Biology subjects

Enge, M.

Publications and source records attributed to Enge, M..

14 recordsLinked to original sources

bioRxiv2026

Principles of subclonal gene dosage across human cancer

Intratumor heterogeneity drives disease progression, but even though subclonal copy number variation (CNV) is a major contributor to this heterogeneity1, its impact on cell phenotype is not fully understood. Here, by applying high-quality joint whole genome sequencing and mRNA profiling in single cells (DNTR-seq2) to solid tumors and leukemias from 57 patients, we have analyzed the in vivo transcriptional effect of subclonal CNVs within a tumor. We found that gene dosage is generally additive in low and moderate copy states, but that cancer-type-specific compensation is common, and core promoter elements are associated with reduced additivity. We find that different classes of subclonal CNV impose varying degrees of transcriptional constraints on a cell, with highly amplified megabase-size regions associated with a strong effect both in cis and trans, while arm level CNVs generally exert a milder effect. We also describe a previously unappreciated class of tumors with transient clonality, where every cell is genetically highly distinct. We find that transient clonality is common in ovarian cancer and soft tissue sarcoma, that it is preceded by a whole genome duplication event, and that gene dosage in these tumors affects transcript abundance to a similar degree as in cancer with stable subclones.

cancer biology↗

bioRxiv2026

ALK R1275Q mutation drives expansion of SCP-like cells during sympathoadrenal commitment and primes neuroblastoma initiation.

Neuroblastoma (NB) is a pediatric malignancy developing in the sympathoadrenal lineage of the neural crest, characterized by clinical heterogeneity ranging from spontaneous regression to poor outcomes. Activating mutations in the receptor tyrosine kinase anaplastic lymphoma kinase (ALK) are frequently observed in both sporadic and familial NB, yet the functional role of ALK in tumor initiation is not fully understood. Using a patient-derived human induced pluripotent stem cell (iPSC) model of sympathoadrenal development, we show that upon sympathoadrenal lineage commitment, ALK R1275Q, the most common hotspot mutation found in familial NB, sustain a proliferative, immature Schwann cell precursor (SCP)-like cell state with elevated ALK signaling and increased susceptibility to MYCN-driven transformation. While ALK-mutant cells alone did not form tumors in vivo, they cooperated with MYCN to accelerate tumor initiation, suggesting that ALK R1275Q creates a permissive but insufficient state for transformation. These findings define an ALK-driven cell progenitor-like state that facilitates the initiation of NB during embryonal development.

cancer biology↗

bioRxiv2025

Single-Cell Integration of Chromatin Accessibility and Transcriptomics Reveals Regulatory Networks in Ovarian Tumor-Infiltrating Adaptive NK Cells

Natural killer (NK) cells are traditionally recognized for their rapid, non-specific responses against virus-infected or malignantly transformed cells, functioning as key effectors of innate immunity. However, a distinct subset known as adaptive NK (aNK) cells has been shown to acquire memory-like properties following viral infections, indicating their capacity for antigen-specific immune recall. Intriguingly, aNK cells have also been identified within the tumor microenvironment, where they can mediate tumor-specific recall responses. Yet, the regulatory mechanisms governing their function in tumor-infiltrating aNK cells remain largely undefined. In this study, we integrated publicly available multiomics datasets from ovarian cancer, including single-cell chromatin accessibility (scATAC-seq) and single-cell RNA sequencing (scRNA-seq), to identify chromatin-accessible regions and construct transcription factors (TF)-gene regulatory networks. To validate and extend these findings, we performed Smart-seq3 on NK cells isolated from ovarian tumors and applied SCENIC analysis to identify TF-driven gene regulation. By integrating results from both analyses, we identified PRDM1 and STAT2 as key TFs, along with their downstream targets CRCP and MTFP1, specifically enriched in tumor-infiltrating aNK cells. The expression levels of CRCP and MTFP1 positively correlated with NK cell infiltration in ovarian cancer tissues, suggesting their potential functions in supporting tumor-specific NK cell memory responses. In addition, external validation using data from the PROMIX clinical trial demonstrated that PRDM1 and STAT2 expression levels are positively associated with both overall survival and aNK cell-associated transcriptional features. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/676040v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@6df026org.highwire.dtl.DTLVardef@1b16dc8org.highwire.dtl.DTLVardef@19e916borg.highwire.dtl.DTLVardef@15d431a_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

