although single-agent AG-120 treatment does not fully eradicate the disease, it increases cycling of rare leukemia stem cells and triggers transcriptional upregulation of the pyrimidine salvage pathway. Accordingly, AG-120 sensitizes IDH1-mutant AML to azacitidine, with the combination of AG-120 and azacitidine showing vastly improved efficacy in vivo. Our data highlight the impact of non-genetic heterogeneity on treatment response and provide a mechanistic rationale for the observed combinatorial effect of AG-120 and azacitidine in patients., Competing Interests: Declaration of interests L.M.K. has received research funding and/or consultancy payments from Agios Pharmaceuticals, Celgene Corporation, and Servier Pharmaceuticals. J.S. has received research funding from BMS/Celgene, Amgen, and Astex Pharmaceuticals Inc., and served on the advisory boards of Astellas, Novartis, Otsuka, and Mundipharma. B.N., S.D., A.E.T., and M.L.H. were Agios employees, and S.D., A.E.T., and M.L.H. are, or were, Servier employees at the time of conducting these studies., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pharmacologic inhibition of epigenetic enzymes can have therapeutic benefit against hematologic malignancies. In addition to affecting tumor cell growth and proliferation, these epigenetic agents may induce antitumor immunity. Here, we discovered a novel immunoregulatory mechanism through inhibition of histone deacetylases (HDAC). In models of acute myeloid leukemia (AML), leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor (HDACi) panobinostat required activation of the type I interferon (IFN) pathway. Plasmacytoid dendritic cells (pDC) produced type I IFN after panobinostat treatment, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated induction of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, whereas combined treatment with panobinostat and IFNα improved outcomes in preclinical models. These discoveries offer a new therapeutic approach for AML and demonstrate that epigenetic rewiring of pDCs enhances antitumor immunity, opening the possibility of exploiting this approach for immunotherapies., Significance: We demonstrate that HDACis induce terminal differentiation of AML through epigenetic remodeling of pDCs, resulting in production of type I IFN that is important for the therapeutic effects of HDACis. The study demonstrates the important functional interplay between the immune system and leukemias in response to HDAC inhibition. This article is highlighted in the In This Issue feature, p. 1397., (©2022 The Authors; Published by the American Association for Cancer Research.)

Purpose: Mitogen-activated protein kinase pathway-activating mutations occur in the majority of colorectal cancer (CRC) cases and show mutual exclusivity. We identified 47 epidermal growth factor receptor/BRAF inhibitor-naive CRC patients with dual RAS hotspot/ BRAF V600E mutations (CRC-DD) from a cohort of 4,561 CRC patients with clinical next-generation sequencing results. We aimed to define the molecular phenotypes of the CRC-DD and to test if the dual RAS hotspot/BRAF V600E mutations coexist within the same cell., Materials and Methods: We developed a single-cell genotyping method with a mutation detection rate of 96.3% and a genotype prediction accuracy of 92.1%. Mutations in the CRC-DD cohort were analyzed for clonality, allelic imbalance, copy number, and overall survival., Results: Application of single-cell genotyping to four CRC-DD revealed the co-occurrence of both mutations in the following percentages of cells per case: NRAS G13D/ KRAS G12C, 95%; KRAS G12D/ NRAS G12V, 48%; BRAF V600E/ KRAS G12D, 44%; and KRAS G12D/ NRAS G13V, 14%, respectively. Allelic imbalance favoring the oncogenic allele was less frequent in CRC-DD (24 of 76, 31.5%, somatic mutations) compared with a curated cohort of CRC with a single-driver mutation (CRC-SD; 119 of 232 mutations, 51.3%; P = .013). Microsatellite instability-high status was enriched in CRC-DD compared with CRC-SD (23% v 11.4%, P = .028). Of the seven CRC-DD cases with multiregional sequencing, five retained both driver mutations throughout all sequenced tumor sites. Both CRC-DD cases with discordant multiregional sequencing were microsatellite instability-high., Conclusion: Our findings indicate that dual-driver mutations occur in a rare subset of CRC, often within the same tumor cells and across multiple tumor sites. Their presence and a lower rate of allelic imbalance may be related to dose-dependent signaling within the mitogen-activated protein kinase pathway., Competing Interests: Ahmet ZehirStock and Other Ownership Interests: Arcus Biosciences, Mirati TherapeuticsHonoraria: Illumina Luciano G. MartelottoPatents, Royalties, Other Intellectual Property: I'm and advisor for OmniScope, a startup tech company that develop test for detecting immune systems responses Rona YaegerConsulting or Advisory Role: Array BioPharma, Natera, Mirati TherapeuticsResearch Funding: Array BioPharma (Inst), Boehringer Ingelheim (Inst), Pfizer (Inst), Mirati Therapeutics (Inst) Britta WeigeltStock and Other Ownership Interests: Repare TherapeuticsConsulting or Advisory Role: Genentech/Roche, Invicro, Ventana Medical Systems, Volition RX, PAIGE, Goldman Sachs, Repare Therapeutics, Lilly, Repare Therapeutics Jorge S. Reis-FilhoLeadership: Grupo OncoclinicasStock and Other Ownership Interests: Repare Therapeutics, PAIGE.AIConsulting or Advisory Role: Genentech/Roche, Invicro, Ventana Medical Systems, Volition RX, Paige.AI, Goldman Sachs, Novartis, Repare Therapeutics, Lilly, Personalis Jaclyn F. HechtmanEmployment: NeoGenomics LaboratoriesStock and Other Ownership Interests: NeoGenomics LaboratoriesHonoraria: WebMD, Illumina, BayerConsulting or Advisory Role: Cor2Ed, Axiom Healthcare Strategies, BayerResearch Funding: Bayer, Lilly, Boehringer IngelheimNo other potential conflicts of interest were reported.

