Cell type signatures in cell-free DNA fragmentation profiles reveal disease biology.

Circulating cell-free DNA (cfDNA) fragments have characteristics that are specific to the cell types that release them. Current methods for cfDNA deconvolution typically use disease tailored marker selection in a limited number of bulk tissues or cell lines. Here, we utilize single cell transcriptome data as a comprehensive cellular reference set for disease-agnostic cfDNA cell-of-origin analysis. We correlate cfDNA-inferred nucleosome spacing with gene expression to rank the relative contribution of over 490 cell types to plasma cfDNA. In 744 healthy individuals and patients, we uncover cell type signatures in support of emerging disease paradigms in oncology and prenatal care. We train predictive models that can differentiate patients with colorectal cancer (84.7%), early-stage breast cancer (90.1%), multiple myeloma (AUC 95.0%), and preeclampsia (88.3%) from matched controls. Importantly, our approach performs well in ultra-low coverage cfDNA datasets and can be readily transferred to diverse clinical settings for the expansion of liquid biopsy.

Evaluating offspring Genomic and Epigenomic alterations after prenatal exposure to Cancer treatment In Pregnancy (GE-CIP): a multicentric observational study.

INTRODUCTION: Around 1 in 1000-2000 pregnancies are affected by a cancer diagnosis. Previous studies have shown that chemotherapy during pregnancy has reassuring cognitive and cardiac neonatal outcomes, and hence has been proposed as standard of care. However, although these children perform within normal ranges for their age, subtle differences have been identified. Given that chemotherapeutic compounds can cross the placenta, the possibility that prenatal chemotherapy exposure mutates the offspring's genome and/or epigenome, with potential deleterious effects later in life, urges to be investigated. METHODS AND ANALYSES: This multicentric observational study aims to collect cord blood, meconium and neonatal buccal cells at birth, as well as peripheral blood, buccal cells and urine from infants when 6, 18 and/or 36 months of age. Using bulk and single-cell approaches, we will compare samples from chemotherapy-treated pregnant patients with cancer, pregnant patients with cancer not treated with chemotherapy and healthy pregnant women. Potential chemotherapy-related newborn genomic and/or epigenomic alterations, such as single nucleotide variants, copy number variants and DNA-methylation alterations, will be identified in mononuclear and epithelial cells, isolated from blood, buccal swabs and urine. DNA from maternal peripheral blood and paternal buccal cells will be used to determine de novo somatic mutations in the neonatal blood and epithelial cells. Additionally, the accumulated exposure of the fetus, and biological effective dose of alkylating agents, will be assessed in meconium and cord blood via mass spectrometry approaches. ETHICS AND DISSEMINATION: The Ethics Committee Research of UZ/KU Leuven (EC Research) and the Medical Ethical Review Committee of University Medical Center Amsterdam have approved the study. Results of this study will be disseminated via presentations at (inter)national conferences, through peer-reviewed, open-access publications, via social media platforms aimed to inform patients and healthcare workers, and through the website of the International Network on Cancer, Infertility and Pregnancy (www.cancerinpregnancy.org).

The serine synthesis pathway drives osteoclast differentiation through epigenetic regulation of NFATc1 expression.

Bone-resorbing osteoclasts are vital for postnatal bone health, as increased differentiation or activity results in skeletal pathologies such as osteoporosis. The metabolism of mature osteoclasts differs from their progenitor cells, but whether the observed metabolic changes are secondary to the altered cell state or actively drive the process of cell differentiation is unknown. Here, we show that transient activation of the serine synthesis pathway (SSP) is essential for osteoclastogenesis, as deletion of the rate-limiting enzyme phosphoglycerate dehydrogenase in osteoclast progenitors impairs their differentiation and results in increased bone mass. In addition, pharmacological phosphoglycerate dehydrogenase inhibition abrogated bone loss in a mouse model of postmenopausal osteoporosis by blocking bone resorption. Mechanistically, SSP-derived α-ketoglutarate is necessary for histone demethylases that remove repressive histone methylation marks at the nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1) gene locus, thereby inducing NFATc1 expression and consequent osteoclast maturation. Taken together, this study reveals a metabolic-epigenetic coupling mechanism that directs osteoclast differentiation and suggests that the SSP can be therapeutically targeted to prevent osteoporotic bone loss.

