Scalable Integration of Multiomic Single Cell Data Using Generative Adversarial Networks.

MOTIVATION: Single cell profiling has become a common practice to investigate the complexity of tissues, organs and organisms. Recent technological advances are expanding our capabilities to profile various molecular layers beyond the transcriptome such as, but not limited to, the genome, the epigenome and the proteome. Depending on the experimental procedure, these data can be obtained from separate assays or from the very same cells. Despite development of computational methods for data integration is an active research field, most of the available strategies have been devised for the joint analysis of two modalities and cannot accommodate a high number of them. RESULTS: We here propose a multiomic data integration framework based on Wasserstein Generative Adversarial Networks (MOWGAN) suitable for the analysis of paired or unpaired data with high number of modalities (>2). At the core of our strategy is a single network trained on all modalities together, limiting the computational burden when many molecular layers are evaluated. AVAILABILITY: Source code of our framework is available at https://github.com/vgiansanti/MOWGAN. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Evolutionary fingerprints of EMT in pancreatic cancers.

Mesenchymal plasticity has been extensively described in advanced and metastatic epithelial cancers; however, its functional role in malignant progression, metastatic dissemination and therapy response is controversial. More importantly, the role of epithelial mesenchymal transition (EMT) and cell plasticity in tumor heterogeneity, clonal selection and clonal evolution is poorly understood. Functionally, our work clarifies the contribution of EMT to malignant progression and metastasis in pancreatic cancer. We leveraged ad hoc somatic mosaic genome engineering, lineage tracing and ablation technologies and dynamic genetic reporters to trace and ablate tumor-specific lineages along the phenotypic spectrum of epithelial to mesenchymal plasticity. The experimental evidences clarify the essential contribution of mesenchymal lineages to pancreatic cancer evolution and metastatic dissemination. Spatial genomic analysis combined with single cell transcriptomic and epigenomic profiling of epithelial and mesenchymal lineages reveals that EMT promotes with the emergence of chromosomal instability (CIN). Specifically tumor lineages with mesenchymal features display highly conserved patterns of genomic evolution including complex structural genomic rearrangements and chromotriptic events. Genetic ablation of mesenchymal lineages robustly abolished these mutational processes and evolutionary patterns, as confirmed by cross species analysis of pancreatic and other human epithelial cancers. Mechanistically, we discovered that malignant cells with mesenchymal features display increased chromatin accessibility, particularly in the pericentromeric and centromeric regions, which in turn results in delayed mitosis and catastrophic cell division. Therefore, EMT favors the emergence of high-fitness tumor cells, strongly supporting the concept of a cell-state, lineage-restricted patterns of evolution, where cancer cell sub-clonal speciation is propagated to progenies only through restricted functional compartments. Restraining those evolutionary routes through genetic ablation of clones capable of mesenchymal plasticity and extinction of the derived lineages completely abrogates the malignant potential of one of the most aggressive form of human cancer.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing.

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks. ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs' position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome. Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final evaluation in the therapeutically relevant setting of interest.

Interferon-inducible phospholipids govern IFITM3-dependent endosomal antiviral immunity.

The interferon-induced transmembrane proteins (IFITM) are implicated in several biological processes, including antiviral defense, but their modes of action remain debated. Here, taking advantage of pseudotyped viral entry assays and replicating viruses, we uncover the requirement of host co-factors for endosomal antiviral inhibition through high-throughput proteomics and lipidomics in cellular models of IFITM restriction. Unlike plasma membrane (PM)-localized IFITM restriction that targets infectious SARS-CoV2 and other PM-fusing viral envelopes, inhibition of endosomal viral entry depends on lysines within the conserved IFITM intracellular loop. These residues recruit Phosphatidylinositol 3,4,5-trisphosphate (PIP3) that we show here to be required for endosomal IFITM activity. We identify PIP3 as an interferon-inducible phospholipid that acts as a rheostat for endosomal antiviral immunity. PIP3 levels correlated with the potency of endosomal IFITM restriction and exogenous PIP3 enhanced inhibition of endocytic viruses, including the recent SARS-CoV2 Omicron variant. Together, our results identify PIP3 as a critical regulator of endosomal IFITM restriction linking it to the Pi3K/Akt/mTORC pathway and elucidate cell-compartment-specific antiviral mechanisms with potential relevance for the development of broadly acting antiviral strategies.