T cell correction pipeline for Inborn Errors of Immunity

CRISPR/Cas9 gene editing technology is a promising tool for correcting pathogenic variants for autologous cell therapies for Inborn Errors of Immunity (IEI). The present IEI correction strategies mainly focus on the knock-in of therapeutic cDNAs, or knockout of the disease-causing gene when feasible. These strategies address many single-gene defects but may disrupt gene expression and require significant optimization for each newly discovered IEI-causing gene, highlighting the need for complementary platforms that can precisely correct diverse pathogenic variants. Here, we present a safe and efficient T cell single nucleotide variant (SNV) correction pipeline based on homology-directed repair (HDR), suitable for diverse monogenic mutations. By using founder mutations of Deficiency of ADA2 (DADA2), Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) and Cartilage Hair Hypoplasia (CHH) as IEI models, we show that our pipeline can achieve up to 80% bi-allelic editing, with resultant functional correction of the disease phenotype in patient T cells. We do not find detectable pre-malignant off-target effects or karyotypic, transcriptomic or proteomic aberrations upon profiling patient T cells with GUIDE-seq, single cell RNA sequencing, PacBio based long-read whole genome sequencing, and high-throughput proteomics. This study demonstrates that HDR-based SNV editing is a safe and effective option for IEI T cell correction and that it could be developed to an autologous T cell therapy, as the presented protocol is scalable for a GMP-compatible workflow. This study is a step towards the development of gene correction platform that targets a broad number of monogenic mutations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/610811v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1f1eb47org.highwire.dtl.DTLVardef@18dbcc2org.highwire.dtl.DTLVardef@63862dorg.highwire.dtl.DTLVardef@1fe1561_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

bioengineering↗

bioRxiv2022

Shared requirement for MYC upstream super-enhancer in tissue regeneration and cancer

Cancer has been characterized as a wound that does not heal. Malignant cells are morphologically distinct from normal proliferating cells, but have extensive similarities to tissues undergoing wound healing and/or regeneration. The mechanistic basis of this similarity has, however, remained enigmatic. Here we show that the genomic region upstream of Myc, which is required for intestinal tumorigenesis, is also required for intestinal regeneration after radiation damage. The region is also critical for growth of adult intestinal cells in 3D organoid culture, indicating that culture conditions that recapitulate most aspects of normal tissue architecture still reprogram normal cells to proliferate using a mechanism similar to that employed by cancer cells. Our results uncover a genetic link between tissue regeneration and tumorigenesis, and establish that normal tissue renewal and regeneration of tissues after severe damage are mechanistically distinct. SummaryMyc regulatory region links regeneration and cancer

cancer biology↗

bioRxiv2022

Single-cell Transcriptome and Accessible Chromatin Dynamics During Endocrine Pancreas Development

Delineating gene regulatory networks that orchestrate cell-type specification is an ongoing challenge for developmental biology studies. Single-cell analyses offer opportunities to address these challenges and accelerate discovery of rare cell lineage relationships and mechanisms underlying hierarchical lineage decisions. Here, we describe the molecular analysis of pancreatic endocrine cell differentiation using single-cell gene expression, chromatin accessibility assays coupled to genetic labeling and cell sorting. We uncover transcription factor networks that delineate {beta}-, - and{delta} -cell lineages. Through genomic footprint analysis we identify transcription factor-regulatory DNA interactions governing pancreatic cell development at unprecedented resolution. Our analysis suggests that the transcription factor Neurog3 may act as a pioneer transcription factor to specify the pancreatic endocrine lineage. These findings could improve protocols to generate replacement endocrine cells from renewable sources, like stem cells, for diabetes therapy.

developmental biology↗

bioRxiv2021

Smart3-ATAC: a highly sensitive method for joint accessibility and full-length transcriptome analysis in single cells

Joint single-cell measurements of gene expression and DNA regulatory element activity holds great promise as a tool to understand transcriptional regulation. Towards this goal we have developed Smart3-ATAC, a highly sensitive method which allows joint mRNA and chromatin accessibility analysis genome wide in single cells. With Smart3-ATAC, we are able to obtain the highest possible quality measurements per cell. The method combines transcriptomic profiling based on the highly sensitive Smart-seq3 protocol on cytosolic mRNA, with a novel low-loss single-cell ATAC (scATAC) protocol to measure chromatin accessibility. Compared to current droplet multiome methods, the yield of both the scATAC protocol and mRNA-seq protocol is markedly higher.