Epigenetic remodelling under hypoxia.

Hypoxia is intrinsic to tumours and contributes to malignancy and metastasis while hindering the efficiency of existing treatments. Epigenetic mechanisms play a crucial role in the regulation of hypoxic cancer cell programs, both in the initial phases of sensing the decrease in oxygen levels and during adaptation to chronic lack of oxygen. During the latter, the epigenetic regulation of tumour biology intersects with hypoxia-sensitive transcription factors in a complex network of gene regulation that also involves metabolic reprogramming. Here, we review the current literature on the epigenetic control of gene programs in hypoxic cancer cells. We highlight common themes and features of such epigenetic remodelling and discuss their relevance for the development of therapeutic strategies.

Cell-free DNA methylation-based preeclampsia prediction: A journey to improve maternal health.

Presymptomatic prediction of preeclampsia (PE) is crucial to enable early prophylactic treatment. Current screening tools are either complex or lack predictive value. We recently demonstrated that cell-free DNA methylation can be leveraged to predict early-onset PE in 57% at a 10% false positive rate. Importantly, this minimally invasive screening test can be implemented as an add-on to current widespread noninvasive prenatal aneuploidy screening. Here, we highlight the pitfalls and promising prospects of this research.

A Metabolic Gene Survey Pinpoints Fucosylation as a Key Pathway Underlying the Suppressive Function of Regulatory T Cells in Cancer.

Forkhead box P3 (Foxp3)-expressing regulatory T cells (Treg) are the guardians of controlled immune reactions and prevent the development of autoimmune diseases. However, in the tumor context, their increased number suppresses antitumor immune responses, indicating the importance of understanding the mechanisms behind their function and stability. Metabolic reprogramming can affect Foxp3 regulation and, therefore, Treg suppressive function and fitness. Here, we performed a metabolic CRISPR/Cas9 screen and pinpointed novel candidate positive and negative metabolic regulators of Foxp3. Among the positive regulators, we revealed that targeting the GDP-fucose transporter Slc35c1, and more broadly fucosylation (Fuco), in Tregs compromises their proliferation and suppressive function both in vitro and in vivo, leading to alteration of the tumor microenvironment and impaired tumor progression and protumoral immune responses. Pharmacologic inhibition of Fuco dampened tumor immunosuppression mostly by targeting Tregs, thus resulting in reduced tumor growth. In order to substantiate these findings in humans, tumoral Tregs from patients with colorectal cancer were clustered on the basis of the expression of Fuco-related genes. FucoLOW Tregs were found to exhibit a more immunogenic profile compared with FucoHIGH Tregs. Furthermore, an enrichment of a FucoLOW signature, mainly derived from Tregs, correlated with better prognosis and response to immune checkpoint blockade in melanoma patients. In conclusion, Slc35c1-dependent Fuco is able to regulate the suppressive function of Tregs, and measuring its expression in Tregs might pave the way towards a useful biomarker model for patients with cancer. See related Spotlight by Silveria and DuPage, p. 1570.

GiRAFR improves gRNA detection and annotation in single-cell CRISPR screens.

Novel methods that combine single cell RNA-seq with CRISPR screens enable high-throughput characterization of transcriptional changes caused by genetic perturbations. Dedicated software is however lacking to annotate CRISPR guide RNA (gRNA) libraries and associate them with single cell transcriptomes. Here, we describe a CRISPR droplet sequencing (CROP-seq) dataset. During analysis, we observed that the most commonly used method fails to detect mutant gRNAs. We therefore developed a python tool to identify and characterize intact and mutant gRNAs, called GiRAFR. We show that mutant gRNAs are dysfunctional, and failure to detect and annotate them leads to an inflated estimate of the number of untransformed cells, attenuated downregulation of target genes, as well as an underestimated multiplet frequency. These findings are mirrored in publicly available datasets, where we find that up to 35% of cells are transduced with a mutant gRNA. Applying GiRAFR hence stands to improve the annotation and quality of single cell CRISPR screens.