All cancers emerge after a period of clonal selection and subsequent clonal expansion. Although the evolutionary principles imparted by genetic intratumour heterogeneity are becoming increasingly clear 1 , little is known about the non-genetic mechanisms that contribute to intratumour heterogeneity and malignant clonal fitness 2 . Here, using single-cell profiling and lineage tracing (SPLINTR)-an expressed barcoding strategy-we trace isogenic clones in three clinically relevant mouse models of acute myeloid leukaemia. We find that malignant clonal dominance is a cell-intrinsic and heritable property that is facilitated by the repression of antigen presentation and increased expression of the secretory leukocyte peptidase inhibitor gene (Slpi), which we genetically validate as a regulator of acute myeloid leukaemia. Increased transcriptional heterogeneity is a feature that enables clonal fitness in diverse tissues and immune microenvironments and in the context of clonal competition between genetically distinct clones. Similar to haematopoietic stem cells 3 , leukaemia stem cells (LSCs) display heritable clone-intrinsic properties of high, and low clonal output that contribute to the overall tumour mass. We demonstrate that LSC clonal output dictates sensitivity to chemotherapy and, although high- and low-output clones adapt differently to therapeutic pressure, they coordinately emerge from minimal residual disease with increased expression of the LSC program. Together, these data provide fundamental insights into the non-genetic transcriptional processes that underpin malignant clonal fitness and may inform future therapeutic strategies., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) are an approved treatment for hormone receptor-positive breast cancer and are currently under evaluation across hundreds of clinical trials for other cancer types. The clinical success of these inhibitors is largely attributed to well-defined tumor-intrinsic cytostatic mechanisms, whereas their emerging role as immunomodulatory agents is less understood. Using integrated epigenomic, transcriptomic, and proteomic analyses, we demonstrated a novel action of CDK4/6 inhibitors in promoting the phenotypic and functional acquisition of immunologic T-cell memory. Short-term priming with a CDK4/6 inhibitor promoted long-term endogenous antitumor T-cell immunity in mice, enhanced the persistence and therapeutic efficacy of chimeric antigen receptor T cells, and induced a retinoblastoma-dependent T-cell phenotype supportive of favorable responses to immune checkpoint blockade in patients with melanoma. Together, these mechanistic insights significantly broaden the prospective utility of CDK4/6 inhibitors as clinical tools to boost antitumor T-cell immunity. SIGNIFICANCE: Immunologic memory is critical for sustained antitumor immunity. Our discovery that CDK4/6 inhibitors drive T-cell memory fate commitment sheds new light on their clinical activity, which is essential for the design of clinical trial protocols incorporating these agents, particularly in combination with immunotherapy, for the treatment of cancer. This article is highlighted in the In This Issue feature, p. 2355 ., (©2021 American Association for Cancer Research.)