Analyzing genomic and epigenetic profiles in single cells by hybrid transposase (scGET-seq).

scGET-seq simultaneously profiles euchromatin and heterochromatin. scGET-seq exploits the concurrent action of transposase Tn5 and its hybrid form TnH, which targets H3K9me3 domains. Here we present a step-by-step protocol to profile single cells by scGET-seq using a 10× Chromium Controller. We describe steps for transposomes preparation and validation. We detail nuclei preparation and transposition, followed by encapsulation, library preparation, sequencing, and data analysis. For complete details on the use and execution of this protocol, please refer to Tedesco et al. (2022)1 and de Pretis and Cittaro (2022).2.

A Whole-Genome Sequencing Study Implicates GRAMD1B in Multiple Sclerosis Susceptibility.

While the role of common genetic variants in multiple sclerosis (MS) has been elucidated in large genome-wide association studies, the contribution of rare variants to the disease remains unclear. Herein, a whole-genome sequencing study in four affected and four healthy relatives of a consanguineous Italian family identified a novel missense c.1801T > C (p.S601P) variant in the GRAMD1B gene that is shared within MS cases and resides under a linkage peak (LOD: 2.194). Sequencing GRAMD1B in 91 familial MS cases revealed two additional rare missense and two splice-site variants, two of which (rs755488531 and rs769527838) were not found in 1000 Italian healthy controls. Functional studies demonstrated that GRAMD1B, a gene with unknown function in the central nervous system (CNS), is expressed by several cell types, including astrocytes, microglia and neurons as well as by peripheral monocytes and macrophages. Notably, GRAMD1B was downregulated in vessel-associated astrocytes of active MS lesions in autopsied brains and by inflammatory stimuli in peripheral monocytes, suggesting a possible role in the modulation of inflammatory response and disease pathophysiology.

Neural precursor cells tune striatal connectivity through the release of IGFBPL1.

The adult brain retains over life endogenous neural stem/precursor cells (eNPCs) within the subventricular zone (SVZ). Whether or not these cells exert physiological functions is still unclear. In the present work, we provide evidence that SVZ-eNPCs tune structural, electrophysiological, and behavioural aspects of striatal function via secretion of insulin-like growth factor binding protein-like 1 (IGFBPL1). In mice, selective ablation of SVZ-eNPCs or selective abrogation of IGFBPL1 determined an impairment of striatal medium spiny neuron morphology, a higher failure rate in GABAergic transmission mediated by fast-spiking interneurons, and striatum-related behavioural dysfunctions. We also found IGFBPL1 expression in the human SVZ, foetal and induced-pluripotent stem cell-derived NPCs. Finally, we found a significant correlation between SVZ damage, reduction of striatum volume, and impairment of information processing speed in neurological patients. Our results highlight the physiological role of adult SVZ-eNPCs in supporting cognitive functions by regulating striatal neuronal activity.

Loss of ribonuclease DIS3 hampers genome integrity in myeloma by disrupting DNA:RNA hybrid metabolism.