Cell-free DNA methylome analysis for early preeclampsia prediction.

Preeclampsia (PE) is a leading cause for peripartal morbidity, especially if developing early in gestation. To enable prophylaxis in the prevention of PE, pregnancies at risk of PE must be identified early-in the first trimester. To identify at-risk pregnancies we profiled methylomes of plasma-derived, cell-free DNA from 498 pregnant women, of whom about one-third developed early-onset PE. We detected DNA methylation differences between control and PE pregnancies that enabled risk stratification at PE diagnosis but also presymptomatically, at around 12 weeks of gestation (range 9-14 weeks). The first-trimester risk prediction model was validated in an external cohort collected from two centers (area under the curve (AUC) = 0.75) and integrated with routinely available maternal risk factors (AUC = 0.85). The combined risk score correctly predicted 72% of patients with early-onset PE at 80% specificity. These preliminary results suggest that cell-free DNA methylation profiling is a promising tool for presymptomatic PE risk assessment, and has the potential to improve treatment and follow-up in the obstetric clinic.

Metabolic profiling stratifies colorectal cancer and reveals adenosylhomocysteinase as a therapeutic target.

The genomic landscape of colorectal cancer (CRC) is shaped by inactivating mutations in tumour suppressors such as APC, and oncogenic mutations such as mutant KRAS. Here we used genetically engineered mouse models, and multimodal mass spectrometry-based metabolomics to study the impact of common genetic drivers of CRC on the metabolic landscape of the intestine. We show that untargeted metabolic profiling can be applied to stratify intestinal tissues according to underlying genetic alterations, and use mass spectrometry imaging to identify tumour, stromal and normal adjacent tissues. By identifying ions that drive variation between normal and transformed tissues, we found dysregulation of the methionine cycle to be a hallmark of APC-deficient CRC. Loss of Apc in the mouse intestine was found to be sufficient to drive expression of one of its enzymes, adenosylhomocysteinase (AHCY), which was also found to be transcriptionally upregulated in human CRC. Targeting of AHCY function impaired growth of APC-deficient organoids in vitro, and prevented the characteristic hyperproliferative/crypt progenitor phenotype driven by acute deletion of Apc in vivo, even in the context of mutant Kras. Finally, pharmacological inhibition of AHCY reduced intestinal tumour burden in ApcMin/+ mice indicating its potential as a metabolic drug target in CRC.

Heterogeneity of Repolarization and Cell-Cell Variability of Cardiomyocyte Remodeling Within the Myocardial Infarction Border Zone Contribute to Arrhythmia Susceptibility.

BACKGROUND: After myocardial infarction, the infarct border zone (BZ) is the dominant source of life-threatening arrhythmias, where fibrosis and abnormal repolarization create a substrate for reentry. We examined whether repolarization abnormalities are heterogeneous within the BZ in vivo and could be related to heterogeneous cardiomyocyte remodeling. METHODS: Myocardial infarction was induced in domestic pigs by 120-minute ischemia followed by reperfusion. After 1 month, remodeling was assessed by magnetic resonance imaging, and electroanatomical mapping was performed to determine the spatial distribution of activation-recovery intervals. Cardiomyocytes were isolated and tissue samples collected from the BZ and remote regions. Optical recording allowed assessment of action potential duration (di-8-ANEPPS, stimulation at 1 Hz, 37 °C) of large cardiomyocyte populations while gene expression in cardiomyocytes was determined by single nuclear RNA sequencing. RESULTS: In vivo, activation-recovery intervals in the BZ tended to be longer than in remote with increased spatial heterogeneity evidenced by a greater local SD (3.5±1.3 ms versus remote: 2.0±0.5 ms, P=0.036, npigs=5). Increased activation-recovery interval heterogeneity correlated with enhanced arrhythmia susceptibility. Cellular population studies (ncells=635-862 cells per region) demonstrated greater heterogeneity of action potential duration in the BZ (SD, 105.9±17.0 ms versus remote: 73.9±8.6 ms; P=0.001; npigs=6), which correlated with heterogeneity of activation-recovery interval in vivo. Cell-cell gene expression heterogeneity in the BZ was evidenced by increased Euclidean distances between nuclei of the BZ (12.1 [9.2-15.0] versus 10.6 [7.5-11.6] in remote; P<0.0001). Differentially expressed genes characterizing BZ cardiomyocyte remodeling included hypertrophy-related and ion channel-related genes with high cell-cell variability of expression. These gene expression changes were driven by stress-responsive TFs (transcription factors). In addition, heterogeneity of left ventricular wall thickness was greater in the BZ than in remote. CONCLUSIONS: Heterogeneous cardiomyocyte remodeling in the BZ is driven by uniquely altered gene expression, related to heterogeneity in the local microenvironment, and translates to heterogeneous repolarization and arrhythmia vulnerability in vivo.

