LifeTime and improving European healthcare through cell-based interceptive medicine.

Here we describe the LifeTime Initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. This mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. The analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. The timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. The application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.

The Smurf transition: new insights on ageing from end-of-life studies in animal models.

PURPOSE OF REVIEW: Over the past 5 years, many articles were published concerning the prediction of high risk of mortality in apparently healthy adults, echoing the first description in 2011 of the Smurf phenotype, a harbinger of natural death in drosophila. RECENT FINDINGS: These recent findings suggest that the end-of-life is molecularly and physiologically highly stereotyped, evolutionarily conserved and predictable. SUMMARY: Taken altogether, these results from independent teams using multiple organisms including humans draw the lines of future directions in ageing research. The ability to identify and study individuals about to die of natural causes with no apparent diseases is a game-changer in this field. In addition, the public health applications are potentially of tremendous impact in our ageing societies and raise important ethical questions.

Establishment of X chromosome inactivation and epigenomic features of the inactive X depend on cellular contexts.

X chromosome inactivation (XCI) is an essential epigenetic process that ensures X-linked gene dosage equilibrium between sexes in mammals. XCI is dynamically regulated during development in a manner that is intimately linked to differentiation. Numerous studies, which we review here, have explored the dynamics of X inactivation and reactivation in the context of development, differentiation and diseases, and the phenotypic and molecular link between the inactive status, and the cellular context. Here, we also assess whether XCI is a uniform mechanism in mammals by analyzing epigenetic signatures of the inactive X (Xi) in different species and cellular contexts. It appears that the timing of XCI and the epigenetic signature of the inactive X greatly vary between species. Surprisingly, even within a given species, various Xi configurations are found across cellular states. We discuss possible mechanisms underlying these variations, and how they might influence the fate of the Xi.

Regional copy number-independent deregulation of transcription in cancer.

Genetic and epigenetic alterations have been identified that lead to transcriptional deregulation in cancers. Genetic mechanisms may affect single genes or regions containing several neighboring genes, as has been shown for DNA copy number changes. It was recently reported that epigenetic suppression of gene expression can also extend to a whole region; this is known as long-range epigenetic silencing. Various techniques are available for identifying regional genetic alterations, but no large-scale analysis has yet been carried out to obtain an overview of regional epigenetic alterations. We carried out an exhaustive search for regions susceptible to such mechanisms using a combination of transcriptome correlation map analysis and array CGH data for a series of bladder carcinomas. We validated one candidate region experimentally, demonstrating histone methylation leading to the loss of expression of neighboring genes without DNA methylation.

Genome architecture marked by retrotransposons modulates predisposition to DNA methylation in cancer.

Epigenetic silencing plays an important role in cancer development. An attractive hypothesis is that local DNA features may participate in differential predisposition to gene hypermethylation. We found that, compared with methylation-resistant genes, methylation-prone genes have a lower frequency of SINE and LINE retrotransposons near their transcription start site. In several large testing sets, this distribution was highly predictive of promoter methylation. Genome-wide analysis showed that 22% of human genes were predicted to be methylation-prone in cancer; these tended to be genes that are down-regulated in cancer and that function in developmental processes. Moreover, retrotransposon distribution marks a larger fraction of methylation-prone genes compared to Polycomb group protein (PcG) marking in embryonic stem cells; indeed, PcG marking and our predictive model based on retrotransposon frequency appear to be correlated but also complementary. In summary, our data indicate that retrotransposon elements, which are widespread in our genome, are strongly associated with gene promoter DNA methylation in cancer and may in fact play a role in influencing epigenetic regulation in normal and abnormal physiological states.

Long non-coding RNAs and human X-chromosome regulation: a coat for the active X chromosome.

In mammals, the genic disequilibrium between males (XY) and females (XX) is resolved through the inactivation of one of the X-chromosomes in females. X-chromosome inactivation (XCI) takes place in all mammalian species, but has mainly been studied in the mouse model where it was shown to be controlled by the interplay of several long non-coding RNA (lncRNA). However, recent data point toward the existence of species divergences among mammals in the strategies used to achieve XCI. The recent discovery of XACT, a novel lncRNA that coats the active X-chromosome specifically in human pluripotent cells, further highlights the existence of human-specific mechanisms of X-chromosome regulation. Here, we discuss the roles of lncRNAs in defining species-specific mechanisms controlling X-inactivation and explore the potential role of large lncRNAs in gene activation.

Multi-modal quantification of pathway activity with MAYA.