The ribonuclease DIS3 is one of the most frequently mutated genes in the hematological cancer multiple myeloma, yet the basis of its tumor suppressor function in this disease remains unclear. Herein, exploiting the TCGA dataset, we found that DIS3 plays a prominent role in the DNA damage response. DIS3 inactivation causes genomic instability by increasing mutational load, and a pervasive accumulation of DNA:RNA hybrids that induces genomic DNA double-strand breaks (DSBs). DNA:RNA hybrid accumulation also prevents binding of the homologous recombination (HR) machinery to double-strand breaks, hampering DSB repair. DIS3-inactivated cells become sensitive to PARP inhibitors, suggestive of a defect in homologous recombination repair. Accordingly, multiple myeloma patient cells mutated for DIS3 harbor an increased mutational burden and a pervasive overexpression of pro-inflammatory interferon, correlating with the accumulation of DNA:RNA hybrids. We propose DIS3 loss in myeloma to be a driving force for tumorigenesis via DNA:RNA hybrid-dependent enhanced genome instability and increased mutational rate. At the same time, DIS3 loss represents a liability that might be therapeutically exploited in patients whose cancer cells harbor DIS3 mutations.

Integrated Multiomic Profiling Identifies the Epigenetic Regulator PRC2 as a Therapeutic Target to Counteract Leukemia Immune Escape and Relapse.

Immune escape represents a major driver of acute myeloid leukemia (AML) reemergence after allogeneic hematopoietic cell transplantation (allo-HCT), with up to 40% of relapses prompted by nongenomic loss of HLA class II expression in leukemia cells. By integrative analysis of gene expression, DNA methylation, and chromatin accessibility in paired diagnosis/relapse primary samples and in the respective patient-derived xenografts (PDX), we identify the polycomb repressive complex 2 (PRC2) as a key epigenetic driver of this immune escape modality. We report that loss of expression of HLA class II molecules is accompanied by a PRC2-dependent reduction in chromatin accessibility. Pharmacologic inhibition of PRC2 subunits rescues HLA class II expression in AML relapses in vitro and in vivo, with consequent recovery of leukemia recognition by CD4+ T cells. Our results uncover a novel link between epigenetics and leukemia immune escape, which may rapidly translate into innovative strategies to cure or prevent AML posttransplantation relapse. SIGNIFICANCE: Loss of HLA class II expression represents a frequent mechanism of leukemia posttransplantation relapse. Here we identify PRC2 as the main epigenetic driver of this immune escape modality and show that its chemical inhibition can reinstate a proficient graft-versus-leukemia effect, providing an innovative rationale for personalized epigenetic immunotherapies. See related commentary by Köhler and Zeiser, p. 1410. This article is highlighted in the In This Issue feature, p. 1397.

Chromatin Velocity reveals epigenetic dynamics by single-cell profiling of heterochromatin and euchromatin.

Recent efforts have succeeded in surveying open chromatin at the single-cell level, but high-throughput, single-cell assessment of heterochromatin and its underlying genomic determinants remains challenging. We engineered a hybrid transposase including the chromodomain (CD) of the heterochromatin protein-1α (HP-1α), which is involved in heterochromatin assembly and maintenance through its binding to trimethylation of the lysine 9 on histone 3 (H3K9me3), and developed a single-cell method, single-cell genome and epigenome by transposases sequencing (scGET-seq), that, unlike single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq), comprehensively probes both open and closed chromatin and concomitantly records the underlying genomic sequences. We tested scGET-seq in cancer-derived organoids and human-derived xenograft (PDX) models and identified genetic events and plasticity-driven mechanisms contributing to cancer drug resistance. Next, building upon the differential enrichment of closed and open chromatin, we devised a method, Chromatin Velocity, that identifies the trajectories of epigenetic modifications at the single-cell level. Chromatin Velocity uncovered paths of epigenetic reorganization during stem cell reprogramming and identified key transcription factors driving these developmental processes. scGET-seq reveals the dynamics of genomic and epigenetic landscapes underlying any cellular processes.

The frequency of somatic mutations in cancer predicts the phenotypic relevance of germline mutations.