Merkel cell carcinomas (MCC) are immunogenic skin cancers associated with viral infection or UV mutagenesis. To study T-cell infiltrates in MCC, we analyzed 58 MCC lesions from 39 patients using multiplex-IHC/immunofluorescence (m-IHC/IF). CD4 + or CD8 + T cells comprised the majority of infiltrating T lymphocytes in most tumors. However, almost half of the tumors harbored prominent CD4/CD8 double-negative (DN) T-cell infiltrates (>20% DN T cells), and in 12% of cases, DN T cells represented the majority of T cells. Flow cytometric analysis of single-cell suspensions from fresh tumors identified DN T cells as predominantly Vδ2 - γδ T cells. In the context of γδ T-cell inflammation, these cells expressed PD-1 and LAG3, which is consistent with a suppressed or exhausted phenotype, and CD103, which indicates tissue residency. Furthermore, single-cell RNA sequencing (scRNA-seq) identified a transcriptional profile of γδ T cells suggestive of proinflammatory potential. T-cell receptor (TCR) analysis confirmed clonal expansion of Vδ1 and Vδ3 clonotypes, and functional studies using cloned γδ TCRs demonstrated restriction of these for CD1c and MR1 antigen-presenting molecules. On the basis of a 13-gene γδ T-cell signature derived from scRNA-seq analysis, gene-set enrichment on bulk RNA-seq data showed a positive correlation between enrichment scores and DN T-cell infiltrates. An improved disease-specific survival was evident for patients with high enrichment scores, and complete responses to anti-PD-1/PD-L1 treatment were observed in three of four cases with high enrichment scores. Thus, γδ T-cell infiltration may serve as a prognostic biomarker and should be explored for therapeutic interventions. See related Spotlight on p. 600 ., (©2021 American Association for Cancer Research.)

Chronic stimulation of CD8 + T cells triggers exhaustion, a distinct differentiation state with diminished effector function. Exhausted cells exist in multiple differentiation states, from stem-like progenitors that are the key mediators of the response to checkpoint blockade, through to terminally exhausted cells. Due to its clinical relevance, there is substantial interest in defining the pathways that control differentiation and maintenance of these subsets. Here, we show that chronic antigen induces the anergy-associated transcription factor EGR2 selectively within progenitor exhausted cells in both chronic LCMV and tumours. EGR2 enables terminal exhaustion and stabilizes the exhausted transcriptional state by both direct EGR2-dependent control of key exhaustion-associated genes, and indirect maintenance of the exhausted epigenetic state. We show that EGR2 is a regulator of exhaustion that epigenetically and transcriptionally maintains the differentiation competency of progenitor exhausted cells.

Aims: The advent of specific ALK-targeting drugs has radically changed the outcome of patients with ALK translocated non-small-cell lung cancer (NSCLC). However, emerging resistance to treatment with ALK inhibitors in these patients remains a major concern. In previous studies, we analysed two ALK+ patient cohorts (TP53 wild-type/TP53 mutated) in terms of copy number alterations. All patients belonging to the TP53 wild-type group had mainly genetically stable genomes, with one exception showing chromosomal instability and amplifications of several gene loci, including TERT. Here, we aimed to determine the prevalence of TERT amplifications in these ALK+ lung cancer patients by analysing an independent cohort of 109 ALK translocated cases. We further analysed the copy numbers of numerous cancer-relevant genes and other genetic aberrations., Methods and Results: The prevalence of TERT amplifications was determined by means of FISH analyses. Copy numbers of 87 cancer-relevant genes were determined by NanoString nCounter ® technology, FoundationOne ® and lung-specific NGS panels in some of these TERT-amplified samples, and clinical data on patients with TERT-amplified tumours were collected. Our data revealed that five (4.6%) of all 109 analysed ALK+ patients harboured amplification of TERT and that these patients had genetically unstable genomes., Conclusions: Our preliminary study shows that ALK+ adenocarcinomas should be evaluated in the context of their genomic background in order to more clearly understand and predict patients' individual course of disease., (© 2020 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Combined inhibition of BRAF, MEK, and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a BRAF V600 mutation. While this triple therapy has potent tumor-intrinsic effects, the impact of this combination on antitumor immunity remains unexplored. Here, using a syngeneic Braf V600E Cdkn2a -/- Pten -/- melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA sequencing analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted proinflammatory macrophages and cross-priming CD103 + dendritic cells, the absence of which correlated with poor overall survival and clinical responses to ICB in patients with melanoma. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses ex vivo While combined BRAF, MEK, and CDK4/6 inhibition demonstrates favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact antitumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK, and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma., (©2020 American Association for Cancer Research.)