Signaling pathways can be activated through various cascades of genes depending on cell identity and biological context. Single-cell atlases now provide the opportunity to inspect such complexity in health and disease. Yet, existing reference tools for pathway scoring resume activity of each pathway to one unique common metric across cell types. Here, we present MAYA, a computational method that enables the automatic detection and scoring of the diverse modes of activation of biological pathways across cell populations. MAYA improves the granularity of pathway analysis by detecting subgroups of genes within reference pathways, each characteristic of a cell population and how it activates a pathway. Using multiple single-cell datasets, we demonstrate the biological relevance of identified modes of activation, the robustness of MAYA to noisy pathway lists and batch effect. MAYA can also predict cell types starting from lists of reference markers in a cluster-free manner. Finally, we show that MAYA reveals common modes of pathway activation in tumor cells across patients, opening the perspective to discover shared therapeutic vulnerabilities.

A benchmark of computational pipelines for single-cell histone modification data.

BACKGROUND: Single-cell histone post translational modification (scHPTM) assays such as scCUT&Tag or scChIP-seq allow single-cell mapping of diverse epigenomic landscapes within complex tissues and are likely to unlock our understanding of various mechanisms involved in development or diseases. Running scHTPM experiments and analyzing the data produced remains challenging since few consensus guidelines currently exist regarding good practices for experimental design and data analysis pipelines. RESULTS: We perform a computational benchmark to assess the impact of experimental parameters and data analysis pipelines on the ability of the cell representation to recapitulate known biological similarities. We run more than ten thousand experiments to systematically study the impact of coverage and number of cells, of the count matrix construction method, of feature selection and normalization, and of the dimension reduction algorithm used. This allows us to identify key experimental parameters and computational choices to obtain a good representation of single-cell HPTM data. We show in particular that the count matrix construction step has a strong influence on the quality of the representation and that using fixed-size bin counts outperforms annotation-based binning. Dimension reduction methods based on latent semantic indexing outperform others, and feature selection is detrimental, while keeping only high-quality cells has little influence on the final representation as long as enough cells are analyzed. CONCLUSIONS: This benchmark provides a comprehensive study on how experimental parameters and computational choices affect the representation of single-cell HPTM data. We propose a series of recommendations regarding matrix construction, feature and cell selection, and dimensionality reduction algorithms.

Evolution of synchronous female bilateral breast cancers and response to treatment.

Synchronous bilateral breast cancer (sBBC) occurs after both breasts have been affected by the same germline genetics and environmental exposures. Little evidence exists regarding immune infiltration and response to treatment in sBBCs. Here we show that the impact of the subtype of breast cancer on levels of tumor infiltrating lymphocytes (TILs, n = 277) and on pathologic complete response (pCR) rates (n = 140) differed according to the concordant or discordant subtype of breast cancer of the contralateral tumor: luminal breast tumors with a discordant contralateral tumor had higher TIL levels and higher pCR rates than those with a concordant contralateral tumor. Tumor sequencing revealed that left and right tumors (n = 20) were independent regarding somatic mutations, copy number alterations and clonal phylogeny, whereas primary tumor and residual disease were closely related both from the somatic mutation and from the transcriptomic point of view. Our study indicates that tumor-intrinsic characteristics may have a role in the association of tumor immunity and pCR and demonstrates that the characteristics of the contralateral tumor are also associated with immune infiltration and response to treatment.

Evaluation of predictive models in daily practice for the identification of patients with Lynch syndrome.

The optimal strategy for identifying patients with Lynch syndrome among patients with newly diagnosed colorectal cancer (CRC) is still debated. Several predictive models (e.g., MMRpredict, PREMM1,2 and MMRpro) combining personal and familial data have recently been developed to quantify the risk that a given patient with CRC carries a Lynch syndrome-causing mutation. Their clinical applicability to patients with CRC from the general population requires evaluation. We studied a consecutive series of 214 patients with newly diagnosed CRC characterized for tumor microsatellite instability (MSI), somatic BRAF mutation, MLH1 promoter methylation and mismatch repair (MMR) gene germline mutation status. The performances of the models for identifying MMR mutation carriers (8/214, 3.7%) were evaluated and compared to the revised Bethesda guidelines and a molecular strategy based on MSI testing in all patients followed by the exclusion of MSI-positive sporadic cases from mutational testing by screening for BRAF mutation and MLH1 promoter methylation. The sensitivities of the three models, at the lowest thresholds proposed, were identical (75%), with similar numbers of probands eligible for further MSI testing (almost half the patients). In our dataset, the prediction models gave no better discrimination than the revised Bethesda guidelines. Both approaches failed to identify two of the eight mutation carriers (the same two patients, aged 67 and 81 years, both with no family history). Thus, like the revised Bethesda guidelines, predictive models did not identify all patients with Lynch syndrome in our series of consecutive CRC. Our results support systematic screening for MMR deficiency in all new CRC cases.

Epigenomic tumor evolution under the spotlight: the promises of single-cell approaches.