Genomic sequence mutations can be pathogenic in both germline and somatic cells. Several authors have observed that often the same genes are involved in cancer when mutated in somatic cells and in genetic diseases when mutated in the germline. Recent advances in high-throughput sequencing techniques have provided us with large databases of both types of mutations, allowing us to investigate this issue in a systematic way. Hence, we applied a machine learning based framework to this problem, comparing multiple models. The models achieved significant predictive power as shown by both cross-validation and their application to recently discovered gene/phenotype associations not used for training. We found that genes characterized by high frequency of somatic mutations in the most common cancers and ancient evolutionary age are most likely to be involved in abnormal phenotypes and diseases. These results suggest that the combination of tolerance for mutations at the cell viability level (measured by the frequency of somatic mutations in cancer) and functional relevance (demonstrated by evolutionary conservation) are the main predictors of disease genes. Our results thus confirm the deep relationship between pathogenic mutations in somatic and germline cells, provide new insight into the common origin of cancer and genetic diseases, and can be used to improve the identification of new disease genes.

Contrast subgraphs allow comparing homogeneous and heterogeneous networks derived from omics data.

BACKGROUND: Biological networks are often used to describe the relationships between relevant entities, particularly genes and proteins, and are a powerful tool for functional genomics. Many important biological problems can be investigated by comparing biological networks between different conditions or networks obtained with different techniques. FINDINGS: We show that contrast subgraphs, a recently introduced technique to identify the most important structural differences between 2 networks, provide a versatile tool for comparing gene and protein networks of diverse origin. We demonstrate the use of contrast subgraphs in the comparison of coexpression networks derived from different subtypes of breast cancer, coexpression networks derived from transcriptomic and proteomic data, and protein-protein interaction networks assayed in different cell lines. CONCLUSIONS: These examples demonstrate how contrast subgraphs can provide new insight in functional genomics by extracting the gene/protein modules whose connectivity is most altered between 2 conditions or experimental techniques.

Nested Stochastic Block Models applied to the analysis of single cell data.

Single cell profiling has been proven to be a powerful tool in molecular biology to understand the complex behaviours of heterogeneous system. The definition of the properties of single cells is the primary endpoint of such analysis, cells are typically clustered to underpin the common determinants that can be used to describe functional properties of the cell mixture under investigation. Several approaches have been proposed to identify cell clusters; while this is matter of active research, one popular approach is based on community detection in neighbourhood graphs by optimisation of modularity. In this paper we propose an alternative and principled solution to this problem, based on Stochastic Block Models. We show that such approach not only is suitable for identification of cell groups, it also provides a solid framework to perform other relevant tasks in single cell analysis, such as label transfer. To encourage the use of Stochastic Block Models, we developed a python library, schist, that is compatible with the popular scanpy framework.

Gene Expression Analysis in Patients with Cocaine-Induced Midline Destructive Lesions.

BACKGROUND AND OBJECTIVES: Cocaine users may present with positive antineutrophil cytoplasmic antibodies (ANCA) and severe midline destructive lesions (CIMDL) which are histologically characterized by massive apoptosis. However, histopathological and laboratory studies suggest that autoimmunity may not be the main pathogenic driver. We analyzed gene expression both in cell lines of nasal mucosa exposed to cocaine and in CIMDL patients to determine whether genetic predisposition might cause such lesions, which are observed in a minority of cocaine abusers. MATERIALS AND METHODS: The genetic expression profile of nasal mucosa exposed to cocaine was analyzed. Rare variants of expressed genes were searched in patients with CIMDL using exome sequencing and bio-informatics. RESULTS: We identified 462 genes that were induced by cocaine, mainly related to apoptosis and autophagy in response to oxidative stress. Under the hypothesis that genes linked to the phenotype are also induced by cocaine itself, a rare variants burden test was performed to select genes that were significantly enriched in rare mutations. Next, 11 cocaine abusers with CIMDL and no other relevant medical comorbidities underwent exome sequencing, and 12 genes that were significantly enriched in the burden test and present in at least 10 patients were identified. An in-depth analysis of these genes revealed their involvement in apoptosis, tissue homeostasis, autophagy, and response to oxidative stress. CONCLUSIONS: Oxidative stress and rare genetic alterations in the response to reactive oxygen species, apoptosis, autophagy, and tissue regeneration are plausible drivers of damage affecting nasal mucosa exposed to cocaine crystals and, consequently, the pathogenic mechanism behind CIMDL.