Methods and applications for single-cell and spatial multi-omics.

The joint analysis of the genome, epigenome, transcriptome, proteome and/or metabolome from single cells is transforming our understanding of cell biology in health and disease. In less than a decade, the field has seen tremendous technological revolutions that enable crucial new insights into the interplay between intracellular and intercellular molecular mechanisms that govern development, physiology and pathogenesis. In this Review, we highlight advances in the fast-developing field of single-cell and spatial multi-omics technologies (also known as multimodal omics approaches), and the computational strategies needed to integrate information across these molecular layers. We demonstrate their impact on fundamental cell biology and translational research, discuss current challenges and provide an outlook to the future.

The pulmonary vasculature in lethal COVID-19 and idiopathic pulmonary fibrosis at single-cell resolution.

AIMS: Severe acute respiratory syndrome coronavirus-2 infection causes COVID-19, which in severe cases evokes life-threatening acute respiratory distress syndrome (ARDS). Transcriptome signatures and the functional relevance of non-vascular cell types (e.g. immune and epithelial cells) in COVID-19 are becoming increasingly evident. However, despite its known contribution to vascular inflammation, recruitment/invasion of immune cells, vascular leakage, and perturbed haemostasis in the lungs of severe COVID-19 patients, an in-depth interrogation of the endothelial cell (EC) compartment in lethal COVID-19 is lacking. Moreover, progressive fibrotic lung disease represents one of the complications of COVID-19 pneumonia and ARDS. Analogous features between idiopathic pulmonary fibrosis (IPF) and COVID-19 suggest partial similarities in their pathophysiology, yet, a head-to-head comparison of pulmonary cell transcriptomes between both conditions has not been implemented to date. METHODS AND RESULTS: We performed single-nucleus RNA-sequencing on frozen lungs from 7 deceased COVID-19 patients, 6 IPF explant lungs, and 12 controls. The vascular fraction, comprising 38 794 nuclei, could be subclustered into 14 distinct EC subtypes. Non-vascular cell types, comprising 137 746 nuclei, were subclustered and used for EC-interactome analyses. Pulmonary ECs of deceased COVID-19 patients showed an enrichment of genes involved in cellular stress, as well as signatures suggestive of dampened immunomodulation and impaired vessel wall integrity. In addition, increased abundance of a population of systemic capillary and venous ECs was identified in COVID-19 and IPF. COVID-19 systemic ECs closely resembled their IPF counterparts, and a set of 30 genes was found congruently enriched in systemic ECs across studies. Receptor-ligand interaction analysis of ECs with non-vascular cell types in the pulmonary micro-environment revealed numerous previously unknown interactions specifically enriched/depleted in COVID-19 and/or IPF. CONCLUSIONS: This study uncovered novel insights into the abundance, expression patterns, and interactomes of EC subtypes in COVID-19 and IPF, relevant for future investigations into the progression and treatment of both lethal conditions.