Cancer evolution was long-reduced to a genetic equation. The latest technological and subsequent conceptual advances, catalyzed by single-cell approaches, now begin to reveal the long-suspected part played by epigenomic and transcriptomic mechanisms in cancer evolution. Lie ahead numerous challenges to integrate multi-modal measurements of individual cancer cells over time and space, while aiming for better disease management and the discovery of therapeutic targets and biomarkers.

L'évolution du cancer a longtemps été réduite à une équation génétique. Les dernières avancées technologiques et conceptuelles, catalysées par les approches unicellulaires, commencent maintenant à révéler le rôle longtemps soupçonné des mécanismes épigénomiques et transcriptomiques dans l'évolution du cancer. De nombreux défis nous attendent pour intégrer les mesures multimodales des cellules cancéreuses individuelles dans le temps et l'espace, tout en visant une meilleure gestion de la maladie et la découverte de cibles thérapeutiques et de biomarqueurs.

H3K27me3 conditions chemotolerance in triple-negative breast cancer.

The persistence of cancer cells resistant to therapy remains a major clinical challenge. In triple-negative breast cancer, resistance to chemotherapy results in the highest recurrence risk among breast cancer subtypes. The drug-tolerant state seems largely defined by nongenetic features, but the underlying mechanisms are poorly understood. Here, by monitoring epigenomes, transcriptomes and lineages with single-cell resolution, we show that the repressive histone mark H3K27me3 (trimethylation of histone H3 at lysine 27) regulates cell fate at the onset of chemotherapy. We report that a persister expression program is primed with both H3K4me3 (trimethylation of histone H3 at lysine 4) and H3K27me3 in unchallenged cells, with H3K27me3 being the lock to its transcriptional activation. We further demonstrate that depleting H3K27me3 enhances the potential of cancer cells to tolerate chemotherapy. Conversely, preventing H3K27me3 demethylation simultaneously to chemotherapy inhibits the transition to a drug-tolerant state, and delays tumor recurrence in vivo. Our results highlight how chromatin landscapes shape the potential of cancer cells to respond to initial therapy.

A high-risk retinoblastoma subtype with stemness features, dedifferentiated cone states and neuronal/ganglion cell gene expression.

Retinoblastoma is the most frequent intraocular malignancy in children, originating from a maturing cone precursor in the developing retina. Little is known on the molecular basis underlying the biological and clinical behavior of this cancer. Here, using multi-omics data, we demonstrate the existence of two retinoblastoma subtypes. Subtype 1, of earlier onset, includes most of the heritable forms. It harbors few genetic alterations other than the initiating RB1 inactivation and corresponds to differentiated tumors expressing mature cone markers. By contrast, subtype 2 tumors harbor frequent recurrent genetic alterations including MYCN-amplification. They express markers of less differentiated cone together with neuronal/ganglion cell markers with marked inter- and intra-tumor heterogeneity. The cone dedifferentiation in subtype 2 is associated with stemness features including low immune and interferon response, E2F and MYC/MYCN activation and a higher propensity for metastasis. The recognition of these two subtypes, one maintaining a cone-differentiated state, and the other, more aggressive, associated with cone dedifferentiation and expression of neuronal markers, opens up important biological and clinical perspectives for retinoblastomas.

Tuning parameters of dimensionality reduction methods for single-cell RNA-seq analysis.

BACKGROUND: Many computational methods have been developed recently to analyze single-cell RNA-seq (scRNA-seq) data. Several benchmark studies have compared these methods on their ability for dimensionality reduction, clustering, or differential analysis, often relying on default parameters. Yet, given the biological diversity of scRNA-seq datasets, parameter tuning might be essential for the optimal usage of methods, and determining how to tune parameters remains an unmet need. RESULTS: Here, we propose a benchmark to assess the performance of five methods, systematically varying their tunable parameters, for dimension reduction of scRNA-seq data, a common first step to many downstream applications such as cell type identification or trajectory inference. We run a total of 1.5 million experiments to assess the influence of parameter changes on the performance of each method, and propose two strategies to automatically tune parameters for methods that need it. CONCLUSIONS: We find that principal component analysis (PCA)-based methods like scran and Seurat are competitive with default parameters but do not benefit much from parameter tuning, while more complex models like ZinbWave, DCA, and scVI can reach better performance but after parameter tuning.

Interactive analysis of single-cell epigenomic landscapes with ChromSCape.