Deletion of a pseudogene within a fragile site triggers the oncogenic expression of the mitotic CCSER1 gene.

The oncogenic role of common fragile sites (CFS), focal and pervasive gaps in the cancer genome arising from replicative stress, remains controversial. Exploiting the TCGA dataset, we found that in most CFS the genes residing within the associated focal deletions are down-regulated, including proteins involved in tumour immune recognition. In a subset of CFS, however, the residing genes are surprisingly overexpressed. Within the most frequent CFS in this group, FRA4F, which is deleted in up to 18% of cancer cases and harbours the CCSER1 gene, we identified a region which includes an intronic, antisense pseudogene, TMSB4XP8. TMSB4XP8 focal ablation or transcriptional silencing elicits the overexpression of CCSER1, through a cis-acting mechanism. CCSER1 overexpression increases proliferation and triggers centrosome amplifications, multinuclearity, and aberrant mitoses. Accordingly, FRA4F is associated in patient samples to mitotic genes deregulation and genomic instability. As a result, cells overexpressing CCSER1 become sensitive to the treatment with aurora kinase inhibitors. Our findings point to a novel tumourigenic mechanism where focal deletions increase the expression of a new class of "dormant" oncogenes.

BAR-Seq clonal tracking of gene-edited cells.

Gene editing by engineered nucleases has revolutionized the field of gene therapy by enabling targeted and precise modification of the genome. However, the limited availability of methods for clonal tracking of edited cells has resulted in a paucity of information on the diversity, abundance and behavior of engineered clones. Here we detail the wet laboratory and bioinformatic BAR-Seq pipeline, a strategy for clonal tracking of cells harboring homology-directed targeted integration of a barcoding cassette. We present the BAR-Seq web application, an online, freely available and easy-to-use software that allows performing clonal tracking analyses on raw sequencing data without any computational resources or advanced bioinformatic skills. BAR-Seq can be applied to most editing strategies, and we describe its use to investigate the clonal dynamics of human edited hematopoietic stem/progenitor cells in xenotransplanted hosts. Notably, BAR-Seq may be applied in both basic and translational research contexts to investigate the biology of edited cells and stringently compare editing protocols at a clonal level. Our BAR-Seq pipeline allows library preparation and validation in a few days and clonal analyses of edited cell populations in 1 week.

FAM46C and FNDC3A Are Multiple Myeloma Tumor Suppressors That Act in Concert to Impair Clearing of Protein Aggregates and Autophagy.

Multiple myeloma is a plasma cell neoplasm characterized by the production of unfolded immunoglobulins, which cause endoplasmic reticulum (ER) stress and sensitivity to proteasome inhibition. The genomic landscape of multiple myeloma is characterized by the loss of several genes rarely mutated in other cancers that may underline specific weaknesses of multiple myeloma cells. One of these is FAM46C that is lost in more than 10% of patients with multiple myeloma. We show here that FAM46C is part of a new complex containing the ER-associated protein FNDC3A, which regulates trafficking and secretion and, by impairing autophagy, exacerbates proteostatic stress. Reconstitution of FAM46C in multiple myeloma cells that had lost it induced apoptosis and ER stress. Apoptosis was preceded by an increase of intracellular aggregates, which was not linked to increased translation of IgG mRNA, but rather to impairment of autophagy. Biochemical analysis showed that FAM46C requires interaction with ER bound protein FNDC3A to reside in the cytoplasmic side of the ER. FNDC3A was lost in some multiple myeloma cell lines. Importantly, depletion of FNDC3A increased the fitness of FAM46C-expressing cells and expression of FNDC3A in cells that had lost it recapitulated the effects of FAM46C, inducing aggregates and apoptosis. FAM46C and FNDC3A formed a complex that modulates secretion routes, increasing lysosome exocytosis. The cellular landscape generated by FAM46C/FNDC3A expression predicted sensitivity to sphingosine kinase inhibition. These results suggest that multiple myeloma cells remodel their trafficking machinery to cope with ER stress. SIGNIFICANCE: This study identifies a new multiple myeloma-specific tumor suppressor complex that regulates autophagy and unconventional secretion, highlighting the sensitivity of multiple myeloma cells to the accumulation of protein aggregates.

Efficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking.

Targeted gene editing in hematopoietic stem cells (HSCs) is a promising treatment for several diseases. However, the limited efficiency of homology-directed repair (HDR) in HSCs and the unknown impact of the procedure on clonal composition and dynamics of transplantation have hampered clinical translation. Here, we apply a barcoding strategy to clonal tracking of edited cells (BAR-Seq) and show that editing activates p53, which substantially shrinks the HSC clonal repertoire in hematochimeric mice, although engrafted edited clones preserve multilineage and self-renewing capacity. Transient p53 inhibition restored polyclonal graft composition. We increased HDR efficiency by forcing cell-cycle progression and upregulating components of the HDR machinery through transient expression of the adenovirus 5 E4orf6/7 protein, which recruits the cell-cycle controller E2F on its target genes. Combined E4orf6/7 expression and p53 inhibition resulted in HDR editing efficiencies of up to 50% in the long-term human graft, without perturbing repopulation and self-renewal of edited HSCs. This enhanced protocol should broaden applicability of HSC gene editing and pave its way to clinical translation.

Fast analysis of scATAC-seq data using a predefined set of genomic regions.

Background: Analysis of scATAC-seq data has been recently scaled to thousands of cells. While processing of other types of single cell data was boosted by the implementation of alignment-free techniques, pipelines available to process scATAC-seq data still require large computational resources. We propose here an approach based on pseudoalignment, which reduces the execution times and hardware needs at little cost for precision. Methods: Public data for 10k PBMC were downloaded from 10x Genomics web site. Reads were aligned to various references derived from DNase I Hypersensitive Sites (DHS) using kallisto and quantified with bustools. We compared our results with the ones publicly available derived by cellranger-atac. Results: We found that kallisto does not introduce biases in quantification of known peaks and cells groups are identified in a consistent way. We also found that cell identification is robust when analysis is performed using DHS-derived reference in place of de novo identification of ATAC peaks. Lastly, we found that our approach is suitable for reliable quantification of gene activity based on scATAC-seq signal, thus allows for efficient labelling of cell groups based on marker genes. Conclusions: Analysis of scATAC-seq data by means of kallisto produces results in line with standard pipelines while being considerably faster; using a set of known DHS sites as reference does not affect the ability to characterize the cell populations.

Identification of novel genetic variants associated with short stature in a Baka Pygmies population.

Human growth is a complex trait determined by genetic factors in combination with external stimuli, including environment, nutrition and hormonal status. In the past, several genome-wide association studies (GWAS) have collectively identified hundreds of genetic variants having a putative effect on determining adult height in different worldwide populations. Theoretically, a valuable approach to better understand the mechanisms of complex traits as adult height is to study a population exhibiting extreme stature phenotypes, such as African Baka Pygmies. After phenotypic characterization, we sequenced the whole exomes of a cohort of Baka Pygmies and their non-Pygmies Bantu neighbors to highlight genetic variants associated with the reduced stature. Whole exome data analysis revealed 29 single nucleotide polymorphisms (SNPs) significantly associated with the reduced height in the Baka group. Among these variants, we focused on SNP rs7629425, located in the 5'-UTR of the Hyaluronidase-2 (HYAL2) gene. The frequency of the alternative allele was significantly increased compared to African and non-African populations. In vitro luciferase assay showed significant differences in transcription modulation by rs7629425 C/T alleles. In conclusion, our results suggested that the HYAL2 gene variants may play a role in the etiology of short stature in Baka Pygmies population.