Lupus, DNA Methylation, and Air Pollution: A Malicious Triad.

The pathogenesis of systemic lupus erythematosus (SLE) remains elusive to this day; however, genetic, epigenetic, and environmental factors have been implicated to be involved in disease pathogenesis. Recently, it was demonstrated that in systemic lupus erythematosus (SLE) patients, interferon-regulated genes are hypomethylated in naïve CD4+ T cells, CD19+ B lymphocytes, and CD14+ monocytes. This suggests that interferon-regulated genes may have been epigenetically poised in SLE patients for rapid expression upon stimulation by different environmental factors. Additionally, environmental studies have identified DNA (hypo)methylation changes as a potential mechanism of environmentally induced health effects in utero, during childhood and in adults. Finally, epidemiologic studies have firmly established air pollution as a crucial SLE risk factor, as studies showed an association between fine particulate matter (PM2.5) and traditional SLE biomarkers related to disease flare, hospital admissions, and an increased SLEDAI score. In this review, the relationship between aberrant epigenetic regulation, the environment, and the development of SLE will be discussed.

Skeletal progenitors preserve proliferation and self-renewal upon inhibition of mitochondrial respiration by rerouting the TCA cycle.

A functional electron transport chain (ETC) is crucial for supporting bioenergetics and biosynthesis. Accordingly, ETC inhibition decreases proliferation in cancer cells but does not seem to impair stem cell proliferation. However, it remains unclear how stem cells metabolically adapt. In this study, we show that pharmacological inhibition of complex III of the ETC in skeletal stem and progenitor cells induces glycolysis side pathways and reroutes the tricarboxylic acid (TCA) cycle to regenerate NAD+ and preserve cell proliferation. These metabolic changes also culminate in increased succinate and 2-hydroxyglutarate levels that inhibit Ten-eleven translocation (TET) DNA demethylase activity, thereby preserving self-renewal and multilineage potential. Mechanistically, mitochondrial malate dehydrogenase and reverse succinate dehydrogenase activity proved to be essential for the metabolic rewiring in response to ETC inhibition. Together, these data show that the metabolic plasticity of skeletal stem and progenitor cells allows them to bypass ETC blockade and preserve their self-renewal.

More of less: Novel multi-ome profiling of single human neurons.

Epigenetic modifications to DNA and chromatin interact to influence gene expression and cellular phenotypes, but defining these omics layers in complex tissues is a daunting task. In this issue of Cell Genomics, Luo et al. describe a novel single-cell multi-omic method, simultaneously profiling transcriptome, DNA methylome, and chromatin accessibility, to shed light on human neurons.

Correction: Integrative analysis of genomic variants reveals new associations of candidate haploinsufficient genes with congenital heart disease.

[This corrects the article DOI: 10.1371/journal.pgen.1009679.].

Integrative analysis of genomic variants reveals new associations of candidate haploinsufficient genes with congenital heart disease.

Numerous genetic studies have established a role for rare genomic variants in Congenital Heart Disease (CHD) at the copy number variation (CNV) and de novo variant (DNV) level. To identify novel haploinsufficient CHD disease genes, we performed an integrative analysis of CNVs and DNVs identified in probands with CHD including cases with sporadic thoracic aortic aneurysm. We assembled CNV data from 7,958 cases and 14,082 controls and performed a gene-wise analysis of the burden of rare genomic deletions in cases versus controls. In addition, we performed variation rate testing for DNVs identified in 2,489 parent-offspring trios. Our analysis revealed 21 genes which were significantly affected by rare CNVs and/or DNVs in probands. Fourteen of these genes have previously been associated with CHD while the remaining genes (FEZ1, MYO16, ARID1B, NALCN, WAC, KDM5B and WHSC1) have only been associated in small cases series or show new associations with CHD. In addition, a systems level analysis revealed affected protein-protein interaction networks involved in Notch signaling pathway, heart morphogenesis, DNA repair and cilia/centrosome function. Taken together, this approach highlights the importance of re-analyzing existing datasets to strengthen disease association and identify novel disease genes and pathways.