Chromatin modifications orchestrate the dynamic regulation of gene expression during development and in disease. Bulk approaches have characterized the wide repertoire of histone modifications across cell types, detailing their role in shaping cell identity. However, these population-based methods do not capture cell-to-cell heterogeneity of chromatin landscapes, limiting our appreciation of the role of chromatin in dynamic biological processes. Recent technological developments enable the mapping of histone marks at single-cell resolution, opening up perspectives to characterize the heterogeneity of chromatin marks in complex biological systems over time. Yet, existing tools used to analyze bulk histone modifications profiles are not fit for the low coverage and sparsity of single-cell epigenomic datasets. Here, we present ChromSCape, a user-friendly interactive Shiny/R application distributed as a Bioconductor package, that processes single-cell epigenomic data to assist the biological interpretation of chromatin landscapes within cell populations. ChromSCape analyses the distribution of repressive and active histone modifications as well as chromatin accessibility landscapes from single-cell datasets. Using ChromSCape, we deconvolve chromatin landscapes within the tumor micro-environment, identifying distinct H3K27me3 landscapes associated with cell identity and breast tumor subtype.

CD44 regulates epigenetic plasticity by mediating iron endocytosis.

CD44 is a transmembrane glycoprotein linked to various biological processes reliant on epigenetic plasticity, which include development, inflammation, immune responses, wound healing and cancer progression. Although it is often referred to as a cell surface marker, the functional regulatory roles of CD44 remain elusive. Here we report the discovery that CD44 mediates the endocytosis of iron-bound hyaluronates in tumorigenic cell lines, primary cancer cells and tumours. This glycan-mediated iron endocytosis mechanism is enhanced during epithelial-mesenchymal transitions, in which iron operates as a metal catalyst to demethylate repressive histone marks that govern the expression of mesenchymal genes. CD44 itself is transcriptionally regulated by nuclear iron through a positive feedback loop, which is in contrast to the negative regulation of the transferrin receptor by excess iron. Finally, we show that epigenetic plasticity can be altered by interfering with iron homeostasis using small molecules. This study reveals an alternative iron-uptake mechanism that prevails in the mesenchymal state of cells, which illuminates a central role of iron as a rate-limiting regulator of epigenetic plasticity.

High-throughput single-cell ChIP-seq identifies heterogeneity of chromatin states in breast cancer.

Modulation of chromatin structure via histone modification is a major epigenetic mechanism and regulator of gene expression. However, the contribution of chromatin features to tumor heterogeneity and evolution remains unknown. Here we describe a high-throughput droplet microfluidics platform to profile chromatin landscapes of thousands of cells at single-cell resolution. Using patient-derived xenograft models of acquired resistance to chemotherapy and targeted therapy in breast cancer, we found that a subset of cells within untreated drug-sensitive tumors share a common chromatin signature with resistant cells, undetectable using bulk approaches. These cells, and cells from the resistant tumors, have lost chromatin marks-H3K27me3, which is associated with stable transcriptional repression-for genes known to promote resistance to treatment. This single-cell chromatin immunoprecipitation followed by sequencing approach paves the way to study the role of chromatin heterogeneity, not just in cancer but in other diseases and healthy systems, notably during cellular differentiation and development.

Identification in daily practice of patients with Lynch syndrome (hereditary nonpolyposis colorectal cancer): revised Bethesda guidelines-based approach versus molecular screening.

BACKGROUND: The identification of individuals who should undergo hereditary nonpolyposis colorectal cancer (HNPCC) genetic testing remains a critical issue. The Bethesda guidelines were developed to preselect patients for microsatellite instability (MSI) testing before germline mutation screening. These criteria have been revised, and a new set of recommendations, the revised Bethesda guidelines, has been proposed. OBJECTIVE: To evaluate the performance of these revised guidelines for identifying patients with HNPCC in a series of unselected consecutive patients and compare this revised guidelines-based approach with a molecular strategy (MSI testing for all tumors, followed by exclusion of MSI-positive sporadic cases from mutational testing). PATIENTS AND METHODS: The study included 214 patients with newly diagnosed colorectal cancer. The MSI analysis was performed for all tumors. Germline testing, guided by immunohistochemical staining for mismatch repair proteins, was performed for patients with MSI-positive tumors. Sporadic MSI-positive tumors were identified by screening for BRAF mutation and MLH1 promoter methylation. RESULTS: Ninety patients (42.1%) met the revised guidelines. Twenty-one patients (9.8%) had MSI-positive tumors. Germline testing identified eight mutations (3.7%) (MSH2 N = 5, MLH1 N = 2, MSH6 N =1). The revised guidelines failed to identify 2 of the 8 probands (aged 67 and 81 yr, both with no family history). In contrast, the molecular strategy identified all patients requiring testing for germline mutation. The percentages of patients selected for germline testing by the revised guidelines and the molecular strategy were 4.2% and 5.1%, respectively. CONCLUSIONS: The revised Bethesda guidelines did not identify all HNPCC cases in our series. The molecular approach identified all HNPCC patients with MSI-positive tumors, increasing the workload for germline testing only slightly.