systems biology↗

bioRxiv2021

Allele-resolved single-cell multi-omics uncovers the dynamics and transcriptional kinetics of X-chromosome upregulation

X-chromosome inactivation (XCI) and upregulation (XCU) are the major opposing chromosome-wide modes of gene regulation that collectively achieve dosage compensation in mammals, but the regulatory link between the two remains elusive. Here, we use allele-resolved single-cell RNA-seq combined with chromatin accessibility profiling to finely dissect the separate effects of XCI and XCU on RNA levels during mouse development. We uncover that balanced X dosage is flexibly attained through expression tuning by XCU in a sex- and lineage-specific manner along varying degrees of XCI and across developmental and cellular states. Male blastomeres achieve XCU upon zygotic genome activation while females experience two distinct waves of XCU, upon imprinted- and random XCI, and ablation of Xist impedes female XCU. Contrary to widely established models of mammalian dosage compensation, naive female embryonic cells carrying two active X chromosomes do not exhibit upregulation but express both alleles at basal level, yet collectively exceeding the RNA output of a single hyperactive allele. We show, in vivo and in vitro, that XCU is kinetically driven by X-specific modulation of transcriptional burst frequency, coinciding with increased compartmentalization of the hyperactive allele. Altogether, our data provide unprecedented insights into the dynamics of mammalian XCU, prompting a revised model of the chain in events of allelic regulation by XCU and XCI in unitedly achieving stable cellular levels of X-chromosome transcripts.

genomics↗

bioRxiv2020

Tuning TPO-R signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia

Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL)) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable, both experimentally and clinically. We have developed a series of surrogate protein-ligands for TPO-R, in the form of diabodies, that homodimerize the TPO-R on the cell surface in different geometries, in effect tuning downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism and antagonism, thus decoupling the dual functions of TPO/TPO-R. Using scRNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in Essential Thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R. Significance StatementThe TPO cytokine, which signals through its receptor c-MPL (or TPO-R), is essential for megakaryocyte differentiation and maintenance of hematopoietic stem cells (HSCs). Its signaling is deregulated in Essential Thrombocythemia (ET). Here, we engineered diabodies (DBs) against the TPO-R as surrogate TPO ligands to manipulate TPO-R signaling, from full to partial to antagonism, thus decoupling the dual functions of TPO/TPO-R (i.e, HSC maintenance versus megakaryopoiesis). We subsequently discovered that partial agonistic DBs, by reducing the strength of the TPO-R signal, not only preserved HSCs in culture, but also blocked oncogenic signaling in ET. This finding has the potential to improve HSC cultures for transplants, as well as serve as a unique therapeutic approach for ET.

cell biology↗

bioRxiv2020

X-chromosome upregulation is dynamically linked to the X-inactivation state

Mammalian X-chromosome dosage balance is regulated by X-chromosome inactivation (XCI) and X-chromosome upregulation (XCU), but the dynamics of XCU as well as the interplay between the two mechanisms remain poorly understood. Here, we mapped XCU throughout early mouse embryonic development at cellular and allelic resolution, revealing sex- and lineage-specific dynamics along key events in X-chromosome regulation. Our data show that XCU is linearly proportional to the degree of XCI, indicating that dosage compensation ensues based on mRNA levels rather than number of active X chromosomes. In line with this, we reveal that the two active X chromosomes in female naive embryonic stem cells are not hyperactive as previously thought. In all lineages, XCU was underlain by increased transcriptional burst frequencies, providing a mechanistic basis in vivo. Together, our results demonstrate unappreciated flexibility of XCU in balancing X-chromosome expression, and we propose a general model for allelic dosage balance, applicable for wider mechanisms of transcriptional regulation.

cell biology↗

bioRxiv2020

Unsupervised cell interaction profiling reveals major architectural differences between small intestinal and colonic epithelial crypts