Novel next-generation sequencing-based methodologies to characterize the mutational consequences of (prenatal) chemotherapy exposure in noncancerous tissue.

PURPOSE OF REVIEW: Although chemotherapeutics are considered as genotoxins for decades, their exact mutagenic impact on the genome of cancerous and normal cells of cancer patients was unknown for a long time. However, this knowledge is necessary to understand the long-term side effects of chemotherapy. A particular condition represents pregnant cancer patients being treated with chemotherapy. Since certain chemotherapeutics can cross the placenta, concerns exist about possible mutational effects on the fetus' genome with potential long-term health consequences. RECENT FINDINGS: Recent advances of next-generation sequencing (NGS) techniques have opened possibilities to explore the exact mutational footprint of chemotherapies in healthy tissue from treated cancer patients. However, the ultra-low frequency of chemotherapy-induced mutations, introduction of technical artefacts, and inaccessibility of normal tissue has posed important limitations. This review discusses five state-of-the-art approaches that were recently designed to overcome these drawbacks. SUMMARY: Results of the latest investigations give valuable insights into the genome-wide genotoxicity profile of frequently applied chemotherapies, with most of these drugs being associated with a signature of random base substitutions and small indels. Though these findings still might be limited to extrapolate to healthy tissue, they pave the way for studies on the origin of long-term chemotherapy-related adverse health effects.

Overcoming immunotherapy resistance in non-small cell lung cancer (NSCLC) - novel approaches and future outlook.

Immunotherapy (IO) has revolutionized the therapy landscape of non-small cell lung cancer (NSCLC), significantly prolonging the overall survival (OS) of advanced stage patients. Over the recent years IO therapy has been broadly integrated into the first-line setting of non-oncogene driven NSCLC, either in combination with chemotherapy, or in selected patients with PD-L1high expression as monotherapy. Still, a significant proportion of patients suffer from disease progression. A better understanding of resistance mechanisms depicts a central goal to avoid or overcome IO resistance and to improve patient outcome.We here review major cellular and molecular pathways within the tumor microenvironment (TME) that may impact the evolution of IO resistance. We summarize upcoming treatment options after IO resistance including novel IO targets (e.g. RIG-I, STING) as well as interesting combinational approaches such as IO combined with anti-angiogenic agents or metabolic targets (e.g. IDO-1, adenosine signaling, arginase). By discussing the fundamental mode of action of IO within the TME, we aim to understand and manage IO resistance and to seed new ideas for effective therapeutic IO concepts.

Systems genetics analysis identifies calcium-signaling defects as novel cause of congenital heart disease.

BACKGROUND: Congenital heart disease (CHD) occurs in almost 1% of newborn children and is considered a multifactorial disorder. CHD may segregate in families due to significant contribution of genetic factors in the disease etiology. The aim of the study was to identify pathophysiological mechanisms in families segregating CHD. METHODS: We used whole exome sequencing to identify rare genetic variants in ninety consenting participants from 32 Danish families with recurrent CHD. We applied a systems biology approach to identify developmental mechanisms influenced by accumulation of rare variants. We used an independent cohort of 714 CHD cases and 4922 controls for replication and performed functional investigations using zebrafish as in vivo model. RESULTS: We identified 1785 genes, in which rare alleles were shared between affected individuals within a family. These genes were enriched for known cardiac developmental genes, and 218 of these genes were mutated in more than one family. Our analysis revealed a functional cluster, enriched for proteins with a known participation in calcium signaling. Replication in an independent cohort confirmed increased mutation burden of calcium-signaling genes in CHD patients. Functional investigation of zebrafish orthologues of ITPR1, PLCB2, and ADCY2 verified a role in cardiac development and suggests a combinatorial effect of inactivation of these genes. CONCLUSIONS: The study identifies abnormal calcium signaling as a novel pathophysiological mechanism in human CHD and confirms the complex genetic architecture underlying CHD.