Unravelling spatio-temporal patterns of gene expression is crucial to understanding core biological principles from embryogenesis to disease. Here we review emerging technologies, providing automated, high-throughput, spatially resolved quantitative gene expression data. Novel techniques expand on current benchmark protocols, expediting their incorporation into ongoing research. These approaches digitally reconstruct patterns of embryonic expression in three dimensions, and have successfully identified novel domains of expression, cell types, and tissue features. Such technologies pave the way for unbiased and exhaustive recapitulation of gene expression levels in spatial and quantitative terms, promoting understanding of the molecular origin of developmental defects, and improving medical diagnostics.

The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development 1-6 . The molecular mechanism that underpins these reprogramming processes remains largely unexplored, which impedes our understanding and limits rational improvements to reprogramming protocols. Here, to address these issues, we reconstruct molecular reprogramming trajectories of human dermal fibroblasts using single-cell transcriptomics. This revealed that reprogramming into primed and naive pluripotency follows diverging and distinct trajectories. Moreover, genome-wide analyses of accessible chromatin showed key changes in the regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets revealed a role for transcription factors associated with the trophectoderm lineage, and the existence of a subpopulation of cells that enter a trophectoderm-like state during reprogramming. Furthermore, this trophectoderm-like state could be captured, which enabled the derivation of induced trophoblast stem cells. Induced trophoblast stem cells are molecularly and functionally similar to trophoblast stem cells derived from human blastocysts or first-trimester placentas 7 . Our results provide a high-resolution roadmap for the transcription-factor-mediated reprogramming of human somatic cells, indicate a role for the trophectoderm-lineage-specific regulatory program during this process, and facilitate the direct reprogramming of somatic cells into induced trophoblast stem cells.

The Mre11-Rad50-Nbs1 complex is a DNA double-strand break sensor that mediates a tumor-suppressive DNA damage response (DDR) in cells undergoing oncogenic stress, yet the mechanisms underlying this effect are poorly understood. Using a genetically inducible primary mammary epithelial cell model, we demonstrate that Mre11 suppresses proliferation and DNA damage induced by diverse oncogenic drivers through a p53-independent mechanism. Breast tumorigenesis models engineered to express a hypomorphic Mre11 allele exhibit increased levels of oncogene-induced DNA damage, R-loop accumulation, and chromosomal instability with a characteristic copy number loss phenotype. Mre11 complex dysfunction is identified in a subset of human triple-negative breast cancers and is associated with increased sensitivity to DNA-damaging therapy and inhibitors of ataxia telangiectasia and Rad3 related (ATR) and poly (ADP-ribose) polymerase (PARP). Thus, deficiencies in the Mre11-dependent DDR drive proliferation and genome instability patterns in p53-deficient breast cancers and represent an opportunity for therapeutic exploitation., Competing Interests: Declaration of Interests G.P.G. has ownership interest (including patents) in and is a consultant/advisory board member for Naveris, Inc., outside the scope of the present study. J.S.R.-F. reports personal/consultancy fees from VolitionRx, Page.AI, Goldman Sachs, Grail, Ventana Medical Systems, Invicro, Roche Diagnostics, and Genentech, outside the scope of the present study. J.H.J.P. is a consultant for Ideaya Biosciences, Novus Biologicals, and Atropos Therapeutics, outside the scope of the present study., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Primary cilia are sensory organelles which co-ordinate several developmental/repair pathways including hedgehog signalling. Studies of human renal allografts suffering acute tubular necrosis have shown that length of primary cilia borne by epithelial cells doubles throughout the nephron and collecting duct, and then normalises as renal function returns. Conversely the loss of primary cilia has been reported in chronic allograft rejection and linked to defective hedgehog signalling. We investigated the fate of primary cilia in renal allografts suffering acute rejection., Results: Here we observed that in renal allografts undergoing acute rejection, primary cilia were retained, with their length increasing 1 week after transplantation and remaining elevated. We used a mouse model of acute renal injury to demonstrate that elongated renal primary cilia in the injured renal tubule show evidence of smoothened accumulation, a biomarker for activation of hedgehog signalling. We conclude that primary cilium-mediated activation of hedgehog signalling is still possible during the acute phase of renal allograft rejection.