Cellular identity in complex multicellular organisms is strictly maintained over the course of life. This control is achieved in part by the organ structure itself, such that neighboring cells influence each others identity. However, large-scale investigation of the cellular interactome has been technically challenging. Here, we develop CIM-seq, an unsupervised and high-throughput method to analyze direct physical cell-cell interactions between every cell type in a given tissue. CIM-seq is based on RNA sequencing of incompletely dissociated cells, followed by computational deconvolution of these into their constituent cell types using machine learning. We use CIM-seq to define the cell interaction landscape of the mouse small intestinal and colonic epithelium, uncovering both known and novel interactions. Specifically, we find that the general architecture of the stem cell niche is radically different between the two tissues. In small intestine, the stem-Paneth cell interaction forms an exceptionally strong and exclusive niche, in which Paneth cells provide Wnt ligands1. In colonic epithelium, no similar compartment exists to support stem cells, and Wnt signaling is provided by a mesenchymal cell layer2,3. However, colonic stem cells are supported by an adjacent, previously unrecognized goblet cell subtype expressing the wound-healing marker Plet1, which is also highly upregulated during regeneration of colon epithelium. These results identify novel cellular interactions specific for the colonic stem cell niche and suggest an additional level of structural control in the colon. CIM-seq is broadly applicable to studies that aim to simultaneously investigate the constituent cell types and the global interaction profile in a specific tissue.

developmental biology↗

bioRxiv2020

A highly scalable method for joint whole genome sequencing and gene expression profiling of single cells

Understanding how genetic variation alters gene expression - how genotype affects phenotype - is a central challenge in biology. To address this question in complex cell mixtures, we developed Direct Nuclear Tagmentation and RNA-sequencing (DNTR-seq), which enables whole genome and mRNA sequencing jointly in single cells. When applied to biobanked leukemia samples, DNTR-seq readily identified minor subclones within patients, as well as cell-type specific gene editing such as T-cell receptor rearrangements. mRNA-seq quality is equal to RNA-only methods, and the high yield combined with low positional bias of the genomic library preparation allows detection of sub-megabase aberrations at ultra low coverage of 0.5-3M read pairs per genome. Since each cell library is individually addressable, rare subpopulations can be re-sequenced at increased depth, allowing multi-tiered study designs where depth of sequencing is informed by previous results. In addition, the direct tagmentation protocol enables coverage-independent estimation of ploidy, which can be used to unambiguously identify cell singlets. Thus, DNTR-seq directly links each cells state to its corresponding genome at a scale enabling routine analysis of heterogeneous tumors and other complex tissues.

genomics↗

bioRxiv2018

Modeling Spatial Correlation of Transcripts With Application to Developing Pancreas

Recently high-throughput image-based transcriptomic methods were developed and enabled researchers to spatially resolve gene expression variation at the molecular level for the first time. In this work, we develop a general analysis tool to quantitatively study the spatial correlations of gene expression in fixed tissue sections. As an illustration, we analyze the spatial distribution of single mRNA molecules measured by in situ sequencing on human fetal pancreas at three developmental time points 80, 87 and 117 days post-fertilization. We develop a density profile-based method to capture the spatial relationship between gene expression and other morphological features of the tissue sample such as position of nuclei and endocrine cells of the pancreas. In addition, we build a statistical model to characterize correlations in the spatial distribution of the expression level among different genes. This model enables us to infer the inhibitory and clustering effects throughout different time points. Our analysis framework is applicable to a wide variety of spatially-resolved transcriptomic data to derive biological insights.

bioinformatics↗

bioRxiv2017

Single cell transcriptome analysis of human pancreas reveals transcriptional signatures of aging and somatic mutation patterns.

As organisms age, cells accumulate genetic and epigenetic changes that eventually lead to impaired organ function or catastrophic transformation such as cancer. Since aging appears to be a stochastic process of increasing disorder1 cells in an organ will be individually affected in different ways - thus rendering bulk analyses of postmitotic adult tissues difficult to characterize. Here we directly measure the effects of aging in primary human tissue by performing single-cell transcriptome analysis of 2544 human pancreas cells from eight donors spanning six decades of life. We find that islet cells from older donors have increased levels of molecular disorder as measured both by noise in the transcriptome and by the number of cells which display inappropriate hormone expression, revealing a transcriptional instability associated with aging. By further analyzing the spectrum of somatic mutations in single cells, we found a specific age-dependent mutational signature characterized by C to A and C to G transversions. These mutations are indicators of oxidative stress and the signature is absent in single cells from human brain tissue or in a tumor cell line. We have used the single cell measurements of transcriptional noise and mutation level to identify molecular pathways correlated with these changes that could influence human disease. Our results demonstrate the feasibility of using single-cell RNA-seq data from primary cells to derive meaningful insights into the genetic processes that operate on aging human tissue and to determine molecular mechanisms coordinated with these processes.

genomics↗